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Doctoral Student Supervision (Jan 2008 - Nov 2019)
There are numerous parallels between aging and obesity, and insulin may play a crucial role in modulating both conditions. For instance, elevated insulin levels are closely associated with obesity, although the causal role of insulin hypersecretion in the development of obesity remains controversial. Interestingly, genetically reducing components of insulin/insulin-like growth factor (IGF)-1 signaling can increase lifespan in invertebrates and mammals. However, impaired insulin-stimulated glucose disposal is a form of decreased insulin signaling that is paradoxically a detrimental feature of mammalian aging, whereas long-living mammals often show enhanced responsiveness to insulin stimulation. Therefore, the role of insulin/IGF-1 signaling for mammalian longevity, and the relative functions of the insulin and IGF-1 ligands, are still unclear. The lifelong effects of moderately decreasing insulin production in mammals had not been directly tested. In this dissertation, the goal was to further delineate effects of lowering insulin levels on obesity and metabolic health across the lifespan of a mammalian model organism, culminating in an evaluation of longevity. We used a model in which the rodent-specific insulin gene was fully inactivated (Ins1-null), and compared mice with full or partial expression of the ancestral insulin gene (Ins2). Male and female Ins1-/-:Ins2+/- and Ins1-/-:Ins2+/+ littermates were fed a chow diet or high fat diet, and were evaluated across their lifetime to determine long-term effects of reducing insulin gene dosage on obesity, glucose homeostasis, and other physiological parameters. The studies herein show that murine insulin levels and metabolic homeostasis might be regulated in a sex-specific, environmentally-dependent manner, since inactivating one Ins2 allele unexpectedly did not cause a consistent reduction of circulating insulin in Ins1-null male mice, and we observed cross-cohort hyper-variability in circulating insulin of male mice. However, limiting insulin hypersecretion in young, growing female mice can confer long-term protection against obesity. Furthermore, we found that lowering circulating insulin has the potential to improve glucose homeostasis and insulin sensitivity in advanced age, as well as lead to lifespan extension in mammals. To our knowledge, these studies are the first to demonstrate that a targeted, moderate reduction of insulin may be sufficient to promote healthier aging and extend lifespan in mammals.
No abstract available.
Obesity is one of the biggest health concerns around the world and is closely associated with insulin hypersecretion. However, the causality relationship between these conditions remains enigmatic. We tested the hypothesis that fasting hyperinsulinemia is necessary for diet-induced obesity by varying the pancreatic-specific Ins1 gene dosage in Ins2-/- mice. Male Ins1+/-:Ins2-/- mice did not exhibit high fat diet-induced fasting hyperinsulinemia, when compared with their Ins1+/+:Ins2-/- littermate controls. This genetic inability to become hyperinsulinemic prevented the expected increase in pancreatic β-cell number, confirming a role for insulin in high fat diet-induced β-cell expansion. Male Ins1+/-:Ins2-/- mice were also protected from diet-induced obesity and hepatic steatosis when compared to high fat fed Ins1+/+:Ins2-/- littermate controls in the absence of sustained changes in glucose homeostasis. Genetic prevention of hyperinsulinemia increased energy expenditure while reducing adipose inflammation and fatty acid spillover. Female control Ins1+/+:Ins2-/- mice did not exhibit hyperinsulinemia or weight gain on the high fat diet we employed, so it was not possible to test the same hypothesis in the female mice. The effects of reducing Ins2 gene dosage on the Ins1 null background were also assessed. Male Ins1-/-:Ins2+/- mice had a phenotype that differed strongly between cohorts. In one cohort of the male mice, Ins2 haploinsufficiency was associated with increased food intake of the high fat diet, relative to Ins1-/-:Ins2+/- mice fed the same diet, but no changes in circulating insulin levels. On the other hand, female Ins1-/-:Ins2+/- mice were partially protected from high fat diet-induced obesity relative to their littermate controls. The differences in the consequences of Ins1 versus Ins2 loss prompted analysis of the tissue expression of both insulin genes, focusing on the central nervous system. We demonstrated that, unlike Ins1, Ins2 is expressed in the brain. High fat feeding reduced Ins2 expression in the brain in a region- and sex-specific manner. Collectively, our data provide genetic evidence that circulating hyperinsulinemia can drive obesity in mammals. These findings may be important for understanding the causes of obesity and eventually the development of approaches to prevent or treat it.
Disrupted pancreatic β-cell function is a key event in the pathogenesis of diabetes mellitus, a metabolic disorder resulting in elevated blood sugar levels. β-cells are responsible for the secretion of insulin, which promotes the uptake of blood glucose into peripheral tissue. Additionally, autocrine insulin signalling contributes to the maintenance of properly functioning β-cells. Upon insulin binding, the insulin receptor tyrosine kinase is activated and recruits insulin receptor substrates to its intracellular domain. These substrates can activate two major signalling branches, the Akt branch and the Ras/Erk branch. Both signalling branches are suggested to be involved in the maintenance of β-cell function and survival. Interestingly, results from experiments in adipose-like cell lines demonstrate, that endocytic vesicles can act as signalling hubs potentially directing insulin receptor signals between the Erk and Akt branches. Endosomes have also been suggested as organelles that are capable of buffering the rapid influx of calcium into β-cells following glucose stimulation thus avoiding calcium-induced β-cell death. These findings highlight endosomes as important organelles involved in the maintenance of β-cell function. This thesis examines the role of endosomes in autocrine insulin signalling and their involvement in calcium homeostasis. To observe the impact of endocytosis on autocrine insulin signalling, a novel fluorescent protein-labeled insulin receptor construct was developed and validated, revealing that tyrosine-phosphorylated caveolin-1 (Cav1) participates in insulin receptor internalization in β-cells. Remarkably, this process was found to bias insulin signalling towards the Erk branch in vitro and in vivo. As a functional consequence, reduction of Cav1 activity inhibited Erk signalling and was associated with increased β-cell apoptosis and decreased β-cell mass in mice lacking Cav1. The role of endosomes in β-cell calcium buffering was elucidated by creating a genetically encoded calcium sensor specifically localized to the lumen of endosomes and estimating calcium levels in defined endosome sub-populations. Indeed, endosomes accumulate calcium during glucose stimulation. Together, this work highlights endosomes as hubs for autocrine insulin signalling and contributors to the calcium homeostasis in the glucose response of β-cells.
Programmed β-cell death plays an important role in both type 1 and type 2 diabetes, but analysis of candidate survival factors has yielded a few hormones and growth factors exhibiting modest β-cell protection against various stresses. Most of what is known about the mechanisms of β-cell death comes from single time-point, single parameter measurements of bulk populations of mixed cells, which are inadequate for studying the heterogeneity in death mechanisms. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with bright-field imaging. This allowed the characterization of the timing and order of molecular events associated with cell death in single β-cells under multiple diabetic stress conditions. Using this approach, we identified several cell death modes where the order of events that typically define apoptosis were not observed. It is becoming increasingly apparent that islets release and respond to more secreted factors than previously thought and systematic analyses of their pro-survival effects can assist in therapeutic developments. Novel putative autocrine/paracrine signalling loops in islets were identified by compiling results from gene expression datasets. Factors best known for their roles in axon guidance, Netrin and Slit families, were further characterized for their pro-survival roles in adult β-cells. With the development of the live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate β-cell death, we screened the Prestwick library of small molecules and a custom library of endogenous factors. Carbamazepine, a Na+ channel inhibitor, down-regulated the pro-apoptotic and ER-stress signalling induced by cytotoxic cytokines pointing to Na+ channels as a novel therapeutic target in diabetes. Whether specific cellular stresses associated with type 1 or type 2 diabetes require specific β-cell survival factors remains unknown. Our comparison of 206 endogenous soluble factors, predicted to act on islet cells, under 5 diabetes-relevant stress conditions revealed unique sets of protective survival factors for each stress and identified a cluster of survival factors that exhibited generalized protective effects. Since diabetes results from a deficiency in functional β-cell mass, these studies are important steps towards developing novel therapies to improve β-cell survival and function.
A decrease in functional β-cell mass is key in the pathogenesis of both type 1 and type 2 diabetes and in the failure of transplanted islet grafts. Knowledge of the endogenous regulators of β-cell proliferation and survival are important for understanding the physiological regulation of β-cell mass. We have shown that physiological concentrations of the insulin hormone act directly on β-cells to promote proliferation and survival, but its mechanisms remain unclear. We hypothesized that Raf-1, a kinase upstream of both ERK1/2 and Bad, is a critical target of insulin in β-cells. To test this hypothesis, we treated primary β-cells and MIN6 β-cells with multiple insulin concentrations and examined putative downstream targets. Low concentrations of insulin rapidly activated Raf-1 and ERK1/2 in primary islets and MIN6 cells. The phosphorylation of ERK1/2 by insulin was eliminated by exposure to a Raf inhibitor or transfection with a dominant-negative Raf-1 mutant. Insulin enhanced the interaction between mitochondrial Raf-1 and Bad, promoting the inactivation of pro-apoptotic Bad. Over-expression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of insulin. We also tested if Raf-1 signalling plays an important role in β-cell survival both in vitro and in vivo. We utilized a Raf inhibitor and dominant-negative Raf-1 mutants to block basal Raf-1 signalling in serum free conditions in vitro and the Cre-lox recombination system to obtain a β-cell specific deletion of the Raf-1 gene in vivo. Our data show that blocking basal Raf-1 signalling in vitro caused apoptosis. Preliminary data indicate that β-cell specific Raf-1 knockout mice are viable, have increased fasting basal blood glucose levels and have impaired glucose tolerance compared to littermate controls, consistent with the concept that Raf-1 plays an important role in β-cell survival. Together, these findings have significant implications for the understanding of insulin signalling pathway in β-cells and the regulation of β-cell mass.
Master's Student Supervision (2010 - 2018)
Pancreatic β cells are selectively destroyed by the host immune system in type 1 diabetes, which results in the inability to regulate glucose homeostasis due to loss of insulin production capacity. Drugs that preserve β cell mass and function therefore have the potential to prevent or slow the progression of this disease. It was recently reported by our group that the use-dependent sodium channel blocker, carbamazepine, protects pancreatic β cells from inflammatory cytokines in vitro. Subsequent experiments found carbamazepine increased insulin gene expression, which corroborated with an increase in insulin content in islets from mice lacking the Nav1.7 voltage gated sodium channel, which was shown to be a target of carbamazepine in β cells. While these in vitro results were promising, it was unclear whether carbamazepine would protect β cells in vivo against a complete immune system. Therefore, we tested the effects of oral treatment in female non-obese diabetic (NOD) mice, achieving serum carbamazepine levels of 14.98 ± 3.19 μM. Remarkably, diabetes incidence over 25 weeks was ~50% lower in carbamazepine treated animals. Partial protection from diabetes in carbamazepine-fed NOD mice was also associated with improved glucose tolerance at 6 weeks of age, prior to the onset of diabetes in our colony. Insulitis was improved in carbamazepine treated NOD mice at 6 weeks of age, but we did not observe differences in CD4⁺ and CD8⁺ T cell composition in the pancreatic lymph node, as well as circulating markers of inflammation. Taken together, our results demonstrate that carbamazepine reduces the development of type 1 diabetes in NOD mice.
Therapeutic benefits of caloric restriction (CR) on clinical outcomes in the treatment of neurodegenerative disease, cancer, cardiovascular disease, and diabetes have been found. Studies indicate the positive health outcomes produced by CR may involve cellular nutrient-sensing pathways including insulin/insulin-like growth factor 1 signalling. CR has been reported to have significant effects on glucose metabolism and body composition: lowering fasting blood glucose and insulin, improving glucose tolerance, insulin sensitivity and decreasing energy expenditure and body fat. However, it is not clear which, if any, of the effects of CR are due to the lowering of circulating insulin. To determine which CR effects are a function of circulating insulin-dependent mechanisms we placed female Ins1+/-:Ins2-/- mice and Ins1+/+:Ins2-/- littermate controls on either a chow diet ad libitum (AL) or on a CR diet where they were fed 60% of what their genotype-matched littermate controls ate daily. All mice were singly housed and CR mice fed at night. With the onset of CR, body mass in both genotypes fell and reached a new equilibrium by 20 weeks of age. As expected, mice on CR had lower fasting, fed plasma glucose and improved glucose tolerance when compared to AL controls. We observed a more rapid return to baseline glucose post-insulin injection in mice on CR and no difference in glucose-stimulated insulin secretion compared to AL littermates. CR was able to prevent an age-dependent decline in fasting insulin of Ins1+/-:Ins2-/- mice. Ins1+/+:Ins2-/- and Ins1+/-:Ins2-/- on CR also exhibited increased plasma leptin, glucose-dependent insulinotropic peptide, subcutaneous white and intrascapular brown adipose tissue size compared to the AL controls. The endocrine milieu created in these very low insulin mice appears to disrupt several well-established effects of CR on body composition, insulin and insulin sensitivity.
Epidemiological studies have reported positive correlation between type 2 diabetes and risk of pancreatic cancer. Hyperinsulinemia occurring during early diabetes has been postulated to be a possible pathophysiological mechanism in promoting pancreatic cancer, but the mechanisms by which elevated insulin levels contribute to pancreatic carcinogenesis are still unknown. Here, the effects of insulin and its associated mechanism on cellular viability were examined in three cell models that are meant to represent three stages in pancreatic cancer progression. Furthermore, using small molecule inhibitors, the role of RAF/ERK pathway and PI3K/AKT pathway on cellular viability were also compared across three cell models. Different stages of pancreatic cancer progression were modeled in vitro with primary human pancreatic ductal cells, an immortalized pre-malignant human pancreatic ductal cell line (HPDE), and an advanced metastatic human pancreatic adenocarcinoma cell line (PANC1). All cell types were serum starved and treated with insulin, IGF1, or small molecule inhibitors targeting RAF1, MEK, AKT, or PI3K, then subjected to cell viability assays, cell death assays, and Western blot analysis for ERK and AKT activation. We observed that cell viability was promoted by exogenous insulin in PANC1 cells, and not in primary cells. In PANC1 cells, the insulin-mediated enhancement of cell viability was associated with sustained AKT activation. Furthermore, insulin-mediated reduction in cell death was not observed. When comparing the roles of RAF/ERK and PI3K/AKT pathways on cell viability, we observed an increase in cell death in primary cells when AKT was inhibited. In contrast, cell death was induced in HPDE and PANC1 cells when the RAF1 or MEK were inhibited. If extrapolated, the data suggest that hyperinsulinemia may not play a role in initiating pancreatic cancer, but high levels of insulin may accelerate the cancer progression.
UBC scientists discover possible “obesity gene” (10 Aug 2015)