James Johnson
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer death in Canada and its incidence is increasing, largely driven by the expanding epidemics of PDAC risk factors including obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased PDAC incidence and mortality. Despite epidemiological data linking hyperinsulinemia to PDAC, there was no direct in vivo evidence of a causal role for endogenous insulin in any cancer type before this work. We studied how reduced insulin production or local insulin action affected the development of pancreatic intraepithelial neoplasia (PanIN) precursor lesions in Ptf1aCreER;KrasLSL-G12D mice. We first generated Ptf1aCreER;KrasLSL-G12D;Ins1+/+;Ins2-/- control and Ptf1aCreER;KrasLSL-G12D;Ins1+/-;Ins2-/- experimental mice. We found high fat diet (HFD)-induced hyperinsulinemia was modestly reduced in experimental mice without affecting glucose homeostasis. Genetically reduced insulin production resulted in ~50% suppression of PanIN. However, in this study, only female mice remained normoglycemic and only the gene dosage of rodent-specific Ins1 alleles was tested. Therefore, we then generated Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/+ control and Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/- experimental mice. Mice with reduced insulin production tended to develop fewer PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia modulated pathways associated with protein translation, MAPK/ERK signaling and PI3K/AKT/mTOR signaling, which were changed in epithelial cells and subsets of immune cells. Finally, we examined whether hyperinsulinemia contributed to PDAC development directly through insulin receptor (INSR) signaling in KrasG12D carrying pancreatic acinar cells. We generated Ptf1aCreER;LSL-KrasG12D;nTnG mice with an Insrwt/wt, Insrwt/f, or Insrf/f genotype to reduce insulin receptor mRNA by 0%, 50%, or 100% in acinar cells. Loss of insulin receptors from acinar cells did not significantly influence body weight, fasting glucose, or insulin levels. Compared to mice with wild-type INSR expression in acinar cells, mice lacking INSR had a 2.7-fold and 5.3-fold significant reduction in PanIN plus tumor area in males and females, respectively. Collectively, these results indicate that hyperinsulinemia and INSR signaling in acinar cells are important for the early stages of pancreatic cancer. Insulin-lowering interventions such as lifestyle management and therapies targeting insulin receptor signaling may be beneficial in preventing and/or treating pancreatic cancer.
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Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have suggested that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined. Particularly, the regulation of insulin receptor (INSR) mRNA levels, protein abundance, cell-surface dynamics, and internalization in the presence and absence of insulin are incompletely understood in muscle cells. To study the direct effects of insulin on INSR in muscle cells, we conducted in vitro studies on C2C12 myotubes and myoblasts, and analyzed publicly available human muscle transcriptomic data. Our in vitro studies established that acute AKT and ERK signalling were attenuated by 16 hours of hyperinsulinemia. RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signalling, FOXO signalling, and glucose metabolism pathways indicative of ‘hyperinsulinemia’ and ‘starvation’ programs. We observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Transcriptomic meta-analysis in >450 human samples demonstrated a reliable negative correlation between fasting insulin and INSR mRNA in skeletal muscle. Bioinformatic modelling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA and JUND, MAX, and MXI as positive regulators of Irs2 mRNA. To study INSR internalization, we used surface labelling and live-cell imaging and observed robust basal internalization of INSR and relatively modest effects of insulin in C2C12 myoblasts. We performed a stringent mass spectrophotometry analysis of INSR interactors to provide clues as to the molecular mechanisms associated with internalization, which identified previously unappreciated interactors such as ANXA2. Mapping these interactors into a protein-protein interaction network pointed to a role for caveolin-mediated endocytosis. Interestingly, INSR interacted with both caveolin and clathrin in mouse skeletal muscle and C2C12 myoblasts, with the interactions modulated by insulin. Together, this work identifies novel mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology of type 2 diabetes.
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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.
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The cardiac ryanodine receptor 2 (RYR2) is a sarcoplasmic reticulum Ca²⁺ release channel central to cardiomyocyte biology. RYR2 Ca²⁺ release has a well-established role in activating cardiomyocyte motor proteins during excitation-contraction coupling and is therefore critical for heart function. RYR2 is also poised to have other important cardiac functions such as setting heart rate, stimulating ATP metabolism, regulating cardiac hypertrophy, and controlling cardiomyocyte survival. In addition, there is evidence that RYR2 dysfunction occurs during heart disease, suggesting that RYR2 may be a driver of cardiac pathology. The research in this thesis seeks to test which aspects of cardiomyocyte biology are regulated by RYR2 signaling and whether RYR2 loss-of-function is pathogenic. Using a heart-specific, inducible gene deletion system in mice we were able to show that loss of Ryr2 caused heart failure and reduced cardiac contraction. In addition, we saw that Ryr2 deletion lead to reduced heart rate, tachycardic arrhythmia, diminished oxidative metabolism, increased cardiac hypertrophy, and increased cell death via a novel mechanism. To test whether the metabolic and heart rate effects persist in the absence of heart failure, we used an inducible, heart-specific 50% Ryr2 deletion model. In this context we did not see heart failure or decreased cardiac function, but still observed a decrease in heart rate and altered oxidative metabolism. Unlike complete Ryr2 knockout, the 50% Ryr2 ablation model did not display a general decrease in oxidative ATP metabolism, but instead a specific decrease in glucose oxidation. This was associated with reduced mitochondrial Ca²⁺ uptake and decreased activation of the pyruvate dehydrogenase complex, a Ca²⁺ sensitive gatekeeper of glucose oxidation. Collectively, these results provide compelling evidence that RYR2 is an essential component of excitation-contraction coupling and a critical driver of cardiac pacemaking. These results also demonstrate that RYR2 is critical for mitochondrial Ca²⁺ uptake and stimulating oxidative metabolism and strongly suggest that RYR2 has a specific role in activating glucose oxidation. This research also shows that loss of Ryr2 recapitulates heart failure and suggests RYR2 may be involved in hypertrophy and cell death. This suggests a model where RYR2 simultaneously regulates a several facets of cardiomyocyte biology.
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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.
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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.
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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.
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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.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Death and dysfunction of insulin producing pancreatic β-cells is a major feature of diabetes. Recently, it was discovered that the eukaryotic initiation factor 2A (EIF2A) – an unconventional translation factor – was uniquely abundant in β-cells compared to other cell types. Past studies using lentiviral transduction to overexpress EIF2A in MIN6 cells experiencing ER stress resulted in increased β-cell survival and reversed the downregulation of global translation that normally occurs as part of the unfolded protein response (UPR), as it seeks to alleviate stress. In this study, we used MIN6 cells and human islets transduced with adenoviruses to induce EIF2A overexpression. Amino acid radiolabeling was used to measure nascent global translation, in combination with immunoprecipitation to isolate and measure proinsulin synthesis. Western blotting was used to quantify changes in steady state protein levels under stressed and unstressed conditions. In MIN6 cells, adenovirus-induced overexpression of GFP-tagged EIF2A did not result in restoration of translation as previously seen, and proinsulin synthesis did not appear to be affected separately. However, EIF2A overexpression appeared to improve proinsulin-insulin conversion under stress. In human islets, regardless of EIF2A overexpression, thapsigargin treatment did not induce a significant repression of translation, nor were we able to measure proinsulin synthesis in any condition using our methods. Western blotting showed that EIF2B subunits were affected differentially by EIF2A overexpression. EIF2B5, which contains the guanine exchange factor catalytic site, was upregulated in EIF2A overexpressing cells in both stressed and unstressed conditions. EIF2B3, which is responsible for GTP binding, was upregulated in EIF2A-overexpressing cells only under stress. The EIF2B2 subunit, which is part of the core that mediates interactions with EIF2S1, showed no trend towards upregulation by EIF2A overexpression. This suggests that EIF2A’s effects on translation may indeed be mediated through EIF2B, but consequently the exact mechanism by which this occurs, and whether or not there is a specific link to insulin synthesis, remain questions in need of further study.
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Diabetes is caused by the dysfunction and/or destruction of insulin-producing pancreatic β-cells, and curative therapy will require their repair, regeneration, or replacement. Beyond insulin, islets have been reported to contain transcripts for hundreds of other ligands and receptors. Some of these intercellular signals may have therapeutic benefit in diabetes, or in efforts to generate insulin-producing cells from stem cells. Over 90% of the small molecules and proteins used to generate stem cell-derived β-like cells (SCβ-cells) are activators or inhibitors of receptors, and modulation of signaling pathways can alter SCβ-cell differentiation. In this study, we systematically mapped intercellular signaling in human islets and stem cell-derived cells, and built prioritized lists of protein ligands and receptors to guide future screening studies. First, we created a custom ligand-receptor database of 422 protein ligands, 349 receptors, and 1552 interactions. We then integrated multiple datasets to rank ligands and receptors in stem cell-derived cells based on gene expression, protein abundance, and differential expression and abundance compared to human islets. We combined the ligand and receptor ranks to rank each interacting ligand-receptor pair. Finally, we presented three prioritized lists to guide future screens: scarce ligands with abundant receptors in stem cell-derived cells, scarce receptors with abundant ligands in stem cell-derived cells, and abundant receptors with abundant ligands in stem cell-derived cells. Collectively, we expect these data will enable future studies to define the roles of ligands and receptors in diabetes and diabetes therapies.
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Insulin resistance in the brain is associated with Alzheimer’s disease, the most common form of dementia with women being more affected than men. Diabetes is a metabolic disorder signified by dysfunctional insulin release and action, and it is a risk factor for Alzheimer’s Disease. Interestingly, administration of insulin to the brain is an effective experimental treatment to improve learning and memory impairments in Alzheimer’s Disease patients. This suggests an important role of insulin action local to the brain. Increasingly more research reports that insulin can be synthesized in the hippocampus, the brain region responsible for learning and memory, and one of the only areas in which new neurons can form throughout adulthood. However, not much is known about how brain-derived insulin effects adult neurogenesis and what factors modulate its expression. This study aimed to confirm insulin production in the hippocampus, assess its modulation by diet and exercise, and identify how brain-derived insulin loss affects adult neurogenesis in female and male mice. I hypothesized that insulin expression is most abundant in the hippocampus, that a high carbohydrate diet decreases insulin expression while exercise increases its expression, and that brain-derived insulin loss decreases adult neurogenesis. To examine these aims I utilized a genetically engineered mouse model with deletion of the Ins2 gene (expressed in the brain) while leaving the Ins1 gene (expressed only in the pancreas), and thus normal glucose homeostasis, intact. In line with my prediction, Ins2 mRNA was high in the hippocampus compared to other regions. Ins2 mRNA was expressed at higher levels in hippocampi of females than males. Contrary to my prediction, Ins2 expression was higher in females eating a high carbohydrate diet and exercise blunted this effect. Surprisingly, the deletion of Ins2 alleles led to increased cell proliferation and this effect was sexually dimorphic. Overall, these results suggest brain-derived insulin differs between sexes and influences adult neurogenesis in mice, highlighting the potential of brain-derived insulin for therapeutic research of Alzheimer’s Disease.
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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of merely 10%. Mouse studies in the past decade have made progress towards a better understanding of how PDAC cellular origin affects tumorigenesis. However, there is little study done on the immune microenvironment differences between acinar and ductal cell-derived precursor lesions and PDAC. Following our previous study that showed loss of Pten with oncogenic KrasG12D mutations in the ductal cells (KPtenΔDuct/+) resulted the formation of intraductal papillary mucinous neoplasias (IPMN) as the precursor lesion in mice, we further found similar mutations in the acinar cells (KPtenΔAcinar/+) formed pancreatic intraepithelial neoplasia (PanIN) instead. I subsequently used the KPtenΔDuct/+and KPtenΔAcinar/+ models to elucidate the effect of cellular origin on the immune microenvironment by performing immunohistochemistry. I focused on immune cell infiltration densities in precursor lesions and PDAC derived from KPtenΔDuct/+ and KPtenΔAcinar/+ mice and found that immune cell population and its changes throughout tumorigenesis are different starting at a precursor lesion stage between these two models. Additionally, macrophages polarized by conditioned media derived from KPtenΔDuct PDAC cells were polarized in less magnitude compared with macrophages polarized by KPtenΔAcinar PDAC cells. This difference in polarization was at least partially due to the lower expression of GM-CSF in KPtenΔDuct PDAC cells. Our study is the first to directly compare immune cell population between acinar- and ductal-derived PDAC with the same genetic background. Our study suggests cellular origin could influence PDAC immune heterogeneity.
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The risk of developing type 2 diabetes (T2D) is ~40% higher in men than in pre-menopausal women. While lifestyle and cultural factors play a role in this male-biased risk of T2D, studies across animal species suggest biological sex contributes significantly to the sex difference in developing T2D. Large-scale gene expression studies suggest sex differences in pancreatic β cells play a role in the differential T2D risk between men and women. Yet, we lack a comprehensive understanding of β cell dysfunction between the sexes in both normal and pathological contexts. Here, we examined scRNA-seq data of human insulin-producing pancreatic β cells from non-diabetic (ND) and T2D men and women, revealing profound sex-specific changes to β cell gene expression in T2D. To gain deeper insight into sex-specific β cell responses in T2D, we sought a detailed understanding of β cell gene expression in normal physiological conditions. Unbiased pathway analysis of our well-powered islet RNAseq dataset from 20-week-old male and female mice with equivalent insulin sensitivity revealed a sex difference in the enrichment of UPR pathway-associated genes under basal conditions. Because female islets had higher expression of genes involved in protein synthesis, folding, and processing compared with males, we hypothesized female islets would be more resilient to acute ER stress induction with thapsigargin. Indeed, we found female islets resolved ER stress-induced protein synthesis repression faster than males and showed less cell death. These differences were significant for β cell function, as female islets maintained better insulin secretion than males in an ER stress context. Given the profound differences that we observed between the sexes in the transcriptional response to thapsigargin and the known links between ER stress and T2D pathogenesis, these findings provide additional insight into potential mechanisms underlying the differential risk of T2D between men and women.
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Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-secreting beta-cells. Genetic variations upstream at the insulin (INS) locus contribute to ~10% of T1D heritable risk. Multiple studies showed an association between rs3842753 C/C genotype and T1D susceptibility. Three small studies reported an association between rs3842753 C allele and increased whole pancreas INS expression. To date, no large-scale studies have looked at the effect of those genetic variations on insulin expression at the single cell level. We aligned all available human pancreatic single cell RNA sequencing datasets using STAR and used Samtools mpileup to genotype rs3842753. Using Seurat, we integrated 2315 beta-cells from 13 A/A donors, 23 A/C heterozygous donors, and 35 C/C at-risk donors. The donors included persons with and without type 2 diabetes, but not T1D. We compared variance using Bartlett’s test or Fligner-Killeen test and means using Wilcox Rank Sum, Student-t, ANOVA, or Kruskal-Wallis tests. Per β-cell INS expression mean and variance were significantly higher in females compared with males. In male cells, INS expression appeared to be significantly lower in T2D compared to non-diabetic cells. Comparing across all cells, we found that rs3842753 A/C genotype had the highest INS expression followed by C/C genotype, lastly by A/A genotype. Donor level comparisons between genotypes were not statistically significant. Conversely, within A/C heterozygous β-cells, A allele specific INS expression was higher. This association was consistent at the donor level. Lastly, we examined whole pancreatic islets from a small subset of donors and found no relationship between insulin protein abundance and rs3842753 genotype. Our analysis suggests that in single β-cells, rs3842753 may affect INS variance and expression. The contribution of these differences to T1D risk remains unclear.
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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.
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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.
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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.
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