Alvin C. Powers

Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets α and δ cells resided at the periphery of the β-cell core. However, human islets were markedly different in that α, β, and δ cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of α, β, and δ cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Reduction in Pancreatic Transcription Factor PDX-1 Impairs Glucose-stimulated Insulin Secretion

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1+/− mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1+/−mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1+/− mice. The in situ perfused pancreas of PDX-1+/− mice secreted about 45% less insulin when stimulated with 16.7 mm glucose. The Km for insulin release was similar in wild type and PDX-1+/− mice. Insulin secretion in response to 20 mm arginine was unchanged; the response to 10 nm glucagon-like peptide-1 was slightly increased. However, insulin secretory responses to 10 mm 2-ketoisocaproate and 20 mm KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca2+) and that PDX-1 is important for normal function of adult pancreatic islets.

Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization, and Function

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and β-cell mass, mice with β-cell–reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of β-cell secretagogues to islets, we showed that reduced insulin output is not a result of β-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Islet cell autoantigen 69 kD (ICA69). Molecular cloning and characterization of a novel diabetes-associated autoantigen.

We have identified a novel 69-kD peptide autoantigen (ICA69) associated with insulin-dependent diabetes mellitus (IDDM) by screening a human islet lambda gt11 cDNA expression library with cytoplasmic islet cell antibody positive sera from relatives of IDDM patients who progressed to the overt disease. The deduced open reading frame of the ICA69 cDNA predicts a 483-amino acid protein. ICA69 shows no nucleotide or amino acid sequence relation to any known sequence in GenBank, except for two short regions of similarity with BSA. The ICA69 cDNA probe hybridizes with a 2-kb mRNA in poly(A+) RNA from human pancreas, brain, heart, thyroid, and kidney, but not with skeletal muscle, placenta, spleen, or ovary. Expression of ICA69 was also detected in beta cells and cell lines, as well as in tumoral tissue of islet cell origin. The native ICA69 molecule migrates to 69 kD in SDS-PAGE as detected with specific antibodies. Serum samples from relatives of IDDM patients specifically reacted with affinity-purified recombinant ICA69 on Western blotting. The structural gene for ICA69 was designated ICA1. A homologue in the mouse, designated Ica-1 was mapped to the proximal end of chromosome 6 (within 6 cM of the Met protooncogene). ICA69 adds a novel autoantigen to the family of identified islet target molecules, and by the manner of its identification and characterization large amounts of antigen are available for development of quantitative, convenient predictive assays for autoantibodies and analysis of the role of this molecule in diabetes autoimmunity, as well as its physiologic function.

Conditional gene targeting in mouse pancreatic β-cells: Analysis of ectopic Cre transgene expression in the brain

OBJECTIVE Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. CONCLUSIONS Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.

Inactivation of specific β cell transcription factors in type 2 diabetes

Type 2 diabetes (T2DM) commonly arises from islet β cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with β cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of β cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key β cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet β cells restored nuclear MAFA, nuclear NKX6.1, and β cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human β cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet β cell function, with loss of MAFA (and/or MAFB) representing an early indicator of β cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

β-Cell Failure in Type 2 Diabetes: Postulated Mechanisms and Prospects for Prevention and Treatment

OBJECTIVE This article examines the foundation of β-cell failure in type 2 diabetes (T2D) and suggests areas for future research on the underlying mechanisms that may lead to improved prevention and treatment. RESEARCH DESIGN AND METHODS A group of experts participated in a conference on 14–16 October 2013 cosponsored by the Endocrine Society and the American Diabetes Association. A writing group prepared this summary and recommendations. RESULTS The writing group based this article on conference presentations, discussion, and debate. Topics covered include genetic predisposition, foundations of β-cell failure, natural history of β-cell failure, and impact of therapeutic interventions. CONCLUSIONS β-Cell failure is central to the development and progression of T2D. It antedates and predicts diabetes onset and progression, is in part genetically determined, and often can be identified with accuracy even though current tests are cumbersome and not well standardized. Multiple pathways underlie decreased β-cell function and mass, some of which may be shared and may also be a consequence of processes that initially caused dysfunction. Goals for future research include to 1) impact the natural history of β-cell failure; 2) identify and characterize genetic loci for T2D; 3) target β-cell signaling, metabolic, and genetic pathways to improve function/mass; 4) develop alternative sources of β-cells for cell-based therapy; 5) focus on metabolic environment to provide indirect benefit to β-cells; 6) improve understanding of the physiology of responses to bypass surgery; and 7) identify circulating factors and neuronal circuits underlying the axis of communication between the brain and β-cells.

Glucose Metabolism In Vivo in Four Commonly Used Inbred Mouse Strains

OBJECTIVE—To characterize differences in whole-body glucose metabolism between commonly used inbred mouse strains. RESEARCH DESIGN AND METHODS—Hyperinsulinemic-euglycemic (∼8.5 mmol/l) and -hypoglycemic (∼3.0 mmol/l) clamps were done in catheterized, 5-h-fasted mice to assess insulin action and hypoglycemic counter-regulatory responsiveness. Hyperglycemic clamps (∼15 mmol/l) were done to assess insulin secretion and compared with results in perifused islets. RESULTS—Insulin action and hypoglycemic counter-regulatory and insulin secretory phenotypes varied considerably in four inbred mouse strains. In vivo insulin secretion was greatest in 129X1/Sv mice, but the counter-regulatory response to hypoglycemia was blunted. FVB/N mice in vivo showed no increase in glucose-stimulated insulin secretion, relative hepatic insulin resistance, and the highest counter-regulatory response to hypoglycemia. In DBA/2 mice, insulin action was lowest among the strains, and islets isolated had the greatest glucose-stimulated insulin secretion in vitro. In C57BL/6 mice, in vivo physiological responses to hyperinsulinemia at euglycemia and hypoglycemia were intermediate relative to other strains. Insulin secretion by C57BL/6 mice was similar to that in other strains in contrast to the blunted glucose-stimulated insulin secretion from isolated islets. CONCLUSIONS—Strain-dependent differences exist in four inbred mouse strains frequently used for genetic manipulation and study of glucose metabolism. These results are important for selecting inbred mice to study glucose metabolism and for interpreting and designing experiments.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

VEGF-A recruits a proangiogenic MMP-9–delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue

Recruitment and retention of leukocytes at a site of blood vessel growth are crucial for proper angiogenesis and subsequent tissue perfusion. Although critical for many aspects of regenerative medicine, the mechanisms of leukocyte recruitment to and actions at sites of angiogenesis are not fully understood. In this study, we investigated the signals attracting leukocytes to avascular transplanted pancreatic islets and leukocyte actions at the engraftment site. Expression of the angiogenic stimulus VEGF-A by mouse pancreatic islets was elevated shortly after syngeneic transplantation to muscle. High levels of leukocytes, predominantly CD11b(+)/Gr-1(+)/CXCR4(hi) neutrophils, were observed at the site of engraftment, whereas VEGF-A-deficient islets recruited only half of the amount of leukocytes when transplanted. Acute VEGF-A exposure of muscle increased leukocyte extravasation but not the levels of SDF-1α. VEGF-A-recruited neutrophils expressed 10 times higher amounts of MMP-9 than neutrophils recruited to an inflammatory stimulus. Revascularization of islets transplanted to MMP-9-deficient mice was impaired because blood vessels initially failed to penetrate grafts, and after 2 weeks vascularity was still disturbed. This study demonstrates that VEGF-A recruits a proangiogenic circulating subset of CD11b(+)/Gr-1(+) neutrophils that are CXCR4(hi) and deliver large amounts of the effector protein MMP-9, required for islet revascularization and functional integration after transplantation.