β-Cell mass expansion is one mechanism by which obese animals compensate for insulin resistance and prevent diabetes. FoxM1 is a transcription factor that can regulate the expression of multiple cell cycle genes and is necessary for the maintenance of adult β-cell mass, β-cell proliferation, and glucose homeostasis. We hypothesized that FoxM1 is up-regulated by nondiabetic obesity and initiates a transcriptional program leading to β-cell proliferation. We performed gene expression analysis on islets from the nondiabetic C57BL/6 Leptinob/ob mouse, the diabetic BTBR Leptinob/ob mouse, and an F2 Leptinob/ob population derived from these strains. We identified obesity-driven coordinated up-regulation of islet Foxm1 and its target genes in the nondiabetic strain, correlating with β-cell mass expansion and proliferation. This up-regulation was absent in the diabetic strain. In the F2 Leptinob/ob population, increased expression of Foxm1 and its target genes segregated with higher insulin and lower glucose levels. We next studied the effects of FOXM1b overexpression on isolated mouse and human islets. We found that FoxM1 stimulated mouse and human β-cell proliferation by activating many cell cycle phases. We asked whether FOXM1 expression is also responsive to obesity in human islets by collecting RNA from human islet donors (body mass index range: 24–51). We found that the expression of FOXM1 and its target genes are positively correlated with body mass index. Our data suggest that β-cell proliferation occurs in adult obese humans in an attempt to expand β-cell mass to compensate for insulin resistance, and that the FoxM1 transcriptional program plays a key role in this process.
Type 1 and Type 2 diabetes (TID and TIID) differ in etiology, but both have decreased functional pancreatic β-cell mass. Genome-wide association studies identified transcription factor 19 (Tcf19) as a potential causal gene for both TID and TIID. Tcf19 is expressed in both humans and rodents, most highly in the pancreatic islet and upregulated in mouse models of non-diabetic obesity. We showed that TCF19 is necessary for β-cell proliferation and survival in INS1 cells. Thus, we hypothesized that TCF19 regulates β-cell mass developmentally and in adaptive response to stress. A germline whole-body knockout (wbTcf19KO) of Tcf19 mouse model was generated. wbTcf19KO and control (C57BL6/N) mice were fed a chow diet. Lean wbTcf19KO are metabolically similar to controls other than significantly higher body weights. Markers of proliferation (Ki67) and β-cell identity (Pdx1, Nkx6.1, Nkx2.2) were significantly decreased while markers of pro-apoptosis (Chop) and DNA damage response (Bak, Gadd45a, Dtx3l) were significantly increased in islets from wbTcf19KO mice. DNA damage is significantly elevated in wbTcf19KO islets as measured by y-H2AX Western blot. Islet size distribution is significantly altered in wbTcf19KO islets, skewed by many very small islets. wbTcf19KO and control male mice were challenged with high fat diet (HFD; 1-week & 10-week) feeding. These mice failed to appropriately upregulate proliferation markers (Ki67, CyclinD2) in islets after 1-week of HFD. Glucose intolerance and elevated fasting glucose develop in these mice after 10-weeks of HFD. Female mice, less susceptible to diet induced hyperglycemia, were stressed with long-term high fat high sucrose diet (HFHS) feeding to induce insulin resistance. After 8-weeks of HFHS feeding wbTcf19KO females became significantly more glucose intolerant than control HFHS fed female mice. Ongoing studies will examine the impact of Tcf19 knockout on β-cell mass during pregnancy in these HFHS-fed females. Overall, loss of Tcf19 reveals changes in proliferation and DNA damage, which may be critical in stress-induced β-cell mass regulation.
Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.
Cholecystokinin (CCK) is a gut peptide hormone that is also produced by pancreatic β-cells under conditions of metabolic stress and obesity. Treatment with exogenous CCK can alleviate obesity-related diabetes in animal models without significant side effects. We have established that CCK is expressed primarily in the pancreatic β-cell and that CCK is highly upregulated in ob/ob mice. We have also shown that elevated cellular cAMP stimulates β-cell CCK expression and secretion. However, the specific islet cell types that express CCK and the regulation of Cck transcription in the islet has not been thoroughly explored. A more recent study from our lab showed that high glucose concentrations can upregulate Cck expression in mouse islets and human islets. We hypothesize that Cck expression is upregulated in response to various diabetogenic conditions and that CCK upregulation by glucose can occur independent of cAMP activation. Islets from 16-week-old C57BL/6J mice were harvested for use in a dose-dependent 24-hour study using different glucose concentrations: No glucose, 2.5mM, 7mM, 11mM & 25mM. After 24-hour treatment, islets were collected for RNA isolation and cDNA synthesis to identify changes in Cck mRNA expression after incubation with different glucose concentrations. Cck mRNA levels were elevated 4-8 fold in islets treated with 25 mM glucose compared to islets treated with 0 to 11mM glucose. Notably, the hypoglycemic treatment of no glucose and 2.5mM showed a 4-fold increase when compared to the 7mM and 11mM treatments. Following this experiment, we wanted to determine if high glucose would still increase Cck mRNA expression after inhibiting cAMP. Islets were pre-incubated for 45 minutes with 10nM sulprostone, which activated prostaglandin E3 (EP3) receptor and inhibits adenylate cyclase. After inhibition of cAMP, we incubated the islets at 2.5mM and 25mM glucose concentrations like the prior experiment and observed changes in Cck mRNA expression. After treatment with sulprostone, we are still able to see a significant increase in Cck mRNA expression in islets treated with 25mM glucose compared to those treated with 2.5mM glucose, even with cAMP inhibition. This data suggests that there are alternative pathways that could lead to CCK upregulation in the pancreatic islet independent of cAMP. It also shows that hypoglycemia may also increase Cckexpression, which suggests that CCK is upregulated in response to several forms of metabolic stress. We can further explore the effectiveness of glucose to increase CCK expression by treating islets with cycloheximide and deoxyglucose to determine if glucose metabolism is required for upregulation of Cck.This information will help with our general understanding of how CCK functions in pancreatic islet and how Cck transcription is regulated through signaling pathways and transcriptional networks that regulate β-cell function and identity under diabetogenic conditions.
Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic β-cells, but only in models of obesity and insulin resistance. Whole body deletion of CCK in obese mice leads to reduced β-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from β-cell apoptosis. To determine the specific role of β-cell-derived CCK in β-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the β-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain β-cell mass when subjected to apoptotic stress, with advanced age, and after streptozotocin treatment. Aged MIP-CCK mice have increased β-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced β-cell apoptosis. Directed CCK overexpression in cultured β-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of β-cells from apoptotic stress. Understanding the role of β-cell CCK adds to the emerging knowledge of classic gut peptides in intraislet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect β-cells from apoptosis.
Context. As catecholamine elevation is a key element in the diagnosis of pheochromocytoma, more commonplace causes of sympathetic excess, such as obstructive sleep apnea (OSA), should be excluded as standard practice prior to diagnosis. This is essential to avoid misdiagnosis of adrenal incidentalomas identified in the estimated 42 million Americans with OSA, with greater than 4 million projected to undergo a computed tomography study annually. Case Description. A 56-year-old woman presented with a several year history of paroxysmal hypertension, palpitations, and diaphoresis. Abdominal/pelvic computed tomography performed during an unrelated hospitalization revealed a 2-cm left-sided adrenal nodule initially quantified at 37 Hounsfield units. Posthospitalization, 24-hour urine normetanephrine level was markedly elevated. Reassessment 2 weeks later revealed continued normetanephrine excess. Following normal thyroid function tests, morning cortisol, aldosterone, and plasma renin activity, laparoscopic adrenalectomy was performed. Surgical pathology identified an adrenal cortical adenoma. As paroxysms continued postoperatively, repeat 24-hour urine metanephrines were measured, demonstrating essentially unchanged normetanephrine elevation. Search for an alternate cause ensued, revealing OSA with progressive continuous positive airway pressure noncompliance over the preceding year. Regular continuous positive airway pressure therapy was resumed, and at the end of 7 weeks, 24-hour urine normetanephrine levels had declined. Conclusion. Pheochromocytomas are rare and sleep apnea is common. However, the overlap of clinical symptoms between these disorders is substantial, as is their ability to produce catecholamine excess. Thus, excluding uncontrolled or undiagnosed OSA in high-risk patients should be standard practice before diagnosing pheochromocytoma.
Abstract Glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) are gut-derived peptide hormones known to play important roles in the regulation of gastrointestinal motility and secretion, appetite, and food intake. We have previously demonstrated that both GLP-1 and CCK are produced in the endocrine pancreas of obese mice. Interestingly, while GLP-1 is well known to stimulate insulin secretion by the pancreatic β-cells, direct evidence of CCK promoting insulin release in human islets remains to be determined. Here, we tested whether islet-derived GLP-1 or CCK is necessary for the full stimulation of insulin secretion. We confirm that mouse pancreatic islets secrete GLP-1 and CCK, but only GLP-1 acts locally within the islet to promote insulin release ex vivo . GLP-1 is exclusively produced in approximately 50% of α-cells in lean mouse islets and 70% of α-cells in human islets, suggesting a paracrine α to β-cell signaling through the β-cell GLP-1 receptor. Additionally, we provide evidence that islet CCK expression is regulated by glucose, but its receptor signaling is not required during glucose-stimulated insulin secretion (GSIS). We also see no increase in GSIS in response to CCK peptides. Importantly, all these findings were confirmed in islets from non-diabetic human donors. In summary, our data suggest no direct role for CCK in stimulating insulin secretion and highlight the critical role of intra-islet GLP-1 signaling in the regulation of human β-cell function.
