Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly prevalent, resulting from lipid accumulation in the liver, leading to impaired hepatocyte function. NAFLD has the potential to develop into non-alcoholic steatohepatitis (NASH), cirrhosis or fibrosis, and hepatocellular carcinoma (HCC). The western diet is linked to insulin resistance and NAFLD development. Murine models are frequently used to study NAFLD, as their livers display similar steps in disease progression as humans; however, the field lacks an appropriate and consistent diet-induced model that mimics human disease pathogenesis. In this study, we examined 2 diets: a high-fat diet with 30% fructose water (HFHF) and a NASH diet (high fructose, fat and cholesterol) compared to a grain-based diet. We also compared these diets in mice with diethylnitrosamine(DEN)-induced HCC. We hypothesized that NASH diet leads to worsened liver phenotype and metabolism compared to HFHF-fed mice. 4-8 male and female mice on each diet were fed for 31 weeks in the NASH model and 26 weeks in the cancer model. HCC mice were injected with 25mg/kg DEN at 2 weeks old. HFHF-fed mice of both sexes gain more weight than their NASH-fed counterparts, although NASH mice have larger livers. Female NASH livers had higher triglyceride content while no difference was seen in males. HFHF mice also develop more prominent metabolic syndrome compared to NASH mice. However, NASH-fed mice display increased inflammatory gene expression (TNF-a, MCP1, IL-1b) and fibrosis, better mimicking a NASH liver phenotype. NASH diet mice had similar cancer phenotypes to HFHF-fed mice. Notably, NASH-fed females had a 100% incidence rate of liver cancer while HFHF-fed females had 57% incidence. Overall the mice on the NASH diet acquired less severe obesity and metabolic characteristics typical of humans with NAFLD, but had more severe liver disease than the HFHF-fed mice. NASH-fed mice appeared to oxidize more fat as fuel, but acquire hepatomegaly. This data supports the growing evidence of the role of dietary cholesterol in exacerbating NAFLD progression, as NASH-fed mice have increased liver damage. There were also notable sex differences in the mouse responses to the diets. While females diverge in body weight very early on, they do not have as pronounced differences in metabolism as seen in males and take longer to acquire liver damage. The NASH-fed HCC model emerges as a reliable method to induce NASH-driven liver cancer in both mouse sexes. Based on these results, researchers can choose the mouse model most suitable for the needs of their study.
The manner in which white adipose tissue (WAT) expands in response to positive energy balance directly impacts the prevalence of obesity-related metabolic abnormalities. Pathologic WAT remodeling is characterized by adipocyte hypertrophy, reduced differentiation capacity and chronic inflammation, results in ectopic fat accumulation and insulin resistance. Conversely, healthy adipogenesis, one of the features of metabolically healthy obesity (MHO), is determined by adipocyte hyperplasia, along with lower degree of inflammation and limited lipid spill-over to other organs. Pan-deletion of the monoacylglycerol (MAG) lipase alpha/beta-domain hydrolase-6 (ABHD6) demonstrated the therapeutic potential of ABHD6 inhibition against obesity and type-2-diabetes (Zhao et al., Cell Reports, 2016). However, the precise depot-specific role of ABHD6 in the AT metabolism and adipogenesis remains unexplored.
Abhd6flox/flox/Adipoq-Cre mice were injected with tamoxifen to obtain AT-ABHD6-KO mice. Control littermates and AT-ABHD6-KO mice were fed normal diet (ND) or high fat diet (HFD) for 12 weeks. Influence of ABHD6 suppression on adipocyte differentiation was assessed using 3T3-L1 pre-adipocytes (± ABHD6 inhibitor WWL70), as well as isolated stromal vascular fraction containing primary pre-adipocytes from WAT of whole-body ABHD6 KO and WT mice.
Our results show that ABHD6 expression level increases in adipocytes during differentiation, and correlates with adiposity in WAT from HFD mice and in visceral fat depot from patients with obesity. AT-ABHD6-KO mice on ND showed similar phenotype as controls, but displayed reduced adipocyte size and increased anti-inflammatory cytokines in the WAT. AT-specific KO mice on HFD displayed healthy-obese characteristics, including improved insulin sensitivity, elevated beta-oxidation in WAT, lower liver TG content, and improved systemic and WAT inflammation. ABHD6 inhibition in vitro, in the initial stages of 3T3-L1 differentiation, led to enhanced expression of adipogenic markers (aP2, GLUT4 and PPARγ) and increased fat deposition into smaller lipid droplets. Similar data were obtained in mouse primary adipocytes, where lipid content and differentiation markers level were higher in the whole-body ABHD6 KO versus WT adipocytes.
The results indicate that AT-ABHD6 deletion promotes a healthy-obese phenotype in HFD fed mice and ABHD6 suppression enhances WAT capacity to recruit new fat cells through differentiation of pre-adipocytes, to sequester fat away from ectopic fat deposition and ameliorate obesity-related complications. These findings provide insight into the pathways that underlie MHO and WAT expandability, suggesting adipose ABHD6 as a target for obesity-related complications.
Introduction: Angiotensin II type 2 receptor (AT2R) deficient mice (AT2RKO) exhibit a spectrum of congenital abnormalities of the kidney and urinary tract. We aimed to study whether AT2R deficiency in dams that also had diabetes mellitus would result in offspring with even more abnormalities in the kidney.
Methods: The offspring (male/female) of non-diabetic and diabetic dams of wild-type (WT) and AT2RKO mice were followed until 20 weeks of age. Systolic blood pressure, insulin sensitivity test (IST), albumin/creatinine ratio (ACR), glomerular filtration rate (GFR), renal morphology, oxidative stress and gene expression including angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), synaptopodin (Synpo) and NADPH oxidase 4 (Nox4) were assessed accordingly. Results: Compared to the age- and sex-matched offspring of non-diabetic and diabetic dams, body weight (BW) differed significantly from each subgroup through life (e.g., male ˃ female; AT2RKO ˃ WT mice; and non-diabetic dams’ ˃ diabetic dams’ offspring). Female offspring from diabetic AT2RKO dams developed insulin resistance, while male diabetic progeny of AT2RKO dams had normal IST. As compared to the offspring of non-diabetic dams, the diabetic progeny of both WT and AT2RKO mice developed more evidence of nephropathy (higher GFR and apparent glomerulosclerosis with podocyte loss) at 20 weeks of age, which were further aggravated in the offspring of AT2RKO diabetic dams, particularly female mice. Moreover, the female offspring of AT2RKO dams had heightened oxidative stress and significantly lower basal expression of ACE2/ACE ratio in their glomeruli, and this impaired ACE2/ACE ratio was completely blunted in the female progeny of diabetic AT2RKO dams.
Conclusions: AT2R deficiency accelerated features of nephropathy in the female progeny of diabetic dams, which might be due to loss of the protective effects of ACE2 expression in the kidney.
Introduction: Puberty is a time of hormonal changes that are associated with insulin resistance (IR). Although insulin sensitivity is restored at the end of puberty in healthy youth, it does not resolve in obese adolescents leading to an increased risk of metabolic disease such as type 2 diabetes. During pregnancy-induced IR, pancreatic β cells increase their functional mass to maintain glucose homeostasis. During puberty, however, the mechanism of pancreatic β-cell compensation to IR and its role in glucose metabolism in adulthood have not been established. In this study we characterized pancreatic β-cell adaptation to pubertal IR in rats and evaluated the effect of metabolic stress during puberty on glucose homeostasis in adult animals.
Methods: Wistar rats were fed a chow or high-fat diet (HFD) during puberty. Body weight, fasted plasma insulin, glucose tolerance were determined from weaning to adulthood. β-cell proliferation was assessed by immunostaining of pancreatic cryosections for Ki67 and insulin or Nkx6.1 to mark β-cells. β-cell mass was measured by morphometric analysis of insulin staining. Isolated islets from male Wistar rats at weaning were exposed during 72h to the serum of prepubertal, pubertal, or postpubertal animals and β-cell proliferation was evaluated by flow cytometry using insulin and EdU antibodies.
Results: During puberty, glucose intolerance was associated with an increase in circulating insulin in both sexes, suggestive of IR. Accordingly, puberty was characterized by a pulse in β-cell proliferation and a progressive rise in β-cell mass. Pubertal but not pre- or post-pubertal rat serum induced β-cell proliferation in isolated islets. HFD led to a decrease in β-cell proliferation during puberty with impaired glucose tolerance and defective insulin secretion in adult animals.
Conclusion: During puberty in rats, β cells proliferate to compensate for IR. Metabolic stress during puberty impairs glucose homeostasis later in life. Future studies will identify the circulating factor(s) that trigger β-cell expansion during puberty.
Bone morphogenetic proteins (BMPs) are critical for embryonic and post-natal homeostasis. Secreted by the liver, BMP9 has been implicated in vascular morphogenesis, yet little is known about its roles on hepatic metabolism. Here, we evaluated how BMP9 signaling affects glucose homeostasis in healthy and diabetic animals and investigated the molecular mechanisms underlying these effects. Adenoviral-mediated delivery of BMP9 in diabetic mice resulted in lowered blood glucose levels for up to 4 weeks after treatment. BMP9 decreased hepatic glucose production through inhibition of gluconeogenesis and decreased hepatic expression of gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). We found that BMP9, through the receptor Alk3, regulates expression of gluconeogenic enzymes through phosphorylation of AKT, which led to FOXO1 phosphorylation and its subsequent transcriptional activity. Collectively, these data show that BMP9 delivery is a potent suppressor of hepatic gluconeogenesis and improves glucose control in diabetic mice and highlight BMP9/Alk3 signaling as a potential target for the development of diabetes-related metabolic complications.
Le cancer est la principale cause de décès au Canada. La protéine Ras est une petite GTPase fréquemment mutée dans les cancers induisant une prolifération non-contrôlée. Le stress occasionné induit de la sénescence via le suppresseur de tumeur p53. Cette réponse, nommée sénescence induite par les oncogène (SIO), protège les cellules à risque contre l’initiation et la progression tumorale, mais les mécanismes impliqués restent mal caractérisés.
Récemment, nous avons identifié ZNF768 comme une nouvelle cible négative de la voie oncogénique Ras. La déplétion de ZNF768 suite à l’activation de Ras permet d’induire la SIO via p53. L’objectif de ce projet consiste à définir les fonctions de ZNF768 in vivo et de confirmer son implication dans le développement et la tumorigenèse. ZNF768 fut inactivé chez la souris par la technique CRISPR. Le développement ainsi que la susceptibilité aux tumeurs ont été étudiés dans ce modèle. De plus, des mouse embryonic fibroblasts (MEFs) ont été isolés à partir d’embryons et caractérisées. Les souris ZNF768 KO sont plus petites que les souris contrôles ce qui indique un défaut de croissance. Les MEFs KO entrent en sénescence réplicative plus rapidement lors du processus d’immortalisation. Ces phénomènes concordent avec notre hypothèse et sont observés dans d’autres modèles de souris KO de régulateurs négatifs de p53. La perte de ZNF768 n’affecte pas la susceptibilité aux tumeurs des souris. Nous avons toutefois observé que les niveaux de ZNF768 sont augmentés dans les tumeurs chez la souris et chez l’humain. Il est donc possible que la surexpression de ZNF768 dans les tumeurs soit un mécanisme acquis pour bloquer la SIO. Les résultats obtenus suggèrent que ZNF768 est un nouveau facteur impliqué dans la mise en place de la SIO induite par Ras et que l’altération de ses niveaux dans les tumeurs est possiblement un mécanisme pour contourner celle-ci.
Introduction. Les patients atteints de la fibrose kystique (FK) sont à risque de carence en vitamine K (VK). La VK joue un rôle dans la coagulation, le métabolisme osseux et glycémique. L’ostéocalcine (OCN), une protéine sécrétée par l’os et dont l’action dépend du statut en VK pourrait être impliquées pour les effets sur la glycémie. La forme circulante décarboxylée de l’OCN (ucOCN) pourrait agir comme marqueur du statut et de l’action de la VK. Peu d'études chez l'humain ont mesuré à la fois les formes totale (tOCN) et décarboxylée de l’OCN, et les méthodes antérieures d’analyses utilisées manquent de fiabilité. L’objectif est d’étudier l’association entre la VK et l’OCN (totale et décarboxylée), la tolérance au glucose et le statut clinique chez des patients adultes atteints de FK. Méthode. Analyse transversale avec les données à l’inclusion menée chez 167 patients adultes de la cohorte de FK de Montréal. La VK et les différentes formes d’OC (méthodes validées) ont été mesurés à partir du sérum. Les résultats du test d’hyperglycémie provoquée par voie orale (2 heures avec mesures de la glycémie et insuline aux 30 minutes) et les données cliniques (ex. fonction pulmonaire et nutritionnel) ont été recueillis. Résultats. Soixante-six pourcent (66%) des patients présentent une concentration sous optimale de VK (<0.28 nmol/L). Les patients avec une VK sous optimal ont des niveaux d’ucOCN plus élevés que ceux avec une concentration de VK normale (10.70±11.06 vs 5.20±5.55 ng/ml, p<0.001). Dans la cohorte totale, l’ucOCN corrèle négativement avec l’âge (r=-0.205, p=0.009), la masse grasse (r=-0.233, p=0.03) et la VK sérique (r=-0.210, p=0.007) et positivement avec le marqueur de remodelage osseux C-Telopeptide (r=0.410, p<0.001). Conclusion. Aucune corrélation significative n’a été observée avec la fonction pulmonaire ou des marqueurs du métabolisme des glucides.
The sterol regulatory element-binding protein 1 (SREBP1) is the master transcription factor regulating the lipid homeostasis in the body by controlling the expression of over 30 genes. The expression and activity of SREBP1 can be modulated by a variety of stimuli including nutritional or hormonal and even associated with stress. However, the molecular and cellular mechanisms underlying these adaptative responses remains to be elucidated. SREBP1 is initially synthesized as a transmembrane precursor form which is inactive in the endoplasmic reticulum (ER). Following changes in various metabolic conditions, SREBP1 is directed towards the Golgi where it is activated by cleavage. The cleaved form can then travel to the nucleus where it dimerizes to stimulate the expression of lipogenic genes, including itself, fatty acid synthetase (FAS) and acetyl-CoA carboxylase (ACC). The acylation of a protein is a post-translational modification that involves a covalent link between a fatty acid and a protein. Acylation increases the hydrophobic character of a protein and thus promotes membrane binding, regulates subcellular localization and stability of proteins and modulates protein-protein interactions. Recent results in our laboratory have shown that a decrease in intracellular oleate concentration leads to a substantial decrease in expression, nuclear accumulation and activity of SREBP1.Preliminary results suggest that this effect could be also mediated by the action of oleate, or one of its metabolites. We hypothesized that SREBP1 could be acylated by oleate (or one of its metabolites) and that this post-translational modification would be involved in its localization and regulation of its activity. Our objective is now to confirm the existence of this acylation and to characterize its impact on the activity of SREBP1. A preliminary bioinformatics analysis identified two potentially acylated residues of SREBP1. The first residue is found on the cleaved form of SREBP1 and could be involved in its dimerization. The second residue is found near the cleavage site of the precursor form of SREBP1, which could thus play a role in the modulation of protein-protein interactions between SREBP1 and the cleavage proteins, therefore influencing the activation of the transcription factor. To identify the nature of the acyl groups and the putative acylation sites of SREBP1, human hepatocytes are incubated with oleate and SREBP1 proteins immunoprecipitated. A part of this precipitate undergo a treatment to detach the acyl fractions. Proteolytic enzymes are used to generate peptides that contain only one potentially acylated residue. The generated peptides will be analyzed by mass spectrometry (MALDI-TOF). We also generated SREBP1 mutant (mutated on one of the acylated residues identified by bioinformatics) to transfect hepatocytes. The localization of mutated SREBP1 will be then evaluated using confocal microscopy at the level of the ER, Golgi and nucleus. In addition, we will determine the transcriptional activity of mutated SREBP1 by measuring the expression of several of its target genes (such as FAS and ACC) by quantitative PCR. The characterization of a new mechanism for regulating the activity of SREBP1 would represent a real advance in the field of lipid metabolism.
Objectif:Le système des endocannabinoïdes(eCB)est connu pour réguler l’activité des neurones de l’aire tegmentale ventrale (VTA). Comme ces neurones sont essentiels au comportement motivé, cette étude a examiné le rôle de la régulation par les eCB des neurones de la VTA sur le comportement alimentaire.
Méthodes:Le domaine 6 de l’enzymes alpha / bêta-hydrolase de la sérine hydrolase (ABHD6) dégrade le eCB 2-arachidonoylglycérol, contrôlant ainsi sa signalisation au niveau du récepteur aux cannabinoïdes 1. L'expression de ABHD6 dans la VTA a été évaluée en utilisant une hybridation in situ à l'ARN fluoréscent. Nous avons cherché à supprimer de manière conditionnelle l'ABHD6 via des microinjections au niveau de la VTA d'un virus AAV délivrant l’enzyme Cre (ou un virus contrôle exprimant la GFP) sous le contrôle d’un promoteur spécifique aux neurones (Syn) à des souris ABHD6-LoxP. Trois semaines après la chirurgie, les souris ont été soumises à un jeûne de 22h, après quoi la prise alimentaire a été mesurée sur 24h. Quatre semaines plus tard, les souris ont été nourries avec un un régime riche en graisses (high-fat diet) pendant 13 semaines, avec mesure de la prise alimentaire et du poids chaque semaine. Au terme des 13 semaines, les souris ont été mises dans des cages métaboliques CLAMS pour mesurer la dépense énergétique, le quotient respiratoire et l'activité locomotrice.
Résultats:ABHD6 est exprimée dans les cellules de la VTA, y compris dans les neurones dopaminergiques. L’injection d'un virus Syn.Cre au niveau de la VTA a augmenté la prise alimentaire cumulative d'un régime riche en graisses par rapport au contrôle (sans affecter les réponses à la réalimentation après un jeûne) et augmenté l'activité locomotrice nocturne sans impact sur le quotient respiratoire ni sur la dépense énergétique.
Conclusions: Ces résultats suggèrent que le système eCB joue un rôle dans la régulation de la consommation d'aliments palatables par les neurones de la VTA, et pavent la voie pour les expériences futures concernantle rôle de la signalisation des eCB dans la VTA dans le comportement motivé.
La pratique d’activité physique (AP) régulière est liée à plusieurs bénéfices pour la santé chez les personnes atteintes de diabète de type 1 (DbT1), incluant l’amélioration de la santé cardiovasculaire et de la qualité de vie. Toutefois, plus de 60 % des patients avec le DbT1 sont sédentaires, ce qui contribue à augmenter les facteurs de risque cardiométabolique. L’augmentation du risque d’hypoglycémie est la barrière la plus importante rapportée par les patients pour la pratique de l’AP. Un apport en glucides est souvent requis pour prévenir les hypoglycémies lors d’une AP prolongée. La quantité de glucides nécessaire peut varier selon le type d’insulinothérapie, le moment où l’AP est effectuée, le type d’AP et la glycémie au début de l’AP. En plus de la quantité de glucides ingérée, le moment où les glucides sont consommés pourrait également avoir un impact sur le contrôle de la glycémie durant l’AP. Une prise de glucides à un moment unique avant une AP pourrait induire une hyperglycémie transitoire, alors que la même quantité de glucides répartie durant l’AP pourrait être associée à un meilleur profil glycémique. Notre objectif est de comparer l’efficacité de 2 stratégies de collation pour maintenir la glycémie dans les cibles glycémiques (4,0 à 10,0 mmol/L) lors d’une AP chez les adolescents et adultes avec le DbT1 traités par multi-injections d’insuline.
Trois heures et demie après avoir consommé leur diner standardisé, les sujets ont participé à 2 interventions durant lesquelles une heure de vélo stationnaire est réalisée à 60% de leur VO2peak. Aléatoirement, ils consommaient un apport en glucides de 0,5g/kg du poids corporel. Cet apport est consommé en une seule fois 5 minutes avant l’AP avec la stratégie de prise unique (SPU) ou de façon répartie avant et pendant l’AP (40% 5 minutes avant l’AP, 30% à la 20eminute et 30% à la 40e minute) avec la stratégie de prise répartie (SPR). La glycémie capillaire est mesurée avant l’AP, puis toutes les 10 minutes durant l’AP.
Trente-trois participants ont complété l’étude. La glycémie de départ moyenne était de 7,8 mmol/L avec la SPU et de 7,3 mmol/L avec la SPR. Quatre participants ont eu une hypoglycémie (< 4,0 mmol/L) durant l’AP avec la SPU comparativement à 6 participants pour la SPR. Une tendance favorable a été observée pour la SPR pendant l'AP pour plusieurs facteurs, mais celle-ci n’est pas statistiquement significative ; pourcentage de temps passé dans les cibles glycémiques (SPU: 75 ± 35%; SPR: 87 ± 26%; P = 0,12), pourcentage de temps passé > 10,0 mmol/L (SPU: 18 ± 33%; SPR: 7 ± 23%; P = 0,11), glycémie moyenne (SPU: 7,6 ± 2,7 mmol/L, SPR: 6,6 ± 2,0 mmol/L; P = 0,12). Il n'y a pas eu de différence observée pour le pourcentage de temps passé <4,0 mmol/L pendant l'AP entre les deux stratégies (SPU: 6 ± 18; SPR: 6 ± 16%, P = 0,90). La glycémie des sujets a augmenté davantage avec la SPU (+1,1 ± 1,3 mmol/L) à comparer de la SPR +0,5 ± 0,7mmol/L (P = 0,01).
Nos résultats suggèrent qu’un apport en glucide de 0.5g/kg du poids corporel semble être une option raisonnable pour une AP d’intensité modérée d’une heure non planifiée à distance du repas et sans ajustement antérieur de l’insuline. Les deux stratégies de collation sont similaires pour prévenir les hypoglycémies lors de l’AP. Cependant, la SPR pourrait être associée à un meilleur profil glycémique d’un point de vue clinique.
Pancreatic β-cells continuously sense blood glucose levels and secrete insulin to maintain normoglycemia. In β-cells, ATP generated by glycolysis promotes the closure of ATP-sensitive K+ channels, thereby increasing intracellular calcium and ultimately insulin secretion. 14-3-3 proteins, and in particular 14-3-3ζ, have been found to regulate ATP synthase and mitochondrial respiration, suggesting that members of this family of scaffold proteins in b-cells may influence glucose-stimulated insulin secretion (GSIS) and glucose homeostasis.
To date, we have identified critical contributions of 14-3-3 proteins to GSIS. In mouse and human islets, pan-inhibition of 14-3-3 proteins with cell-permeable inhibitors potentiated ex-vivo GSIS. This was associated with increased mitochondrial function, as oxygen consumption and ATP synthesis rates were significantly enhanced. Moreover, increased ATP production was confirmed with luciferase-based assays.
Of the seven isoforms, we previously reported critical metabolic roles of 14-3-3ζ in glucose homeostasis, and to understand its role in β-cells, β-cell-specific knockout mice (Ins1CreThor:14-3-3ζfl/fl, β-KO) were generated. When compared to control mice, no differences in body weights or glucose and insulin tolerance were observed, but β-KO mice displayed significantly enhanced insulin secretion following i.p. glucose (2 g/kg), and ex vivo islet perifusion studies revealed enhanced 2nd-phase secretion of GSIS from β-KO islets. Similar to pan-14-3-3 inhibition, increased mitochondrial activity and ATP synthesis were detected in islets of β-KO mice.
In summary, these results demonstrate critical functions of 14-3-3ζ and its related proteins in mitochondrial activity in β-cells and the regulation of GSIS. These data also suggest that 14-3-3ζ inhibition may represent a promising target to enhance pancreatic β-cell function in the context of diabetes.
Funding: CIHR, Canada Research Chairs Program
Introduction: Attrition in pediatric weight management programs is notoriously high. Greater understanding of its determinants is needed to inform retention strategies. To this end, we identified determinants of attrition in the CIRCUIT program, a healthy behaviour intervention for youth at risk of cardiovascular disease.
Methods: Intervention study of children aged 4-18 years who initiated the CIRCUIT program before July 2015. We defined attrition, the outcome, as attending the baseline visit but ceasing attendance prior to the 1-year follow-up. Potential determinants of dropout included the child’s age, sex, ethnicity, body mass index (BMI) z-score, family socio-demographic characteristics, and estimated driving time to the program, all measured at baseline. Associations were estimated bivariately, using chi-square- and t-tests, and simultaneously in a multivariable logistic regression model.
Results: Of the 403 participants who started the program, 198 (49%) dropped out within 12 months of enrollment. Youth who dropped out were older (mean age 12.8y vs. 11.3y; p<0.01), were less likely to live with both parents (62% vs. 71%; p=0.05), and to have mothers who had completed high school (79% vs. 88%; p=0.01). No group differences were observed for sex, ethnicity, baseline BMI z-score, fathers’ education, or driving time to the program. In multivariate modeling, only older age at initiation of the intervention (OR:1.2; CI:1.1,1.3) and lower maternal education (OR:2.1; CI:1.1,4.0) were associated with dropout.
Conclusion: Improved tailoring of interventions to older pediatric patients and to families of lower maternal education may help reduce attrition in CIRCUIT and similar behavioural intervention programs.
Background: Non-alcoholic fatty liver disease (NAFLD) is associated with type II diabetes (T2D) and has become the main cause of cirrhosis. Both NAFLD and T2D are associated with cognitive and neurological impairments. T2D has been established as a risk factor for first-time development of overt hepatic encephalopathy (HE) in cirrhotic patients. The onset of HE in diabetic patients with cirrhosis develops earlier compared to cirrhosis patients without T2D. However it remains unclear whether NAFLD-induced cirrhosis increases the risk for HE. The present study aims to address the association between NAFLD, T2D and HE. Methods: Our retrospective study includes 102 cirrhotic patients on the liver transplant list at the Liver Unit of the Montreal University Hospital Center. Patients were classified by etiology of cirrhosis; 1) NAFLD and 2) non-NAFLD. Demographic data, blood biochemistry, clinical information on T2D-related comorbidities and cirrhosis complications (including number and severity of HE episodes) were collected. These factors were statistically associated with HE episodes. Results: Our cohort comprised 20 (19%) NAFLD and 82 (79%) non-NAFLD patients presenting similar MELD and Child-Pugh scores. The prevalence of T2D was higher in NAFLD vs non-NAFLD cirrhotics (15 (75%) vs 24 (29%) respectively) and was associated to co-morbidities such as cardiac disease, dyslipidemia, hypertension and obesity. Among non-NAFLD cirrhotics, 47 (57%) patients had a history of HE whereas 8 (40%) were found in the NAFLD cirrhotics (p>0.05). Since T2D is already known as a risk factor for HE, we subdivided both NAFLD and non-NAFLD groups into non-T2D and T2D subgroups. HE was significantly more prevalent in patients with T2D: in the NAFLD group, 5 (25%) T2D patients had developed an episode of HE compared to 3 (15%) patients without T2D (p<0.05); in the non-NAFLD group, 16 (67%) patients had T2D and HE compared to 31 (53%) HE patients without T2D (p<0.001). Fasting glycemia levels analysis in the 4 sub-groups of patients revealed increased levels in patients with history of HE and T2D, regardless of NAFLD etiology; in the NAFLD group 8.60 ± 0.84 mmol/l in patients with HE and T2D vs 6.00 ± 1.35 mmol/l in patients with HE without T2D (p<0.01); in the non-NAFLD group: 9.23 ± 0.93 mmol/l in patients with HE and T2D vs 5.82 ± 0.27 mmol/l in patients with HE without T2D (p<0.001). Conclusion: Our results sustain the association between T2D and HE and suggest high glucose might play a pathological role in the development of cognitive decline. NAFLD is not a risk factor for the development of HE. These interesting results provide new insights in the role of T2D in the development of HE and further studies are required to understand the underlying mechanisms. Furthermore, identifying patients who are at higher risk of developing HE is imperative to initiate early treatment strategies to protect neurological decline in patients with cirrhosis.
In tandem with the rise in obesity and the aging population, the prevalence of type 2 diabetes mellitus (T2D) has also been rising steadily. In addition, as a manifestation of metabolic syndrome, approximately 70% of patients with T2D will develop non-alcoholic fatty liver disease (NAFLD), a chronic disease that ranges from simple steatosis to severe cirrhosis characterized by inflammation and fibrosis. Previous studies have demonstrated that circulating DPP4 has a direct pro-inflammatory role in the activation of immune cells. Our recent work demonstrated that increases in circulating DPP4 in the setting of metabolic disease derive from hepatocytes. Additionally, genetic elimination of Dpp4 from hepatocytes reduced markers of inflammation in both liver and adipose. Therefore, this study aims to evaluate the efficacy of eliminating hepatic Dpp4 in the progression of liver disease. Mice containing LoxP sites in Dpp4 were aged for one year, injected with adeno-associated adenovirus serotype 8 (AAV8) containing a hepatocyte-specific promoter (thyroxine-binding globulin) driving Cre recombinase (Dpp4 Hep-/- mice) or control - GFP (Dpp4GFPmice). They were then fed a high fat, high cholesterol diet for 24 weeks to induce features of human liver disease. Dpp4 Hep-/- mice had a greater than 90% reduction of Dpp4 mRNA expression within the liver compared with Dpp4GFP mice. This was associated with 70% reduction in plasma DPP4 activity and a 50% reduction in plasm DPP4 protein in Dpp4 Hep-/- mice. Despite the significant changes in DPP4 protein and activity, both biochemical analysis and examination of liver histology demonstrated no differences in lipid accumulation or fibrosis between Dpp4 Hep-/- and Dpp4GFP. At endpoint, protein markers of inflammation in adipose tissue, liver and plasma were equivalent between genotypes. Additionally, high-throughput Nanostring mRNA expression analysis no differences in inflammation between liver tissue and in isolated Kupffer cells Taken together, these data suggest that elimination of hepatocyte-specific Dpp4 does not improve markers of liver disease in aged, high-fat, high cholesterol fed mice.
Growth arrest-specific 6 (GAS6) is a secreted γ-carboxylated protein that acts as a ligand for the TAM tyrosine kinase receptor family, which includes: TYRO3, AXL, and MERTK. GAS6 and its receptors play a critical role in the immune system, tumor progression, and cancer metastasis. More recent studies found that circulating GAS6 levels or SNPs in GAS6 are associated with obesity and insulin resistance in humans. However, the mechanism by which GAS6 influences these metabolic disorders is not understood.
We therefore decided to 1) investigate the role of GAS6 in the development of insulin resistance and diabetes in vivo; and 2) determine the signalling pathways involved.
We first noticed in wild-type mice that the serum levels of GAS6 are higher in feeding condition compared to fasting, suggesting that the secretion of this factor is regulated by the nutritional and energetic status of the animals. Next, we characterize the function of GAS6 in energy metabolism, by analyzing the metabolic phenotype of 3-month-old Gas6-/- male mice fed a normal diet. We found that these mice have improved glucose tolerance and insulin sensitivity, but normal insulin secretion. This phenotype was also observed at 16 months of age and in young mice fed a high-fat diet, suggesting that the absence of GAS6 protects from age- or diet-induced insulin resistance. Conversely, in a transgenic gain-of-function model (ApoE-Gas6Tg), increased GAS6 circulating levels is sufficient to cause reduced insulin sensitivity.
TAM receptors gene expression analysis in insulin-sensitive tissues revealed that AXL is highly expressed in skeletal muscle and white adipose tissue. We further show that AXL is activated by GAS6 in myotubes (C2C12) and adipocytes (3T3-L1) in culture. We next characterized the GAS6/AXL-dependent transcriptome in C2C12 myotubes following treatment with recombinant GAS6 or a pharmacological inhibitor of TAM receptors (LDC1267). Interestingly, RNA sequencing analysis revealed that GAS6/AXL signalling regulates the expression of genes encoding proteins involved in signalling downstream of the insulin receptor (e.g., Irs2 and Eif4ebp1) and in mevalonate biosynthesis (e.g., Hgmcs1, Hmgcr, Nsdhl, Fdft1, etc.). We further show that several of these genes are up-regulated in the skeletal muscle of Gas6-/- mice at 16 months of age. In addition, we found a decrease in the cholesterol levels, produced from the mevalonate pathway, in myotubes treated with GAS6 in vitro. In the presence of an inhibitor of AXL (R428), the effect of GAS6 is blunted, suggesting that GAS6 inhibits cholesterol synthesis in an AXL-dependent manner in muscle cells. Finally, co-immunoprecipation experiments suggest that AXL physically interacts with the insulin receptors at the plasma membrane.
Several studies show that statins, which are inhibitors of the mevalonate pathway, can cause insulin resistance and promote diabetes. Our results suggest that GAS6 may decrease insulin sensitivity through a similar mechanism, i.e., by inhibiting the mevalonate pathway.
Glycerol-3-phosphate is a key metabolite that regulates flux of various metabolic pathways and in particular the glycerolipid/fatty acid (GL/FA) cycle associated with obesity, type-2-diabetes, and cardiometabolic disorders. Recently, we have identified a glycerol-3-phosphate phosphatase (G3PP) in mammalian cells, capable of hydrolyzing glycerol-3-phosphate to glycerol. The function of G3PP in vivo in various organisms is poorly defined and its implication in intermediate metabolism, stress responses and aging has not been examined yet. Since most biological processes including metabolic pathways are highly evolutionarily conserved, we used Caenorhabditis elegans (C. elegans) as a genetic model to screen for function (s) of G3PP. In this study, we demonstrate that G3PP plays a critical role in glycerol synthesis and contributes to various stress responses and healthy aging in the worm. Using blast alignment, we identified three PGP-like homologs/G3PP genes in C. elegans, pgph-1, pgph-2, and pgph-3. We found that pgph transcripts are upregulated during hyperosmotic stress and glucose feeding. Glycerol accumulates in animals exposed to hyperosomotic stress and high glucose levels in a PGPH-dependent manner, demonstrating a critical role of PGPH in glycerol synthesis. To elaborate the role of PGPH in vivo, we generated double and triple PGPH deletion mutant animals using CRISPR-CAS9. Importantly, double (pgph-2; pgph-3) and triple (pgph-1; pgph-2; pgph-3) mutant animals are highly sensitive to hyperosmotic stress demonstrating an important role of PGPH in rapid adaptation to hyperosmotic stress. We further show that loss of pgph-2 alone and not pgph-3 strongly decreased the survival to hyperosmotic stress to a similar extent to the double and triple mutant nematodes suggesting that PGPH-2 is the major enzyme involved in glycerol synthesis. Loss of PGPH enzymes in the double and triple mutant animals increased fat deposition, exacerbated glucotoxicity, decreased resistance to various stresses, shortened median lifespan and decreased healthspan parameters. Importantly, the overexpression of pgph-2 led to a small but significant extension of median lifespan and improved healthspan parameters in aging animals under excess glucose conditions. Metabolomics studies are currently being performed to assess changes in important metabolites including glycerol-3-P in the double and triple mutant PGPH animals in comparison to control animals at basal, hyperosmotic and glucose excess conditions. Finally, using an RNAi-based transcription factor screen, we identified ELT-2/GATA4 and six other transcription factors that regulate the expression of PGPH-2 and PGPH-3 enzymes in response to salt stress. Overall, the results suggest that G3PP/PGP is an evolutionary conserved regulator of glucose and fat metabolism that act to protect against nutrient and environmental stresses and is possibly involved in healthy aging.
Introduction: Blood glucose is tightly regulated by pancreatic islets hormone secretion. Changes in blood glucose concentrations are the main triggers for the insulin-secreting β cells, glucagon-secreting α cells and somatostatin-secreting δ cells. In addition, endogenous fatty acids modulate islet hormone release. Gpr120/FFA4 is a G protein-coupled receptor activated by long-chain fatty acids (FA), including omega-3 polyunsaturated FA (PUFA). Activation of Gpr120 in islets stimulates the secretion of insulin and glucagon and inhibits somatostatin (SST) release. Gpr120 mRNA is predominantly expressed in δ cells but also in β and α cells, but the respective roles of Gpr120 signaling in these different cell types on its insulinotropic and glucagonotropic are unknown. Objective: We aimed to explore the contribution of δ-cell Gpr120 signaling to the regulation of insulin, glucagon and SST secretion in mouse islets. Methods: We used isolated islets from C57BL/6N male mice with either global (GPR120KO) or δ-cell-specific (δGPR120KO) deletion of Gpr120. The latter was generated by crossing mice expressing the Cre-recombinase under the control of the SST promoter with mice expressing a floxed Gpr120 allele. GPR120KO islets were compared to wild-type (WT) littermates. δGPR120KO islets were studied alongside WT, Cre, or floxed littermates. Insulin and SST secretion were measured in 1-h static incubations in response to glucose (2,8 and 16,7 mM). Glucagon and SST secretion was measured in response to arginine (10mM). Results: In WT islets, two distinct pharmacological agonists of Gpr120 dose-dependently increased insulin and decreased SST secretion in response to glucose, and increased glucagon secretion. Both global and δ-cell specific deletion of Gpr120 prevented the modulation of hormone secretion by Gpr120 agonists. Conclusion: Our results indicate that the primary site of action of Gpr120 in islets is the δ cell, where it inhibits SST secretion leading to increased glucagon and insulin release.
Background: Studies using flow cytometry identified macrophage and T-cells within adipose tissue (AT) that contribute to an inflammatory microenvironment. However, the immune cell profiles within different AT regions are not well characterized.
Objctive: Our objective was to compare the immune cell profiles of abdominal subcutaneous AT (SAT) and visceral AT (VAT) in women undergoing bariatric surgery.
Methods: Eleven women undergoing bariatric surgery were recruited from Hospital Sacre-Coeur in Montreal, QC. The average age and BMI was 40.6 ± 8.0 yand 47.1 ± 6.6 kg/m2. AT biopsies were taken from VAT and SAT during surgery, via excision. Samples were digested using a collagenase/hepes solution and the stromovascular fraction was isolated. Flow cytometry (BD FACSVerse, BD Biosciences) was used to characterize immune cells. The cell markers examined were; CD206, CD68, CD3, CD4, CD8, CD45, CD16, CD56, CD19, CD11c and CD11b.
Results: SAT mass was greater (p<0.01) than VAT, 36.5kg ± 8.6 vs 1.5kg ± 0.56, respectively. There was no difference in the number of CD68+ (macrophage) cells between VAT and SAT however, the number of CD68+CD206+ (M2-macrophage) was greater (p=0.03) in VAT (1903.3 ± 535.5 cells/g tissue) than SAT (448.1 ± 197.5 cells/g tissue). VAT had more (p<0.01) CD3+ (T-cells) (4061.7 ± 810.2 cells/g tissue) compared to SAT (1260.5 ± 240.4 cells/g tissue). The number of CD3+CD8+ (cytotoxic T-cells) and CD3+CD4+ (T-helper cells) was greater (p<0.01 and p=0.02, respectively) in VAT vs SAT (1500.0 ± 288.1 cells/g tissue vs 494.84 ± 93.7 cells/g tissue and 1579.37 ± 444.1 cells/g tissue vs 556.4 ± 145.53 cells/g tissue, respectively. Interestingly the proportion of CD3+ cells that were CD4+ and CD8+ was not different between depots. There was no difference in the number or proportion of CD45+CD16+ (natural killer cells), CD45+CD56+ (natural killer T-cells), CD45+CD19+ (B cells) or CD11c+CD11b+ (myeloid dendritic cells).
Conclusion: In women undergoing bariatric surgery, VAT has a lower mass and greater presence of M2-macrophages and pro-inflammatory T-cells than SAT. The composition of the T cell population is similar in both depots. Understanding the immune cell profiles in regions of AT in individuals with obesity can help in understanding how each depot contributes differently to inflammation.
Contexte et objectifs. Le prédiabète et le diabète de type 2 (T2D) sont en hausse chez l’enfant et ses facteurs de risque peu documentés. Cette étude vise à 1) décrire l’histoire naturelle du développement du T2D de l’enfance jusqu’à la fin de l’adolescence 2) identifier les caractéristiques à l’enfance qui augmentent le risque de prédiabète et T2D.
Méthodes. Nous avons utilisé les données des trois cycles d’évaluation (8-10, 10-12 et 15-17 ans) de la cohorte QUALITY d’enfants caucasiens avec un parent obèse (n=630). Un test d’hyperglycémie orale provoquée a été fait à chaque visite après un jeûne de 12h. Le glucose plasmatique et l’insuline ont été mesurés à 0/30/60/90/120 minutes après l’ingestion de 1.75g/kg (max 75g) de glucose. Le prédiabète (anomalie du glucose à jeun, intolérance au glucose) et le T2D ont été évalués avec les critères de l’American Diabetes Association (n=350). Des trajectoires de sensibilité et de sécrétion d’insuline et de fonction de cellule bêta ont été estimées avec des modèles additifs généralisés ajustés pour adiposité et stade pubertaire. Une analyse de moyennage de modèles de régression logistique basé sur l’Aikake Information Criterion a été réalisée chez les enfants avec tolérance normale au glucose (NGT) à 8-10 ans. Les facteurs investigués incluaient des caractéristiques biologiques (sexe, adiposité, glucose, ratio leptine-adiponectine, etc.), les habitudes de vie et le revenu et l’éducation des parents.
Résultats. Trente et un des 317 enfants avec tolérance normale au glucose (NGT) à 8-10 ans ont développé un prédiabète et 2 ont développé un T2D au cours du suivi. De ceux-ci, 14 sont revenus NGT au 2e suivi. Chez les 33 enfants avec prédiabète à 8-10 ans, 24 sont revenus NGT. La sensibilité à l’insuline diminue temporairement entre 8 et 14 ans. La fonction de cellule bêta réduit de 20% entre 8 et 17 ans. Seuls le glucose à jeun (OR=4,5; IC 95%=1,1-18,8) et le glucose 2-h (OR=1,7; IC 95%=1,1-2,7) à 8-10 ans augmentaient significativement le risque de développer un prédiabète ou T2D.
Conclusions. Plusieurs enfants ont développé un prédiabète ou T2D, mais 55-72% d’entre eux ont normalisé leur tolérance au glucose en cours d’adolescence. Peu de facteurs de risque ont été identifiés à 8-10 ans, outre un glucose à jeun et à 2-h plus élevés bien que considérés dans les valeurs normales. Une meilleure compréhension de la variation de l’homéostasie du glucose et des facteurs de risque durant cette période est essentielle pour développer des stratégies préventives et thérapeutiques optimales du T2D pédiatrique.
L’obésité est un fléau mondial d’autant plus préoccupante plus qu’elle est associée à de nombreuses comorbidités telles que le diabète de type 2, les dyslipidémies et les maladies cardiovasculaires. De plus, le foie développe des désordres métaboliques menant à la stéatose hépatique, une condition qui favorise la fibrose, la cirrhose et le cancer. L’expression des gènes est hautement régulée par divers mécanismes moléculaires impliquant les facteurs de transcription. Ces derniers ont un rôle dans une variété de processus biologiques et représentent donc de potentielles cibles thérapeutiques pour le traitement des pathologies associées à l’obésité.
L’objectif de ce projet est d’identifier et de caractériser de nouveaux facteurs de transcription qui pourraient servir de cible thérapeutique pour améliorer la santé des personnes obèses.
Nous avons identifié Zinc Finger Protein X (ZfpX) comme un facteur de transcription majoritairement exprimé par le foie. L’expression hépatique de ZfpX est modulée en contexte d’obésité chez la souris (ob/ob, db/db et diète riche en gras). Nous avons trouvé que l’expression de ZfpX est fortement associée à la stéatose hépatique. Nous avons montré que ZFPX interagit avec TRIM28 pour réprimer l’expression de gènes cibles. Pour conclure, nous avons identifié ZfpX, un nouveau facteur de transcription exprimé en réponse à la stéatose hépatique, et qui pourrait participer à l’apparition de pathologies associées à l’obésité. D’autres investigations permettront de préciser les fonctions de ZFPX dans le métabolisme et les mécanismes régulant son expression.
Introduction: Obesity is a serious health problem that drives multiple diseases including type 2 diabetes and cardiovascular diseases. It is characterized by the excessive accumulation of white adipose tissue (WAT). The expansion of WAT in obesity is linked to a rise in cell size (hypertrophy) and cell number (hyperplasia). The factors that control the hyperplastic growth of WAT are not yet well characterized. Recently, we have identified V-set and transmembrane domain containing 2a (VSTM2A) as a protein produced by pre-adipocytes that promotes adipogenic commitment. Interestingly, in vitro experiments showed that VSTM2A depletion impairs adipogenesis, while its overexpression induces it1. Yet, the mechanisms regulating the expression of VSTM2A are still elusive.
Objective: Identify the regulatory factors controlling expression of VSTM2A.
Methods: In order to identify the signaling pathways and transcriptional regulators controlling VSTM2A transcription, we have tested the impact of various kinase inhibitors and small molecules on the basal expression of VSTM2A in 3T3-L1 cell line.
Results: VSTM2A mRNA was significantly reduced upon treatment with inhibitors targeting the Phosphoinositide 3 kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. We also found a diverse effect of the adipogenic mixture on VSTM2A expression where the synthetic glucocorticoid dexamethasone reduced the expression of VSTM2A. In contrast, VSTM2A expression was increased several folds upon treatment with IBMX, a molecule that promotes adipogenic commitment. The analysis of these results coupled to a literature review suggested that signal transducer and activator of transcription 3 (STAT3) could be a potential regulator of VSTM2A expression. Supporting this hypothesis, we found that STAT3 inhibition reduced the expression of VSTM2A. We also observed consistent changes in STAT3 phosphorylation in response to all the small molecules initially identified as regulators of VSTM2A expression. In addition, we showed that STAT3 inhibition was sufficient to block the increase in VSTM2A expression brought by IBMX. Lastly, we were able to show that IL6 treatment, a well-known STAT3 activator, activated VSTM2A expression as well.
Conclusion: Collectively, these results strongly suggest that STAT3 is a key transcription factor involved in the regulation of VSTM2A expression. Also, our results support the involvement of STAT3 in the regulation of adipogenesis and the adipogenic commitment. Finally, identifying and targeting the factors regulating VSTM2A expression could be a promising way to regulate adipose tissue expansion.
1-Secco, Blandine, et al. "Amplification of adipogenic commitment by VSTM2A." Cell reports 18.1 (2017): 93-106.
Objectif: Pour les patients atteints de diabète de type 1 (DT1), la gestion des apports en glucides est complexe (calcul des glucides et des doses d’insuline). Les diètes faibles en glucides (FG) qui sont aussi élevées en gras sont populaires pour réduire ce fardeau. Toutefois, peu d’informations sont disponibles sur les ajustements des doses d’insuline nécessaires, en particulier pour éviter les hypoglycémies et/ou les fluctuations de la glycémie (objectif principal). De plus, l’impact potentiel d’une diète FG sur les facteurs de risques cardiométaboliques (ex. : cardiovasculaire et hépatique) n’est pas établi (objectifs secondaires).
Méthode: Étude pilote (n=30) randomisée en chassé-croisé pour comparer deux types de diète: la diète actuellement recommandée (glucides : 50%, lipides : 35%, protéines : 15% de l'énergie) et la FG (glucides : 10%, lipides : 75%, protéines : 15% de l’énergie). L’étude comprend 4 phases : 1) Optimisation des doses d’insuline; 2) Adoption d’une des deux diètes pour 6 semaines (ordre randomisé); 3) Retour aux habitudes alimentaires pour 4 semaines; 4) Adoption de la 2e diète. La variabilité de la glycémie sera évaluée à l'aide d'un moniteur de glycémie en continu. Les doses d’insuline, les épisodes de cétose et le profil pharmacologique seront collectés. Les paramètres suivants seront mesurés à l’inclusion et après chaque diète : profil lipidique, profil hépatique (IRM), composition corporelle (DEXA) et métabolisme de repos (calorimétrie indirecte).
Résultats: Le recrutement a débuté en octobre 2019. Les résultats permettront aux professionnels de la santé de guider les patients atteints de DT1 qui décident d’adhérer à une diète FG et donneront des données préliminaires pour la sécurité cardiométabolique.
La NAFLD (non-alcoholic fatty liver disease) ou stéatose hépatique est un regroupement de conditions liées à une accumulation de lipides au foie sans être influencée par la consommation excessive d'alcool. Cette accumulation de gras est généralement corrélée avec plusieurs dérèglements liés aux maladies cardiovasculaires et dyslipidémies associées à l'augmentation de la concentration de cholestérol dans la circulation (LDL-C). Ma recherche a pour objectif principal d’utiliser un modèle animal pour mieux comprendre le rôle du foie dans la régulation du cholestérol circulant en ciblant particulièrement l’impact d’une accumulation de lipides au foie. Notre hypothèse est que l’accumulation des lipides au foie est un élément déterminant qui affecte la régulation du cholestérol par le foie. Nous avançons également l’hypothèse que la régulation hépatique de cholestérol est altérée selon que la stéatose est induite par un apport important de lipides exogènes ou d’un apport en fructose exogène connu pour stimuler la lipogenèse. Afin de valider ces éléments, nous avons mis en place un protocole utilisant des rats Wistar (n = 60), répartis en 6 groupes soumis à 3 différentes diètes: diète chow (témoin), diète riche en lipides (60% fat) et diète Western (ajout de fructose). De plus, la durée du protocole fut de 2 ou 6 semaines afin de nous permettre de jauger l’effet de la sévérité de la stéatose hépatique sur la régulation hépatique du cholestérol. Les deux diètes riches en lipides ont résulté en une accumulation de TG et de cholestérol dans le foie et ce même après 2 semaines. Les données préliminaires nous indiquent que l’expression géniques du récepteur des LDL ainsi que PCSK9, SREBP-2, ABCG5 et ABCG8 diminuent particulièrement avec la diète Western et ce après seulement 2 semaines. Ces résultats soutiennent l’hypothèse que l’activité métabolique liée aux lipides influence la régulation hépatique du cholestérol.
Diabetic peripheral arterial disease (PAD) is a source of high morbidity and cost, and amputation is one of the most feared complications among patients. Peripheral vascular pathology is characterized by obstructive atherosclerosis disease reducing distal blood flow and perfusion pressure. Collateral vessel formation process in response to artery occlusion is seriously impaired in patients with diabetes rendering the tissue downstream more susceptible to critical ischemia. Several abnormalities in the angiogenic response to ischemia have been documented in the diabetic state involving complex interactions of vascular endothelial growth factor (VEGF) with vascular cells and is believed to be the major risk factors for the development of micro- and macrovascular diseases. Our group has published that activation of protein kinase C delta in diabetes induced the expression and activity of the tyrosine phosphatase SHP-1 in the ischemic muscle of diabetic mice as well as in cultured endothelial cells exposed to high glucose levels. These data indicate that activation of SHP-1 and the consequent inhibition of VEGF activity contribute to the reduction of blood flow reperfusion following ischemia. However, the definitive proof that enhanced SHP-1 activity in diabetes causes vascular growth factor unresponsiveness and poor angiogenic response in the diabetic PAD remain unresolved.
Methods: Cultured bovine aortic endothelial cells (BAEC) were exposed to normal or high glucose (HG) levels with or without hypoxia. BAEC were stimulated with VEGF and cell proliferation, cell migration and tubule formation were evaluated. The expression of p-Akt, p-ERK, p-VEGFR2 and SHP-1 was assessed by immunoblot analyses. Interaction of SHP-1 with VEGFR2 and its activity was evaluated by co-immunoprecipitation and phosphatase assay. Adenoviral vector containing a dominant-negative form of SHP-1 was used to assess the role of SHP-1 by an inhibition of its catalytic activity.
Results: The phosphatase activity of SHP-1 and its interaction with VEGFR2 were significantly increased in BAEC exposed to HG levels in both normoxia and hypoxia. A reduction of the activation of VEGFR2 was observed in HG levels. A reduction of VEGF effects on Akt activation in HG levels was observed in both normoxia (-66%) and hypoxia (-54%). The exposition of BAEC to HG levels decreased VEGF effects on ERK activation in normoxia (-76%) and hypoxia (-87%). HG levels reduced VEGF pro-angiogenic effects on cell proliferation (-100%), cell migration (-98%) and tube formation (-93%). HG levels also decreased VEGF-induced cell proliferation (-100%), cell migration (-100%) and tube formation (-75%) in hypoxia. The overexpression of the dominant-negative form of SHP-1 significantly restored VEGF mitogenic effects (on proliferation, migration and tubule formation) through the activation of Akt and ERK in HG levels.
Conclusion: Our results suggest that elevated SHP-1 activity and interaction with VEGFR2 induced by hyperglycemia inhibits the pro-angiogenic properties of VEGF in BAEC. Transgenic mice exhibiting a specific deletion of SHP-1 in endothelial cells will be generated to confirm the implication of SHP-1 in poor collateral vessel formation. Ischemia of lower leg will be caused by ligation of the femoral artery and blood flow reperfusion will be assess every week for 4 weeks. Changes in VEGF expression and activation of downstream signalling proteins will be evaluated in isolated muscle tissues.
Problématique: Le diabète de type 1 (T1D) est une maladie auto-immune causée par la déficience des mécanismes de tolérance immunitaire, résultant en la destruction des cellules β productrices d’insuline. Afin de développer de nouveaux traitements pour les maladies du système immunitaire, plusieurs groupes de recherche se penchent sur l’étude de lymphocytes régulateurs, capables de prévenir les réactions auto-immunes.
Les lymphocytes T CD4-CD8- (DN, double négatif) sont une population de cellules immunorégulatrices, et le laboratoire du Dre Lesage a préalablement démontré qu’un seul transfert de ces cellules dans des modèles murins est suffisant pour diminuer l’incidence du T1D. L’utilisation des cellules T DN présente donc un grand potentiel thérapeutique pour le traitement des maladies auto-immunes, puisqu’elles inhibent les réponses immunitaires de façon antigène spécifique, diminuant ainsi le risque d’effets secondaires.
Hypothèse: Le potentiel immunorégulateur des cellules T DN humaines peut être exploité afin de créer une thérapie cellulaire pouvant être administrée de façon efficace et sécuritaire chez les patients atteints du T1D.
Objectifs: Dans le but de créer un produit de thérapie cellulaire qui est sécuritaire et efficace, nous avons d’abord optimisé notre protocole d’expansion cellulaire grâce à un milieu sans sérum et diverses cytokines (IL-2, IL-7, IL-15 et IL-1β). Par la suite, nous avons procédé à une caractérisation phénotypique par cytométrie en flux des cellules T DN humaines provenant de donneurs sains et de donneurs atteints du T1D, en mesurant l’expression de marqueurs d’activation (CD69, KLRG1, CD160), de mémoire (CD45Ro, CD127) et d’épuisement (Lag3, Tim-3, PD-1), afin de voir si les cellules ayant subi notre protocole d’expansion sont activées de façon non aberrante et ne sont pas épuisées. Finalement, nous avons étudié l’activité fonctionnelle des cellules T DN dans des essais de cytotoxicité ayant pour cible des lignées cellulaires cancéreuses (Jurkat, Nalm et Raji), afin de voir si elles ont une activité cytotoxique similaire à celle cellules T CD8+ classiques, par relâche de perforine et granzyme, et si l’activité des cellules T DN humaines varie en fonction de l’état de santé du donneur.
Conclusion et impact: L’analyse de ces résultats nous permettra de mieux comprendre le phénotype et l’activité ces cellules humaines immunorégulatrices T DN dans le contexte du diabète de type 1, afin d’éventuellement pouvoir une développer une thérapie cellulaire pour cette pathologie dans le futur.
In the brain, the medio-basal hypothalamus (MBH) is the regulatory center of energetic balance. Neurons of the HMB detect circulating nutritional signals including fatty acids (FA) to subsequently control energetic homeostasis. Alterations in these mechanisms lead to obesity. Adipocyte Triglyceride Lipase (ATGL) is a lipase involved in the first step of degradation of triglycerides (TG) and generates energetic substrates (FA) that also play a role as signaling molecules. Results from our group demonstrate the expression of ATGL in neurons of the HMB and modifications to its expression in response to metabolic challenges. Objective: We propose that ATGL controls the dynamic of TG and lipid droplets (LD) in neurons of the HMB and contributes to the regulation of energetic homeostasis. Method: Targeted viral strategies are used to induce a loss or a gain of function of ATGL specifically in anorexigenic (POMC) or orexigenic (AgRP) neurons of the HMB. We also test the effect of pharmacologically inhibiting ATGL in neurons in vitro or in neurons of the C.Elegans worm on the content in TG, LD and on the intracellular FA profile using lipidomics. Results: Inhibition of ATGL by ATGListatin in cultured hypothalamic neurons induces an accumulation of TG and LD. Inactivation of ATGL in neurons of the C.Elegans induces an increase in adiposity. The loss of function of ATGL in neurons of the HMB induces an obesity phenotype in mice. Our results of ATGL over-expression specifically in POMC or AgRP neurons (viral Cre-Flex strategy) of mice suggest an antagonizing role of the lipase in these populations on energetic balance. Conclusion: Neuronal TG and their mobilization via ATGL play an important role in the hypothalamic regulation of energetic homeostasis. Studies are ongoing to characterize the intracellular signaling pathways involved.
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver biochemistry in North America. It is now estimated that 10-22% of hepatocellular carcinoma (HCC) cases are attributed to fatty liver. However, the mechanisms underlying the relationship between diet-related hepatic metabolic disease and cancer development are poorly understood. PGC-1α is a master regulator of metabolism and its role in carcinogenesis remains controversial. We aim to determine whether low hepatic PGC-1α, when combined with a western diet, potentiates the development of liver cancer.
We probed the TCGA databases related to liver cancer to determine whether expression levels of PGC-1α correlate with patient prognosis. We developed a mouse model of diet-associated liver cancer by combining a high-fat/high-fructose diet (HFHF) with a “second hit” of diethylnitrosamine (DEN). Mice expressing either one (LH, liver heterozygotes) or two (LKO, liver knockouts) floxed PGC-1α alleles under the control of the albumin promoter were subjected to the protocol. Liver tumour multiplicity and maximum size were quantified. Liver tissues and primary hepatocytes were used to investigate pathways known to influence nutrient metabolism, oxidative stress, inflammation, and hepatocarcinogenesis.
We found that low PGC-1α correlated with reduced survival of subjects with HCC. Our data showed that LKO male mice exhibit increased liver tumour number and maximum size. In vivo and in vitro data suggested that cells expressing low levels of PGC-1α fail to induce cellular damage response as shown by attenuated phosphorylation of H2AX (DNA damage response) and cleaved caspase 3 (apoptosis). This is associated with a raise in p-ERK (survival) in both liver tissues and primary hepatocytes of LKO mice. Also, loss of hepatic PGC-1α resulted in increased expression of cancer stem cells markers and reduction in E-cadherin. Finally, we successfully generated a gain-of-function model using CRISPR-Cas9 technology which is protected from diet-associated liver cancer and restores cellular damage response.
In conclusion, our data implicates PGC-1α as an important mitigating factor in the development of diet-associated liver cancer.
Epidemiological studies have consistently reported an association between exposure to persistent organic pollutants and diabetes risk in humans. We have previously shown that a single high-dose of the highly persistent pollutant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) supresses fasting and glucose-induced plasma insulin levels in male and female mice in vivo, however transient hyperglycemia was only observed in the females. We have also shown that chronic low dose TCDD exposure combined with high fat diet (HFD) feeding caused severe hyperglycemia and insulin insufficiency compared to control HFD mice in females only. These results suggest that females may be more susceptible to developing diabetes following TCDD exposure, especially if faced with an additional metabolic stressor. The present study investigated whether there are lasting effects of transient chronic low-dose TCDD (20 ng/kg/d) exposure on dam metabolism, and whether subsequent HFD feeding would exacerbate the effects of TCDD. Female mice were injected with corn oil (vehicle) or TCDD 2x/week during mating, pregnancy, and lactation, and then tracked for 10 weeks following the last TCDD exposure (at weaning). At 10 weeks post-TCDD, dams were either transferred to a HFD or remained on chow diet and then tracked for another 10 weeks. TCDD-exposed dams were transiently hypoglycemic at birth and had a modest increase in body weight starting at 5 weeks after the last TCDD injection. When transferred to a HFD, TCDD-exposed dams rapidly developed obesity, hyperglycemia, and dysregulated insulin secretion. Additionally, dams exposed to both TCDD/HFD had decreased islet size compared to vehicle/HFD mice and had a lower proportion of MafA+ mature beta cells. Taken together, our data supports our previous findings that TCDD increases diabetes susceptibility in females, with rapid disease progression when combined with a HFD.
RAS est une petite protéine Rho-GTPase à la tête d’un réseau de signalisation prolifératif important. Les sentiers activés par RAS incluent les Mitogen-Activated proteins Kinases (MAPK) et la voie Phosphoinositide-3-kinase (PI3K)/Mechanistic Target of Rapamycin (mTOR). Bien que de nombreuses évidences soutiennent une forte implication de RAS dans la carcinogenèse, les mécanismes moléculaires précis liant RAS et prolifération cellulaire ne sont pas tous élucidés. En utilisant des données publiques de phosphoprotéomique, notre équipe a identifié Zinc Finger Protein 768 (ZNF768) comme une nouvelle cible de RAS essentielle à la croissance et à la prolifération. ZNF768 est un facteur de transcription qui est déstabilisé au niveau post-traductionnel par les voies MAPK et mTOR. La déplétion aigue de ZNF768 induit prématurément une entrée en sénescence, un état caractérisé par un arrêt irréversible du cycle cellulaire, et souvent mis en place en réponse au stress. Nos études montrent que ZNF768 est dégradé durant ce phénomène. De plus, la surexpression de ZNF768 réduit l’entrée en sénescence, via un mécanisme dépendant de TP53. Ce facteur de transcription joue un rôle clé dans la sénescence et interagit avec ZNF768. L’ensemble de ces résultats suggère que les voies MAPK et mTOR, toutes deux activées par RAS, déstabilisent ZNF768 afin de renforcer la sénescence prématurée. De manière intéressante, les niveaux de ZNF768 sont élevés dans certaines tumeurs humaines. Ainsi, il est envisageable que des niveaux élevés de ZNF768 servent à soutenir la carcinogenèse en réduisant la sénescence normalement induite par les oncogènes.
Context and objective. The postnatal obesogenic environment in which children evolve has been suggested to predict the long-term risk to develop metabolic disorders associated with obesity in adulthood. The objective of this project was to evaluate the preventive effect of a polyphenol-rich cranberry extract (CE) administered to high-fat high-sucrose (HFHS) diet-induced obese dams during the preconception, gestational and lactation period, on the metabolic health of their offspring, and the potential role of the gut microbiota using a cross-fostering approach.
Methods. Dams were fed a HFHS diet and daily gavage with the vehicle (Veh) or the CE for a period of 14 to 19 weeks. Dams were mated and within 48 hours of birth about half of their litter was cross-fostered, that is, from a Veh mother to a mother receiving the CE and from a mother receiving the CE to a Veh mother. Offspring were then weaned by their adoptive mother. Mice in the second half of the litter were weaned by their biological mothers. After the weaning period, mice were housed individually and fed an HFHS diet. Analysis of body mass and an oral glucose tolerance test were performed after 8 weeks of treatment. Feces were collected for subsequent analysis.
Results. CE administration was associated with improved metabolic parameters and glucose homeostasis in HFHS-fed dams. However, CE treatment of dams was associated with a deleterious phenotype in female offspring, mainly on the weight of adipose tissues and on glucose metabolism, while no effects were observed in male offspring. 16S rRNA sequencing analysis revealed increased α-diversity in male and female offspring weaned by a CE dam but no effect on β-diversity. We identified Akkermansia genus as an important responder to the nursing mother following the weaning in both male and female offspring. Moreover, we observed a strong sexual dimorphism in offspring which strongly correlate in female offspring with different parameters associated with body weight and glucose homeostasis compare to male offspring.
Conclusions. These results show for the first time the beneficial health properties associated with cranberry polyphenols consumption in dams. However, CE treatment of dams promote sex-specific metabolic and microbiota alterations in offspring.
Insulin-secreting beta-like cells produced from pluripotent stem cells (PSCs) are a promising cell source to replace those destroyed by type 1 diabetes. Currently, the directed differentiation protocols used to generate functional pancreatic cells require a transition from 2D adherent cultures towards 3D cell aggregates. In utero, the structural development of the pancreas begins with the budding PDX1+ posterior foregut cells (PFCs) from the primitive gut tube, followed by ductal branching and delamination of NKX6.1+ pancreatic endocrine cells (PECs). Therefore, we hypothesized that biomechanical influences associated with pancreatic bud formation could guide endocrine differentiation. To test this, we microengineered bioinspired platforms to capture the geometry changes associated with pancreas budding morphogenesis using hPSC-derived PFCs as a model.
PFCs were derived from hPSCs using an established 7-day differentiation protocol and then seeded onto our microengineered platforms and further differentiated for 3 days towards PEC fate. To simulate these biomechanics, hPSC-derived PPs were geometrically confined within cell-adhesive micropatterns. The confined PPCs showed increased nuclear expression of PDX1 and NKX6.1 in 150 µm patterns but not in larger, 500 µm patterns. Finite element models (FEMs) were constructed and predicted that smaller micropatterns contain high stress gradients while larger patterns had low stress gradients. The observed increases in nuclear PDX1 and NKX6.1 in 150 µm patterns were correlated with 3D actin cytoskeletal reorganization, which resembled pancreatic bud morphogenesis. Inhibition of the mechanosensitive ROCK pathway using Y-26732 in 150 µm patterns prevented actin reorganization and abrogated increases in nuclear PDX1 expression from confined culture. To study the effect of mechanics involved in dynamic morphological changes on pancreatic specification, a deformable, pressure-actuated, PDMS membrane-based device was also fabricated. Using this device, a 5% strain was applied to circular patches of a confluent PFC monolayer during the transition from PFCs to PECs. Our preliminary results suggest that mechanical applied stress from the device resulted in downregulated PDX1 expression and this observation was independent of cell density and clustering.
In summary, our results suggest that biomechanics play a critical role during pancreatic specification. Recreating these forces during differentiation could yield improved pancreatic differentiation protocols which may ultimately lead to novel bioprocessing strategies to alleviate the donor supply issues for pancreatic cell therapies.
Chronic high-fat feeding induces alterations in murine intestinal nutrient metabolism and there is intense debate whether this is the result of an adaptation or obesity-induced dysregulation. These changes include a shortening of the small intestine and a decreased secretion rate of intestinally-derived triglyceride-rich lipoproteins. Supplementation of the citrus flavonoid nobiletin to a high-fat, high-cholesterol (HFHC) diet in Ldlr -/- mice prevents obesity, insulin resistance, hepatic steatosis and dyslipidemia. Additionally, nobiletin supplementation prevents HFHC-induced shortening of the small intestine, which may be related to its metabolic protection. In this study, we hypothesized that nobiletin prevents insulin resistance and postprandial lipemia by correcting obesity-induced intestinal dysregulation. Ldlr-/- mice were fed a HFHC (42% kcal fat, 0.2% cholesterol), or HFHC + nobiletin (0.3%w/w; n=16/group) for 10 weeks. Following a 6-hr fast, mice were challenged with either an i.p. or oral glucose tolerance test (GTT). Nobiletin improved glucose tolerance following both glucose challenge methods. The i.p. GTT caused a 60-fold increase in plasma insulin secretion in HFHC fed mice, which was attenuated by nobiletin 3-fold. The oral GTT caused a significant increase in plasma insulin, which was completely normalized by nobiletin. Plasma GLP-1 did not differ between groups; however, HFHC-fed mice had significantly elevated fasting (3-fold) and 15 min post-gavage (2-fold) plasma GIP, both of which were corrected by nobiletin. Similarly, HFHC-feeding increased the size of insulin-positive islets and β-cell mass, which were corrected by nobiletin. A fasting/2 hr refeeding protocol revealed that nobiletin decreased jejunal mTORC1 phosphorylation (p) and increasing pFoxO1, indicating improved insulin sensitivity. Nobiletin decreased jejunal FA-synthesis 1.8-fold and TG-synthesis 2.3-fold, while FA-oxidation was unchanged. Postprandial experiments employing a poloxamer-407 injection (i.p.) and a 3H-TG-containing olive oil gavage showed that nobiletin increased secretion of TG mass and radioactivity into plasma compared to HFHC alone. Electron micrographs of jejunal enterocytes 2-hours post-gavage revealed that nobiletin reduced the accumulation of large cytoplasmic lipid droplets. Plasma chylomicron-triglycerides remained low because nobiletin significantly increased chylomicron-triglyceride clearance 2.3-fold. In conclusion, nobiletin prevented hyperinsulinemia and postprandial lipemia by protecting the gut from HFHC-diet-induced nutrient-sensing dysregulation and insulin resistance.
CONTEXT: Obesity substantially enhances the threat of mood disorders such as anxiety and depression. We showed that chronic intake of a saturated high-fat diet (sHFD) leading to obesity promotes anxiodepressive behaviours in a manner involving nucleus accumbens (NAc) inflammation. The NAc is a brain region controlling motivated behaviour and emotional states that is predominantly composed of medium spiny neurons (MSN) expressing the dopamine receptor type 1 (D1) or 2. NAc D1 MSN optogenetic activation was shown to promote resiliency to stress-induced depression paradigm. While previous observations suggest that NAc D1R expression and function is modulated by a sHFD, the direct contribution of D1 MSNs in diet-induced anxiodepressive behaviours remained to be assessed.
OBJECTIVE: We aimed to determine the role of NAc D1 MSNs activity in the expression of anxiodepressive behaviours consequent to a sHFD.
METHODS: Adult male C57Bl6 mice expressing Cre recombinase in D1R neurons (D1RCre) were fed a sHFD or a control low-fat diet (LFD) for 12 weeks. For chemogenetic activation of NAc D1 MSNs, D1RCre mice received a bilateral NAc injection of an adeno-associated virus expressing the hM3Dq receptor or control mCherry protein (n=5-7/group) in a Cre-dependent manner. Over 5 days, mice underwent testing for anxiety (elevated-plus maze; EPM & open field; OFT) and depressive-like behaviours (sucrose preference; SPT & forced swim test; FST) in a counter-balanced manner following daily intraperitoneal injections of the hM3Dq ligand clozapine-N-oxide (CNO; 1mg/kg).
RESULTS: Mice fed the sHFD and injected with mCherry sHFDmCherry showed a blunted preference for sucrose in the SPT (p<0.005) and enhanced immobility time in the FST (p<0.01) relative to LFDmCherry animals. sHFDhM3Dq mice displayed greater sucrose preference in the SPT (p<0.01) and decreased immobility time in the FST (p<0.01) compared to sHFDmCherry. However, sHFDhM3Dq also spent less time in the open arms of the EPM (p<0.01) and centre of the OFT (p<0.05) relative to LFDmCherry animals. No differences were observed for distance travelled across conditions and CNO treatment did not alter body weight.
CONCLUSIONS: These results suggest the pro-depressant effect of a sHFD is mediated by NAc D1R MSN and highlight a bimodal modulation of anxiety and depressive-like behaviours within the NAc. On-going biochemical analyses aim to identify molecular changes associated with behavioural endpoints observed.
SGLT2 is expressed in the renal proximal tubules and reabsorbs >90% of glucose filtered in the kidneys. Multiple clinical trials with type 2 diabetes have shown that SGLT2 inhibitors improve the cardiac and renal outcome. Currently, several clinical trials are ongoing to evaluate their renoprotective effects in non-diabetic condition. However, the underlying regulatory mechanisms for SGLT2 gene expression remain unclear.
We hypothesized that angiotensin II (Ang II) generated from renal angiotensinogen (Agt) may increase SGLT2 expression. We aimed to assess if canagliflozin, an SGLT2 inhibitor, could ameliorate Ang II-induced hypertensive renal injury in mice with increased SGLT2 expression.
Our lab previously generated transgenic mice overexpressing Agt in their renal proximal tubular cells (Agt-Tg). Control wild-type (WT) and Agt-Tg mice were euthanized at 16 weeks of age, and renal SGLT2 expression was assessed by immunofluorescence. Next, we administered adult (14-16 weeks old) WT mice with Ang II (1000 ng/kg/min), and treated with or without canagliflozin (15 mg/kg/day in drinking water) for 4 weeks (N=7-8 per group). Human immortalized renal proximal tubular cells (HK2) were used as in vitromodel.
SGLT2-immunopositive staining was increased in Agt-Tg mice compared to WT. Ang II infusion in WT mice significantly increased systolic blood pressure, which was unaltered by canagliflozin co-treatment (WT 112±4.1, Ang II 159±8.4, Ang II-cana 156±3.5 mmHg). Ang II infusion caused glomerulosclerosis and tubulointerstitial fibrosis, which were attenuated by canagliflozin. Urinary albumin was increased by Ang II infusion, which was reduced by canagliflozin. In vitro, Ang II treatment stimulated SGLT2 mRNA expression in HK2 cells.
Our data demonstrate that renal Ang II upregulates SGLT2 gene expression. Moreover, SGLT2 inhibition ameliorates Ang II-induced hypertensive renal injury, independent of blood pressure. Our data provide an important foundation for future clinical studies of SGLT2 inhibitors in non-diabetic kidney disease.
Background: Cystic Fibrosis-related diabetes (CFRD) affects about 50% of adults with cystic fibrosis (CF) and is preceded by a decline in clinical status (weight and lung function). Thus, annual screening for glucose abnormalities is recommended using a 2-hr Oral Glucose Tolerance Test (OGTT). Diagnostic criteria for fasting and 2-hr OGTT levels are based on Type 2 Diabetes criteria (risk of retinopathy). However, CFRD is distinct as the most critical complications are pulmonary and nutritional. The aim is to investigate which OGTT parameters most strongly correlate to significant clinical markers in CF: lung function and nutritional status.
Methods: Transversal analysis at baseline (n=293) and observational prospective analysis (n=187; follow-up of 7.5 years) of data from the Montreal cohort with an OGTT and clinical data collection every 18 months. Glycemic: fasting, 2-hr, maximum, and area under the curve (G0; G120; Gmax; GAUC) and insulinemic (I0; I120; Imax; IAUC) parameters were compared with lung function (Forced expiratory volume in 1s: FEV1), body mass index (BMI), and risk of developing CFRD.
Results: FEV1 is inversely correlated with Gmax and I0 (rs=-0.175, p=0.003; rs=0.121, p=0.045), but not G120 & GAUC. I0, Imax, and IAUC are significantly positively correlated to BMI. Prospective analyses show a significant inverse correlation between baseline Gmax and FEV1 decline. This is not observed for G120 nor insulinemic parameters. A Gmax ≥ 8 mmol/L increased the risk of developing CFRD (p=0.024).
Conclusions: In adult patients with CF, Gmax is more strongly associated with FEV1 evolution than the current diagnostic criteria G120.
Objective:Liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. PGC-1αis a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promotersthat coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1αintegrates extracellular signals to balance metabolic and anti-inflammatory outcomes.
Methods:Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1αproteins in regulating hepatic metabolism and inflammatory signaling downstream of TNFα. Gene expression and signaling analysis was combined with biochemical measurement of apoptosis in gain- and loss-of-function in vitroand in vivo.
Results:Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4,which microarray analysis revealed had common and isoform-specific functions linked to metabolism and inflammation compared to canonical PGC-1α1. While PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFαsignaling revealed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFαor LPS. This was in contrast to PGC-1α1, which reduced the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines.
Conclusions:PGC-1αvariants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation and we identify PGC-1α4 as an important mitigator of apoptosis.
Introduction: Type 2 diabetes (T2D) is a chronic disease characterized by insulin resistance, lipid metabolism dysregulation and impaired glucose homeostasis. This pathology is also associated with other metabolic disorders and chronic health problems such as obesity, hypertension, cardiovascular diseases, nephropathy and retinopathy. In T2D and obesity, decreased insulin secretion and/or suppression of insulin action lead to increased hepatic glucose production (HGP) and decreased glucose uptake by peripheral tissues, leading to hyperglycemia. Several anti-diabetic drugs lower blood glucose levels, such as metformin, sulfonylureas and thiazolidinediones. However, these therapies each have their benefits and limitations. In this context, developing new treatments allowing a better glycemic control could have a major impact in patients with T2D.
Methods: To identify new factors implicated in HGP, we used FAO cells and assumed that the capacity of glucose production within this cell population is normally distributed, meaning that some cells within the population would produce either low, average or high amounts of glucose. To test this hypothesis, we performed serial dilutions of our initial population in order to isolate new clonal lines of FAO. We then tested glucose production of 36 lines. Confirming our hypothesis, we found that some FAO clonal lines produced low amounts of glucose (Low Lines) whereas other produced much more (High Lines). In order to define the genes meditating these differences, we next performed a microarray between Low Lines and High Lines, and identified 150 genes differentially expressed among these two groups. A scoring matrix was next built to identify priority candidates. Briefly, genes with unknown function that were previously reported in GeoProfile to be increased in the liver of fasted mice were prioritized. This analysis led to the identification of HSDL2 (Hydroxysteroid dehydrogenase like 2) as a high priority candidate. HSDL2 expression is upregulated in High Lines and highly induced in the liver of fasted mice.
Results: HSDL2 belongs to the family of short-chain dehydrogenase/reductase (SDRs), contains an N-terminal catalytic domain and a C-terminal sterol carrier protein type 2 domain (SCP-2). However, its regulation, function and substrate remain unknown. qRT-PCR analyzes shows that HSDL2 expression decreases in a dose-dependent manner when exposed to insulin and an increases following treatment with fibrates, assuming a role of PPARα in HSDL2 regulation. In vitro, fasting increases HSDL2 expression as well as the inhibition of PI3K, mTORC1 and mTORC2. In vivo, Hsdl2 mRNA increases with fasting, obesity and diabetes. Here, we report that HSDL2 localizes to the mitochondria, suggesting a possible role of this enzyme in oxidative metabolism.
Conclusion: Our preliminary results suggests that HSDL2 is induced by fasting, sensitive to insulin and likely regulated by PPARα. Overexpression and knock-down studies are in progress to better define the role of HSDL2 in hepatic metabolism.
Background: Obesity is the most prevalent risk factor for cardiovascular disease (CVD) in children. We developed a 2-year lifestyle intervention for youth at risk of CVD, and assessed changes in key cardiometabolic outcomes, physical fitness and capacity among those who completed the program.
Methods: The CIRCUIT program is a multidisciplinary, healthy behaviour intervention for children 4-18y at risk of CVD. A personalized plan for increasing physical activity and reducing sedentary behaviours is developed. Both at baseline and two-year follow-up, we measured body mass index (BMI) z-scores, blood pressure (BP) z-scores, adiposity (%body and %trunk fat), fasting plasma glucose and lipid profile, aerobic (VO2max) and anaerobic (5-m shuttle run test) fitness and physical capacity indicators. Differences between baseline and follow-up were examined using paired t-tests (for age-sex standardized outcomes) and multivariable mixed-effect models, adjusted for age and sex (for other outcomes).
Results: Among the 106 participants (53 females) who completed the 2-year program, mean age at baseline was 10.9 years (SD=3.2). BMI z-score and diastolic BP z-score decreased at year 2 compared to baseline by 0.30SD (95% CI: -0.44,-0.16) and 0.42SD (95% CI: -0.65,-0.23), respectively. Participants improved %body and %trunk fat, lipid profile, aerobic and anaerobic fitness levels and physical capacity after 2 years (p<0.02). No changes in systolic BP z-score nor in fasting plasma glucose were observed.
Conclusion: Preliminary findings showing improved cardiometabolic outcomes among children at risk of CVD suggest that CIRCUIT is a promising intervention program.
Type 2 diabetes (T2D) is characterized by peripheral insulin resistance and pancreatic β-cell dysfunction. T2D occurs when β cells become unable to increase their functional mass to compensate for insulin resistance. The identification of molecules capable of stimulating β-cell replication would therefore be of major therapeutic interest. Numerous studies have identified factors that stimulate β-cell proliferation, including glucose, harmine (DYRK1A kinase inhibitor) and the growth factor HB-EGF. However, the reported effect of these compounds on human β cell replication is variable between studies. Objective: To assess the human β cell proliferative response to glucose, harmine and HB-EGF. Methods: We received 14 male and 7 female human islet batches from multiple centers covering a range of donor ages (18-66 years) and BMI (16.4-38.5). Islets were kept intact or dispersed into single cells and cultured in the presence of glucose (2.8 or 16.7 mM), harmine (10 μM) or HB-EGF (100 ng/ml). Proliferation was measured using two independent methods: 1- Immunohistochemistry, by counting the number of double EdU- and Nkx6.1-positive cells in dispersed and intact islets; and 2- Flow cytometry, by counting double EdU- and C-peptide-positive cells. We also measured the proliferation of non-β endocrine cells and ductal cells. Results: Harmine and HB-EGF weakly stimulated β-cell proliferation. Proliferation in response to glucose was observed by immunohistochemistry but not by flow cytometry. Harmine and HB-EGF potently stimulated proliferation of Nkx6.1-negative and cytokeratin 19-positive duct cells. Conclusion: Rigorous assessment of β-cell mitogens requires complementary approaches and careful identification of cell identity. (FRSQ and NIH)
Les hypoglycémies et la crainte d’avoir des hypoglycémies font parties des principales barrières à l’autogestion adéquate du diabète de type 1 (DT1). Les nouvelles thérapies (ex. nouvelles insulines et glucagon nasal) et les technologies (lecteurs de la glycémie en continu et pompes à insuline) ont le potentiel d’améliorer la gestion de la glycémie et de réduire la fréquence de l’hypoglycémie, mais il existe peu de formation accessible aux patients afin d’optimiser leur utilisation. L’objectif de l’étude est d’évaluer si une plateforme de formation en ligne (SUPPORT) pourrait permettre de diminuer la fréquence ou la crainte de l’hypoglycémie après 6 mois d’utilisation chez des adultes vivant avec le DT1. Méthode : La création de la plateforme SUPPORT est guidée par la roue de changement du comportement. Le contenu du programme est divisé en 6 catégories : médication, surveillance de la glycémie, hypo- et hyperglycémie, alimentation, activité physique, santé et autres particularités. La formation est personnalisée en fonction du mode de traitement (multiples injections ou pompe à insuline) et de la méthode de surveillance de la glycémie. Les participants (n=568) seront recrutés via le registre de patients vivant avec le diabète de type 1 (registre BETTER), puis randomisés dans un groupe d’intervention (accès immédiat) ou de contrôle (accès différé de 6 mois) suivant un ratio de 2 :1. L’intervention se fera sur une période de 6 mois avec des mesures avant et après et la comparaison sera faite entre les groupes d’intervention et de contrôle. Une partie des données sera collectée via le registre BETTER. Résultats : L’objectif principal est d’évaluer l’impact sur la fréquence des hypoglycémies (nombre d’hypoglycémies dans les derniers 3 jours) et sur la crainte des hypoglycémies (Hypoglycemia fear survey-II). Conclusion : La plateforme de formation en ligne SUPPORT devrait permettre aux adultes vivant avec le DT1 de s’auto-former pour optimiser leur traitement avec l’objectif de diminuer la fréquence ou la crainte de l’hypoglycémie.
Today, more than 30% of Canadians live with diabetes or pre-diabetes, a chronic disease characterized by dysregulation of blood glucose and insufficient insulin action. Glycemic control can be achieved in patients through potentiation of the signaling by incretin hormones: glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both peptide hormones have been traditionally characterized to be secreted by distinct enteroendocrine cells within the gut in response to nutrients. Signaling through the incretin receptors corrects islet hormone release by potentiating glucose-stimulated insulin secretion from the β-cell and decreasing glucagon secretion from the α-cell.
GLP-1 mediated effects on glucose have been proposed to be through activation of the GLP-1R on the β-cell, as well as GLP-1R found throughout the central nervous and vagal system. However, bioactivity of both GLP-1 and GIP is controlled by post-translational, N-terminal cleavage by the widely expressed serine protease dipeptidyl peptidase-4 (DPP4). As such, DPP4 inhibitors (DPP4i) have been successfully used to treat millions of patients with T2D. DPP4i target the catalytic active site of DPP4 and prevent the cleavage of the incretin hormones, thus prolonging their action. In addition to GLP-1 mediated potentiation of insulin secretion, DPP4 inhibitors also regulate glycemia through the lowering of glucagon-stimulated hepatic glucose production. Therefore, with the current study, we sought to assess whether β-cell DPP4 is an important target for the regulation of glycemia.
Treatment of Glp1r/Giprβ-cell-/- mice with the DPP4 inhibitor, sitagliptin, demonstrated that β-cell incretin receptor signaling is required to mediate the beneficial effects of this class of drugs on glucose homeostasis. Additionally, Dpp4-/- mice exhibit a significant reduction in hepatic glucose production during hyperinsulinemic-euglycemic clamps. Dpp4 mRNA, DPP4 protein and activity are present in isolated mouse islets, further supporting the islet as an important potential site of DPP4i action. Both DPP4i-treated wildtype islets and islets isolated from Dpp4β-cell-/- mice exhibit increased glucose-stimulated insulin secretion during perifusion. However, genetic elimination of Dpp4 from β-cells did not impact whole-body glucose tolerance, response to DPP4i, insulin tolerance or body weight in male mice fed chow or a high-fat diet. Therefore, we provide evidence for islet-derived DPP4 to control local hormonal responses to glucose; however, this effect does not impact systemic glucose metabolism
Background: Obesity is a risk factor for type 2 diabetes (T2D) as it contributes to insulin resistance and pancreatic beta-cell failure due to exhaustion of insulin stores and fuel surfeit mediated glucolipotoxicity in various tissues. Alterations of mitochondrial metabolism and in the glycerolipid / fatty acid (GL/FA) cycle (lipogenesis followed by lipolysis), which links glucose and lipid metabolism are thought to be implicated in the beta-cell dysfunction in T2D. Glycerol-3-phosphate (Gro3P), formed from glucose during glycolysis, is at the crossroads of glucose and lipid metabolism and is one of the starting substrates for the GL/FA cycle. Gro3P is thought to either undergo esterification via lipogenesis or oxidation via glycolysis. Direct hydrolysis of Gro3P to release glycerol, was not known to occur in mammalian cells. Recently, a glycerol-3-phosphate phosphatase (G3PP) was identified and characterized in mammalian cells by the Prentki lab and was shown to hydrolyze glycolysis-derived Gro3P to glycerol. G3PP, by regulating cytosolic Gro3P levels, was shown to control energy and intermediary metabolism, insulin secretion and the response to metabolic stress in pancreatic islet beta-cells in vitro (Mugabo et al. PNAS 2016). To understand the role of pancreatic beta-cells G3PP in the control of fuel stimulated insulin secretion and glucose and fat metabolism in vivo, we developed an inducible beta-cell specific G3PP-KO mouse model using the Cre recombinase system.
Methods: Experimental animals included Mip-Cre-ERT/+;G3PP flox/flox (BKO) and C57BL/6N mice (WT), G3PP flox/flox (fl/fl), Mip-Cre-ERT (MipC) littermates as controls. Tamoxifen was injected IP for 5 consecutive days at 8 weeks of age. The specificity of G3PP deletion was confirmed by Western Blot. Mice were kept on normal diet (ND) for 20 weeks and food intake and body weight were monitored weekly. We performed OGTT, IPGTT, and ITT at 12, 16, and 19 weeks of age, respectively. We also monitored glucose stimulated insulin secretion (GSIS) ex vivo using isolated islets.
Results: Similar results in term of GSIS and other ex vivo and in vivo parameters were seen with fl/fl mice as WT mice. Thus, we considered the MipC mice as the true control for the BKO mice in all experiments. BKO mice showed a modest increase in body weight gain in comparison with control mice after 20 weeks on ND, with no difference in their food intake and glycemia and insulinemia. Also, BKO mice showed no difference in OGTT in term of glycemia and insulinemia in comparison with control mice, both groups showing normal glucose tolerance. IPGTT after 16 weeks on ND showed that BKO mice tend to be more glucose intolerant with higher plasma insulin levels. ITT after 19 weeks showed no difference between BKO and MipC mice. Ex vivo GSIS in isolated islets at 12 weeks of age showed a higher response to 16 mM glucose by BKO islets vs control islets, with no difference in islet insulin contents.
Conclusion: G3PP-BKO mice show a modest increased body weight, and also enhanced insulin secretion response to glucose, as seen in IPGTT. GSIS response ex vivo is also elevated in isolated islets from BKO mice. The results suggest that deletion of G3PP in beta cells increases Gro3P levels, which drive the GL/FA cycle and mitochondrial ATP production, thereby, enhancing insulin secretion. We currently study metabolic signalling pathways in the BKO mice as well as their response to metabolic stress.
Angiotensin II (Ang II), a key vasoactive peptide, is known to play an important role in the pathophysiology of vascular diseases. A heightened activation of Ang II-induced signaling pathways that promote proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMCs) has been suggested to contribute to vascular dysfunctions. We have shown earlier that Ang II enhances the expression of early growth response protein-1 (Egr-1), a zinc transcription factor, which is upregulated in animal models of vascular diseases. Histone deacetylases (HDACs) remove acetyl groups of lysine residues from histone and non-histone proteins and elevated activation of class IIa HDACs is reported in several vascular pathologies. Recent studies have demonstrated that the phosphorylation and the nuclear export of HDAC5, a member of class IIa HDACs, is associated with vascular remodeling through their ability to interact with transcription factors linked to cell proliferation, migration and hypertrophy. However, the involvement of HDAC5 in Ang II-induced Egr-1 expression remain unexplored. Here, we showed that the specific pharmacological inhibition of class IIa HDACs by MC1568 or TMP-195 as well as siRNA-mediated silencing of HDAC5 attenuated Ang II induced Egr-1 expression in VSMCs. In addition, the blockade of the nuclear export of HDAC5 by using nuclear transport inhibitors leptomycin B or KPT-330 decreased Ang II-induced Egr-1 expression. We also demonstrated that siRNA-induced silencing of HDAC5 or Egr-1 prevented VSMC hypertrophy in response to Ang II stimulation. In summary, our results show that Ang II-induced Egr-1 expression is regulated by the activation of HDAC5 and Ang II-induced cell hypertrophy is mediated by HDAC5 and Egr-1.
Chromosomal instability (CIN) is a major form of genomic instability and many proofs support that the CIN is a key driver of the tumorigenesis and the resistance to the chemotherapy. The primary mechanism of the evolution of CIN is abnormalities in mitosis or cytokinesis. Nevertheless, most of the genes directly involved in controlling mitosis are essential and rarely found mutated in human cancers; arguing that changes in the expression or activity of mitotic proteins likely account for the high prevalence of chromosome segregation defects observed in tumors. On the other hand, mutations in RAS or BRAF are frequently found in high fatality cancers that demonstrate CIN, suggesting a possible link between deregulated signaling through the RAS signaling pathway and CIN. Under normal, physiological conditions, RAS signaling is activated by growth factors to promote cell proliferation and survival. However, during tumorigenesis growing evidence indicates that unrestrained signaling through the RAS oncogenic pathway exacerbates CIN, although the mechanisms linking RAS to CIN remain poorly understood. Defining the relationship between unchecked RAS activation and CIN will provide novel insights into cancer pathogenesis, and may lead to the development of new therapeutic avenues for the treatment of cancer.
Our team identified a largely uncharacterized zinc finger protein (ZNF768) as a novel nuclear phosphoprotein targeted by the RAS signaling pathway. Remarkably, there is currently only few references of ZNF768 in the literature. We find that ZNF768 is destabilized in response to the activation of key RAS effectors. In several cell lines, we consistently observed that the protein levels of ZNF768 vary throughout the cell cycle, with the largest change being observed during mitosis. BioID experiments showed that a part of the ZNF768 interactome includes proteins involved in the formation of kinetochores and the mitotic spindle, and proteins essential for the successful execution of mitosis. Transcriptomic analyses reveal that ZNF768 controls the expression of many genes of the cell cycle. Supporting the idea that ZNF768 contributes to CIN downstream of RAS signaling, we found that loss of ZNF768 impaired chromosomal alignment, induced mitotic cell death, and severely attenuated cell proliferation.
Altogether, these results support the notion that ZNF768 is an essential protein that plays key roles at the interface between oncogenic RAS signaling and the development of CIN. This project may provide novel therapeutic targets for the treatment of tumors with RAS mutations, which are common in several high fatality cancers.
Organ functions decline during aging, and the most profound aging-related changes occur in the kidney. Indicators of kidney dysfunction are detected in over 50% of elderly subjects. This dysfunction is further exaggerated by diabetes. Injury to the proximal tubule caused by oxidative stress is a major contributor to common forms of progressive chronic kidney diseases, such as diabetic nephropathy, affecting up to 40% of Type 1 or Type 2 diabetic patients. The polarized epithelium of the proximal tubule is particularly vulnerable to stress. To date, the aging-associated changes in proximal tubule physiology are not well-defined. Using cellular models of aging, our research is directed towards a better understanding of these changes. The proper response to stress is crucial for cell and organ survival. The formation of cytoplasmic stress granules (SGs) is a conserved response that helps eukaryotic cells to survive stress. Aging impairs the stress response, but little is known about the underlying mechanisms. It is our goal to define how aging compromises the kidney’s ability to cope with stress. To this end, we have developed two models of renal proximal tubule cell senescence. They are based on the chemical or pharmacological induction of senescence. We demonstrated that both model systems display hallmarks of aging. Using these models, we assessed SG formation and stress-induced signaling in a quantitative fashion. We showed that aging impairs SG assembly. Moreover, our studies uncovered the underlying molecular mechanisms that contribute to these changes. Taken together, our research provides a better understanding of the senescence-dependent changes in kidney physiology. We identified new biomarkers that can score the stress response in kidney proximal tubule cells. Long-term, this information will facilitate the development of new diagnostic and therapeutic tools to evaluate the complications of diabetic nephropathy and other forms of kidney dysfunction.
SHP1, also known as Protein Tyrosine Phosphatase Non-Receptor Type 6 (PTPN6), plays an important role in regulating glucose homeostasis by controlling insulin signaling. Our laboratory has shown that mice with a liver specific Shp1 knock out (Ptpn6h-ko) on high-fed diet (HFD) exhibit improved fasting glucose levels, reduced insulin resistance and lower inflammation than their flox/flox littermates despite being more prone to hepatic steatosis. Microarray analysis of HFD-fed Ptpn6h-ko versus Ptpn6fl/fl livers revealed elevated levels of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor involved in the regulation of adipogenesis. In the present study, we investigated the various mechanisms by which SHP1 can modulate PPARγ activity.
Molecular modeling analyses suggested that SHP1 interaction with PPARγ depends on the tyrosine phosphorylation status of PPARγ. Using co-immunoprecipitation experiments, we found that SHP1 binds to PPARγ mainly via its N-terminal SH2-domains. We confirmed PPARγ tyrosine-phosphorylation and demonstrated that this phosphorylation could be reduced by SHP1 in vitro. Using mass spectrometry, we identified 6 phosphorylated tyrosine residues in PPARγ. Mutagenesis experiments showed that PPARγ significantly lost its activity when three of these sites were mutated to the non-phosphorylatable phenylalanine. Additionally, we generated SHP1 knockout HepG2 cells using CRISPR-Cas9 technology. We found that expression of several PPARγ regulated transcripts was upregulated in SHP1-KO cells and SHP1-KO cells showed higher de novo lipid synthesis as compared to control cells. Altogether, we show that PPARγ is a novel substrate of SHP1. Our results reveal a selective regulation of PPARγ activity by the protein-tyrosine phosphatase SHP1.
Osteocalcin (OCN) is an osteoblasts-derived hormone regulating glucose and energy metabolism. It favors insulin secretion by pancreatic beta-cells and insulin sensitivity in peripheral tissues. OCN endocrine functions are regulated at least by two post-translational modifications (PTMs), gamma-carboxylation and endoproteolytic cleavage. OCN gamma-carboxylation by the gamma-carboxylase (GGCX) occurs on three of its glutamic acid and requires vitamin K as cofactor. Genetics studies support the notion that carboxylation inhibits OCN endocrine function in mouse. OCN endoproteolytic cleavage is mediated by the proprotein convertase (PC) furin and is critical to convert inactive pro-OCN into mature active OCN. Interestingly, LC-MS proteomics analysis on OCN purified from primary mouse osteoblasts supernatant reveals that OCN is subjected to O-glycosylation. However, the impact of this novel PTM on OCN endocrine functions and whether it regulates its gamma-carboxylation or processing by furin are still unknown.
Hypothesis: OCN O-glycosylation regulates its endocrine functions.
Methods and results: OCN apparent molecular weight is reduced when expressed in CHO-Ldld cells, which are defective in O-glycosylation due to a deficiency in UDP-Gal/UDP-GalNAc 4-epimerase, compared to parental CHO cells. The supplementation of CHO-Ldld cells with N-acetylgalactosamine and galactose blunted this difference. Treatment of bone homogenate of wildtype mice with O-glycosidase and neuraminidase also decreases OCN apparent molecular weight, suggesting that OCN is indeed O-glycosylated in vivo in mice. Pharmacological inhibition of GalNAc-Ts in osteoblasts does not affect OCN processing and gamma-carboxylation, while OCN is still O-glycosylated in osteoblasts when gamma-carboxylation and processing are inhibited. Site directed mutagenesis of OCN serine 8 totally abolished OCN O-glycosylation without affecting its gamma-carboxylation and cleavage. Thus, OCN O-glycosylation occurs independently of its gamma-carboxylation and processing. Moreover, our data shows that O-glycosylated OCN produced in mammalian cells has a longer half-life in plasma ex vivo and in vivo compared to recombinant non-glycosylated OCN produced in bacteria. Interestingly, human OCN sequence does not contain any serine or threonine residue and is not O-glycosylated when expressed in osteoblasts. Mouse OCN O-glycosylation occurs on serine 8 within the PSPDP sequence, a consensus glycosylation motif for GalNAc-T1 and 2. This serine residue is not conserved in human or any other vertebrate species. Amino acid alignment shows that serine 8 correspond to a tyrosine (Y12) in the human protein, while the proline residues are conserved. A single amino acid mutation (Y12S) is sufficient to induce O-glycosylation of human OCN, but does not affect its processing. This artificially O-glycosylated human OCN has an increased half-life in mouse and human plasma compared to non-glycosylated human OCN.
Conclusion: OCN O-glycosylation is a mouse-specific modification which is not conserved in human or any other vertebrate species. O-glycosylation increases the half-life of mouse OCN ex vivo and in vivo. Human osteocalcin O-glycosylation, through a single amino acid substitution, confers human osteocalcin a longer half-life in plasma. Our findings provide a promising approach to improve human OCN half-life in future human therapeutic applications for diabetes and obesity.
The body’s ability to absorb nutrients through the small intestine is a crucial factor in health and survival. Therefore, identifying proteins that regulate and influence how the gut adapts to support increased metabolic demand is of interest. Signaling by the preproglucagon products glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2(GLP-2) is tightly linked to nutrient absorption and utilization. However, the necessity of these peptide hormones to mediate gut adaptation in the face of increased metabolic demand has been incompletely explored. We hypothesize that the incretin hormones GLP-1and GLP-2 are released in proportion to food intake needs to enable efficient nutrient absorption and gut adaptation. We evaluated physical changes in the intestinal epithelium, notably villi length, intestinal circumference, cell composition, crypt depth and changes in mice exposed to chronic cold or thermoneutral conditions. We observed increased crypt to villus height and increased intestinal circumference in mice housed under constant cold. Associated with these morphometric changes, we observed increased significant increases in both fasting and fed concentrations of the incretin hormones, GLP-1 and glucose-dependent insulinotropic protein (GIP). Food intake was increased 2-fold in mice exposed to cold; however, elevated GLP-1 and GIP persisted during a consistent, mixed meal oral challenge suggesting more complex regulatory mechanisms. Further studies will determine the necessity of the preproglucagon peptides with the associated morphometric changes in the gut in response to chronic cold exposure.
Background: Vitamin K (VK) is essential for the conversion of glutamic acid residues (Glu) into g-carboxyglutamic acid (Gla) residues in particular proteins. Recent findings suggest a role for VK in glucose metabolism and diabetes; however, the particular tissue(s) and Gla protein(s) involved remain to be identified. Preliminary data we have gathered indicate that the pancreas is one organ implicated in the beneficial effects of VK on glucose metabolism, since mice lacking Ggcx in pancreas are characterized by a reduced insulin secretion and a decreased β-cell mass. None of the known Gla proteins are expressed in β-cells, suggesting the involvement of a yet to be identified g-carboxylated protein. Hence, the goal of this study is to identify new Gla proteins expressed in β-cells which affect their function.
Results: Western blot analysis using anti-Gla antibodies developed by our group suggest that hepatocytes and pancreatic islets express the same intracellular g-carboxylated proteins. Strikingly, these Gla proteins were increased in pancreatic islets taken from diabetic Ins2-Akita, ob/ob, and db/db mouse. Gla proteins were therefore immunoprecipitated (IP) from liver extracts and identified using a proteomic approach. This led to the identification of two novel carboxylated proteins encoded by the same gene: aspartyl/asparaginyl β-hydroxylase (ASPH) and humbug, a shorter isoform of ASPH, which lacks its catalytic domain. ASPH catalyzes the addition of a hydroxyl group to aspartate and asparagine residues in epidermal growth factor-like domains, while Humbug may act as a Ca2+-sensing ER protein. Quantitative PCR (qPCR) and RNA sequencing on mouse islets confirmed that both ASPH and humbug are highly expressed in pancreatic endocrine cells. Deletion of a 192 amino acid long Glu rich domain (39 glutamic acid residues) shared by both ASPH and humbug completely abrogated carboxylation of both ASPH and humbug as seen by Western blotting with the anti-Gla antibody. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and serial deletion analysis revealed the Gla residues to be clustered around the N- and C-terminal regions of the Glu rich domain. Importantly, neither ASPH nor humbug were g-carboxylated in the presence of warfarin, an inhibitor of ùvk oxidoreductase, demonstrating they are subjected to VK-dependent g-carboxylation. Preliminary results show that Gas6, a secreted protein which is β-hydroxylated by ASPH, may be less β-hydroxylated in the presence of VK as seen by LC-MS/MS analysis. This suggests g-carboxylation may negatively regulate ASPH enzymatic activity.
Conclusion: We have identified two novel Gla proteins expressed in pancreatic islets: ASPH and humbug. Our current work is aimed at determining if g-carboxylation directly controls the functions of ASPH and humbug using cellular and biochemical approaches. We are also generating a mouse line lacking either of these two proteins to assess their role in β-cell function in vivo.
Obesity is a global epidemic and the identification of factors which affect energy balance is essential in the search for therapeutics. Thermogenesis is known to combat obesity through energy expenditure. A novel thermogenic pathway has been uncovered in which creatine cycling plays a key role; determining the main factors in the regulation of this pathway will lead to new potential therapeutic targets against obesity. We have now identified creatine kinase, brain type (CKB) as the key candidate for mediating this effect in brown adipocytes. Out of the known creatine kinase isoforms in brown adipocytes, CKB depletion was uniquely capable of blunting respiration. In addition, this effect was also selective to brown adipocytes, as CKB knockdown has no effect on respiration of white adipocytes, preadipocytes, or mouse embryonic fibroblasts. To begin to examine these seemingly unique aspects of CKB in brown fat, we have purified CKB from brown adipocytes and have shown it to have higher specific activity than CKB purified from other cell types. Previous work (mainly studies on the muscle variant of creatine kinase) has shown that creatine kinase activity is modulated in two key ways in vivo, complex formation and post translational modifications (PTM). Through co-immunoprecipitation followed by proteomics, we have elucidated binding partners, as well as previously uncharacterized PTMs of CKB that are specific to thermogenically activated brown adipocytes. The regulation of CKB in brown fat by these interacting proteins and PTMs represent potential factors which can explain the specificity of CKB for the control of respiration. Future work will uncover the mechanism behind CKB regulation in the context of creatine dependent thermogenesis.
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of the beta cells of the pancreas. This autoimmune reaction is primarily driven by autoreactive CD4 and CD8 T cells that escape negative selection in the thymus. In addition to thymic dendritic and epithelial cells, a recent report suggested that class-switched thymic B cells contribute to thymic negative selection in C57BL/6 mice. To determine whether class-switched thymic B cells correlate with thymic selection beyond the C57BL/6 strain, we immunophenotyped eight different mouse strains. We demonstrate that the proportion of class-switched thymic B cells positively correlates with negative selection. Interestingly, the strain with the lowest percentage of class-switched thymic B cells is the NOD mice, which spontaneously develop autoimmune diabetes. To determine if isotype switching contributes to the maintenance of T cell tolerance, we used NOD.Aid-/-mice, where a deficiency in Aid completely prevents isotype switching. We find a greater proportion of self-reactive T cells in NOD.Aid-/-mice when compared to NOD mice, in agreement with the higher incidence of diabetes in NOD.Aid-/-mice. Altogether, our work demonstrates that B cell class switching contributes to the maintenance of T cell tolerance, likely by facilitating negative selection of autoreactive thymocytes. Understanding how class switching in the thymus modulates the T cell repertoire could lead to new immunotherapies against various autoimmune diseases.
Background: In non-diabetic obese individuals, β-cell mass accretion maintains glucose homeostasis by balancing levels of circulating insulin and insulin resistance. Type 2 diabetes appears when these compensatory mechanisms fail. Deciphering the pathways controlling β-cell proliferation has thus become a major research goal in the hope to identify therapeutic targets to expand β-cell mass and prevent or delay the onset of diabetes. Previously we demonstrated that fatty acids potentiate glucose induced β-cell proliferation in response to nutrient surfeit in rats. In recent studies we identified the mono-unsaturated fatty-acid oleate as a major fatty acid driving β-cell proliferation in rat islets ex vivo.
Aim: To identify the transcriptional mechanisms underlying oleate-induced β-cell proliferation in rats.
Method: Rat islets were exposed to 16.7mM glucose +/- oleate (0.5mM) for 48h. Proliferation was assessed by flow cytometry using c-peptide antibodies and EdU. Single cell cDNA libraries were generated using 10X Genomics technology and sequenced on the Illumina platform. Bioinformatic analyses were performed using the Cell Ranger pipeline, Seurat and Monocle.
Results: Oleate induced a 3.1+/-0.4 fold increase (p<0.01; n=4) in β-cell proliferation. Following single-cell RNA sequencing, t-SNE plots revealed several β-cell sub-populations, including proliferating β-cells. Comparison between proliferative and non-proliferative β-cells identified more than 300 differentially expressed genes. Gene ontology suggested an increase in mitochondrial activity and reactive oxygen species (ROS) in proliferating β-cells. Pseudotime ordering of β cells revealed that ER-stress related genes are up-regulated prior to proliferation in response to oleate.
Conclusion: Our study suggests an involvement of ROS and ER-stress in oleate-induced β-cells proliferation. Further functional analyses will substantiate the importance of these stress responses.
After their isolation, primary hepatocytes, which are an important model for the analysis of liver metabolism, differentiate within few days into fibroblasts and lose their hepatic functionality. This is a major limitation of this in vitro model. Previous studies showed that primary human hepatocytes (PHH) can be cultured up to two months by using chemicals that modulate signaling pathways responsible for the expression of hepatocyte specific genes. Those studies showed that PHH kept their morphology, albumin and urea production as well as the enzymatic activity of Cytochrome P450 after several days in culture. However, it is not known whether these long-term cultured hepatocytes retained their metabolic functionality such as insulin signaling and glucose production. Here, we show that primary mouse hepatocytes obtained by liver perfusion and cultured for 7 days in the presence of four chemicals : Forskolin (Adenylyl cyclase activator), DAPT (Notch pathway inhibitor), LDN (Bone morphogenetic protein signaling inhibitor) and IWP2 (Wnt pathway inhibitor), kept the typical morphology of hepatocytes and the metabolic response to insulin and glucagon. Hepatic glucose production has been verified at different time points. Insulin signaling was determined by western blot by measuring Akt Ser473 and Thr308 phosphorylation. Immunofluorescence analysis was done to verify the hepatic marker albumin and the fibroblast marker vimentin to evaluate differentiation capacity. Albumin expression was also verified by Western Blot. This work confirms that hepatocytes kept most of their metabolic functionality compared to freshly isolated hepatocytes after 7 days of culture in the presence of the chemicals making this a promising model for long-term studies in primary hepatocytes.
Objective: To identify baseline characteristics that are associated with a reduction in body mass index z-score (zBMI) among youth enrolled in a healthy behaviour intervention.
Methods: CIRCUIT is a behaviour intervention using personalized strategies to increase physical activity (PA) among youth aged 5-18y at risk for cardiovascular disease. Participation (n=211; 46% female; mean zBMI=3.3±3.1) was considered successful if a reduction in zBMI was achieved over a one-year period. Differences in baseline characteristics between the success and non-success groups were explored using chi-squared- and t-tests. Baseline characteristics included sociodemographic (age, sex, ethnicity, maternal education, family structure), psychosocial (based on indicators of maternal support for PA), zBMI at initiation of the program, and features of the residential built environment, based on 1-km road network buffers.
Results: Participants in the success group (n=120) were younger (mean: 10.3y vs 12.2y, p<0.01) compared to participants in the non-success group (n=91). They also were more likely to report having mothers who enjoyed doing PA (46% vs 27%, p=0.06) and planned PA for outings (40% vs 22%, p=0.05). Neighbourhoods of the success group also had higher land use mix (0.41 vs 0.38, p=0.09) and were more likely to have a supermarket (52% vs 37%, p=0.08).
Conclusion: CIRCUIT was more likely to be successful among participants who were younger, had greater maternal support for PA, and lived in neighbourhoods with more diverse features and greater access to supermarkets. These observations further underscore the importance of both family and neighborhood environments in the treatment of childhood obesity.