Increased NKX6.1 expression and decreased ARX expression in alpha cells accompany reduced beta-cell volume in human subjects

Pancreatic islet cells have plasticity, such as the abilities to dedifferentiate and transdifferentiate. Islet cell conversion to other characteristic cell is largely determined by transcription factors, but significance of expression patterns of these transcription factors in human islet cells remained unclear. Here, we present the NKX6.1-positive ratio of glucagon-positive cells (NKX6.1+/GCG+ ratio) and the ARX-negative ratio of glucagon-positive cells (ARX−/GCG+ ratio) in 34 patients who were not administered antidiabetic agents. Both of NKX6.1+/GCG+ ratio and ARX−/GCG+ ratio negatively associated with relative beta cell area. And these ratios did not have significant correlation with other parameters including age, body mass index, hemoglobin A1c, fasting plasma glucose level or relative alpha-cell area. Our data demonstrate that these expression ratios of transcription factors in glucagon-positive cells closely correlate with the reduction of beta-cell volume in human pancreas.

3D quantification of changes in pancreatic islets in mouse models of diabetes type I and II

ABSTRACTDiabetes is characterized by rising levels of blood glucose and is often associated with a progressive loss of insulin-producing beta cells. Recent studies have demonstrated that it is possible to regenerate new beta cells through proliferation of existing beta cells or trans-differentiation of other cell types into beta cells, raising hope that diabetes can be cured through restoration of functional beta cell mass. Efficient quantification of beta cell mass and islet characteristics is needed to enhance drug discovery for diabetes. Here, we report a 3D quantitative imaging platform for unbiased evaluation of changes in islets in mouse models of type I and II diabetes. To determine whether the method can detect pharmacologically induced changes in beta cell volume, mice were treated for 14 days with either vehicle or the insulin receptor antagonist S961 (2.4 nmol/day) using osmotic minipumps. Mice treated with S961 displayed increased blood glucose and insulin levels. Light-sheet imaging of insulin and Ki67 (also known as Mki67)-immunostained pancreata revealed a 43% increase in beta cell volume and 21% increase in islet number. S961 treatment resulted in an increase in islets positive for the cell proliferation marker Ki67, suggesting that proliferation of existing beta cells underlies the expansion of total beta cell volume. Using light-sheet imaging of a non-obese diabetic mouse model of type I diabetes, we also characterized the infiltration of CD45 (also known as PTPRC)-labeled leukocytes in islets. At 14 weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. These results demonstrate how quantitative light-sheet imaging can capture changes in individual islets to help pharmacological research in diabetes.

Determination of secretory granule maturation times in pancreatic islet beta-cells by serial block face scanning electron microscopy

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting beta cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the beta cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It is found that 7.2±1.2% (±st. dev.) of the beta cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96±12 hours, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of beta cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135±14 minutes, and the immature transforming granules for an average time of 130±17 minutes.

Possible Prevention of Diabetes with a Gluten-Free Diet

Gluten seems a potentially important determinant in type 1 diabetes (T1D) and type 2 diabetes (T2D). Intake of gluten, a major component of wheat, rye, and barley, affects the microbiota and increases the intestinal permeability. Moreover, studies have demonstrated that gluten peptides, after crossing the intestinal barrier, lead to a more inflammatory milieu. Gluten peptides enter the pancreas where they affect the morphology and might induce beta-cell stress by enhancing glucose- and palmitate-stimulated insulin secretion. Interestingly, animal studies and a human study have demonstrated that a gluten-free (GF) diet during pregnancy reduces the risk of T1D. Evidence regarding the role of a GF diet in T2D is less clear. Some studies have linked intake of a GF diet to reduced obesity and T2D and suggested a role in reducing leptin- and insulin-resistance and increasing beta-cell volume. The current knowledge indicates that gluten, among many environmental factors, may be an aetiopathogenic factors for development of T1D and T2D. However, human intervention trials are needed to confirm this and the proposed mechanisms.

Long-Term Fgf23 Deficiency Does Not Influence Aging, Glucose Homeostasis, or Fat Metabolism in Mice with a Nonfunctioning Vitamin D Receptor

It is still controversial whether the bone-derived hormone fibroblast growth factor-23 (FGF23) has additional physiological functions apart from its well-known suppressive actions on renal phosphate reabsorption and vitamin D hormone synthesis. Here we analyzed premature aging, mineral homeostasis, carbohydrate metabolism, and fat metabolism in 9-month-old male wild-type (WT) mice, vitamin D receptor mutant mice (VDRΔ/Δ) with a nonfunctioning vitamin D receptor, and Fgf23−/−/VDRΔ/Δ compound mutant mice on both a standard rodent chow and a rescue diet enriched with calcium, phosphorus, and lactose. Organ atrophy, lung emphysema, and ectopic tissue or vascular calcifications were absent in compound mutants. In addition, body weight, glucose tolerance, insulin tolerance, insulin secretory capacity, pancreatic beta cell volume, and retroperitoneal and epididymal fat mass as well as serum cholesterol and triglycerides were indistinguishable between vitamin D receptor and compound mutants. In contrast to VDRΔ/Δ and Fgf23−/−/VDRΔ/Δ mice, which stayed lean, WT mice showed obesity-induced insulin resistance. To rule out alopecia and concomitantly elevated energy expenditure present in 9-month-old VDRΔ/Δ and Fgf23−/−/VDRΔ/Δ mice as a confounding factor for the lacking effect of Fgf23 deficiency on fat mass, we analyzed whole-body composition in WT, Fgf23−/−, VDRΔ/Δ, and Fgf23−/−/VDRΔ/Δ mice at the age of 4 wk, when the coat in VDRΔ/Δ mice is still normal. Whole-body fat mass was reduced in Fgf23−/− mice but almost identical in WT, VDRΔ/Δ, and Fgf23−/−/VDRΔ/Δ mice. In conclusion, our data indicate that Fgf23 has no molecular vitamin D-independent role in aging, insulin signaling, or fat metabolism in mice.

Glucose-inducible hypertrophy and suppression of anion efflux in rat beta cells

Hypertrophy of beta cells from obese fa/fa rats is associated with increased sensitivity to basal glucose. Exposure to glucose in culture distorts insulin secretion more in beta cells from large than small islets from fa/fa rats. The aim of the present study is to investigate whether increased beta cell volume is associated with both glucose hypersensitivity and altered activity of the glucose-sensitive anion conductance. Beta cells from fa/fa rats had increased volume compared with those from lean rats after 24 h culture. Three-day exposure to 25 mM glucose in culture induced 10-15% hypertrophy in beta cells from lean rats and basal secretion from intact islets was increased tenfold. Estimates of ion channel activity were made from measurement of radiolabeled ion efflux. Taurine efflux, a marker of glucose-regulated anion channel activity, was reduced after high glucose exposure but no alterations in glucose-dependent K+ efflux were detected. The reverse hemolytic plaque assay was used to determine the contributions of the number of secreting cells (recruitment) versus secretion per cell in beta cells from enlarged (>250 microm diameter), intermediate (125-250 microm) and small (<125 microm) islets from lean and obese rats exposed to conditions mimicking hyperglycemia. After overnight culture, basal secretion was twofold greater from beta cells of large fa/fa islets compared with all other groups. Recruitment at low glucose was increased in all lean or fa/fa beta cells derived from >125 microm islets. When beta cells from small islets were exposed to supra-physiological glucose for 3 days, recruitment was increased at basal glucose and blunted at high glucose. Glucose exposure converts the recruitment profile of beta cells from small islets to resemble that of beta cells from large islets while inducing cellular hypertrophy and reduced anion conductance. However, hypertrophy alone did not predict functional characteristics of overnight-cultured beta cells from fa/fa rats.