<p>Previous studies suggest that nicotine impairs pancreatic function, which may explain the increased risk of T2DM in smokers. We have previously shown that nicotine exposure results in decreased beta cell function, an effect which appears to be mediated via increased beta cell oxidative stress. The goal of this study is to determine whether folic acid, an antioxidant, can prevent nicotine-induced beta cell dysfunction in the beta cell.</p> <p>INS 1E cells, a rat pancreatic beta cell line, were treated with nicotine or vehicle ± 10µM folic acid for 48 hours. Nicotine treatment decreased both basal and glucose stimulated insulin secretion, but had no effect on insulin content, mitochondrial function or markers of apoptosis. Expression of oxidative stress/damage markers (HSP70 and 4-HNE), antioxidant enzymes (Cu/ZnSOD, MnSOD and CAT), insulin gene transcription factor PDX1 and K_ATPchannel subunit kir_6.2 were determined by western blot analysis. Expression of HSP70, 4-HNE and MnSOD were significantly increased with nicotine treatment (p=0.002, 0.05 and 0.03 respectively). Cu/ZnSOD and CAT expression remained unchanged with nicotine treatment. The addition of folic acid significantly reduced HSP70 expression, 4-HNE expression, CAT expression, but did not alter the expression of MnSOD. There was a significant (p6.2expression (p=0.019) which showed a trend toward reduced expression following treatment with folic acid (p=0.067).</p> <p>Nicotine treatment significantly increases markers of oxidative stress and oxidative damage in pancreatic beta cells; an effect which was reversed by folic acid administration. Nicotine and folic acid treatment increased insulin content, likely mediated through an increase in the insulin gene transcription factor, PDX1. Furthermore, nicotine treatment increased expression of kir_6.2,suggesting a defect in the insulin secretory mechanism. This effect was reversed with folic acid treatment.Although many studies suggest that Canadians are meeting or exceeding recommended folate levels, this is not true in smokers. Our data suggest that additional folate supplementation in smokers may prevent nicotine-induced damage to the pancreas and thus reduce the risk of type 2 diabetes.</p> / Master of Science (MSc)

The objective of this thesis was to determine the association of leg length (LL), a marker of early childhood conditions, with metabolic abnormalities underlying type 2 diabetes. Utilizing data from a population at-risk for diabetes, the associations of LL with i) insulin resistance (IR) and beta-cell dysfunction and ii) a continuous metabolic syndrome risk score (MetScore) were analyzed. Results showed that shorter LL was associated with IR and beta-cell dysfunction, and that the combination of short legs and large waist (a marker of adult obesogenic conditions) was associated with the greatest IR. Height, a marker of overall childhood conditions, was found to be inversely associated with the MetScore. Therefore, both adverse childhood conditions and early-late life mismatched conditions may increase the risk for diabetes through differing pathways. Improving childhood conditions (i.e. nutritionally or economically) may be an important strategy to prevent diabetes.

The objective of this thesis was to determine the association of leg length (LL), a marker of early childhood conditions, with metabolic abnormalities underlying type 2 diabetes. Utilizing data from a population at-risk for diabetes, the associations of LL with i) insulin resistance (IR) and beta-cell dysfunction and ii) a continuous metabolic syndrome risk score (MetScore) were analyzed. Results showed that shorter LL was associated with IR and beta-cell dysfunction, and that the combination of short legs and large waist (a marker of adult obesogenic conditions) was associated with the greatest IR. Height, a marker of overall childhood conditions, was found to be inversely associated with the MetScore. Therefore, both adverse childhood conditions and early-late life mismatched conditions may increase the risk for diabetes through differing pathways. Improving childhood conditions (i.e. nutritionally or economically) may be an important strategy to prevent diabetes.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

Chronic hyperlipidemia (lipotoxicity) and hyperglycemia (glucotoxicity) have recently been shown to induce Endoplasmic Reticulum (ER) stress, which may contribute to pancreatic beta-cell dysfunction in type 2 diabetes. This thesis examined the involvement of ER stress in beta-cell lipotoxicity and glucotoxicity. Although chronic treatment with saturated free fatty acids (FFA) in vitro induced ER stress, altering ER stress by increasing or knocking-down GRP78 chaperone expression had no effect on apoptosis induction. Conversely, overexpression of ER chaperones rescued the reduction in proinsulin protein levels caused by chronic exposure to high glucose, although it had no effect on the decreased insulin mRNA levels and proinsulin translation rate. Thus, ER stress is likely not the main mechanism involved in saturated FFA-induced beta-cell apoptosis in vitro, but it may contribute to glucotoxic effects on proinsulin levels. These findings have increased our understanding of the link between ER stress and beta-cell dysfunction in type 2 diabetes.

Hhip (Hedgehog interacting protein), un antagoniste de la voie de signalisation Hegehog (Hh) a était devouverte comme un antagoniste des 3 ligands Hh, soit Sonic (Shh), Indian (Ihh) et Desert (Dhh). La protéines Hhip régularise la fonction cellulaire autant par voie (Hh) canonique que non-canonique. Elle est formée de 700 acides aminés et est fortement exprimée dans les tissus riches en cellules endothéliales, comme les reins et le pancréas. Toutefois, son rôle dans le fonctionnement des cellules bêta matures soit en condition de bonne santé ou de maladie comme dans des conditions d’obésité provoquée par une diète riche en gras ainsi que son role dans les maladies chronique du rein et la dysfonction rénale. Les souris en déficience de Hhip (Hhip-/-) ont une malformation des ilots pancréatiques (une diminution de 45% des ilots et de 40% de la prolifération des cellules beta) et un problème pulmonaire qui cause la mort post-natale. L’objectif de notre étude initiale était de démontrer le role de Hhip dans le pancréas, en utilisant un KO corporel entier en réponse à une diète riche en gras (HFD) et la dysfonction des cellules beta in vivo et ex vivo sur des souris hétérozygotes pour Hhip (Hhip+/-) et des souris contrôles (Hhip +/+) Suite à une HFD, toutefois, les souris mâles et femelles HFD-Hhip+/+ ont développé une intolérance sévère au glucose (IPGTT) et cette intolérance a été améliorée chez les souris HFD-Hhip+/-. Associé a cette intolérance, les males HFD-Hhip+/- démontraient une hyperinsulinémie et leur taux d’insuline plasmatique (phase 1 et 2), contrairement aux souris males HFD-Hhip+/+, augmentait de façon significative. Dans les îlots de souris Hhip+/+, l’augmentation de Hhip induite par une HFD a été observée principalement dans les cellules bêta mais aucunement dans les cellules alpha. Sans varier le nombre total d’îlots et la quantité de cellules bêta, les souris mâles HFD-Hhip+/+ avaient un nombre supérieur de gros îlots dans lesquels le taux d’insuline était diminué. La structure de ces îlots était désorganisée, démontrant une évidente invasion des cellules alpha au coeur des îlots bêta, le stress oxidatif (8-OHdG et NADPH oxidase 2 (Nox 2)) est aussi augmentée. En revanche, chez les souris mâles HFD-Hhip+/-, il a été possible d’observer une augmentation du nombre de petits îlots, de la prolifération des cellules bêta, et aussi de la sécrétion d’insuline stimulée par le glucose (GSIS), une amélioration du stress oxidatif et un maintien de l’intégrité des îlots ont été démontré. In vitro, la protéine recombinante Hhip (rHhip) a accentué le stress oxidatif (Nox2 et l’activité de NADPH oxidase 2) et a causé une diminution du nombre de cellules bêta ; par contre, le siRNA-Hhip augmente le GSIS et abolit la stimulation de l’expression du gène Nox2 induite par le palmitate de sodium (PA)-BSA. Grace a ces observations, il est démontré que les genes Hhip pancréatiques inhibe la sécrétion d’insuline en altérant la structure des ilots et en favorisant l’expression du gene Nox2 dans les ilots en réponse à la dysfonction des cellules beta suite a une diète riche en gras HFD. Le diabète engendre des risques élevés de complication tel que des problèmes chroniques des reins caractérisés par une perte graduelle des fonctions rénales. Cette situation a été récemment reliée au taux élevé d’obésité. On a aussi démontré dans notre modèle de diabète gestationnel que l’augmentation de Hhip causait des irrégularités durant la néphrogénèse des rejetons [127]. Ensuite, nos données récentes démontrent que, chez les souris adultes, l’hyperglycémie a provoqué une forte expression du gene Hhip rénales causant ainsi l’apoptose des cellules épithéliales des glomérules et la transition endothéliale à mésenchymateuse (EndoMT) - liée à fibrose rénale [128]. Dans l’étude présente, on a établi que la surexpression de Hhip dans les cellules des tubules proximaux rénaux contribuait au développement initial des problèmes chroniques des reins suite a une HFD de 14 semaines. Un gain de poids significatif a été observé chez les souris du groupe HFD comparativement aux groupes ND. Les souris du groupe HFD ont développé une intolérance au glucose mais sans changement apparent à la sensibilité à l’insuline ni à l’hypertension (pression arterielle) même si ces souris mâles avaient des légers dépôts du gras périrénal. Les fonctions rénales telle que mesurées par le taux de filtration glomérulaire restaient normales dans tous les groupes révélant ainsi que ces deux facteurs (HFD et surexpression de Hhip) n’avaient aucune influence sur l’hyperfiltration rénale. Néanmoins, la morphologie rénale a révélé que les souris du groupe HFD présentaient une lésion infraclinique et des signes de vacuolisation tubulaire et des lésions par rapport aux souris ND. Cette pathologie de lésion tubulaire et de vacuolisation était plus prononcée chez les souris transgéniques (Hhip-Tg) que chez les souris non-Tg, ce qui favorisait l'apoptose des cellules tubulaires bénignes et un stress oxydatif accru. En conclusion, l'obésité provoquée par l'HFD a eu des effets néfastes sur la tolérance au glucose et de légères modifications morphologiques des reins, caractérisées par la présence d'une néphrose osmotique, une augmentation du stress oxydatif rénal et une apoptose pouvant être induites par une augmentation de la FABP4 rénale. Cela a été exacerbé par la surexpression de Hhip dans les tubules rénaux proximaux. / Hedgehog interacting protein (Hhip), a signaling molecule in the Hedgehog Hh pathway, was originally discovered as a putative antagonist of all 3 secreted Hh ligands, i.e., Sonic (Shh), Indian (Ihh), and Desert (Dhh). Hhip regulates cell function via either canonical- or non-canonical Hh pathway. Hhip encodes a protein of 700 amino acids, and is abundantly expressed in vascular endothelial cell-rich tissues, including the pancreas, and kidneys. To date, less is known about Hhip’s expression pattern in mature islet cells, and its function under normal and/or disease conditions, such as diet induced-obesity, as well as its role in chronic kidney disease, and kidney dysfunction. Hhip null mice (Hhip-/-) display markedly impaired pancreatic islet formation (45% reduction of islet mass with a decrease of beta cell proliferation by 40%), however Hhip-/- mice die shortly after birth mainly due to lung defects. In our first study, we systemically studied the role of pancreatic Hhip expression by using a whole body knock out in response to 8 weeks high fat diet (HFD) insult, and HFD-mediated beta cell dysfunction in vivo, ex vivo and in vitro using heterozygous (Hhip+/-) vs. wild type (Hhip+/+) mice. Both HFD-fed Hhip+/+ male and female mice developed severe glucose intolerance (IPGTT), which was ameliorated in male and female HFD-Hhip+/- mice. Associated with this glucose intolerance, was hyperinsulinemia, which was observed only in HFD-fed male Hhip+/- mice. HFD-fed Hhip+/- mice had high levels of circulating plasma insulin in both insulin secretion phases compared to HFD fed Hhip+/+ mice. In the pancreas, Hhip expression was increased in the islets of HFD-Hhip+/+ mice, mainly co-localized in beta cells and none in alpha cells. While maintaining the total islet number, and beta cell mass, male HFD-Hhip+/+ mice had a higher number of larger islets, in which insulin content was reduced; islet architecture was disoriented, with evident invasion of alpha cells into the central core of beta cells; and an evident increase in oxidative stress markers (8-OHdG and NADPH oxidase 2 (Nox 2)). In contrast, male HFD-Hhip+/- mice had a higher number of smaller islets, with increased beta cell proliferation, pronounced glucose stimulated insulin secretion (GSIS), ameliorated oxidative stress and preserved islet integrity. In vitro, recombinant Hhip (rHhip) dose-dependently increased oxidative stress (Nox2 and NADPH activity), and decreased the number of insulin-positive beta cells, while siRNA-Hhip enhanced GSIS, and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We believe our data highlights a novel finding as to how pancreatic Hhip gene inhibits insulin secretion, by altering islet integrity, and promoting Nox2 gene expression in beta cells in response to HFD-mediated beta cell dysfunction. Diabetes presents high risk factors associated with complications such as chronic kidney disease (CKD) characterized by a gradual loss in kidney function. The increased incidence of diabetic related kidney complications has been recently correlated with increase rate of obesity. We recently established that impaired nephrogenesis in kidneys of offsprings of our murine model of maternal diabetes was associated with upregulation of Hhip gene expression [127]. Subsequently, our recent data also shows that hyperglycemia induced increased renal Hhip gene expression in adult murine kidneys leading to apoptosis of glomerular epithelial cells and endothelial to mesenchymal transition (Endo-MT) - related renal fibrosis [128]. In this current study, we demonstrated how Hhip overexpression in renal proximal tubular cells, contributes to early development of chronic kidney disease after 14 weeks of HFD. Mice in HFD-fed groups showed significantly greater weight gain as compared to mice in ND fed groups. IPGTT revealed that HFD fed mice also developed glucose intolerance, with no apparent changes in insulin sensitivity. HFD did not impact hypertension, even though we had a modest trend of increase in perirenal fat deposit in the HFD fed subgroups. Renal function as measured by the glomerular filtration rate was normal in all four subgroups, indicating that neither HFD, nor Hhip overexpression promoted renal hyperfiltration. Nonetheless, renal morphology revealed HFD kidneys had subclinical injury, presented signs of tubular vacuolization and damage compared to ND fed mice. This pathology of tubular damage and vacuolization was more pronounced in HFD-fed transgenic (Hhip-Tg) mice compared to non-Tg mice, and this promoted mild tubular cell apoptosis and enhanced oxidative stress. In conclusion, HFD feeding-induced obesity led to detrimental effects on glucose toleranc,e and mild morphological changes in kidneys, characterized by the presence of osmotic nephrosis, increased renal oxidative stress, and apoptosis which might be mediated by an increase in renal FABP4. This was exacerbated by the over-expression of Hhip in the renal proximal tubules.