Up-regulation of the expression of activins in the pancreatic duct by reduction of the beta-cell mass

Activins expressed in progenitor cells of the pancreas regulate differentiation of endocrine cells during development. Neogenesis of beta-cells takes place in adult animals under some conditions, and beta-cells are thought to arise from precursors locating in the pancreatic duct. In the present study, we investigated whether or not activins are expressed in the duct where beta-cell neogenesis is initiated. mRNA for the beta(A)- and beta(B)-subunits was expressed in isolated mouse pancreatic ducts. Immunohistochemically, the beta(A)-subunit was detected in the pancreatic duct and colocalized with cytokeratin, a marker of ductal cells. The beta(A)-subunit was also expressed in nestin-positive cells in the duct. Likewise, the beta(B)-subunit was detected in the pancreatic duct. In addition, mRNA for the type II and type IIB activin receptors was expressed in the duct. Expression of mRNA for two activin subunits was markedly increased after streptozotocin injection. Similarly, the mRNA expression was up-regulated after partial pancreatectomy. These results indicate that activins are expressed in the pancreatic duct and are up-regulated shortly after the reduction of the beta-cell mass. Induction of activins in the duct may be a critical step in the initiation of beta-cell neogenesis.     

Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells

Glucagon-like peptide-1 (GLP-1) is an intestinal incretin hormone, derived from the processing of proglucagon, that exerts insulinotropic actions on insulin-producing pancreatic islet beta-cells. Recently GLP-1 was shown to stimulate the growth and differentiation (neogenesis) of beta-cells and appears to do so by inducing the expression of the homeodomain protein IDX-1 (islet duodenum homeobox-1; also known as PDX-1, pancreatic and duodenal homeobox gene; and as IPF-1, insulin promoter factor), which is required for pancreas development and the expression of beta-cell-specific genes. Earlier we identified multipotential progenitor cells in the islet and ducts of the pancreas, termed nestin-positive islet-derived progenitor cells (NIPs). Here we report the expression of functional GLP-1 receptors on NIPs and that GLP-1 stimulates the differentiation of NIPs into insulin-producing cells. Furthermore, confluent NIP cultures express the proglucagon gene and secrete GLP-1. These findings suggest a model of islet development in which pancreatic progenitor cells express both GLP-1 receptors and proglucagon with the formation of GLP-1. Locally produced GLP-1 may act as an autocrine/paracrine developmental morphogen on receptors on NIPs, resulting in the activation of IDX-1 and the expression of the proinsulin gene conferring a beta-cell phenotype. GLP-1 may be an important morphogen both for the embryonic development of the pancreas and for the neogenesis of beta-cells in the islets of the adult pancreas.     

Cell therapy for type 2 diabetes: is it desirable and can we get it?

The functional mass of beta-cells is decreased in type 2 diabetes. Replacing missing beta-cells or triggering their regeneration may thus allow for improved treatment of type 2 diabetes, to the extent that this is combined with therapy for improved insulin sensitivity. Although progress has been made in deriving beta-cell-like cells from stem or precursor cells in vitro, these cannot yet be obtained in sufficient quantities or well enough differentiated to envisage their therapeutic use in beta-cell replacement therapy. Likewise, our very limited understanding of beta-cell regeneration in adult man does not yet allow for development of a valid strategy for kick-starting such a process in individuals with type 2 diabetes, whether by bona fide neogenesis or self-replication of existing beta-cells. Regardless of how beta-cell mass is restored in type 2 diabetes, it will be important to prevent any renewed decrease thereafter. Current understanding suggests that islet inflammation as well as signals from (insulin-resistant/inflamed) adipose tissue and skeletal muscle contribute towards decreased beta-cell mass in type 2 diabetes. It will likely be important to protect newly formed or implanted beta-cells from these negative influences to ensure their long-term survival.     

��-Zell-Erhalt durch inkretinbasierte Therapien

Preservation of pancreatic beta-cells in patients with type 2 diabetes would be a substantial therapeutic improvement as the disease is associated with a progressive loss of insulin-producing beta-cells. In various preclinical studies glucagon-like peptide 1 (GLP-1) led to preservation of beta-cell mass by inducing beta-cell proliferation and neogenesis as well as inhibiting apoptosis. These results cannot readily be translated to the situation in humans and further clinical evidence is needed.     

GLP-1 receptor signaling: effects on pancreatic beta-cell proliferation and survival

Type 2 diabetes is a metabolic disorder characterized by insulin resistance as well as a progressive deterioration of pancreatic beta-cell mass and function. Glucagon-like peptide 1 (GLP-1), an incretin hormone secreted by intestinal L cells, is a promising therapeutic agent in the treatment of diabetes. GLP-1 analogs and enhancers constitute a novel class of anti-diabetes medications which address both the insulin secretion defect as well as the decline in beta-cell mass. GLP-1 improves glucose-stimulated insulin secretion, restores glucose competence in glucose-resistant beta-cells, and stimulates insulin gene expression and biosynthesis. Furthermore, GLP-1 acts as a growth factor by promoting beta-cell proliferation, survival and neogenesis. This review focuses on the molecular mechanisms by which GLP-1 signaling induces beta-cell mass expansion.     

Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells

In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase beta-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing beta-cells. Here, we investigated the effect of exendin 4 in the growth and differentiation of beta-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases. Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining. The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls. We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats. In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide. In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation. Thus, exendin 4 induces functional maturation of fetal beta-cells in response to glucose. In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells. We found a significant increase in beta-cell number, compared with the control rats. Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal beta-cells from undifferentiated precursor cells. It also accelerates the functional maturation of fetal beta-cells as evidenced by their glucose-stimulated insulin secretion.     

Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats

Glucose homeostasis in mammals is maintained by insulin secretion from the beta-cells of the islets of Langerhans. Type 2 diabetes results either from primary beta-cell failure alone and/or a failure to secrete enough insulin to overcome insulin resistance. Here, we show that continuous infusion of glucagon-like peptide-1 (7-36) (GLP-1; an insulinotropic agent), to young and old animals, had effects on the beta-cell of the pancreas other than simply on the insulin secretory apparatus. Our previous studies on a rodent model of glucose intolerance, the aging Wistar rat, show that a plateau in islet size, insulin content, and beta-cell mass is reached at 13 months, despite a continuing increase in body weight. Continuous sc infusion of GLP-1 (1.5 pM/kg x min), over 5 days, resulted in normal glucose tolerance. Our current results in both young and old rats demonstrate that treatment caused an up-regulation of pancreatic-duodenum homeobox-1 (PDX-1) expression in islets and total pancreas, induced pancreatic cell proliferation, and beta-cell neogenesis. The effects on levels of PDX-1 messenger RNA were abrogated by simultaneous infusion of Exendin (9-39), a specific antagonist of GLP-1. PDX-1 protein levels increased 4-fold in whole pancreata and 6-fold in islets in response to treatment. Beta-cell mass increased to 7.2 +/- 0.58 from 4.88 +/- 0.38 mg, treated vs. control, respectively, P < 0.02. Total pancreatic insulin content also increased from 0.55 +/- 0.02 to 1.32 +/- 0.11 microg/mg total pancreatic protein. Therefore, GLP-1 would seem to be a unique therapy that can stimulate pancreatic cell proliferation and beta-cell differentiation in the pancreas of rodents.     

Adult pancreatic alpha-cells: a new source of cells for beta-cell regeneration

Beta-cell deficit is the major pathological feature in type 1 and type 2 diabetes patients, and plays a key role in disease progression. In principle, beta-cell regeneration can occur by replication of pre-existing beta-cells, or by beta-cell neogenesis from stem/progenitors. Unfortunately, beta-cell replication is limited by the almost complete absence of beta-cells in patients with type 1 diabetes, and the increasing recognition that the beta-cell replicative capacity declines severely with age. Therefore, beta-cell neogenesis has received increasing interest. Many different cell types within the pancreas have been suggested as potential beta-cell stem/progenitor cells, but the data have been conflicting. In some cases, this may be due to different regeneration models. On the other hand, different results have been obtained with similar regeneration models, leading to confusion about the nature and existence of beta-cell neogenesis in adult animals. Here, we review the major candidates for adult regeneration pathways, and focus on the recent discovery that alpha-cells can function as a novel beta-cell progenitor. Of note, this is a pathway that appears to be unique to beta-cell neogenesis in the adult, as the embryonic pathway of beta-cell neogenesis does not proceed through a glucagon-positive intermediate. We conclude that beta-cell neogenesis from alpha-cells is a new pathway of potential therapeutic significance, making it of high importance to elucidate the molecular events in alpha- to beta-cell conversion.     

Maturation of in vitro-generated human islets after transplantation in nude mice

The long-term function of human pancreatic islet grafts may depend on the neogenesis of beta cells from epithelial precursors within the grafted tissue. We have developed an in vitro model for human islet neogenesis. In this study, we have investigated the morphological signs of maturation in cultivated human islet buds (CHIBs) before and after transplantation. Clusterin is a molecule associated with beta-cell differentiation in rodents. In adult human islets, clusterin expression was located only in alpha- and PP-cells, but in CHIBs and human fetal islets, it was distributed in all four types of endocrine cells. Some immature endocrine cells in the CHIBs co-expressed insulin and glucagon. After transplantation, CHIBs became mature with one type of hormone per endocrine cell, and clusterin expression became restricted in alpha-cells. Cells co-expressing endocrine markers and cytokeratin 19, as a sign of ductal to endocrine cell transition, were frequently detected in both fresh islets and CHIBs after transplantation. We conclude that clusterin may be involved in the development of islets, and the in vitro-derived islets become mature after transplantation into nude mice. Ductal cell differentiation into endocrine cells may be an important factor in sustaining the long-term function of islet transplants.     

Evidence of unrestrained beta-cell proliferation and neogenesis in a patient with hyperinsulinemic hypoglycemia after gastric bypass surgery

ABSTRACT

Hyperinsulinemic hypoglycemia syndrome (HIHG) is a rare complication of roux-en-Y gastric bypass surgery. The pathology is associated with an excessive function of pancreatic beta-cells, and requires pancreas resection in patients that are recalcitrant to nutritional and pharmacological interventions. The exact prevalence is not clearly understood and the underlying mechanisms not yet fully characterized. We herein sought to perform histological and molecular examination of pancreatic sections obtained from a patient who developed HIHG as a complication of gastric bypass compared to 3 weight-matched controls. We studied markers of cellular replication and beta-cell differentiation by immunohistochemistry and immunofluorescence. HIHG after gastric bypass was characterized by a profound increase in beta-cell mass. Cellular proliferation was increased in islets and ducts compared to controls, suggesting unrestrained proliferation in HIHG. We also detected beta-cell differentiation markers in duct cells and occasional duct cells displaying both insulin and glucagon immunoreactivity. These histological observations suggest that beta-cell differentiation from ductal progenitor cells could also underly beta-cell mass expansion in HIHG. Altogether, our results can be construed to demonstrate that HIHG after gastric bypass is characterized by abnormal beta-cell mass expansion, resulting from both unrestrained beta-cell replication and neogenesis.     

The effects of glucagon-like peptide-1 on the beta cell

Type 2 diabetes is a progressive disease characterized by insulin resistance and impaired beta-cell function. Treatments that prevent further beta-cell decline are therefore essential for the management of type 2 diabetes. Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is known to stimulate glucose-dependent insulin secretion. Furthermore, GLP-1 appears to have multiple positive effects on beta cells. However, GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), which limits the clinical relevance of GLP-1 for the treatment of type 2 diabetes. Two main classes of GLP-1-based therapies have now been developed: DPP-4 inhibitors and GLP-1 receptor agonists. Liraglutide and exenatide are examples of GLP-1 receptor agonists that have been developed to mimic the insulinotropic characteristics of endogenous GLP-1. Both have demonstrated improved beta-cell function in patients with type 2 diabetes, as assessed by homoeostasis model assessment-B analysis and proinsulin : insulin ratio. Additionally, liraglutide and exenatide are able to enhance first- and second-phase insulin secretion and are able to restore beta-cell sensitivity to glucose. Preclinical studies have shown that both liraglutide and exenatide treatment can increase beta-cell mass, stimulate beta-cell proliferation, increase beta-cell neogenesis and inhibit beta-cell apoptosis. Clinical studies are needed to confirm these findings in humans. Replication of these data in humans could have important clinical implications for the treatment of type 2 diabetes.     

Type 2 diabetes - a matter of failing beta-cell neogenesis? Clues from the GK rat model

Now that reduction in beta-cell mass has been clearly established in humans with type 2 diabetes mellitus (T2D), the debate focuses on the possible mechanisms responsible for decreased beta-cell number. Appropriate inbred rodent models are essential tools for this purpose. The information available from the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2D, is reviewed in such a perspective. We propose that the defective beta-cell mass in the GK model reflects mostly a persistently decreased beta-cell neogenesis. The data discussed in this review are consistent with the notion that poor proliferation and/or survival of the endocrine precursor cells during GK foetal life will result in a decreased pool of endocrine precursors in the pancreas, and hence an impaired capacity of beta-cell neogenesis (either primary in the foetus or compensatory in the newborn and the adult). As we also demonstrated that beta-cell neogenesis can be pharmacologically reactivated in the GK model, our work supports, on a more prospective basis, the concept that facilitation of T2D treatment may be obtained through beta-cell mass expansion after stimulation of beta-cell regeneration/neogenesis in diabetic patients.     

Molecular control of cell cycle progression in the pancreatic beta-cell

Type 1 and type 2 diabetes both result from inadequate production of insulin by the beta-cells of the pancreatic islet. Accordingly, strategies that lead to increased pancreatic beta-cell mass, as well as retained or enhanced function of islets, would be desirable for the treatment of diabetes. Although pancreatic beta-cells have long been viewed as terminally differentiated and irreversibly arrested, evidence now indicates that beta-cells can and do replicate, that this replication can be enhanced by a variety of maneuvers, and that beta-cell replication plays a quantitatively significant role in maintaining pancreatic beta-cell mass and function. Because beta-cells have been viewed as being unable to proliferate, the science of beta-cell replication is undeveloped. In the past several years, however, this has begun to change at a rapid pace, and many laboratories are now focused on elucidating the molecular details of the control of cell cycle in the beta-cell. In this review, we review the molecular details of cell cycle control as they relate to the pancreatic beta-cell. Our hope is that this review can serve as a common basis and also a roadmap for those interested in developing novel strategies for enhancing beta-cell replication and improving insulin production in animal models as well as in human pancreatic beta-cells.     

Existence of islet regenerating factors within the pancreas

Reduction in the functional mass of beta-cells is a common denominator in most forms of diabetes. Since the replicative potential of beta-cells is limited, the search for factors that trigger islet neogenesis becomes imperative. Here we tested the hypothesis that regenerating factors for the pancreas are either secreted by or present within the pancreatic milieu itself. For this purpose, we intraperitoneally injected pancreatic cell culture supernatant (PCCS), from normal pancreas, into streptozotocin (STZ)-induced diabetic mice for 15 consecutive days. The PCCS-treated mice showed sustained reversal in 77.77% of experimental diabetic mice as evidenced by restoration of normoglycemia, increase in serum insulin levels and occurrence of neo islets in histopathological studies during a two month follow up, as opposed to the control diabetic mice which remained hyperglycemic throughout. In order to examine the potential of PCCS to bring about the regeneration of islets, we treated intra-islet precursor cells with PCCS in vitro, which led to the neogenesis of islets as evidenced by dithiozone and insulin immunostaining. These findings substantiate our hypothesis and make the search for regenerative factors converge towards the pancreas and its immediate surroundings. Such regenerative approaches, in combination with other therapeutic strategies to promote islet neogenesis may, in future, provide a cure and/or better means for the control and management of diabetes.     

A role for beta-cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release

Pancreatic beta-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic beta-cell mass. These effects are mediated via stimulation of cAMP through beta-cell GLP-1 receptors. We report that the Galpha(s)-coupled receptor GPR119 is largely restricted to insulin-producing beta-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A(y) mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion.     

Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.     

Detrimental effect of protracted hyperglycaemia on beta-cell neogenesis in a mouse murine model of diabetes

AIMS/HYPOTHESIS: Previous studies have shown that new beta cells differentiate from intra-islet precursors in pancreatic islets of mice in which diabetes is induced by injecting a high dose of the beta-cell toxin streptozotocin. Moreover, the re-establishment of euglycaemia by insulin therapy 1 day after streptozotocin treatment improved the process of regeneration. We sought to assess whether a 1-week delay in the restoration of euglycaemia would affect beta-cell regeneration. METHODS: Adult CD-1 mice were injected with 200 mg/kg of streptozotocin. One group of mice remained hyperglycaemic throughout the experiment while a second group became normoglycaemic following the administration of insulin therapy 1 week after the injection of streptozotocin. Pancreata removed at different times after treatment were processed for visualization ofbeta precursor-cell markers and insulin by confocal microscopy. RESULTS: New beta cells appeared in islets of streptozotocin-treated mice after restoration of normoglycaemia. Like islets of streptozotocin mice in which blood glucose concentrations were rapidly restored, islets of mice that became normoglycaemic 1 week after streptozotocin treatment also had two potential insulin precursor cell types. Protracted hyperglycaemia however, had several harmful effects on insulin cell neogenesis, such as a reduction in the number of euglycaemic mice with successful beta-cell regeneration and a decrease in the number and survival of the newly differentiated insulin-containing cells. CONCLUSION/INTERPRETATION: These results indicate that islets gradually lose their regenerative potential when they are exposed to high circulating glucose concentrations for an extended period of time.     

Exercise and dexamethasone oppositely modulate beta-cell function and survival via independent pathways in 90% pancreatectomized rats

Long-term dexamethasone (DEX) treatment is well known for its ability to increase insulin resistance in liver and adipose tissues leading to hyperinsulinemia. On the other hand, exercise enhances peripheral insulin sensitivity. However, it is not clear whether DEX and/or exercise affect beta-cell mass and function in diabetic rats, and whether their effects can be associated with the modulation of the insulin/IGF-I signaling cascade in pancreatic beta-cells. After an 8-week study, whole body glucose disposal rates in 90% pancreatectomized (Px) and sham-operated male rats decreased with a high dose treatment of DEX (0.1mg DEX/kg body weight/day)(HDEX) treatment, while disposal rates increased with exercise. First-phase insulin secretion was decreased and delayed by DEX via the impairment of the glucose-sensing mechanism in beta-cells, while exercise reversed the impairment of first-phase insulin secretion caused by DEX, suggesting ameliorated beta-cell functions. However, exercise and DEX did not alter second-phase insulin secretion except for the fact that HDEX decreased insulin secretion at 120 min during hyperglycemic clamp in Px rats. Unlike beta-cell functions, DEX and exercise exhibited increased pancreatic beta-cell mass in two different pathways. Only exercise, through increased proliferation and decreased apoptosis, increased beta-cell mass via hyperplasia, which resulted from an enhanced insulin/IGF-I signaling cascade by insulin receptor substrate 2 induction. By contrast, DEX expanded beta-cell mass via hypertrophy and neogenesis from precursor cells, rather than increasing proliferation and decreasing apoptosis. In conclusion, the improvement of beta-cell function and survival via the activation of an insulin/IGF-I signaling cascade due to exercise has a crucial role in preventing the development and progression of type 2 diabetes.