Genetics and diet shape the relationship between islet function and whole-body metabolism.

Despite the fact that genes and the environment are known to play a central role in islet function, our knowledge of how these parameters interact to modulate insulin secretory function remains relatively poor. Presently, we performed ex vivo glucose-stimulated insulin secretion and insulin content assays in islets of 213 mice from 13 inbred mouse strains on chow, western diet, and ketogenic diet. Strikingly, among these 13 strains, islets from the commonly used C57BL/6J mouse strain were the least glucose responsive. Using matched metabolic phenotyping data, we performed correlation analyses of inter-islet parameters and found a positive correlation between basal and glucose-stimulated insulin secretion, but no relationship between insulin secretion and insulin content. Using in vivo metabolic measures, we found that metabolic health determines the relationship between ex vivo islet insulin secretion and fasting plasma insulin. Finally, we showed that islet glucose-stimulated insulin secretion decreased with ketogenic diet in almost all strains, concomitant with broader phenotypic changes, such as increased adiposity and glucose intolerance. This is an important finding as it should caution against the application of the ketogenic diet for beta-cell health. Together these data offer key insights into the intersection of diet and genetic background on islet function and whole-body metabolism.

Erratum: Proteomic pathways to metabolic disease and type 2 diabetes in the pancreatic islet.

[This corrects the article DOI: 10.1016/j.isci.2021.103099.].

What the BTBR/J mouse has taught us about diabetes and diabetic complications.

Human and mouse genetics have delivered numerous diabetogenic loci, but it is mainly through the use of animal models that the pathophysiological basis for their contribution to diabetes has been investigated. More than 20 years ago, we serendipidously identified a mouse strain that could serve as a model of obesity-prone type 2 diabetes, the BTBR (Black and Tan Brachyury) mouse (BTBR T+ Itpr3tf/J, 2018) carrying the Lepob mutation. We went on to discover that the BTBR-Lepob mouse is an excellent model of diabetic nephropathy and is now widely used by nephrologists in academia and the pharmaceutical industry. In this review, we describe the motivation for developing this animal model, the many genes identified and the insights about diabetes and diabetes complications derived from >100 studies conducted in this remarkable animal model.

ß-cell function is regulated by metabolic and epigenetic programming of islet-associated macrophages, involving Axl, Mertk, and TGFß receptor signaling.

We have exploited islet-associated macrophages (IAMs) as a model of resident macrophage function, focusing on more physiological conditions than the commonly used extremes of M1 (inflammation) versus M2 (tissue remodeling) polarization. Under steady state, murine IAMs are metabolically poised between aerobic glycolysis and oxidative phosphorylation, and thereby exert a brake on glucose-stimulated insulin secretion (GSIS). This is underpinned by epigenetic remodeling via the metabolically regulated histone demethylase Kdm5a. Conversely, GSIS is enhanced by engaging Axl receptors on IAMs, or by augmenting their oxidation of glucose. Following high-fat feeding, efferocytosis is stimulated in IAMs in conjunction with Mertk and TGFß receptor signaling. This impairs GSIS and potentially contributes to ß-cell failure in pre-diabetes. Thus, IAMs serve as relays in many more settings than currently appreciated, fine-tuning insulin secretion in response to dynamic changes in the external environment. Intervening in this nexus might represent a means of preserving ß-cell function during metabolic disease.

Insulin sensitivity is preserved in mice made obese by feeding a high starch diet.

Obesity is generally associated with insulin resistance in liver and muscle and increased risk of developing type 2 diabetes, however there is a population of obese people that remain insulin sensitive. Similarly, recent work suggests that mice fed high carbohydrate diets can become obese without apparent glucose intolerance. To investigate this phenomenon further, we fed mice either a high fat (Hi-F) or high starch (Hi-ST) diet and measured adiposity, glucose tolerance, insulin sensitivity, and tissue lipids compared to control mice fed a standard laboratory chow. Both Hi-ST and Hi-F mice accumulated a similar amount of fat and tissue triglyceride compared to chow-fed mice. However, while Hi-F diet mice developed glucose intolerance as well as liver and muscle insulin resistance (assessed via euglycaemic/hyperinsulinaemic clamp), obese Hi-ST mice maintained glucose tolerance and insulin action similar to lean, chow-fed controls. This preservation of insulin action despite obesity in Hi-ST mice was associated with differences in de novo lipogenesis and levels of C22:0 ceramide in liver and C18:0 ceramide in muscle. This indicates that dietary manipulation can influence insulin action independently of the level of adiposity and that the presence of specific ceramide species correlates with these differences.

Targeting the insulin granule for modulation of insulin exocytosis.

The pancreatic ß-cells control insulin secretion in the body to regulate glucose homeostasis, and ß-cell stress and dysfunction is characteristic of Type 2 Diabetes. Pharmacological targeting of the ß-cell to increase insulin secretion is typically utilised, however, extended use of common drugs such as sulfonylureas are known to result in secondary failure. Moreover, there is evidence they may induce ß-cell failure in the long term. Within ß-cells, insulin secretory granules (ISG) serve as compartments to store, process and traffic insulin for exocytosis. There is now growing evidence that ISG exist in multiple populations, distinct in their protein composition, motility, age, and capacity for secretion. In this review, we discuss the implications of a heterogenous ISG population in ß-cells and highlight the need for more understanding into how unique ISG populations may be targeted in anti-diabetic therapies.

Islet Biology and Metabolism.

This Special Issue, Islet Biology and Metabolism, was intended as a collection of studies highlighting the importance of the pancreatic islet-in both form and function-to our growing understanding of metabolic physiology and disease [...].

Proteomic pathways to metabolic disease and type 2 diabetes in the pancreatic islet.

Pancreatic islets are essential for maintaining physiological blood glucose levels, and declining islet function is a hallmark of type 2 diabetes. We employ mass spectrometry-based proteomics to systematically analyze islets from 9 genetic or diet-induced mouse models representing a broad cross-section of metabolic health. Quantifying the islet proteome to a depth of >11,500 proteins, this study represents the most detailed analysis of mouse islet proteins to date. Our data highlight that the majority of islet proteins are expressed in all strains and diets, but more than half of the proteins vary in expression levels, principally due to genetics. Associating these varied protein expression levels on an individual animal basis with individual phenotypic measures reveals islet mitochondrial function as a major positive indicator of metabolic health regardless of strain. This compendium of strain-specific and dietary changes to mouse islet proteomes represents a comprehensive resource for basic and translational islet cell biology.

Inside the Insulin Secretory Granule.

The pancreatic ß-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy ß-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing ß-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

Machine Learning Algorithms, Applied to Intact Islets of Langerhans, Demonstrate Significantly Enhanced Insulin Staining at the Capillary Interface of Human Pancreatic ß Cells.

Pancreatic ß cells secrete the hormone insulin into the bloodstream and are critical in the control of blood glucose concentrations. ß cells are clustered in the micro-organs of the islets of Langerhans, which have a rich capillary network. Recent work has highlighted the intimate spatial connections between ß cells and these capillaries, which lead to the targeting of insulin secretion to the region where the ß cells contact the capillary basement membrane. In addition, ß cells orientate with respect to the capillary contact point and many proteins are differentially distributed at the capillary interface compared with the rest of the cell. Here, we set out to develop an automated image analysis approach to identify individual ß cells within intact islets and to determine if the distribution of insulin across the cells was polarised. Our results show that a U-Net machine learning algorithm correctly identified ß cells and their orientation with respect to the capillaries. Using this information, we then quantified insulin distribution across the ß cells to show enrichment at the capillary interface. We conclude that machine learning is a useful analytical tool to interrogate large image datasets and analyse sub-cellular organisation.

Impact of dietary carbohydrate type and protein-carbohydrate interaction on metabolic health.

Reduced protein intake, through dilution with carbohydrate, extends lifespan and improves mid-life metabolic health in animal models. However, with transition to industrialised food systems, reduced dietary protein is associated with poor health outcomes in humans. Here we systematically interrogate the impact of carbohydrate quality in diets with varying carbohydrate and protein content. Studying 700 male mice on 33 isocaloric diets, we find that the type of carbohydrate and its digestibility profoundly shape the behavioural and physiological responses to protein dilution, modulate nutrient processing in the liver and alter the gut microbiota. Low (10%)-protein, high (70%)-carbohydrate diets promote the healthiest metabolic outcomes when carbohydrate comprises resistant starch (RS), yet the worst outcomes were with a 50:50 mixture of monosaccharides fructose and glucose. Our findings could explain the disparity between healthy, high-carbohydrate diets and the obesogenic impact of protein dilution by glucose-fructose mixtures associated with highly processed diets.

Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes.

AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.

Pancreatic ß-Cell-Specific Deletion of VPS41 Causes Diabetes Due to Defects in Insulin Secretion.

Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic ß-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in ß-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

Enhanced structure and function of human pluripotent stem cell-derived beta-cells cultured on extracellular matrix.

The differentiation of human stem cells into insulin secreting beta-like cells holds great promise to treat diabetes. Current protocols drive stem cells through stages of directed differentiation and maturation and produce cells that secrete insulin in response to glucose. Further refinements are now needed to faithfully phenocopy the responses of normal beta cells. A critical factor in normal beta cell behavior is the islet microenvironment which plays a central role in beta cell survival, proliferation, gene expression and secretion. One important influence on native cell responses is the capillary basement membrane. In adult islets, each beta cell makes a point of contact with basement membrane protein secreted by vascular endothelial cells resulting in structural and functional polarization. Interaction with basement membrane proteins triggers local activation of focal adhesions, cell orientation, and targeting of insulin secretion. This study aims to identifying the role of basement membrane proteins on the structure and function of human embryonic stem cell and induced pluripotent stem cell-derived beta cells. Here, we show that differentiated human stem cells-derived spheroids do contain basement membrane proteins as a diffuse web-like structure. However, the beta-like cells within the spheroid do not polarize in response to this basement membrane. We demonstrate that 2D culture of the differentiated beta cells on to basement membrane proteins enforces cell polarity and favorably alters glucose dependent insulin secretion.

A fluorescent timer reporter enables sorting of insulin secretory granules by age.

Within the pancreatic ß-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in ß-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from ß-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the ß-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in ß-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 ß-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the ß-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

Arp2/3 nucleates F-actin coating of fusing insulin granules in pancreatic ß cells to control insulin secretion.

F-actin dynamics are known to control insulin secretion, but the point of intersection with the stimulus-secretion cascade is unknown. Here, using multiphoton imaging of ß cells isolated from Lifeact-GFP transgenic mice, we show that glucose stimulation does not cause global changes in subcortical F-actin. Instead, we observe spatially discrete and transient F-actin changes around each fusing granule. This F-actin remodelling is dependent on actin nucleation and is observed for granule fusion induced by either glucose or high potassium stimulation. Using GFP-labelled proteins, we identify local enrichment of Arp3, dynamin 2 and clathrin, all occurring after granule fusion, suggesting early recruitment of an endocytic complex to the fusing granules. Block of Arp2/3 activity with drugs or shRNA inhibits F-actin coating, traps granules at the cell membrane and reduces insulin secretion. Block of formin-mediated actin nucleation also blocks F-actin coating, but has no effect on insulin secretion. We conclude that local Arp2/3-dependent actin nucleation at the sites of granule fusion plays an important role in post-fusion granule dynamics and in the regulation of insulin secretion.

Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 ß-cells.

A threonine-to-Isoleucine (Thr52Ile) mutation within the pro-domain of the Sorcs1 gene was positionally cloned as the gene underlying a quantitative trait locus that affects fasting insulin levels in mice. In humans, genome-wide association studies and linkage studies have shown that SORCS1 is associated with diabetes and all of diabetes complications. We have recently shown that deletion of Sorcs1 in mice made obese with the leptinob mutation results in diabetes and an insulin granule stability defect. This present study investigates the functional consequence of the Sorcs1 Thr52Ile mutation in the rat INS1 ß-cell line expressing either the wildtype or mutant Sorcs1 allele. We find that Sorcs1 Thr52Ile mutation is associated with increased basal insulin secretion, reduced glucose-stimulated insulin secretion and decreased insulin content in INS1 cells. Moreover, expression of Thr52Ile causes differential processing of the Sorcs1 protein resulting in the formation of an additional 90 kDa mutant form of the protein. The mutant form of the protein is localised to the ER, retains its pro-domain, and concurrently reduces expression of the functional mature 130 kDa Sorcs1 protein. These findings provide a mechanistic clue to why this specific allelic variation in Sorcs1 was associated with reduced insulin levels and type 2 diabetes.

ABHD15 regulates adipose tissue lipolysis and hepatic lipid accumulation.

OBJECTIVE: Insulin suppresses adipose tissue lipolysis after a meal, playing a key role in metabolic homeostasis. This is mediated via the kinase Akt and its substrate phosphodiesterase 3B (PDE3B). Once phosphorylated and activated, PDE3B hydrolyses cAMP leading to the inactivation of cAMP-dependent protein kinase (PKA) and suppression of lipolysis. However, several gaps have emerged in this model. Here we investigated the role of the PDE3B-interacting protein, α/ß-hydrolase ABHD15 in this process. METHODS: Lipolysis, glucose uptake, and signaling were assessed in ABHD15 knock down and knock out adipocytes and fat explants in response to insulin and/or ß-adrenergic receptor agonist. Glucose and fatty acid metabolism were determined in wild type and ABHD15-/- littermate mice. RESULTS: Deletion of ABHD15 in adipocytes resulted in a significant defect in insulin-mediated suppression of lipolysis with no effect on insulin-mediated glucose uptake. ABHD15 played a role in suppressing PKA signaling as phosphorylation of the PKA substrate Perilipin-1 remained elevated in response to insulin upon ABHD15 deletion. ABHD15-/- mice had normal glucose metabolism but defective fatty acid metabolism: plasma fatty acids were elevated upon fasting and in response to insulin, and this was accompanied by elevated liver triglycerides upon ß-adrenergic receptor activation. This is likely due to hyperactive lipolysis as evident by the larger triglyceride depletion in brown adipose tissue in these mice. Finally, ABHD15 protein levels were reduced in adipocytes from mice fed a Western diet, further implicating this protein in metabolic homeostasis. CONCLUSIONS: Collectively, ABHD15 regulates adipocyte lipolysis and liver lipid accumulation, providing novel therapeutic opportunities for modulating lipid homeostasis in disease.