Impaired β-cell regeneration after partial pancreatectomy in the adult Goto-Kakizaki rat, a spontaneous model of type II diabetes

In the Goto-Kakizaki (GK) rat, a genetic model of type II diabetes, there is a restriction of the beta-cell mass as early as fetal age, which is maintained reduced in the adult animal. In order to investigate the beta-cell growth potential in the adult hyperglycemic GK rat, and to determine whether it differs from non-diabetic Wistar (W) rats, we have performed 90% pancreatectomy (Px) in 8- to 10-week-old male animals. Spontaneous beta-cell regeneration and involvement of beta-cell replication, beta-cell neodifferentiation from ductal precursor, and beta-cell apoptosis were evaluated by immunocytochemistry and morphometry at different time points: day 0 (D0), D2, D7, and D14 after Px. In GK rats, deterioration of the diabetic state with severe and chronic hyperglycemia was evident as soon as D2, while in W/Px, normoglycemia to moderate hyperglycemia was observed. In W/Px rats, the total beta-cell mass gradually increased on D2, D7, and D14, as compared to non-Px W rats. By contrast, in GK/Px rats, there was only a non-significant tendency to increased total beta-cell mass, as compared to related non-Px group. Adult GK rats displayed lower beta-cell proliferation rates compared to W. In response to Px, early increase of beta-cell proliferation was present in both W/Px and GK/Px rats on D2, but it returned to non-Px values in GK rats on D7 and D14, while in W/Px rats beta-cell proliferation was maintained increased as compared to non-Px W rats. The very low apoptotic beta-cell frequency on D0, D2, D7, and D14, in both W and GK, either non-Px or Px, did not allow us to conclude that any significant differences exist between the different groups. beta-cell neoformation from ducts, and more specifically from foci of regeneration, was found to be less activated in GK/Px rats as compared to W/Px. Together, these results suggest that in the adult hyperglycemic GK rat undergoing Px, beta-cells still have the capacity to regenerate, but with a lower efficiency as compared to non-diabetic W rats. This defect in the GK rat is the result of both genetic predisposition contributing to an altered beta-cell neogenesis potential already present in the neonatal period, and environmental factors (chronic hyperglycemia) leading to a reduced beta-cell proliferative capacity specific to the adult animals.     

Exercise improves glucose homeostasis that has been impaired by a high-fat diet by potentiating pancreatic beta-cell function and mass through IRS2 in diabetic rats.

In this study, we investigated the effects of a high-fat diet and exercise on pancreatic beta-cell function and mass and its molecular mechanism in 90% pancreatectomized male rats. The pancreatectomized diabetic rats were given control diets (20% energy) or a high-fat (HF) diet (45% energy) for 12 wk. Half of each group was given regular exercise on an uphill treadmill at 20 m/min for 30 min 5 days/wk. HF diet lowered first-phase insulin secretion with glucose loading, whereas exercise training reversed this decrease. However, second-phase insulin secretion did not differ among the groups. Exercise increased pancreatic beta-cell mass. This resulted from stimulated beta-cell proliferation and reduced apoptosis, which is associated with potentiated insulin or IGF-I signaling through insulin receptor substrate-2 (IRS2) induction. Although the HF diet resulted in decreased proliferation and accelerated apoptosis by weakened insulin and IGF-I signaling from reduction of IRS2 protein, beta-cell mass was maintained in HF rats just as much as in control rats via increased individual beta-cell size and neogenesis from precursor cells. Consistent with the results of beta-cell proliferation, pancreas duodenal homeobox-1 expression increased in the islets of rats in the exercise groups, and it was reduced the most in rats fed the HF diet. In conclusion, exercise combined with a moderate fat diet is a good way to maximize beta-cell function and mass through IRS2 induction to alleviate the diabetic condition. This study suggests that dietary fat contents and exercise modulate beta-cell function and mass to overcome insulin resistance in two different pathways.     

Impaired beta-cell regeneration in perinatally malnourished rats: a study with STZ.

We investigated the mechanisms implicated in beta-cell mass reduction observed during late fetal and early postnatal malnutrition in the rat. Beta-cell regeneration, including proliferation and neogenesis, was studied after neonatal beta-cell destruction by streptozotocin (STZ). STZ was injected at birth and maternal food restriction was continued until weaning. Beta-cell mass, proliferation, and islet number were quantified by morphometrical measurements on pancreatic sections in STZ-injected normal (C-STZ) and malnourished (R-STZ) rats, with noninjected C and R rats as controls. At day 4, only 20% of the beta cell-mass remained in C-STZ rats. It regenerated to 50% that of noninjected controls, mainly through active neogenesis, as shown by the entire recovery of islet number/cm(2), and also through moderately increased beta-cell proliferation. In contrast, beta-cell mass from R-STZ animals poorly regenerated, despite a dramatic increase of beta-cell proliferation, because islet number/cm(2) recovered insufficiently. In conclusion, perinatal malnutrition impairs neogenesis and the capacity of beta-cell regeneration by neogenesis but preserves beta-cell proliferation, which remains the elective choice to increase beta-cell mass. These results provide an explanation for the impaired capacity of malnourished animals to adapt their beta-cell mass during aging or pregnancy, which aggravate glucose tolerance.     

Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet beta-cell mass

A cDNA termed reg was recently isolated by differential screening of a library prepared from regenerating islets isolated from pancreatic remnants of rats subjected to 90% pancreatectomy and nicotinamide treatment. This led to speculation that this gene may be involved in expansion of beta-cell mass. In the current study we have measured reg expression after implantation and resection of a solid insulinoma tumor into rats, maneuvers known, respectively, to reduce and reexpand the volume of beta-cells in the islet. Animals with an implanted insulinoma tumor became profoundly hypoglycemic. Islet beta-cells declined from the normal 75% of total islet volume to less than 30%, in concert with a marked reduction in the reg mRNA level. Removal of the tumor resulted in a sharp increase in beta-cell replication, as measured by [3H]thymidine incorporation and a return to normal beta-cell volume within 4 days of tumor resection. This was associated with a transient induction in reg expression compared to that in tumor-bearing animals, effectively returning the amount of reg mRNA to the levels found in normal animals within 48 h; at later time points after tumor removal (3-7 days) reg expression declined, but then rose toward normal. In situ hybridization analysis localized the initial induction in reg mRNA expression to the exocrine pancreas. Continuous infusion of insulin into normal rats for 4 days, a maneuver that does not significantly reduce beta-cell mass, resulted in dramatically reduced insulin mRNA in islets, but no change in the levels of reg mRNA. We conclude that the diminution in pancreatic beta-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced reg gene expression and that the increase in beta-cell replication after resection of the tumor is preceded by return of reg gene expression toward normal.     

TNF-Like Weak Inducer of Apoptosis (TWEAK) Promotes Beta Cell Neogenesis from Pancreatic Ductal Epithelium in Adult Mice

Aim/Hypothesis

The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice.

Methods

C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER^TM: R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px.

Results

TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion.

Conclusions/Interpretation

We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice.     

Increased IRS-2 content and activation of IGF-I pathway contribute to enhance beta-cell mass in fetuses from undernourished pregnant rats

We have previously shown that fetuses from undernourished (U) pregnant rats exhibited an increased beta-cell mass probably related to an enhanced IGF-I replicative response. Because IGF-I signaling pathways have been implicated in regulating beta-cell growth, we investigated in this study the IGF-I transduction system in U fetuses. To this end, an in vitro model of primary fetal islets was developed to characterize glucose/IGF-I-mediated signaling that specially influences beta-cell proliferation. We found that U fetal islets showed a greater replicative response to glucose and IGF-I than controls. Furthermore, insulin receptor substrate (IRS)-2 protein and its association with p85 were also increased. In the complete absence of IGF-I or stimulatory glucose, U islets presented an increased basal phosphorylation of downstream signals of the phosphatidylinositol 3-kinase (PI3K) pathway such as PKB, glycogen synthase kinase (GSK)3alpha/beta, PKCzeta, and mammalian target of rapamycin (mTOR). Similarly, phosphorylation of these proteins (except GSK3alpha/beta) by glucose and IGF-I was augmented even though total protein content remained unchanged. Downstream of PKB, direct glucose activation of mTOR was increased as well. In contrast, ERK1/2 phosphorylation was unaffected by undernutrition, but ERK activation seemed to be required to induce a higher proliferative response in U islets. In conclusion, we have demonstrated that fetal U islets show increased IRS-2 content and an enhancement in both basal and glucose/IGF-I activations of the IRS-2/PI3K/PKB pathway. These molecular changes may be responsible for the greater glucose/IGF-I islet replication and contribute to the increased beta-cell mass found in these fetuses.     

Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis

Development of diabetes generally reflects an inadequate mass of insulin-producing beta-cells. beta-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of beta-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired beta-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal beta-cell number and function.     

Effects of high-fat diet exposure during fetal life on type 2 diabetes development in the progeny

Nutrition during fetal life is a critical factor contributing to diabetes development in adulthood. The aim of our study was to verify: 1) whether a high-fat (HF) diet in young adult mice induces alterations in beta-cell mass, proliferation, neogenesis, and apoptosis, as well as insulin sensitivity and secretion; 2) whether these alterations may be reversible after HF diet suspension; 3) the effects in a first (F1) and second generation (F2) of mice without direct exposure to a HF diet after birth. Type 2 diabetes developed in adult mice on a HF diet, in F1 mice that were HF diet-exposed during fetal or neonatal life, and in F2 mice whose mothers were HF diet-exposed during their fetal life. beta-cell mass, replication, and neogenesis were high in HF diet-exposed mice and decreased after diet suspension. beta-cell mass and replication remained high in F1 mice and decreased in F2 mice whose mothers were exposed to a HF diet. beta-cell neogenesis was present in adult mice on a HF diet and in F1 mice that were HF diet-exposed during fetal and/or neonatal life. We conclude that a HF diet during fetal life, particularly if combined with the same insult during the suckling period, can induce the type 2 diabetes phenotype, which can be directly transmitted to the progeny even in the absence of additional dietary insults.     

Protein calorie restriction has opposite effects on glucose metabolism and insulin gene expression in fetal and adult rat endocrine pancreas

We previously demonstrated that fetuses from undernourished pregnant rats exhibited increased beta-cell mass and hyperinsulinemia, whereas keeping food restriction until adult age caused reduced beta-cell mass, hypoinsulinemia, and decreased insulin secretion. Because these alterations can be related to insulin availability, we have now investigated early and long-term effects of protein calorie food restriction on insulin mRNA levels as well as the possible mechanisms that could modulate the endogenous insulin mRNA content. We used fetuses at 21.5 days of gestation proceeding from food-restricted rats during the last week of pregnancy and 70-day-old rats undernourished from day 14 of gestation until adult age and with respective controls. Insulin mRNA levels, glucose transporters, and total glycolysis and mitochondrial oxidative fluxes were evaluated. We additionally analyzed undernutrition effects on signals implicated in glucose-mediated insulin gene expression, especially pancreatic duodenal homeobox-1 (PDX-1), stress-activated protein kinase-2 (p38/SAPK2), and phosphatidylinositol 3-kinase. Undernourished fetuses showed increased insulin mRNA, oxidative glucose metabolism, and p38/SAPK2 levels, whereas undernutrition until adult age provoked a decrease in insulin gene expression, oxidative glucose metabolism, and PDX-1 levels. The results indicate that food restriction caused changes in insulin gene expression and content leading to alterations in glucose-stimulated insulin secretion. The molecular events, increased p38/SAPK2 levels in fetuses and decreased PDX-1 levels in adults, seem to be the responsible for the altered insulin mRNA expression. Moreover, because PDX-1 activation appears to be regulated by glucose-derived metabolite(s), the altered glucose oxidation caused by undernutrition could in some manner affect insulin mRNA expression.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

Activation of the Reg family genes by pancreatic-specific IGF-I gene deficiency and after streptozotocin-induced diabetes in mouse pancreas

We have recently reported that Pdx1-Cre-mediated whole pancreas inactivation of IGF-I gene [in pancreatic-specific IGF-I gene-deficient (PID) mice] results in increased beta-cell mass and significant protection against both type 1 and type 2 diabetes. Because the phenotype is unlikely a direct consequence of IGF-I deficiency, the present study was designed to explore possible activation of proislet factors in PID mice by using a whole genome DNA microarray. As a result, multiple members of the Reg family genes (Reg2, -3alpha, and -3beta, previously not known to promote islet cell growth) were significantly upregulated in the pancreas. This finding was subsequently confirmed by Northern blot and/or real-time PCR, which exhibited 2- to 8-fold increases in the levels of these mRNAs. Interestingly, these Reg family genes were also activated after streptozotocin-induced beta-cell damage and diabetes (wild-type T1D mice) when islet cells were undergoing regeneration. Immunohistochemistry revealed increased Reg proteins in exocrine as well as endocrine pancreas and suggested their potential role in beta-cell neogenesis in PID or T1D mice. Previously, other Reg proteins (Reg1 and islet neogenesis-associated protein) have been shown to promote islet cell replication and neogenesis. These uncharacterized Reg proteins may play a similar but more potent role, not only in normal islet cell growth in PID mice, but also in islet cell regeneration after T1D.     

Pancreatic endocrine and exocrine cell ontogeny from renal capsule transplanted embryonic stem cells in streptozocin-injured mice.

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia.

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.     

A general and islet cell-enriched overexpression of IGF-I results in normal islet cell growth, hypoglycemia, and significant resistance to experimental diabetes

Insulin-like growth factor I (IGF-I) is normally produced from hepatocytes and various other cells and tissues, including the pancreas, and is known to stimulate islet cell replication in vitro, prevent Fas-mediated beta-cell destruction and delay the onset of diabetes in nonobese diabetic mice. Recently, however, the notion that IGF-I stimulates islet cell growth has been challenged by the results of IGF-I and receptor gene targeting. To test the effects of a general, more profound increase in circulating IGF-I on islet cell growth and glucose homeostasis, we have characterized MT-IGF mice, which overexpress the IGF-I gene under the metallothionein I promoter. In early reports, a 1.5-fold-elevated serum IGF-I level caused accelerated somatic growth and pancreatic enlargement. We demonstrated that the transgene expression, although widespread, was highly concentrated in the beta-cells of the pancreatic islets. Yet, islet cell percent and pancreatic morphology were unaffected. IGF-I overexpression resulted in significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance but normal insulin secretion and sensitivity. Pyruvate tolerance test indicated significantly suppressed hepatic gluconeogenesis, which might explain the severe hypoglycemia after fasting. Finally, due to a partial prevention of beta-cell death against onset of diabetes and/or the insulin-like effects of IGF-I overexpression, MT-IGF mice (which overexpress the IGF-I gene under the metallothionein I promoter) were significantly resistant to streptozotocin-induced diabetes, with diminished hyperglycemia and prevention of weight loss and death. Although IGF-I might not promote islet cell growth, its overexpression is clearly antidiabetic by improving islet cell survival and/or providing insulin-like effects.     

Enhanced beta-cell mass without increased proliferation following chronic mild glucose infusion

The physiological mechanisms underlying pancreatic beta-cell mass (BCM) homeostasis are complex and not fully resolved. Here we examined the factors contributing to the increased BCM following a mild glucose infusion (GI) whereby normoglycemia was maintained through 96 h. We used morphometric and immunochemical methods to investigate enhanced beta-cell growth and survival in Sprague-Dawley rats. BCM was elevated >2.5-fold over saline-infused control rats by 48 h and increased modestly thereafter. Unexpectedly, increases in beta-cell proliferation were not observed at any time point through 4 days. Instead, enhanced numbers of insulin(+) cell clusters and small islets (400-12,000 microm(2); approximately 23- to 124-microm diameter), mostly scattered among the acini, were observed in the GI rats by 48 h despite no difference in the numbers of medium to large islets. We previously showed that increased beta-cell growth in rodent models of insulin resistance and pancreatic regeneration involves increased activated Akt/PKB, a key beta-cell signaling intermediate, in both islets and endocrine cell clusters. GI in normal rats also leads to increased Akt activation in islet beta-cells, as well as in insulin(+) and insulin(-) cells in the common duct epithelium and endocrine clusters. This correlated with strong Pdx1 expression in these same cells. These results suggest that mechanisms other than proliferation underlie the rapid beta-cell growth response following a mild GI in the normal rat and involve Akt-regulated enhanced beta-cell survival potential and neogenesis from epithelial precursors.     

Overexpression of a dominant negative GIP receptor in transgenic mice results in disturbed postnatal pancreatic islet and beta-cell development

The expression of a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) under the control of the rat pro-insulin gene promoter induces severe diabetes mellitus in transgenic mice. This study aims to gain further insight into the effect of the expression of a dominant negative GIPR on glucose homeostasis and postnatal development of the endocrine pancreas. The diabetic phenotype of GIPRdn transgenic animals was first observed between 14 and 21 days of age (urine glucose>1000 mg/dl). After onset of diabetes, serum glucose was significantly higher and insulin values were significantly lower in GIPRdn transgenic mice vs. non-transgenic littermate controls. Morphometric studies of pancreatic islets and their endocrine cell types were carried out at 10, 30 and 90 days of age. The total islet and total beta-cell volume of transgenic mice was severely reduced as compared to control mice, irrespective of the age at sampling (p<0.05). The total volume of isolated insulin positive cells that were not contained within established islets was significantly reduced in transgenic mice, indicating disturbed islet neogenesis. These findings demonstrate in vivo evidence that intact signaling of G-protein coupled receptors is involved in postnatal islet and beta-cell development and neogenesis of the pancreatic islets.     

Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.