Dysregulation of cholesterol homeostasis is an early signal of beta cell proteotoxicity characteristic of type 2 diabetes.

Type 2 diabetes (T2D) is a common metabolic disease due to insufficient insulin secretion by pancreatic beta cells in the context of insulin resistance. Islet molecular pathology reveals a role for protein misfolding in beta cell dysfunction and loss with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed and co-secreted with insulin. The most toxic form of misfolded IAPP is intracellular membrane disruptive toxic oligomers present in beta cells in T2D and in beta cells of mice transgenic for human IAPP (hIAPP). Prior work revealed a high degree of overlap of transcriptional changes in islets from T2D and pre-diabetic 9-10-week-old mice transgenic for hIAPP with most changes being pro-survival adaptations and therefore of limited therapeutic guidance. Here we investigated islets from hIAPP transgenic mice at an earlier age (6 weeks) to screen for potential mediators of hIAPP toxicity that precede predominance of pro-survival signaling. We identified early suppression of cholesterol synthesis and trafficking along with aberrant intra-beta cell cholesterol and lipid deposits, and impaired cholesterol trafficking to cell membranes. These findings align with comparable lipid deposits present in beta cells in T2D and increased vulnerability to develop T2D in individuals taking medications that suppress cholesterol synthesis.

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.

Efficacy of IAPP suppression in mouse and human islets by GLP-1 analogue conjugated antisense oligonucleotide.

Insulin resistance is the major risk factor for Type 2 diabetes (T2D). In vulnerable individuals, insulin resistance induces a progressive loss of insulin secretion with islet pathology revealing a partial deficit of beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP). IAPP is co-expressed and secreted with insulin by beta cells, expression of both proteins being upregulated in response to insulin resistance. If IAPP expression exceeds the threshold for clearance of misfolded proteins, beta cell failure occurs exacerbated by the action of IAPP toxicity to compromise the autophagy lysosomal pathway. We postulated that suppression of IAPP expression by an IAPP antisense oligonucleotide delivered to beta cells by the GLP-1 agonist exenatide (eGLP1-IAPP-ASO) is a potential disease modifying therapy for T2D. While eGLP1-IAPP-ASO suppressed mouse IAPP and transgenic human IAPP expression in mouse islets, it had no discernable effects on IAPP expression in human islets under the conditions studied. Suppression of transgenic human IAPP expression in mouse islets attenuated disruption of the autophagy lysosomal pathway in beta cells, supporting the potential of this strategy.

IAPP-induced beta cell stress recapitulates the islet transcriptome in type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by islet amyloid and toxic oligomers of islet amyloid polypeptide (IAPP). We posed the questions, (1) does IAPP toxicity induce an islet response comparable to that in humans with type 2 diabetes, and if so, (2) what are the key transcriptional drivers of this response? METHODS: The islet transcriptome was evaluated in five groups of mice: beta cell specific transgenic for (1) human IAPP, (2) rodent IAPP, (3) human calpastatin, (4) human calpastatin and human IAPP, and (5) wild-type mice. RNA sequencing data was analysed by differential expression analysis and gene co-expression network analysis to establish the islet response to adaptation to an increased beta cell workload of soluble rodent IAPP, the islet response to increased expression of oligomeric human IAPP, and the extent to which the latter was rescued by suppression of calpain hyperactivation by calpastatin. Rank-rank hypergeometric overlap analysis was used to compare the transcriptome of islets from human or rodent IAPP transgenic mice vs humans with prediabetes or type 2 diabetes. RESULTS: The islet transcriptomes in humans with prediabetes and type 2 diabetes are remarkably similar. Beta cell overexpression of soluble rodent or oligomer-prone human IAPP induced changes in islet transcriptome present in prediabetes and type 2 diabetes, including decreased expression of genes that confer beta cell identity. Increased expression of human IAPP, but not rodent IAPP, induced islet inflammation present in prediabetes and type 2 diabetes in humans. Key mediators of the injury responses in islets transgenic for human IAPP or those from individuals with type 2 diabetes include STAT3, NF-κB, ESR1 and CTNNB1 by transcription factor analysis and COL3A1, NID1 and ZNF800 by gene regulatory network analysis. CONCLUSIONS/INTERPRETATION: Beta cell injury mediated by IAPP is a plausible mechanism to contribute to islet inflammation and dedifferentiation in type 2 diabetes. Inhibition of IAPP toxicity is a potential therapeutic target in type 2 diabetes.

The ß-cell glucose toxicity hypothesis: Attractive but difficult to prove.

ß cells in the hyperglycemic environment of diabetes have marked changes in phenotype and function that are largely reversible if glucose levels can be returned to normal. A leading hypothesis is that these changes are caused by the elevated glucose levels leading to the concept of glucose toxicity. Support for the glucose toxicity hypothesis is largely circumstantial, but little progress has been made in defining the responsible mechanisms. Then questions emerge that are difficult to answer. In the very earliest stages of diabetes development, there is a dramatic loss of glucose-induced first-phase insulin release (FPIR) with only trivial elevations of blood glucose levels. A related question is how impaired insulin action on target tissues such as liver, muscle and fat can cause increased insulin secretion. The existence of a sophisticated feedback mechanism between insulin secretion and insulin action on peripheral tissues driven by glucose has been postulated, but it has been difficult to measure increases in blood glucose levels that might have been expected. These complexities force us to challenge the simplicity of the glucose toxicity hypothesis and feedback mechanisms. It may turn out that glucose is somehow driving all of these changes, but we must develop new questions and experimental approaches to test the hypothesis.

Live-cell imaging of glucose-induced metabolic coupling of ß and α cell metabolism in health and type 2 diabetes.

Type 2 diabetes is characterized by ß and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in ß cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in ß cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.

Visualizing insulin vesicle neighborhoods in ß cells by cryo-electron tomography.

Subcellular neighborhoods, comprising specific ratios of organelles and proteins, serve a multitude of biological functions and are of particular importance in secretory cells. However, the role of subcellular neighborhoods in insulin vesicle maturation is poorly understood. Here, we present single-cell multiple distinct tomogram acquisitions of ß cells for in situ visualization of distinct subcellular neighborhoods that are involved in the insulin vesicle secretory pathway. We propose that these neighborhoods play an essential role in the specific function of cellular material. In the regions where we observed insulin vesicles, a measurable increase in both the fraction of cellular volume occupied by vesicles and the average size (diameter) of the vesicles was apparent as sampling moved from the area near the nucleus toward the plasma membrane. These findings describe the important role of the nanometer-scale organization of subcellular neighborhoods on insulin vesicle maturation.

Activation of the HIF1α/PFKFB3 stress response pathway in beta cells in type 1 diabetes.

AIMS/HYPOTHESIS: The conserved hypoxia inducible factor 1 α (HIF1α) injury-response pro-survival pathway has recently been implicated in early beta cell dysfunction but slow beta cell loss in type 2 diabetes. We hypothesised that the unexplained prolonged prediabetes phase in type 1 diabetes may also be, in part, due to activation of the HIF1α signalling pathway. METHODS: RNA sequencing (RNA-Seq) data from human islets with type 1 diabetes or after cytokine exposure in vitro was evaluated for activation of HIF1α targets. This was corroborated by immunostaining human pancreases from individuals with type 1 diabetes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), the key effector of HIF1α-mediated metabolic remodelling, and by western blotting of islets and INS-1 832/13 cells exposed to cytokines implicated in type 1 diabetes. RESULTS: HIF1α signalling is activated (p = 4.5 × 10-9) in islets from individuals with type 1 diabetes, and in human islets exposed in vitro to cytokines implicated in type 1 diabetes (p = 1.1 × 10-14). Expression of PFKFB3 is increased fivefold (p < 0.01) in beta cells in type 1 diabetes and in human and rat islets exposed to cytokines that induced increased lactate production. HIF1α attenuates cytokine-induced cell death in beta cells. CONCLUSIONS/INTERPRETATION: The conserved pro-survival HIF1α-mediated injury-response signalling is activated in beta cells in type 1 diabetes and likely contributes to the relatively slow rate of beta cell loss at the expense of early defective glucose-induced insulin secretion.

Pancreatic alpha-cell mass across adult human lifespan.

AIM: To establish pancreatic alpha-cell mass in lean, non-diabetic humans over the adult lifespan, performed as a follow-up study to beta-cell mass across the adult human lifespan. METHODS: We examined human pancreatic autopsy tissue from 66 lean, non-diabetic individuals aged from 30 to 102 years, grouped into deciles: 3rd (30-39 years), 4th (40-49 years), 5th (50-59 years), 6th (60-69 years), 7th (70-79 years), 8th (80-89 years) and 9th deciles (90+ years). Sections of pancreas were immunostained for glucagon and analyzed for fractional alpha-cell area. Population-based pancreatic volume data were used to calculate alpha-cell mass. RESULTS: With advanced age, the exocrine pancreas undergoes atrophy demonstrated by increased fat area (as % exocrine area) (0.05 ± 0.01 vs 1.6 ± 0.7% fat area of total exocrine pancreas, 3rd vs 9th decile, P < 0.05). Consequently, islet density increases with age (2.7 ± 0.4 vs 10.5 ± 3.3 islets/mm2, 3rd vs 9th decile, P < 0.05). Alpha-cell fractional area increases with advanced age (0.34 ± 0.05% vs 0.73 ± 0.26%, 3rd vs 9th decile, P < 0.05). However, alpha-cell mass remains constant at ~190 mg throughout the adult lifespan in lean, non-diabetic humans. Within islets, alpha-cell distribution between mantle and core is unchanged across deciles (1862 ± 220 vs 1945 ± 200 vs 1948 ± 139 alpha cells in islet mantle/mm2, 3rd vs 6th vs 9th decile, P = 0.93 and 1912 ± 442 vs 1449 ± 123 vs 1514 ± 168 alpha cells in islet core/mm2, 3rd vs 6th vs 9th decile, P = 0.47), suggesting that human islets retain their structural organization in the setting of age-related exocrine atrophy. CONCLUSIONS: Consistent with our previous findings for beta-cell mass, alpha-cell mass remains constant in humans, even with advanced age. Pancreatic endocrine cells are much more robustly preserved than exocrine cells in aged humans, and islets maintain their structural integrity throughout life.

Publisher Correction: IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.

Pregnancy in human IAPP transgenic mice recapitulates beta cell stress in type 2 diabetes.

AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP) misfolding and toxic oligomers contribute to beta cell loss and stress in type 2 diabetes. Pregnancy-related diabetes predicts subsequent risk for type 2 diabetes but little is known about the impact of pregnancy on beta cell mass, turnover and stress. Availability of human pancreas tissue in pregnancy is limited and most widely used mouse models of type 2 diabetes do not develop pregnancy-related diabetes, possibly because rodent IAPP is not prone to form toxic oligomers. We hypothesised that mice transgenic for human IAPP (hIAPP) are prone to pregnancy-related diabetes with beta cell responses reflective of those in type 2 diabetes. METHODS: We evaluated the impact of a first and second pregnancy on glucose homeostasis, beta cell mass and turnover and markers of beta cell stress in hIAPP transgenic (hTG) mice. RESULTS: Pregnancy induced both endoplasmic reticulum stress and oxidative stress and compromised autophagy in beta cells in hTG mice, which are characteristic of beta cells in type 2 diabetes. Beta cell stress persisted after pregnancy, resulting in subsequent diabetes before or during a second pregnancy. CONCLUSIONS/INTERPRETATION: High expression of hIAPP in response to pregnancy recapitulates mechanisms contributing to beta cell stress in type 2 diabetes. We hypothesise that, in individuals prone to type 2 diabetes, pregnancy-induced increased expression of IAPP inflicts beta cell damage that persists and is compounded by subsequent additive stress such as further pregnancy. The hTG mouse model is a novel model for pregnancy-related diabetes.

Low Grade Islet but Marked Exocrine Pancreas Inflammation in an Adult with Autoimmune Pre-Diabetes.

There is unexplained deficit in size and function of the exocrine pancreas in type 1 diabetes (T1D). We obtained pancreas from an individual with pre-T1D obtained at surgery and addressed the question, what is the relative inflammation in the exocrine and endocrine pancreas in pre-T1D in the absence of the potential confounding changes at autopsy or in brain dead organ donors. Pancreas was removed surgically from a 36 year woman for benign neuroendocrine tumors (NET). The patient had gestational diabetes at age 29 and has a 23 year old sister with T1D. Pre-operative fasting glucose of 109 mg/dl and HbA1C 5.8% revealed prediabetes with an anti-GAD 1,144 (5-250 U/ml) together with family history implying pre-T1D. There was patchy low grade immune infiltration in some, but not all, islets that met criteria for autoimmune insulitis. The exocrine pancreas showed more abundant inflammation with areas of chronic pancreatitis and acinar to ductal metaplasia, and with other areas of atrophy and fatty infiltration. In pre-T1D inflammation may be more prominent in the exocrine than the endocrine pancreas, calling into question the sequence of events and assumed islet centric basis of autoimmunity leading to T1D.

An Increase in Chromogranin A-Positive, Hormone-Negative Endocrine Cells in Pancreas in Cystic Fibrosis.

We sought to establish whether an increase in chromogranin A-positive, hormone-negative (CPHN) endocrine cells occurs in the pancreas of patients with cystic fibrosis (CF), as potential evidence of neogenesis. Pancreata were obtained at autopsy from nondiabetic patients with CF (n = 12) and age-matched nondiabetic control subject (CS) individuals without CF (n = 12). In addition, pancreas from three diabetic patients with CF was obtained. Pancreas sections were stained for chromogranin A, insulin, and a cocktail of glucagon, somatostatin, pancreatic polypeptide, and ghrelin and evaluated for the frequency of CPHN cells. There was a higher frequency of CPHN cells in islets of the patients with CF compared with the CS group. Moreover, CPHN cells occurring as single cells or clusters scattered in the exocrine pancreas were also more frequent in patients with CF. The increased frequency of CPHN cells in pancreas of patients with CF may indicate an attempt at endocrine cell regeneration.

In the setting of ß-cell stress, the pancreatic duct gland transcriptome shows characteristics of an activated regenerative response.

The pancreatic duct gland (PDG) compartment has been proposed as a potential stem cell niche based on its coiled tubular structure embedded in mesenchyme, its proliferation and expansion in response to pancreatic injury, and the fact that it contains endocrine and exocrine epithelial cells. Little is known of the molecular signature of the PDG compartment in either a quiescent state or the potentially activated state during ß-cell stress characteristic of diabetes. To address this, we performed RNA sequencing on RNA obtained from PDGs of wild-type vs. prediabetic HIP rats, a model of type 2 diabetes. The transcriptome of the PDG compartment, compared with a library of 84 tissue types, placed PDGs midpoint between the exocrine and endocrine pancreas and closely related to seminiferous tubules, consistent with a role as a stem cell niche for the exocrine and endocrine pancreas. Standard differential expression analysis (permissive threshold P < 0.005) identified 245 genes differentially expressed in PDGs from HIP rats vs. WT rats, with overrepresentation of transcripts involved in acute inflammatory responses, regulation of cell proliferation, and tissue development, while pathway analysis pointed to enrichment of cell movement-related pathways. In conclusion, the transcriptome of the PDG compartment is consistent with a pancreatic stem cell niche that is activated by ongoing ß-cell stress signals. The documented PDG transcriptome provides potential candidates to be exploited for lineage tracing studies of this as yet little investigated compartment. NEW & NOTEWORTHY The pancreatic duct gland (PDG) compartment has been proposed as a potential stem cell niche. Transcriptome analysis of the PDG gland placed it midpoint between exocrine and endocrine tissues with adaptation toward response to inflammation and increased cell movement in a model of type 2 diabetes with ongoing ß-cell apoptosis. These findings support the proposal that PDGs may act as a pancreatic stem cell niche.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment.

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

Increased Chromogranin A-Positive Hormone-Negative Cells in Chronic Pancreatitis.

Context: Chronic pancreatitis (CP) is characterized by inflammation, fibrosis, and a loss of pancreatic acinar cells, which can result in exocrine and eventually endocrine deficiency. Pancreatitis has been reported to induce formation of new endocrine cells (neogenesis) in mice. Our recent data have implicated chromogranin A-positive hormone-negative (CPHN) cells as potential evidence of neogenesis in humans. Objective: We sought to establish if CPHN cells were more abundant in CP in humans. Design, Setting, and Participants: We investigated the frequency and distribution of CPHN cells and the expression of the chemokine C-X-C motif ligand 10 (CXCL10) and its receptor chemokine C-X-C motif receptor 3 in pancreas of nondiabetic subjects with CP. Results: CPHN cell frequency in islets was increased sevenfold in CP [2.1% ± 0.67% vs 0.35% ± 0.09% CPHN cells in islets, CP vs nonpancreatitis (NP), P < 0.01], as were the CPHN cells found as scattered cells in the exocrine areas (17.4 ± 2.9 vs 4.2 ± 0.6, CP vs NP, P < 0.001). Polyhormonal endocrine cells were also increased in CP (2.7 ± 1.2 vs 0.1 ± 0.04, CP vs NP, % of polyhormonal cells of total endocrine cells, P < 0.01), as was expression of CXCL10 in α and ß cells. Conclusion: There is increased islet endogenous expression of the inflammation marker CXCL10 in islets in the setting of nondiabetic CP and an increase in polyhormonal (insulin-glucagon expressing) cells. The increase in CPHN cells in CP, often in a lobular distribution, may indicate foci of attempted endocrine cell regeneration.