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Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+into β-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca2+dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: 1) 8 mM glucose; 2) 8 mM glucose plus amino acids; 3) 8 mM glucose, amino acids, plus 10 nM GIP; and 4) 2 mM glucose. These stimuli interrogate β-cell function, α-cell to β-cell signaling, and incretin responses. We then correlated components of the Ca2+waveforms to islet protein abundances in the same strains used for the Ca2+measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated SNPs in human GWAS. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominated novel regulators of islet Ca2+oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.

Insulin secretion from pancreatic β cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that β cell-specific deletion of Zfp148 (β-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from β‑Zfp148KO and control mice fed both a chow and a Western-style diet. β-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. β-Zfp148KO islets also exhibited elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of β-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, β-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving β cell function that are robust to the metabolic challenge imposed by a Western diet.

Genetic susceptibility to type 2 diabetes is primarily due to β cell dysfunction. However, a genetic study to directly interrogate β cell function ex vivo has never been previously performed. We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged greater than 1000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion quantitative trait loci (QTL) from mice are associated with diabetes-related SNPs in human genome-wide association studies. We report on 3 genes, Ptpn18, Hunk, and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These 3 genes encode a nonreceptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in nondiabetic individuals.

Gain-of-function (GOF) mutations in the ATP-sensitive potassium (KATP) channels cause neonatal diabetes. Despite the well-established genetic root of the disease, pathways modulating disease severity and treatment effectiveness remain poorly understood. Patient phenotypes can vary from severe diabetes to remission, even in individuals with the same mutation and within the same family, suggesting that subtle modifiers can influence disease outcome. We have tested the underlying mechanism of transient vs. permanent neonatal diabetes in KATP-GOF mice treated for 14 days with glibenclamide. Some KATP-GOF mice show remission of diabetes and enhanced insulin sensitivity long after diabetes treatment has ended, while others maintain severe insulin-resistance. However, insulin sensitivity is not different between the two groups before or during diabetes induction, suggesting that improved sensitivity is a consequence, rather than the cause of, remission, implicating other factors modulating glucose early in diabetes progression. Leptin, glucagon, insulin, and glucagon-like peptide-1 are not different between remitters and nonremitters. However, liver glucose production is significantly reduced before transgene induction in remitter, relative to nonremitter and nontreated, mice. Surprisingly, while subsequent remitter animals exhibited normal serum cytokines, nonremitter mice showed increased cytokines, which paralleled the divergence in blood glucose. Together, these results suggest that systemic inflammation may play a role in the remitting versus non-remitting outcome. Supporting this conclusion, treatment with the anti-inflammatory meloxicam significantly increased the fraction of remitting animals. Beyond neonatal diabetes, the potential for inflammation and glucose production to exacerbate other forms of diabetes from a compensated state to a glucotoxic state should be considered.

Assessing the response of pancreatic islet cells to glucose stimulation is important for understanding β-cell function. Zebrafish are a promising model for studies of metabolism in general, including stimulus-secretion coupling in the pancreas. We used transgenic zebrafish embryos expressing a genetically-encoded Ca2+ sensor in pancreatic β-cells to monitor a key step in glucose induced insulin secretion; the elevations of intracellular [Ca2+]i. In vivo and ex vivo analyses of [Ca2+]i demonstrate that β-cell responsiveness to glucose is well established in late embryogenesis and that embryonic β-cells also respond to free fatty acid and amino acid challenges. In vivo imaging of whole embryos further shows that indirect glucose administration, for example by yolk injection, results in a slow and asynchronous induction of β-cell [Ca2+]i responses, while intravenous glucose injections cause immediate and islet-wide synchronized [Ca2+]i fluctuations. Finally, we demonstrate that embryos with disrupted mutation of the CaV1.2 channel gene cacna1c are hyperglycemic and that this phenotype is associated with glucose-independent [Ca2+]i fluctuation in β-cells. The data reveal a novel central role of cacna1c in β-cell specific stimulus-secretion coupling in zebrafish and demonstrate that the novel approach we propose – to monitor the [Ca2+]i dynamics in embryonic β-cells in vivo – will help to expand the understanding of β-cell physiological functions in healthy and diseased states.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.

Zebrafish have recently gained popularity as model organisms for the study of insulin secretory control and diabetes, and their beta-cells (b-cells) have remarkable genetic and physiologic conservation to mammals [1]. Zebrafish have significant advantages over mammalian models, due to their translucent embryos and large number of progeny, allowing for large-scale genetic and pharmacologic screens. However, monitoring glucose levels in fish requires either time-consuming blood draws or sacrificing of the animal, and attempts by multiple groups at directly measuring insulin secretion have been unsuccessful. To optimize zebrafish as a model organism, we are developing approaches for less invasive determinations of glucose levels and for quantitative imaging of glucose stimulated b-cell activity. Insulin secretion is regulated by ATP-sensitive potassium (KATP) channels. At low plasma [glucose], KATP channels are open and the membrane is hyperpolarized, thus inhibiting insulin secretion. Increased glucose metabolism raises the intracellular ATP, closing KATP channels. This leads to membrane depolarization, calcium influx, and ultimately triggers insulin release. Genetic disruption of this pathway can lead to neonatal diabetes or congenital hyperinsulinism. We have generated b-cell specific GCaMP transgenic fish to allow detection of intracellular [Ca2+]. We demonstrate a 2-fold increase in intracellular [Ca2+] when switching from 2mM glucose to 20mM. This increase is fully inhibited when incubated in 20 mM glucose with 250 uM diazoxide. To assess secretion, islets were labeled with a fluorescent zinc indicator [2] and exposed to increasing [glucose]. Fluorescence increases 10-25% over baseline in response to high glucose concentration, indicating secretion of zinc-containing insulin vesicles. Increase in fluorescence is fully inhibited with 250 uM diazoxide. We are also evaluating a genetically encoded glucose indicator, created by our collaborators through modification of a bacterial glucose binding protein and GFP. When expressed in Cos cells, a cytosolic form shows a 2-fold fluorescence increase in the range of 10-30 mM glucose. We have generated transgenic lines for this biosensor allowing detection of real time, in vivo changes in [glucose] without sacrificing the animal. Establishment of these techniques will facilitate the use of zebrafish as a model organism for the study of disorders of insulin secretion and identify novel treatment approaches. 1. Emfinger, C.H., et al., Expression and function of ATP-dependent potassium channels in zebrafish islet beta-cells. R Soc Open Sci, 2017. 4(2): p. 160808. 2. Li, D., et al., Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR). Proc Natl Acad Sci U S A, 2011. 108(52): p. 21063-8.

Recovery of functional β-cell mass continues to be an ongoing challenge in treating diabetes. Initial work studying β-cells suggested apoptotic β-cell death as a main contributor for the loss of β-cell mass in diabetes. Restoration of β-cells either by transplant or stimulating proliferation of remaining β-cells or precursors would then logically be a viable therapeutic option for diabetes. However, recent work has highlighted the inherent β-cell plasticity and the critical role of loss of β-cell identity in diabetes, and has suggested that β-cells fail to maintain a fully differentiated glucose-responsive and drug-responsive state, particularly in diabetic individuals with poorly controlled, long-lasting periods of hyperglycaemia. Understanding the underlying mechanisms of loss of β-cell identity and conversion in other cell types, as well as how to regain their mature differentiated functional state, is critical to develop novel therapeutic strategies to prevent or reverse these processes. In this review, we discuss the role of plasticity and loss of β-cell identity in diabetes, the current understanding of mechanisms involved in altering this mature functional β-cell state and potential progresses to identify novel therapeutic targets providing better opportunities for slowing or preventing diabetes progression.

Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.

Gain-of-function (GOF) mutations in the ATP-sensitive potassium (K(ATP)) channels cause neonatal diabetes. Despite the well-established genetic root of the disease, pathways modulating disease severity and treatment effectiveness remain poorly understood. Patient phenotypes can vary from severe diabetes to remission, even in individuals with the same mutation and within the same family, suggesting that subtle modifiers can influence disease outcome. We have tested the underlying mechanism of transient vs. permanent neonatal diabetes in K(ATP)-GOF mice treated for 14 days with glibenclamide. Some K(ATP)-GOF mice show remission of diabetes and enhanced insulin sensitivity long after diabetes treatment has ended, while others maintain severe insulin-resistance. However, insulin sensitivity is not different between the two groups before or during diabetes induction, suggesting that improved sensitivity is a consequence, rather than the cause of, remission, implicating other factors modulating glucose early in diabetes progression. Leptin, glucagon, insulin, and glucagon-like peptide-1 are not different between remitters and nonremitters. However, liver glucose production is significantly reduced before transgene induction in remitter, relative to nonremitter and nontreated, mice. Surprisingly, while subsequent remitter animals exhibited normal serum cytokines, nonremitter mice showed increased cytokines, which paralleled the divergence in blood glucose. Together, these results suggest that systemic inflammation may play a role in the remitting versus non-remitting outcome. Supporting this conclusion, treatment with the anti-inflammatory meloxicam significantly increased the fraction of remitting animals. Beyond neonatal diabetes, the potential for inflammation and glucose production to exacerbate other forms of diabetes from a compensated state to a glucotoxic state should be considered.

In clinical trials, inhibition of cholesteryl ester transfer protein (CETP) raises HDL cholesterol levels but does not robustly improve cardiovascular outcomes. Approximately two-thirds of trial participants are obese. Lower plasma CETP activity is associated with increased cardiovascular risk in human studies, and protective aspects of CETP have been observed in mice fed a high-fat diet (HFD) with regard to metabolic outcomes. To define whether CETP inhibition has different effects depending on the presence of obesity, we performed short-term anacetrapib treatment in chow- and HFD-fed CETP transgenic mice. Anacetrapib raised HDL cholesterol and improved aspects of HDL functionality, including reverse cholesterol transport, and HDL’s antioxidative capacity in HFD-fed mice was better than in chow-fed mice. Anacetrapib worsened the anti-inflammatory capacity of HDL in HFD-fed mice. The HDL proteome was markedly different with anacetrapib treatment in HFD- versus chow-fed mice. Despite benefits on HDL, anacetrapib led to liver triglyceride accumulation and insulin resistance in HFD-fed mice. Overall, our results support a physiologic importance of CETP in protecting from fatty liver and demonstrate context selectivity of CETP inhibition that might be important in obese subjects.

ATP-sensitive potassium channels (K ATP channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, K ATP channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism–secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet β-cells express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish K ATP using diazoxide, a specific K ATP channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That β-cell K ATP channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes.

CETP inhibition has been used as a strategy to raise HDL cholesterol, but trials with two CETP inhibitors torcetrapib and dalcetrapib failed to prevent cardiovascular events for high-risk individuals. Of note, approximately 2/3 of patients in these clinical trials were obese. Obesity and fatty liver are known to significantly impact aspects of liver cholesterol metabolism and HDL biology. We propose that high-fat diet feeding (HFD) might adversely impact the effects of CETP inhibition on HDL composition and function. Mice naturally lack CETP expression, so we used CETP transgenic mice to evaluate the effects of administering the CETP inhibitor torcetrapib on HDL composition in chow- and HFD-fed CETP-transgenic mice. Because of the very short half-life of torcetrapib we used acute intravenous infusion of torcetrapib. In both diet groups torcetrapib infusion suppressed plasma CETP activity and raised HDL cholesterol content. In chow-fed CETP mice we found that torcetrapib resulted in changes in HDL composition that would be predicted to be favorable, including increases in apoA1, apoM and apoE protein levels for 2.5-fold ( P P P

The development of insulin resistance in the liver is a key event that drives dyslipidemia and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor-α (ERα). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ERα signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ERα-knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared with their wild-type floxed (fl/fl) littermates during hyperinsulinemic euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of Akt-Ser473and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin-stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ERα in the liver helps prevent whole body and hepatic insulin resistance associated with HFD feeding in males. Augmenting hepatic estrogen signaling through ERα may lessen the impact of obesity on diabetes and cardiovascular risk in males.

Cholesteryl ester transfer protein (CETP) shuttles lipids between lipoproteins, culminating in cholesteryl ester delivery to liver and increased secretion of cholesterol as bile. Since gut bile acids promote insulin sensitivity, we aimed to define if CETP improves insulin sensitivity with high-fat feeding. CETP and nontransgenic mice of both sexes became obese. Female but not male CETP mice had increased ileal bile acid levels versus nontransgenic littermates. CETP expression protected female mice from insulin resistance but had a minimal effect in males. In liver, female CETP mice showed activation of bile acid-sensitive pathways including Erk1/2 phosphorylation and Fxr and Shp gene expression. In muscle, CETP females showed increased glycolysis, increased mRNA for Dio2, and increased Akt phosphorylation, known effects of bile acid signaling. These results suggest that CETP can ameliorate insulin resistance associated with obesity in female mice, an effect that correlates with increased gut bile acids and known bile-signaling pathways.

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.