Identification of type 2 diabetes- and obesity-associated human ß-cells using deep transfer learning.

Diabetes affects >10% of adults worldwide and is caused by impaired production or response to insulin, resulting in chronic hyperglycemia. Pancreatic islet ß-cells are the sole source of endogenous insulin and our understanding of ß-cell dysfunction and death in type 2 diabetes (T2D) is incomplete. Single-cell RNA-seq data supports heterogeneity as an important factor in ß-cell function and survival. However, it is difficult to identify which ß-cell phenotypes are critical for T2D etiology and progression. Our goal was to prioritize specific disease-related ß-cell subpopulations to better understand T2D pathogenesis and identify relevant genes for targeted therapeutics. To address this, we applied a deep transfer learning tool, DEGAS, which maps disease associations onto single-cell RNA-seq data from bulk expression data. Independent runs of DEGAS using T2D or obesity status identified distinct ß-cell subpopulations. A singular cluster of T2D-associated ß-cells was identified; however, ß-cells with high obese-DEGAS scores contained two subpopulations derived largely from either non-diabetic or T2D donors. The obesity-associated non-diabetic cells were enriched for translation and unfolded protein response genes compared to T2D cells. We selected DLK1 for validation by immunostaining in human pancreas sections from healthy and T2D donors. DLK1 was heterogeneously expressed among ß-cells and appeared depleted from T2D islets. In conclusion, DEGAS has the potential to advance our holistic understanding of the ß-cell transcriptomic phenotypes, including features that distinguish ß-cells in obese non-diabetic or lean T2D states. Future work will expand this approach to additional human islet omics datasets to reveal the complex multicellular interactions driving T2D.

RIPK3 promotes islet amyloid-induced ß-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes.

OBJECTIVE: Aggregation of human islet amyloid polypeptide (hIAPP), a ß-cell secretory product, leads to islet amyloid deposition, islet inflammation and ß-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and ß-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced ß-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. METHODS: We quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3-/-, hIAPP and hIAPP; Ripk3-/- mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3-/- bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, ß-cell loss and glucose homeostasis in vivo. RESULTS: We found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3-/- islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3-/- BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased ß-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3-/- mice were protected from these impairments. CONCLUSIONS: In conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced ß-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and ß-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

Small molecule glucagon release inhibitors with activity in human islets.

Purpose: Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet ß-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Results: Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 ß-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Conclusion: Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Therapeutic Strategies Targeting Pancreatic Islet ß-Cell Proliferation, Regeneration, and Replacement.

Diabetes results from insufficient insulin production by pancreatic islet ß-cells or a loss of ß-cells themselves. Restoration of regulated insulin production is a predominant goal of translational diabetes research. Here, we provide a brief overview of recent advances in the fields of ß-cell proliferation, regeneration, and replacement. The discovery of therapeutic targets and associated small molecules has been enabled by improved understanding of ß-cell development and cell cycle regulation, as well as advanced high-throughput screening methodologies. Important findings in ß-cell transdifferentiation, neogenesis, and stem cell differentiation have nucleated multiple promising therapeutic strategies. In particular, clinical trials are underway using in vitro-generated ß-like cells from human pluripotent stem cells. Significant challenges remain for each of these strategies, but continued support for efforts in these research areas will be critical for the generation of distinct diabetes therapies.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

The Pancreatic ß-cell Response to Secretory Demands and Adaption to Stress.

Pancreatic ß cells dedicate much of their protein translation capacity to producing insulin to maintain glucose homeostasis. In response to increased secretory demand, ß cells can compensate by increasing insulin production capability even in the face of protracted peripheral insulin resistance. The ability to amplify insulin secretion in response to hyperglycemia is a critical facet of ß-cell function, and the exact mechanisms by which this occurs have been studied for decades. To adapt to the constant and fast-changing demands for insulin production, ß cells use the unfolded protein response of the endoplasmic reticulum. Failure of these compensatory mechanisms contributes to both type 1 and 2 diabetes. Additionally, studies in which ß cells are "rested" by reducing endogenous insulin demand have shown promise as a therapeutic strategy that could be applied more broadly. Here, we review recent findings in ß cells pertaining to the metabolic amplifying pathway, the unfolded protein response, and potential advances in therapeutics based on ß-cell rest.

High-Throughput Screening for Insulin Secretion Modulators.

The application of forward chemical genetics to insulin secretion in high-throughput has been uncommon because of high costs and technical challenges. However, with the advancement of secreted luciferase tools, it has become feasible for small laboratories to screen large numbers of compounds for effects on insulin secretion. The purpose of this chapter is to outline the methods involved in high-throughput screening for small molecules that chronically impact pancreatic beta cell function. Attention is given to specific points in the protocol that help to improve the dynamic range and reduce variability in the assay. Using this approach in 384-well format, at least 48 and as many as 144 plates can theoretically be processed per week. This protocol serves as a guideline and can be modified as required for alternate stimulation paradigms and improved upon as new technologies become available.

α2-Adrenergic Disruption of ß Cell BDNF-TrkB Receptor Tyrosine Kinase Signaling.

Adrenergic signaling is a well-known input into pancreatic islet function. Specifically, the insulin-secreting islet ß cell expresses the Gi/o-linked α2-adrenergic receptor, which upon activation suppresses insulin secretion. The use of the adrenergic agonist epinephrine at micromolar doses may have supraphysiological effects. We found that pretreating ß cells with micromolar concentrations of epinephrine differentially inhibited activation of receptor tyrosine kinases. We chose TrkB as an example because of its relative sensitivity to the effects of epinephrine and due to its potential regulatory role in the ß cell. Our characterization of brain-derived neurotrophic factor (BDNF)-TrkB signaling in MIN6 ß cells showed that TrkB is activated by BDNF as expected, leading to canonical TrkB autophosphorylation and subsequent downstream signaling, as well as chronic effects on ß cell growth. Micromolar, but not nanomolar, concentrations of epinephrine blocked BDNF-induced TrkB autophosphorylation and downstream mitogen-activated protein kinase pathway activation, suggesting an inhibitory phenomenon at the receptor level. We determined epinephrine-mediated inhibition of TrkB activation to be Gi/o-dependent using pertussis toxin, arguing against an off-target effect of high-dose epinephrine. Published data suggested that inhibition of potassium channels or phosphoinositide-3-kinase signaling may abrogate the negative effects of epinephrine; however, these did not rescue TrkB signaling in our experiments. Taken together, these results show that (1) TrkB kinase signaling occurs in ß cells and (2) use of epinephrine in studies of insulin secretion requires careful consideration of concentration-dependent effects. BDNF-TrkB signaling in ß cells may underlie pro-survival or growth signaling and warrants further study.

Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy.

The exocrine pancreas expresses the highest concentrations of fibroblast growth factor 21 (FGF21) in the body, where it maintains acinar cell proteostasis. Here, we showed in both mice and humans that acute and chronic pancreatitis is associated with a loss of FGF21 expression due to activation of the integrated stress response (ISR) pathway. Mechanistically, we found that activation of the ISR in cultured acinar cells and mouse pancreata induced the expression of ATF3, a transcriptional repressor that directly bound to specific sites on the Fgf21 promoter and resulted in loss of FGF21 expression. These ATF3 binding sites are conserved in the human FGF21 promoter. Consistent with the mouse studies, we also observed the reciprocal expression of ATF3 and FGF21 in the pancreata of human patients with pancreatitis. Using three different mouse models of pancreatitis, we showed that pharmacologic replacement of FGF21 mitigated the ISR and resolved pancreatitis. Likewise, inhibition of the ISR with an inhibitor of the PKR-like endoplasmic reticulum kinase (PERK) also restored FGF21 expression and alleviated pancreatitis. These findings highlight the importance of FGF21 in preserving exocrine pancreas function and suggest its therapeutic use for prevention and treatment of pancreatitis.

Chromomycin A2 potently inhibits glucose-stimulated insulin secretion from pancreatic ß cells.

Modulators of insulin secretion could be used to treat diabetes and as tools to investigate ß cell regulatory pathways in order to increase our understanding of pancreatic islet function. Toward this goal, we previously used an insulin-linked luciferase that is cosecreted with insulin in MIN6 ß cells to perform a high-throughput screen of natural products for chronic effects on glucose-stimulated insulin secretion. In this study, using multiple phenotypic analyses, we found that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion by at least three potential mechanisms: disruption of Wnt signaling, interference of ß cell gene expression, and partial suppression of Ca2+ influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even after washout, but it did not inhibit glucose-stimulated generation of ATP or Ca2+ influx. However, by using the KATP channel opener diazoxide, we uncovered defects in depolarization-induced Ca2+ influx that may contribute to the suppressed secretory response. Glucose-responsive ERK1/2 and S6 phosphorylation were also disrupted by chronic CMA2 treatment. By querying the FUSION bioinformatic database, we revealed that the phenotypic effects of CMA2 cluster with a number of Wnt-GSK3 pathway-related genes. Furthermore, CMA2 consistently decreased GSK3ß phosphorylation and suppressed activation of a ß-catenin activity reporter. CMA2 and a related compound, mithramycin, are known to have DNA interaction properties, possibly abrogating transcription factor binding to critical ß cell gene promoters. We observed that CMA2 but not mithramycin suppressed expression of PDX1 and UCN3. However, neither expression of INSI/II nor insulin content was affected by chronic CMA2. The mechanisms of CMA2-induced insulin secretion defects may involve components both proximal and distal to Ca2+ influx. Therefore, CMA2 is an example of a chemical that can simultaneously disrupt ß cell function through both noncytotoxic and cytotoxic mechanisms. Future therapeutic applications of CMA2 and similar aureolic acid analogues should consider their potential effects on pancreatic islet function.

Mechanisms of the amplifying pathway of insulin secretion in the ß cell.

Pancreatic islet ß cells secrete insulin in response to nutrient secretagogues, like glucose, dependent on calcium influx and nutrient metabolism. One of the most intriguing qualities of ß cells is their ability to use metabolism to amplify the amount of secreted insulin independent of further alterations in intracellular calcium. Many years studying this amplifying process have shaped our current understanding of ß cell stimulus-secretion coupling; yet, the exact mechanisms of amplification have been elusive. Recent studies utilizing metabolomics, computational modeling, and animal models have progressed our understanding of the metabolic amplifying pathway of insulin secretion from the ß cell. New approaches will be discussed which offer in-roads to a more complete model of ß cell function. The development of ß cell therapeutics may be aided by such a model, facilitating the targeting of aspects of the metabolic amplifying pathway which are unique to the ß cell.

Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic ß cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 ß cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence ß-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in ß cells will have implications for diabetes and other emergent fields involving these receptors.

Insulin promoter-driven Gaussia luciferase-based insulin secretion biosensor assay for discovery of ß-cell glucose-sensing pathways.

High throughput screening of insulin secretion is intractable with current methods. We developed a secreted insulin-luciferase system (Ins-GLuc) in ß cells that is rapid, inexpensive, and amenable to 96- and 384-well formats. We treated stable Ins-GLuc-expressing MIN6 cells overnight with 6298 marine natural product fractions. The cells were then washed to remove media and chemicals, followed by stimulation with glucose in the diazoxide paradigm. These conditions allowed the discovery of many insulin secretion suppressors and potentiators. The mechanisms of action of these natural products must be long-lasting given the continuance of secretory phenotypes in the absence of chemical treatment. We anticipate that these natural products and their target pathways will lead to a greater understanding of glucose-stimulated insulin secretion.

Isoxazole Alters Metabolites and Gene Expression, Decreasing Proliferation and Promoting a Neuroendocrine Phenotype in ß-Cells.

Novel strategies are needed to modulate ß-cell differentiation and function as potential ß-cell replacement or restorative therapies for diabetes. We previously demonstrated that small molecules based on the isoxazole scaffold drive neuroendocrine phenotypes. The nature of the effects of isoxazole compounds on ß-cells was incompletely defined. We find that isoxazole induces genes that support neuroendocrine and ß-cell phenotypes and suppresses genes important for proliferation. Isoxazole alters ß-cell metabolites and protects glucose-responsive signaling pathways under lipotoxic conditions. Finally, we show that isoxazole improves glycemia in a mouse model of ß-cell regeneration. Isoxazole is a prime candidate to alter cell fate in different contexts.

Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet ß cells.

The maintenance of whole-body glucose homeostasis is critical for survival, and is controlled by the coordination of multiple organs and endocrine systems. Pancreatic islet ß cells secrete insulin in response to nutrient stimuli, and insulin then travels through the circulation promoting glucose uptake into insulin-responsive tissues such as liver, skeletal muscle and adipose. Many of the genes identified in human genome-wide association studies of diabetic individuals are directly associated with ß cell survival and function, giving credence to the idea that ß-cell dysfunction is central to the development of type 2 diabetes. As such, investigations into the mechanisms by which ß cells sense glucose and secrete insulin in a regulated manner are a major focus of current diabetes research. In particular, recent discoveries of the detailed role and requirements for reorganization/remodeling of filamentous actin (F-actin) in the regulation of insulin release from the ß cell have appeared at the forefront of islet function research, having lapsed in prior years due to technical limitations. Recent advances in live-cell imaging and specialized reagents have revealed localized F-actin remodeling to be a requisite for the normal biphasic pattern of nutrient-stimulated insulin secretion. This review will provide an historical look at the emergent focus on the role of the actin cytoskeleton and its regulation of insulin secretion, leading up to the cutting-edge research in progress in the field today.

A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic ß cells.

Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ∼80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 ß cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 ß cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release.

Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms.

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet ß-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and ß-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ∼20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in ß-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.

Doc2b is a key effector of insulin secretion and skeletal muscle insulin sensitivity.

Exocytosis of intracellular vesicles, such as insulin granules, is carried out by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. An additional regulatory protein, Doc2b (double C2 domain), has recently been implicated in exocytosis from clonal ß-cells and 3T3-L1 adipocytes. Here, we investigated the role of Doc2b in insulin secretion, insulin sensitivity, and the maintenance of whole-body glucose homeostasis. Doc2b heterozygous (Doc2b(+/-)) and homozygous (Doc2b(-/-)) knockout mice exhibited significant whole-body glucose intolerance and peripheral insulin resistance, compared with wild-type littermates. Correspondingly, Doc2b(+/-) and Doc2b(-/-) mice exhibited decreased responsiveness of pancreatic islets to glucose in vivo, with significant attenuation of both phases of insulin secretion ex vivo. Peripheral insulin resistance correlated with ablated insulin-stimulated glucose uptake and GLUT4 vesicle translocation in skeletal muscle from Doc2b-deficient mice, which was coupled to impairments in Munc18c-syntaxin 4 dissociation and in SNARE complex assembly. Hence, Doc2b is a key positive regulator of Munc18c-syntaxin 4-mediated insulin secretion as well as of insulin responsiveness in skeletal muscle, and thus a key effector for glucose homeostasis in vivo. Doc2b's actions in glucose homeostasis may be related to its ability to bind Munc18c and/or directly promote fusion of insulin granules and GLUT4 vesicles in a stimulus-dependent manner.