Connective Tissue Growth Factor Modulates Adult β-Cell Maturity and Proliferation to Promote β-Cell Regeneration in Mice

Stimulation of endogenous β-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic β-cells and in adult β-cells during periods of expansion. We discovered that in embryos CTGF is necessary for β-cell proliferation, and increased CTGF in β-cells promotes proliferation of immature (MafA⁻) insulin-positive cells. CTGF overexpression, under nonstimulatory conditions, does not increase adult β-cell proliferation. In this study, we tested the ability of CTGF to promote β-cell proliferation and regeneration after partial β-cell destruction. β-Cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased β-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature β-cells but promotes proliferation of both mature and immature β-cells. A shortened β-cell replication refractory period is also observed. CTGF treatment upregulates positive cell-cycle regulators and factors involved in β-cell proliferation, including hepatocyte growth factor, serotonin synthesis, and integrin β1. Ex vivo treatment of whole islets with recombinant human CTGF induces β-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote β-cell replication. Thus, CTGF can induce replication of adult mouse β-cells given a permissive microenvironment.     

Nutrient Excess Stimulates β-Cell Neogenesis in Zebrafish

Persistent nutrient excess results in a compensatory increase in the β-cell number in mammals. It is unknown whether this response occurs in nonmammalian vertebrates, including zebrafish, a model for genetics and chemical genetics. We investigated the response of zebrafish β-cells to nutrient excess and the underlying mechanisms by culturing transgenic zebrafish larvae in solutions of different nutrient composition. The number of β-cells rapidly increases after persistent, but not intermittent, exposure to glucose or a lipid-rich diet. The response to glucose, but not the lipid-rich diet, required mammalian target of rapamycin activity. In contrast, inhibition of insulin/IGF-1 signaling in β-cells blocked the response to the lipid-rich diet, but not to glucose. Lineage tracing and marker expression analyses indicated that the new β-cells were not from self-replication but arose through differentiation of postmitotic precursor cells. On the basis of transgenic markers, we identified two groups of newly formed β-cells: one with nkx2.2 promoter activity and the other with mnx1 promoter activity. Thus, nutrient excess in zebrafish induces a rapid increase in β-cells though differentiation of two subpopulations of postmitotic precursor cells. This occurs through different mechanisms depending on the nutrient type and likely involves paracrine signaling between the differentiated β-cells and the precursor cells.     

Assessment of β-Cell Mass and α- and β-Cell Survival and Function by Arginine Stimulation in Human Autologous Islet Recipients

We used intravenous arginine with measurements of insulin, C-peptide, and glucagon to examine β-cell and α-cell survival and function in a group of 10 chronic pancreatitis recipients 1–8 years after total pancreatectomy and autoislet transplantation. Insulin and C-peptide responses correlated robustly with the number of islets transplanted (correlation coefficients range 0.81–0.91; P < 0.01–0.001). Since a wide range of islets were transplanted, we normalized the insulin and C-peptide responses to the number of islets transplanted in each recipient for comparison with responses in normal subjects. No significant differences were observed in terms of magnitude and timing of hormone release in the two groups. Three recipients had a portion of the autoislets placed within their peritoneal cavities, which appeared to be functioning normally up to 7 years posttransplant. Glucagon responses to arginine were normally timed and normally suppressed by intravenous glucose infusion. These findings indicate that arginine stimulation testing may be a means of assessing the numbers of native islets available in autologous islet transplant candidates and is a means of following posttransplant α- and β-cell function and survival.     

Disruption of Growth Factor Receptor–Binding Protein 10 in the Pancreas Enhances β-Cell Proliferation and Protects Mice From Streptozotocin-Induced β-Cell Apoptosis

Defects in insulin secretion and reduction in β-cell mass are associated with type 2 diabetes in humans, and understanding the basis for these dysfunctions may reveal strategies for diabetes therapy. In this study, we show that pancreas-specific knockout of growth factor receptor–binding protein 10 (Grb10), which is highly expressed in pancreas and islets, leads to elevated insulin/IGF-1 signaling in islets, enhanced β-cell mass and insulin content, and increased insulin secretion in mice. Pancreas-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high-fat diet and protected mice from streptozotocin-induced β-cell apoptosis and body weight loss. Our study has identified Grb10 as an important regulator of β-cell proliferation and demonstrated that reducing the expression level of Grb10 could provide a novel means to increase β-cell mass and reduce β-cell apoptosis. This is critical for effective therapeutic treatment of both type 1 and 2 diabetes.To maintain glucose homeostasis, pancreatic β-cells are able to adapt their insulin- secretory capacity in response to altered physiological and pathological demands. A key mechanism contributing to this adaptability is enhanced insulin secretion from existing β-cells and increased β-cell mass (1,2). β-Cell mass is the sum of β-cell size, the rate of cell proliferation/differentiation, and the difference between β-cell neogenesis and apoptosis (3). Reduction in β-cell mass is associated with type 2 diabetes in humans (4). Therefore, expansion of the β-cell mass from endogenous sources, either in vivo or in vitro, represents a highly significant research area for developing specific treatment of human diabetes.Growth factor receptor–binding protein 10 (Grb10) is a pleckstrin homology and Src homology 2 (SH2) domain-containing protein that interacts with several receptor tyrosine kinases, including the insulin receptor (IR) and the IGF-1 receptor (IGF1R) (5,6). The interaction between Grb10 and these receptors negatively regulates insulin and IGF-1 signaling in cultured cells (7–9) and in vivo (10,11). Grb10 negatively regulates insulin or IGF-1 signaling by binding to the kinase domain of the tyrosine-phosphorylated IR or IGF1R, mainly via its SH2 domain and partly a region between the pleckstrin homology domain and the SH2 domain (BPS) (9,12–14). Grb10 is expressed in insulin target tissues such as fat and muscle, but the highest expression of this protein is in pancreas of adult mice (11). Although disruption of the Grb10 gene in all tissues (except the brain) significantly increases the size of pancreas in mice (11,15), the underlying mechanisms are unknown. It is also unclear whether Grb10 has an autonomous role in regulating pancreatic cell mass and β-cell function. In this study, we report the establishment and characterization of the first tissue-specific Grb10 knockout mice. In addition, we demonstrate that pancreas-specific deletion of the Grb10 gene enhanced β-cell proliferation and improved glucose tolerance under high-fat diet (HFD) feeding condition. Knockout of Grb10 in the pancreas protects mice from streptozotocin (STZ)-induced apoptosis, suggesting that suppressing the expression levels of Grb10 in the pancreas could be a promising approach for increasing β-cell mass and improving β-cell function.     

Systemic Regulation of the Age-Related Decline of Pancreatic β-Cell Replication

The frequency of pancreatic β-cell replication declines dramatically with age, potentially contributing to the increased risk of type 2 diabetes in old age. Previous studies have shown the involvement of cell-autonomous factors in this phenomenon, particularly the decline of polycomb genes and accumulation of p16/INK4A. Here, we demonstrate that a systemic factor found in the circulation of young mice is able to increase the proliferation rate of old pancreatic β-cells. Old mice parabiosed to young mice have increased β-cell replication compared with unjoined old mice or old mice parabiosed to old mice. In addition, we demonstrate that old β-cells transplanted into young recipients have increased replication rate compared with cells transplanted into old recipients; conversely, young β-cells transplanted into old mice decrease their replication rate compared with young cells transplanted into young recipients. The expression of p16/INK4A mRNA did not change in heterochronic parabiosis, suggesting the involvement of other pathways. We conclude that systemic factors contribute to the replicative decline of old pancreatic β-cells.Aging lowers the replicative potential of most cells and the capacity for tissue regeneration (1,2). The mechanisms responsible for this phenomenon are intensely investigated but are still poorly understood. In some cases, the decline in proliferation reaches an irreversible state called cellular senescence, which is characterized by a total lack of cell divisions and altered cellular morphology, gene expression profile, and chromatin organization (3). The gradual decrease in the rate of cell division seen already in early postnatal life, before any evidence of senescence, is even less well-understood.The prevalence of replicating pancreatic β-cells declines dramatically with age in both rodents and humans (4,5). The potential of β-cells to enter the cell division cycle in response to injury also becomes restricted in old age (6,7); however, we have recently found that a residual replicative potential is retained even in β-cells of very old mice (8). The molecular mechanisms that underlie these phenotypes are usually thought to converge on enhanced expression of the p16/INK4A seen in old age. The p16 deficiency reduces and p16 overexpression enhances the age-related decline in β-cell replication (9). The increase in β-cell p16/INK4A expression was found to result from an age-related decline in the expression of polycomb genes Ezh2 and Bmi1 levels, which act to repress p16/INK4A in young age, and a decrease in the platelet-derived growth factor receptor (10,11). Beyond an increase in p16/INK4A expression, multiple other cyclin kinase inhibitors are upregulated with old age (7), but the functional significance of these genes is less clear. The increase in the expression of cell-cycle inhibitors, including p16/INK4A and others, was linked to upregulated p38MAPK signaling with age (12).Notably, these studies suggest that the age-related decline in β-cell proliferation stems from a cell-autonomous increase in β-cell cycle inhibitors. However, outside the β-cell field, accumulating evidence suggests that systemic circulating factors are important determinants of aging in general and age-related decline of cell replication in particular. Some striking examples include muscle satellite cells (13), liver, skin (14), and neuronal stem cells (15). In all these cases, it was shown that factors present in the circulation of young animals are able to rejuvenate old cells and bring the rate of proliferation to the levels seen in young animals. The nature of the factors mediating the systemic effect of replication in old age remains largely unknown. Roles for Notch and transforming growth factor-β signaling were proposed in satellite muscle cells (13,16), and circulating chemokines were implicated in the age-related decline of neurogenesis (15). More generally, it is proposed that blood-borne factors altered in age may control cell replication via modulation of stem cell niches (16). A particularly powerful methodology used in many of these studies is heterochronic parabiosis, whereby the circulation of young and old mice is joined, allowing for the study of the effect of systemic factors on aging (13).Recent studies have provided indirect evidence for the importance of systemic factors in the age-related decline of β-cell replication. In one study, both young and old islets were grafted into a hyperglycemic mouse. This work showed that when exposed to high levels of glucose, both young and old β-cells replicate at a similar rate (17). Additionally, when human islets were exposed to high levels of glucose in NOD-SCID mice, no connection was found between donor age and the β-cell proliferation rate (18). Most recently, an article by El Ouaamari et al. (19) used parabiosis to demonstrate the presence of a systemic factor emanating from the liver in the liver-specific insulin receptor knockout model of β-cell hyperplasia. To directly investigate the role of systemic factors in the age-related decline of β-cell proliferation, we established two experimental models in which old β-cells were exposed to a young environment. Using both heterochronic parabiosis and heterochronic islet transplantation, we showed the presence of a circulating factor that regulates the age-related β-cell proliferation rate.     

111In-exendin Uptake in the Pancreas Correlates With the β-Cell Mass and Not With the α-Cell Mass

Targeting of the GLP-1 receptor with ¹¹¹In-labeled exendin is an attractive approach to determine the β-cell mass (BCM). Preclinical studies as well as a proof-of-concept study in type 1 diabetic patients and healthy subjects showed a direct correlation between BCM and radiotracer uptake. Despite these promising initial results, the influence of α-cells on the uptake of the radiotracer remains a matter of debate. In this study, we determined the correlation between pancreatic tracer uptake and β- and α-cell mass in a rat model for β-cell loss. The uptake of ¹¹¹In-exendin (% ID/g) showed a strong positive linear correlation with the BCM (Pearson r = 0.82). The fraction of glucagon-positive cells in the total endocrine mass was increased after alloxan treatment (26% ± 4%, 43% ± 8%, and 69% ± 21% for 0, 45, and 60 mg/kg alloxan, respectively). The uptake of ¹¹¹In-exendin showed a negative linear correlation with the α-cell fraction (Pearson r = −0.76). These data clearly indicate toward specificity of ¹¹¹In-exendin for β-cells and that the influence of the α-cells on ¹¹¹In-exendin uptake is negligible.     

Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes

Apoptosis is the major cause of death of insulin-producing β-cells in type 1 diabetes mellitus (T1DM). Klotho is a recently discovered antiaging gene. We found that the Klotho gene is expressed in pancreatic β-cells. Interestingly, halplodeficiency of Klotho (KL^+/−) exacerbated streptozotocin (STZ)-induced diabetes (a model of T1DM), including hyperglycemia, glucose intolerance, diminished islet insulin storage, and increased apoptotic β-cells. Conversely, in vivo β-cell–specific expression of mouse Klotho gene (mKL) attenuated β-cell apoptosis and prevented STZ-induced diabetes. mKL promoted cell adhesion to collagen IV, increased FAK and Akt phosphorylation, and inhibited caspase 3 cleavage in cultured MIN6 β-cells. mKL abolished STZ- and TNFα-induced inhibition of FAK and Akt phosphorylation, caspase 3 cleavage, and β-cell apoptosis. These promoting effects of Klotho can be abolished by blocking integrin β1. Therefore, these cell-based studies indicated that Klotho protected β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage. In an autoimmune T1DM model (NOD), we showed that in vivo β-cell–specific expression of mKL improved glucose tolerance, attenuated β-cell apoptosis, enhanced insulin storage in β-cells, and increased plasma insulin levels. The beneficial effect of Klotho gene delivery is likely due to attenuation of T-cell infiltration in pancreatic islets in NOD mice. Overall, our results demonstrate for the first time that Klotho protected β-cells in T1DM via attenuating apoptosis.     

SH2B1 in β-Cells Regulates Glucose Metabolism by Promoting β-Cell Survival and Islet Expansion

IGF-1 and insulin promote β-cell expansion by inhibiting β-cell death and stimulating β-cell proliferation, and the phosphatidylinositol (PI) 3-kinase/Akt pathway mediates insulin and IGF-1 action. Impaired β-cell expansion is a risk factor for type 2 diabetes. Here, we identified SH2B1, which is highly expressed in β-cells, as a novel regulator of β-cell expansion. Silencing of SH2B1 in INS-1 832/13 β-cells attenuated insulin- and IGF-1–stimulated activation of the PI 3-kinase/Akt pathway and increased streptozotocin (STZ)-induced apoptosis; conversely, overexpression of SH2B1 had the opposite effects. Activation of the PI 3-kinase/Akt pathway in β-cells was impaired in pancreas-specific SH2B1 knockout (PKO) mice fed a high-fat diet (HFD). HFD-fed PKO mice also had increased β-cell apoptosis, decreased β-cell proliferation, decreased β-cell mass, decreased pancreatic insulin content, impaired insulin secretion, and exacerbated glucose intolerance. Furthermore, PKO mice were more susceptible to STZ-induced β-cell destruction, insulin deficiency, and hyperglycemia. These data indicate that SH2B1 in β-cells is an important prosurvival and proproliferative protein and promotes compensatory β-cell expansion in the insulin-resistant state and in response to β-cell stress.     

Cytoplasmic-Nuclear Trafficking of G1/S Cell Cycle Molecules and Adult Human β-Cell Replication: A Revised Model of Human β-Cell G1/S Control

Harnessing control of human β-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human β-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human β-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating β-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in β-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein–tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human β-cell. In addition to known obstacles to β-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human β-cell expansion.In another article in this issue of Diabetes (1), we developed a novel human β-cell G1/S molecule atlas that reveals that essentially all of the G1/S molecules are present not only in the human islet but also in the human β-cell. Surprisingly, although the G1/S molecules are widely considered to be nuclear proteins, we encountered them principally in the cytoplasm, where they presumably would be unable to direct cell cycle progression. The only G1/S molecules encountered in the nucleus of the human β-cell were cell cycle inhibitors pRb, p57, and, variably, p21. In contrast, all of the cell cycle–activating cyclins and cdks were restricted to the cytoplasm. These studies were performed in quiescent human β-cells and shed no light on the functional activities of G1/S molecules during cell cycle progression.In this report, we explored whether G1/S molecules might be able to be induced to shuttle from the cytoplasm to the nuclear compartment in association with activation of cell cycle progression. We found that several cell cycle inhibitors and activators do actively traffic from the cytoplasm to the nucleus in association with activation of proliferation. These results lead to a substantially altered model of G1/S trafficking and its control in the human β-cell.     

Limited Recovery of β-Cell Function After Gastric Bypass Despite Clinical Diabetes Remission

The mechanisms responsible for the remarkable remission of type 2 diabetes after Roux-en-Y gastric bypass (RYGBP) are still puzzling. To elucidate the role of the gut, we compared β-cell function assessed during an oral glucose tolerance test (OGTT) and an isoglycemic intravenous glucose clamp (iso-IVGC) in: 1) 16 severely obese patients with type 2 diabetes, up to 3 years post-RYGBP; 2) 11 severely obese normal glucose-tolerant control subjects; and 3) 7 lean control subjects. Diabetes remission was observed after RYGBP. β-Cell function during the OGTT, significantly blunted prior to RYGBP, normalized to levels of both control groups after RYGBP. In contrast, during the iso-IVGC, β-cell function improved minimally and remained significantly impaired compared with lean control subjects up to 3 years post-RYGBP. Presurgery, β-cell function, weight loss, and glucagon-like peptide 1 response were all predictors of postsurgery β-cell function, although weight loss appeared to be the strongest predictor. These data show that β-cell dysfunction persists after RYGBP, even in patients in clinical diabetes remission. This impairment can be rescued by oral glucose stimulation, suggesting that RYGBP leads to an important gastrointestinal effect, critical for improved β-cell function after surgery.Roux-en-Y gastric bypass (RYGBP) remits type 2 diabetes in ∼40–80% of cases (1,2); however, mechanisms surrounding this remarkable improvement are still elusive. Although caloric restriction and weight loss are important contributors, evidence suggests that altered gut physiology, including bypass of the proximal small intestine, may also contribute. Bolus delivery of oral glucose elicits significantly lower plasma glucose excursions compared with intravenous (IV) bolus delivery, and an isoglycemic IV glucose clamp (iso-IVGC) leads to significantly lower insulin excursions than an oral glucose tolerance test (OGTT) (3). These experiments highlight the importance of gut-mediated factors in the regulation of glucose metabolism and insulin secretion. The difference in postprandial insulin excursion, or incretin effect, is severely blunted in diabetes and normalized shortly after RYGBP, in parallel with a marked increase in the incretin hormone glucagon-like peptide 1 (GLP-1) (4), which has been shown to improve glucose tolerance, insulin secretion, and β-cell glucose sensitivity (BCGS) (5–8). In fact, GLP-1 agonists are used for diabetes management.β-Cell function, often evaluated using BCGS, relating insulin secretion to plasma glucose levels, and the disposition index (DI), which also adds an insulin sensitivity component, has been shown to be an optimal predictor of diabetes risk (9–11). β-Cell function is impaired in diabetes (12,13) and significantly improved after RYGBP (13–15); evidence suggests this could be GLP-1–mediated (8,16,17). However, the contribution of the gastrointestinal tract to improvement in β-cell function after RYGBP has not been directly tested. To investigate this, we examined change in β-cell function up to 3 years after RYGBP in severely obese individuals with type 2 diabetes who experienced clinical diabetes remission post-RYGBP (OB-DM) and compared them to both nonoperated, obese normal glucose-tolerant (OB-NGT) and lean NGT (LEAN) subjects. To assess if improvements in β-cell function after RYGBP were mediated by the gut, we compared measures of β-cell function during an oral and isoglycemic glucose challenge. Lastly, we studied predictors of β-cell function and glucose control after RYGBP.