Alena Shostak

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.

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.

Islet Microenvironment, Modulated by Vascular Endothelial Growth Factor-A Signaling, Promotes β Cell Regeneration

Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and β cell mass, we transiently increased VEGF-A production by β cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to β cell loss. After withdrawal of the VEGF-A stimulus, β cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing β cells. Bone marrow-derived macrophages (MΦs) recruited to the site of β cell injury were crucial for the β cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated β cells will improve strategies aimed at β cell regeneration and expansion.

Pancreatic Islet Vasculature Adapts to Insulin Resistance Through Dilation and Not Angiogenesis

Pancreatic islets adapt to insulin resistance through a complex set of changes, including β-cell hyperplasia and hypertrophy. To determine if islet vascularization changes in response to insulin resistance, we investigated three independent models of insulin resistance: ob/ob, GLUT4+/−, and mice with high-fat diet–induced obesity. Intravital blood vessel labeling and immunocytochemistry revealed a vascular plasticity in which islet vessel area was significantly increased, but intraislet vessel density was decreased as the result of insulin resistance. These vascular changes were independent of islet size and were only observed within the β-cell core but not in the islet periphery. Intraislet endothelial cell fenestration, proliferation, and islet angiogenic factor/receptor expression were unchanged in insulin-resistant compared with control mice, indicating that islet capillary expansion is mediated by dilation of preexisting vessels and not by angiogenesis. We propose that the islet capillary dilation is modulated by endothelial nitric oxide synthase via complementary signals derived from β-cells, parasympathetic nerves, and increased islet blood flow. These compensatory changes in islet vascularization may influence whether β-cells can adequately respond to insulin resistance and prevent the development of diabetes.

Vascular Endothelial Growth Factor-A and Islet Vascularization Are Necessary in Developing, but Not Adult, Pancreatic Islets

Pancreatic islets are highly vascularized mini-organs, and vascular endothelial growth factor (VEGF)-A is a critical factor in the development of islet vascularization. To investigate the role of VEGF-A and endothelial cells (ECs) in adult islets, we used complementary genetic approaches to temporally inactivate VEGF-A in developing mouse pancreatic and islet progenitor cells or in adult β-cells. Inactivation of VEGF-A early in development dramatically reduced pancreatic and islet vascularization, leading to reduced β-cell proliferation in both developing and adult islets and, ultimately, reduced β-cell mass and impaired glucose clearance. When VEGF-A was inactivated in adult β-cells, islet vascularization was reduced twofold. Surprisingly, even after 3 months of reduced islet vascularization, islet architecture and β-cell gene expression, mass, and function were preserved with only a minimal abnormality in glucose clearance. These data show that normal pancreatic VEGF-A expression is critical for the recruitment of ECs and the subsequent stimulation of endocrine cell proliferation during islet development. In contrast, although VEGF-A is required for maintaining the specialized vasculature observed in normal adult islets, adult β-cells can adapt and survive long-term reductions in islet vascularity. These results indicate that VEGF-A and islet vascularization have a lesser role in adult islet function and β-cell mass.

Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles

Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets.

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.

Stress-impaired transcription factor expression and insulin secretion in transplanted human islets

Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity.

Age-dependent human β cell proliferation induced by glucagon-like peptide 1 and calcineurin signaling

Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.