Islet Metabolism Research Articles

The role of mu-opioid receptors in pancreatic islet α-cells.

Diabetes is a complex disease that impacts more than 500 million people across the world. Many of these individuals will develop diabetic neuropathy as a comorbidity, which is historically treated with exogenous opioids like morphine, oxycodone, or tramadol. Although these opioids are effective analgesics, growing evidence indicates that they may directly impact the endocrine pancreas function in patients. One common feature of these exogenous opioid ligands is their preference for the mu opioid receptor (MOPR), so we aimed to determine if endogenous MOPRs directly regulate pancreatic islet metabolism and hormone secretion. We show that pharmacological antagonism of MOPRs enhances glucagon secretion, but not insulin secretion, from human islets under high glucose conditions. This increased secretion is accompanied by increased cAMP signaling. mRNA expression of MOPRs is robust in non-diabetic human islets, but downregulated in islets from T2D donors, suggesting a link between metabolism and MOPR expression. Conditional genetic knockout of MOPRs in murine α-cells increases glucagon secretion under high glucose conditions without increasing glucagon content. Consistent with downregulation of MOPRs during metabolic disease, conditional MOPR knockout mice treated with a high fat diet show impaired glucose tolerance, increased glucagon secretion, increased insulin content, and increased islet size. Together, these results demonstrate a direct mechanism of action for endogenous opioid regulation of endocrine pancreas.

Twenty years of islet-on-a-chip: microfluidic tools for dissecting islet metabolism and function.

Pancreatic islets are metabolically active micron-sized tissues responsible for controlling blood glucose through the secretion of insulin and glucagon. A loss of functional islet mass results in type 1 and 2 diabetes. Islet-on-a-chip devices are powerful microfluidic tools used to trap and study living ex vivo human and murine pancreatic islets and potentially stem cell-derived islet organoids. Devices developed over the past twenty years offer the ability to treat islets with controlled and dynamic microenvironments to mimic in vivo conditions and facilitate diabetes research. In this review, we explore the various islet-on-a-chip devices used to immobilize islets, regulate the microenvironment, and dynamically detect islet metabolism and insulin secretion. We first describe and assess the various methods used to immobilize islets including chambers, dam-walls, and hydrodynamic traps. We subsequently describe the surrounding methods used to create glucose gradients, enhance the reaggregation of dispersed islets, and control the microenvironment of stem cell-derived islet organoids. We focus on the various methods used to measure insulin secretion including capillary electrophoresis, droplet microfluidics, off-chip ELISAs, and on-chip fluorescence anisotropy immunoassays. Additionally, we delve into the various multiparametric readouts (NAD(P)H, Ca2+-activity, and O2-consumption rate) achieved primarily by adopting a microscopy-compatible optical window into the devices. By critical assessment of these advancements, we aim to inspire the development of new devices by the microfluidics community and accelerate the adoption of islet-on-a-chip devices by the wider diabetes research and clinical communities.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Lack of vitamin D signalling shifts skeletal muscles towards oxidative metabolism.

Mice lacking vitamin D receptor (VDR) exhibit a glycogen storage disorder, disrupting carbohydrate utilization in muscle. Here, we asked if the defective carbohydrate metabolism alters the fat utilization by the skeletal muscles of vdr-/- mice. To check the effect of high-fat-containing diets on muscle mass and metabolism of vdr-/- mice, we subjected them to two different milk fat-based diets (milk fat diet with 60% of energy from milk fat and milk-based diet [MBD] with 37% of energy from milk fat) and lard-based high-fat diet (HFD) containing 60% of energy from lard fat. Skeletal muscles and pancreas from these mice were analysed using RNA sequencing, quantitative reverse transcription polymerase chain reaction and western blot to understand the changes in signalling and metabolic pathways. Microscopic analyses of cryosections stained with haematoxylin and eosin, BODIPY, succinate dehydrogenase and periodic acid-Schiff reagent were performed to understand changes in morphology and metabolism of muscle fibres and pancreatic islets. Transcriptomic analyses showed that the skeletal muscles of vdr-/- mice exhibit upregulation of the fatty acid oxidation pathways, suggesting a shift towards increased lipid utilization even in a carbohydrate-enriched regular chow diet (chow). Two different milk fat-enriched diets restored body weight (12.01±0.33g in chowvs. 17.99±0.62g in MBD) and muscle weights (38.58±3.84mg in chow vs. 110.72±1.96mg in MBD for gastrocnemius [GAS]) of vdr-/- mice. Muscle ATP levels (0.56±0.18μmol in chow vs. 1.48±0.08μmol in MBD) and protein synthesis (0.25±0.04A.U. in chow vs. 2.02±0.06A.U. in MBD) were upregulated by MBD. However, despite increasing muscle energy levels, HFD failed to restore the muscle mass and cross-sectional area to that of wild-type (WT) mice (104.95±2.6mg for WT mice on chow vs. 77.26±1.7mg for vdr-/- mice on HFD for GAS). Moreover, HFD disrupted glucose homeostasis in vdr-/- mice, while MBD restored it. We further analysed insulin response and pancreatic insulin levels of these mice to show that HFD led to reduced insulin levels in pancreatic beta cells of vdr-/- mice (mean intensity of 1.5×10-8 for WT mice on chow vs. 4.3×10-9 for vdr-/- mice on HFD). At the same time, MBD restored glucose-stimulated pancreatic insulin response (mean intensity of 9.2×10-9 ). Skeletal muscles of vdr-/- mice are predisposed to utilize fatty acids as their primary energy source to circumvent their defective carbohydrate utilization. Thus, HFDs could restore energy levels in the skeletal muscles of vdr-/- mice. This study reveals that when mice are subjected to a lard-based HFD, VDR signalling is essential for maintaining insulin levels in pancreatic islets. Our data show a critical role of VDR in muscle metabolic flexibility and pancreatic insulin response.

Effect of Low-Dose Progesterone on Glycemic Metabolism, Morphology and Function of Adipose Tissue and Pancreatic Islets in Diet-Induced Obese Female Mice.

Obesity is a worldwide concern due to its global rapid expansion and remarkable impact on individual's health by predisposing to several other diseases. About twice as many women as men suffer from severe obesity and, in fact, there are stages in a woman's life when weight gain and adiposity can result in greater damage to health. For example, obesity triples the chance of a woman developing gestational diabetes. Many hormones promote the metabolic adaptations of pregnancy, including progesterone, whose role in female obesity is still not well known despite being involved in many physiological and pathological processes. Here we investigated whether progesterone treatment at low dose can worsen the glucose metabolism and the morpho functional aspects of adipose tissue and pancreas in obese females. Mice were assigned into four groups: normocaloric diet control (NO-CO), high-fat and -fructose diet control (HFF-CO), normocaloric diet plus progesterone (NO-PG) and high-fat and -fructose diet plus progesterone (HFF-PG) for 10 weeks. Infusion of progesterone (0.25 mg/kg/day) was done by osmotic minipump in the last 21 days of protocol. Animals fed a hypercaloric diet exhibited obesity with increased body weight (p < 0.0001), adipocyte hypertrophy (p < 0.0001), hyperglycemia (p = 0.03), and glucose intolerance (p = 0.001). HFF-CO and HFF-PG groups showed lower adiponectin concentration (p < 0.0001) and glucose-stimulated insulin secretion (p = 0.03), without differences in islet size. Progesterone attenuated glucose intolerance in the HFF-PG group (p = 0.03), however, did not change morphology or endocrine function of adipose tissue and pancreatic islets. Taken together, our results showed that low dose of progesterone does not worsen the effects of hypercaloric diet in glycemic metabolism, morphology and function of adipose tissue and pancreatic islets in female animals. These results may improve the understanding of the mechanisms underlying the pathogenesis of obesity in women and eventually open new avenues for therapeutic strategies and better comprehension of the interactions between progesterone effects and obesity.

Aberrant metabolite trafficking and fuel sensitivity in human pluripotent stem cell-derived islets

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature.

Application of fluorescence lifetime imaging microscopy to monitor glucose metabolism in pancreatic islets in vivo.

Glucose stimulated insulin secretion is mediated by glucose metabolism via oxidative phosphorylation generating ATP that triggers membrane depolarization and exocytosis of insulin. In stressed beta cells, glucose metabolism is remodeled, with enhanced glycolysis uncoupled from oxidative phosphorylation, resulting in the impaired glucose-mediated insulin secretion characteristic of diabetes. Relative changes in glycolysis and oxidative phosphorylation can be monitored in living cells using the 3-component fitting approach of fluorescence lifetime imaging microscopy (FLIM). We engrafted pancreatic islets onto the iris to permit in vivo FLIM monitoring of the trajectory of glucose metabolism. The results show increased oxidative phosphorylation of islet cells (∼90% beta cells) in response to hyperglycemia; in contrast red blood cells traversing the islets maintained exclusive glycolysis as expected in the absence of mitochondria.

235-OR: Myc Activity Is Required for Mitochondrial Function and Glucose-Stimulated Insulin Secretion (GSIS) in Pancreatic ß-Cells

Glucose enhances mitochondrial function, insulin secretion and Myc expression in β-cells. β-cell-specific Myc knockout mice show glucose intolerance, hypoinsulinemia and lack of adaptive β-cell mass expansion following high-fat diet feeding. However, whether Myc regulates GSIS and mitochondrial function in β-cells is unknown. Here, we tested the effects of the Myc activity inhibitor 10058-F4 (1RH) in GSIS, mitochondrial function and metabolism in islets using islet perifusion, Seahorse and metabolomics/transcriptomics approaches. Mouse and human islets incubated 6h with 40µM 1RH displayed impaired phase 1 and phase 2 insulin secretion induced by 11mM glucose. Insulin secretion was not affected in the presence of 25mM KCl suggesting that Myc is required for glucose- but not membrane depolarization-induced insulin secretion. Since adequate mitochondrial function is essential for GSIS, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to analyze mitochondrial respiration and glycolytic flux in these islets. 1RH significantly reduced OCR, ECAR and glucose-induced ATP production. Conversely, the Myc inducer harmine (10µM), increased basal OCR, ATP production, and ECAR and partially reversed 1RH effects on mitochondrial function. RNAseq of mouse islets treated with 1RH in 11mM glucose revealed reduced expression of both oxidative phosphorylation and β-cell signature genes but enhanced gene expression of glycolysis, ROS, fatty acid metabolism and autophagy/mitophagy pathways. Metabolomics analysis of mouse islets treated with 1RH in 11mM glucose confirmed the reduction in ATP and highlighted the accumulation of L-palmitoylcarnitine and palmitic acid suggesting inefficient β-oxidation. In conclusion, Myc is required for GSIS and mitochondrial function in β-cells. Impaired Myc action can lead to unbalanced metabolism and autophagy/mytophagy leading to β-cell dysfunction. Disclosure G.Lu: None. R.Kang: None. P.Diaz-pozo: None. J.Lee: None. M.Li: None. V.M.Victor: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding National Institutes of Health (R01DK126450)

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD). Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression. A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease. The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

Islet-on-a-chip device reveals first phase glucose-stimulated respiration is substrate limited by glycolysis independent of Ca2+ activity.

Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work has revealed the importance of glycolytic flux in triggering insulin secretion. However, it is unclear whether the triggered (‘first phase’) secretion is further amplified by Ca2+-stimulation of mitochondrial NADH production and/or oxidative phosphorylation (OxPhos). Although commercially available tools have been developed to explore islet metabolism, these methods often overlook islet variability and have poor spatiotemporal resolution. To tease apart first phase glucose-stimulated respiration, we designed an islet-on-a-chip microfluidic device to simultaneously measure O2-consumption rate (OCR) and Ca2+-activity of individual islets with high temporal resolution. We used finite element analysis to optimize placement of sensor in optically clear microwells on a thin glass coverslip. The microfluidic channels were subsequently fabricated using O2-impermeable plastic to limit outside-in diffusion and push islets against the microsensor. We validated our device using living mouse islets and well-established modulators of respiration. By inhibiting glycolysis and mitochondrial pyruvate transport, we show that islet OxPhos is limited by NADH-substrate rather than ADP in low and high glucose. We subsequently imaged glucose-stimulated OCR and Ca2+-influx simultaneously to reveal a biphasic respiratory response that is determined by glycolytic flux through pyruvate kinase (PKM2) and independent of Ca2+. These data demonstrate the unique utility of our modular and optically clear O2-sensor to simultaneously measure glucose-stimulated OCR and Ca2+ activity of multiple individual islets.

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

Identification of Novel Regulatory Regions Induced by Intrauterine Growth Restriction in Rat Islets.

Intrauterine growth restriction (IUGR) leads to the development of type 2 diabetes in adulthood, and the permanent alterations in gene expression implicate an epigenetic mechanism. Using a rat model of IUGR, we performed TrueSeq-HELP Tagging to assess the association of DNA methylation changes and gene dysregulation in islets. We identified 511 differentially methylated regions (DMRs) and 4377 significantly altered single CpG sites. Integrating the methylome and our published transcriptome data sets resulted in the identification of pathways critical for islet function. The identified DMRs were enriched with transcription factor binding motifs, such as Elk1, Etv1, Foxa1, Foxa2, Pax7, Stat3, Hnf1, and AR. In silico analysis of 3-dimensional chromosomal interactions using human pancreas and islet Hi-C data sets identified interactions between 14 highly conserved DMRs and 35 genes with significant expression changes at an early age, many of which persisted in adult islets. In adult islets, there were far more interactions between DMRs and genes with significant expression changes identified with Hi-C, and most of them were critical to islet metabolism and insulin secretion. The methylome was integrated with our published genome-wide histone modification data sets from IUGR islets, resulting in further characterization of important regulatory regions of the genome altered by IUGR containing both significant changes in DNA methylation and specific histone marks. We identified novel regulatory regions in islets after exposure to IUGR, suggesting that epigenetic changes at key transcription factor binding motifs and other gene regulatory regions may contribute to gene dysregulation and an abnormal islet phenotype in IUGR rats.

Loss of growth hormone signaling in the mouse germline or in adulthood reduces islet mass and alters islet function with notable sex differences.

In the endocrine pancreas, growth hormone (GH) is known to promote pancreatic islet growth and insulin secretion. In this study, we show that GH receptor (GHR) loss in the germline and in adulthood impacts islet mass in general but more profoundly in male mice. GHR knockout (GHRKO) mice have enhanced insulin sensitivity and low circulating insulin. We show that the total cross-sectional area of isolated islets (estimated islet mass) was reduced by 72% in male but by only 29% in female GHRKO mice compared with wild-type controls. Also, islets from GHRKO mice secreted ∼50% less glucose-stimulated insulin compared with size-matched islets from wild-type mice. We next used mice with a floxed Ghr gene to knock down the GHR in adult mice at 6 mo of age (6mGHRKO) and examined the impact on glucose and islet metabolism. By 12 mo of age, female 6mGHRKO mice had increased body fat and reduced islet mass but had no change in glucose tolerance or insulin sensitivity. However, male 6mGHRKO mice had nearly twice as much body fat, substantially reduced islet mass, and enhanced insulin sensitivity, but no change in glucose tolerance. Despite large losses in islet mass, glucose-stimulated insulin secretion from isolated islets was not significantly different between male 6mGHRKO and controls, whereas isolated islets from female 6mGHRKO mice showed increased glucose-stimulated insulin release. Our findings demonstrate the importance of GH to islet mass throughout life and that unique sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose metabolism.NEW & NOTEWORTHY Growth hormone (GH) is important for more than just growth. GH helps to maintain pancreatic islet mass and insulin secretion throughout life. Sex-specific adaptations to the loss of GH signaling allow mice to maintain normal glucose regulation despite losing islet mass.

Glucose Response by Stem Cell-Derived β Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis

SUMMARYStem cell-derived β (SC-β) cells could provide unlimited human β cells toward a curative diabetes treatment. Differentiation of SC-β cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-β cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-β cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-β cells. This activity can be rescued by challenging SC-β cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.

Isolated pancreatic islet yield and quality is inversely related to organ donor age in rats.

Pancreatic islets consist of several endocrine cell types that maintain glucose homeostasis. Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin producing beta cells in pancreatic islets. Islet transplantation is a treatment for certain individuals with T1D. Islet transplantation in rodents, as an experimental model of the clinical scenario, requires consistency of islet quantity and quality to obtain reproducible results. In this study, we investigated the yield and function of the isolated islets from rats of different ages. Pancreata were harvested from young (10-20 week-old), intermediate (21-40 week-old) and old (>41 week-old) male rats and islets were isolated using a standard protocol. Islet number, morphometry, viability, function, and metabolism were characterized. Islet yield, normalized to body weight, decreased as a function of increasing donor age. Islets from pancreata from young animals were larger and less fragmented compared to islets from organs from intermediate and older animals. Islet viability following overnight culture was the same for islets derived from young and intermediate aged donors but less for islets from old donors. Glucose-stimulated insulin secretion was decreased in islets from older donors. Islet metabolism following glucose challenge, as measured by oxygen consumption, revealed that islets from old donors were metabolically slower and lagged in response to glucose-stimuli. These data demonstrate that increasing donor age has a negative impact on isolated islet yield and quality.

Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function.

Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin.

Design of Islet-On-A-Chip Devices to Dynamically Measure Glucose-Stimulated Metabolism and Insulin Secretion in Individual Pancreatic Islets

Design of Islet-On-A-Chip Devices to Dynamically Measure Glucose-Stimulated Metabolism and Insulin Secretion in Individual Pancreatic Islets

The discovery of novel predictive biomarkers and early-stage pathophysiology for the transition from gestational diabetes to type 2 diabetes.

Gestational diabetes mellitus (GDM) affects up to 20% of pregnancies, and almost half of the women affected progress to type 2 diabetes later in life, making GDM the most significant risk factor for the development of future type 2 diabetes. An accurate prediction of future type 2 diabetes risk in the early postpartum period after GDM would allow for timely interventions to prevent or delay type 2 diabetes. In addition, new targets for interventions may be revealed by understanding the underlying pathophysiology of the transition from GDM to type 2 diabetes. The aim of this study is to identify both a predictive signature and early-stage pathophysiology of the transition from GDM to type 2 diabetes. We used a well-characterised prospective cohort of women with a history of GDM pregnancy, all of whom were enrolled at 6-9weeks postpartum (baseline), were confirmed not to have diabetes via 2 h 75 g OGTT and tested anually for type 2 diabetes on an ongoing basis (2years of follow-up). A large-scale targeted lipidomic study was implemented to analyse ~1100 lipid metabolites in baseline plasma samples using a nested pair-matched case-control design, with 55 incident cases matched to 85 non-case control participants. The relationships between the concentrations of baseline plasma lipids and respective follow-up status (either type 2 diabetes or no type 2 diabetes) were employed to discover both a predictive signature and the underlying pathophysiology of the transition from GDM to type 2 diabetes. In addition, the underlying pathophysiology was examined in vivo and in vitro. Machine learning optimisation in a decision tree format revealed a seven-lipid metabolite type 2 diabetes predictive signature with a discriminating power (AUC) of 0.92 (87% sensitivity, 93% specificity and 91% accuracy). The signature was highly robust as it includes 45-fold cross-validation under a high confidence threshold (1.0) and binary output, which together minimise the chance of data overfitting and bias selection. Concurrent analysis of differentially expressed lipid metabolite pathways uncovered the upregulation of α-linolenic/linoleic acid metabolism (false discovery rate [FDR] 0.002) and fatty acid biosynthesis (FDR 0.005) and the downregulation of sphingolipid metabolism (FDR 0.009) as being strongly associated with the risk of developing future type 2 diabetes. Focusing specifically on sphingolipids, the downregulation of sphingolipid metabolism using the pharmacological inhibitors fumonisin B1 (FB1) and myriocin in mouse islets and Min6 K8 cells (a pancreatic beta-cell like cell line) significantly impaired glucose-stimulated insulin secretion but had no significant impact on whole-body glucose homeostasis or insulin sensitivity. We reveal a novel predictive signature and associate reduced sphingolipids with the pathophysiology of transition from GDM to type 2 diabetes. Attenuating sphingolipid metabolism in islets impairs glucose-stimulated insulin secretion.

CPT1a-Dependent Long-Chain Fatty Acid Oxidation Contributes to Maintaining Glucagon Secretion from Pancreatic Islets

SummaryGlucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the KATP channel, but instead due to reduced operation of the Na+-K+ pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na+-K+ pump is an important ATP-dependent regulator of α cell function.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

Loss-of-function mutations of β-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+] i ), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI β-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.