Human Islet Microtissues as an In Vitro and an In Vivo Model System for Diabetes

Loss of pancreatic β-cell function is a critical event in the pathophysiology of type 2 diabetes. However, studies of its underlying mechanisms as well as the discovery of novel targets and therapies have been hindered due to limitations in available experimental models. In this study we exploited the stable viability and function of standardized human islet microtissues to develop a disease-relevant, scalable, and reproducible model of β-cell dysfunction by exposing them to long-term glucotoxicity and glucolipotoxicity. Moreover, by establishing a method for highly-efficient and homogeneous viral transduction, we were able to monitor the loss of functional β-cell mass in vivo by transplanting reporter human islet microtissues into the anterior chamber of the eye of immune-deficient mice exposed to a diabetogenic diet for 12 weeks. This newly developed in vitro model as well as the described in vivo methodology represent a new set of tools that will facilitate the study of β-cell failure in type 2 diabetes and would accelerate the discovery of novel therapeutic agents.     

Cell-Free DNA Fragments as Biomarkers of Islet β-Cell Death in Obesity and Type 2 Diabetes

Type 2 diabetes (T2D) typically occurs in the setting of obesity and insulin resistance, where hyperglycemia is associated with decreased pancreatic β-cell mass and function. Loss of β-cell mass has variably been attributed to β-cell dedifferentiation and/or death. In recent years, it has been proposed that circulating epigenetically modified DNA fragments arising from β cells might be able to report on the potential occurrence of β-cell death in diabetes. Here, we review published literature of DNA-based β-cell death biomarkers that have been evaluated in human cohorts of islet transplantation, type 1 diabetes, and obesity and type 2 diabetes. In addition, we provide new data on the applicability of one of these biomarkers (cell free unmethylated INS DNA) in adult cohorts across a spectrum from obesity to T2D, in which no significant differences were observed, and compare these findings to those previously published in youth cohorts where differences were observed. Our analysis of the literature and our own data suggest that β-cell death may occur in subsets of individuals with obesity and T2D, however a more sensitive method or refined study designs are needed to provide better alignment of sampling with disease progression events.     

β-Cell Pathophysiology: A Review of Advanced Optical Microscopy Applications

β-cells convert glucose (input) resulting in the controlled release of insulin (output), which in turn has the role to maintain glucose homeostasis. β-cell function is regulated by a complex interplay between the metabolic processing of the input, its transformation into second-messenger signals, and final mobilization of insulin-containing granules towards secretion of the output. Failure at any level in this process marks β-cell dysfunction in diabetes, thus making β-cells obvious potential targets for therapeutic purposes. Addressing quantitatively β-cell (dys)function at the molecular level in living samples requires probing simultaneously the spatial and temporal dimensions at the proper resolution. To this aim, an increasing amount of research efforts are exploiting the potentiality of biophysical techniques. In particular, using excitation light in the visible/infrared range, a number of optical-microscopy-based approaches have been tailored to the study of β-cell-(dys)function at the molecular level, either in label-free mode (i.e., exploiting intrinsic autofluorescence of cells) or by the use of organic/genetically-encoded fluorescent probes. Here, relevant examples from the literature are reviewed and discussed. Based on this, new potential lines of development in the field are drawn.     

Metformin Preserves β-Cell Compensation in Insulin Secretion and Mass Expansion in Prediabetic Nile Rats

Prediabetes is a high-risk condition for type 2 diabetes (T2D). Pancreatic β-cells adapt to impaired glucose regulation in prediabetes by increasing insulin secretion and β-cell mass expansion. In people with prediabetes, metformin has been shown to prevent prediabetes conversion to diabetes. However, emerging evidence indicates that metformin has negative effects on β-cell function and survival. Our previous study established the Nile rat (NR) as a model for prediabetes, recapitulating characteristics of human β-cell compensation in function and mass expansion. In this study, we investigated the action of metformin on β-cells in vivo and in vitro. A 7-week metformin treatment improved glucose tolerance by reducing hepatic glucose output and enhancing insulin secretion. Although high-dose metformin inhibited β-cell glucose-stimulated insulin secretion in vitro, stimulation of β-cell insulin secretion was preserved in metformin-treated NRs via an indirect mechanism. Moreover, β-cells in NRs receiving metformin exhibited increased endoplasmic reticulum (ER) chaperones and alleviated apoptotic unfold protein response (UPR) without changes in the expression of cell identity genes. Additionally, metformin did not suppress β-cell mass compensation or proliferation. Taken together, despite the conflicting role indicated by in vitro studies, administration of metformin does not exert a negative effect on β-cell function or cell mass and, instead, early metformin treatment may help protect β-cells from exhaustion and decompensation.     

Mangiferin Facilitates Islet Regeneration and β-Cell Proliferation through Upregulation of Cell Cycle and β-Cell Regeneration Regulators

Mangiferin, a xanthonoid found in plants including mangoes and iris unguicularis, was suggested in previous studies to have anti-hyperglycemic function, though the underlying mechanisms are largely unknown. This study was designed to determine the therapeutic effect of mangiferin by the regeneration of β-cells in mice following 70% partial pancreatectomy (PPx), and to explore the mechanisms of mangiferin-induced β-cell proliferation. For this purpose, adult C57BL/6J mice after 7–14 days post-PPx, or a sham operation were subjected to mangiferin (30 and 90 mg/kg body weight) or control solvent injection. Mangiferin-treated mice exhibited an improved glycemia and glucose tolerance, increased serum insulin levels, enhanced β-cell hyperplasia, elevated β-cell proliferation and reduced β-cell apoptosis. Further dissection at the molecular level showed several key regulators of cell cycle, such as cyclin D1, D2 and cyclin-dependent kinase 4 (Cdk4) were significantly up-regulated in mangiferin-treated mice. In addition, critical genes related to β-cell regeneration, such as pancreatic and duodenal homeobox 1 (PDX-1), neurogenin 3 (Ngn3), glucose transporter 2 (GLUT-2), Forkhead box protein O1 (Foxo-1), and glucokinase (GCK), were found to be promoted by mangiferin at both the mRNA and protein expression level. Thus, mangiferin administration markedly facilitates β-cell proliferation and islet regeneration, likely by regulating essential genes in the cell cycle and the process of islet regeneration. These effects therefore suggest that mangiferin bears a therapeutic potential in preventing and/or treating the diabetes.     

Mitochondrial Dysfunction as a Driver of Cognitive Impairment in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most frequent cause of age-related neurodegeneration and cognitive impairment, and there are currently no broadly effective therapies. The underlying pathogenesis is complex, but a growing body of evidence implicates mitochondrial dysfunction as a common pathomechanism involved in many of the hallmark features of the AD brain, such as formation of amyloid-beta (Aβ) aggregates (amyloid plaques), neurofibrillary tangles, cholinergic system dysfunction, impaired synaptic transmission and plasticity, oxidative stress, and neuroinflammation, that lead to neurodegeneration and cognitive dysfunction. Indeed, mitochondrial dysfunction concomitant with progressive accumulation of mitochondrial Aβ is an early event in AD pathogenesis. Healthy mitochondria are critical for providing sufficient energy to maintain endogenous neuroprotective and reparative mechanisms, while disturbances in mitochondrial function, motility, fission, and fusion lead to neuronal malfunction and degeneration associated with excess free radical production and reduced intracellular calcium buffering. In addition, mitochondrial dysfunction can contribute to amyloid-β precursor protein (APP) expression and misprocessing to produce pathogenic fragments (e.g., Aβ1-40). Given this background, we present an overview of the importance of mitochondria for maintenance of neuronal function and how mitochondrial dysfunction acts as a driver of cognitive impairment in AD. Additionally, we provide a brief summary of possible treatments targeting mitochondrial dysfunction as therapeutic approaches for AD.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

The Role of Oxidative Stress in Pancreatic β Cell Dysfunction in Diabetes

Diabetes is a chronic metabolic disorder characterized by inappropriately elevated glucose levels as a result of impaired pancreatic β cell function and insulin resistance. Extensive studies have been conducted to elucidate the mechanism involved in the development of β cell failure and death under diabetic conditions such as hyperglycemia, hyperlipidemia, and inflammation. Of the plethora of proposed mechanisms, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and oxidative stress have been shown to play a central role in promoting β cell dysfunction. It has become more evident in recent years that these 3 factors are closely interrelated and importantly aggravate each other. Oxidative stress in particular is of great interest to β cell health and survival as it has been shown that β cells exhibit lower antioxidative capacity. Therefore, this review will focus on discussing factors that contribute to the development of oxidative stress in pancreatic β cells and explore the downstream effects of oxidative stress on β cell function and health. Furthermore, antioxidative capacity of β cells to counteract these effects will be discussed along with new approaches focused on preserving β cells under oxidative conditions.     

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene,a Major Active Metabolite of Bisphenol A,Triggers Pancreatic β-Cell Death via a JNK/AMPKα Activation-Regulated Endoplasmic Reticulum Stress-Mediated Apoptotic Pathway

4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a major active metabolite of bisphenol A (BPA), is generated in the mammalian liver. Some studies have suggested that MBP exerts greater toxicity than BPA. However, the mechanism underlying MBP-induced pancreatic β-cell cytotoxicity remains largely unclear. This study demonstrated the cytotoxicity of MBP in pancreatic β-cells and elucidated the cellular mechanism involved in MBP-induced β-cell death. Our results showed that MBP exposure significantly reduced cell viability, caused insulin secretion dysfunction, and induced apoptotic events including increased caspase-3 activity and the expression of active forms of caspase-3/-7/-9 and PARP protein. In addition, MBP triggered endoplasmic reticulum (ER) stress, as indicated by the upregulation of GRP 78, CHOP, and cleaved caspase-12 proteins. Pretreatment with 4-phenylbutyric acid (4-PBA; a pharmacological inhibitor of ER stress) markedly reversed MBP-induced ER stress and apoptosis-related signals. Furthermore, exposure to MBP significantly induced the protein phosphorylation of JNK and AMP-activated protein kinase (AMPK)α. Pretreatment of β-cells with pharmacological inhibitors for JNK (SP600125) and AMPK (compound C), respectively, effectively abrogated the MBP-induced apoptosis-related signals. Both JNK and AMPK inhibitors also suppressed the MBP-induced activation of JNK and AMPKα and of each other. In conclusion, these findings suggest that MBP exposure exerts cytotoxicity on β-cells via the interdependent activation of JNK and AMPKα, which regulates the downstream apoptotic signaling pathway.     

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

tRNA Biology in the Pathogenesis of Diabetes: Role of Genetic and Environmental Factors

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes.     

Acetone Ingestion Mimics a Fasting State to Improve Glucose Tolerance in a Mouse Model of Gestational Hyperglycemia

Gestational diabetes mellitus results, in part, from a sub-optimal β-cell mass (BCM) during pregnancy. Artemisinins were reported to increase BCM in models of diabetes by α- to β-cell conversion leading to enhanced glucose tolerance. We used a mouse model of gestational glucose intolerance to compare the effects of an artemisinin (artesunate) on glycemia of pregnant mice with vehicle treatment (acetone) or no treatment. Animals were treated daily from gestational days (GD) 0.5 to 6.5. An intraperitoneal glucose tolerance test was performed prior to euthanasia at GD18.5 or post-partum. Glucose tolerance was significantly improved in both pregnant and non-pregnant mice with both artesunate and vehicle-alone treatment, suggesting the outcome was primarily due to the acetone vehicle. In non-pregnant, acetone-treated animals, improved glucose tolerance was associated with a higher BCM and a significant increase in bihormonal insulin and glucagon-containing pancreatic islet cells, suggesting α- to β-cell conversion. BCM did not differ with treatment during pregnancy or post-partum. However, placental weight was higher in acetone-treated animals and was associated with an upregulation of apelinergic genes. Acetone-treated animals had reduced weight gain during treatment despite comparable food consumption to non-treated mice, suggesting transient effects on nutrient uptake. The mean duodenal and ileum villus height was reduced following exposure to acetone. We conclude that acetone treatment may mimic transient fasting, resulting in a subsequent improvement in glucose tolerance during pregnancy.