Destruction of pancreatic beta-cells by transgenic induction of prostaglandin E2 in the islets

Type 2 diabetes mellitus is characterized by insulin resistance of peripheral tissues and dysfunction of pancreatic beta-cells. Furthermore, the number of pancreatic beta-cells decreases as a secondary effect of advanced type 2 diabetes, although the molecular mechanism has not been elucidated. Recently, it has been shown that hyperglycemic conditions induce the expression of cyclooxygenase-2 in pancreatic islets and increase the downstream product prostaglandin E(2) (PGE(2)). To investigate whether high glucose-induced PGE(2) has an adverse effect on pancreatic beta-cells, we generated transgenic mice (RIP-C2mE) that express cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in their beta-cells using the rat insulin-2 gene promoter (RIP). The homozygous RIP-C2mE (Tg/Tg) mice showed severe hyperglycemia from six weeks of age. Although the heterozygous RIP-C2mE (Tg/-) mice showed normal blood glucose levels throughout their lifetime, this level increased significantly compared with that of wild-type mice when glucose was loaded. The relative number of beta-cells to the total islet cell number was reduced to 54 and 14% in the RIP-C2mE (Tg/-) and (Tg/Tg) mice, respectively, whereas that in the wild-type mice was 84%. Importantly, the proliferation rate in the islets of the RIP-C2mE (Tg/Tg) mice at four weeks of age decreased significantly in comparison to that in the wild-type mice. Because beta-cells replicate not only during the postnatal period but also in the adult pancreas at a basal level, it is possible that increased PGE(2) signaling thus contributes to the reduction of the pancreatic beta-cell mass through inhibition of proliferation, thereby aggravating diabetes further.     

Differential sensitivity of GLUT1- and GLUT2-expressing beta cells to streptozotocin.

Streptozotocin injection in animals destroys pancreatic beta cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2(-/-) mice reexpressing either GLUT1 or GLUT2 in their beta cells under the rat insulin promoter (RIPG1 x G2(-/-) and RIPG2 x G2(-/-) mice, respectively). We demonstrated that injection of STZ into RIPG2 x G2(-/-) mice induced hyperglycemia (>20 mM) and an approximately 80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 x G2(-/-) islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 x G2(-/-) mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 x G2(-/-) islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward beta cells depends on the expression of GLUT2.     

GLUT2 in pancreatic and extra-pancreatic gluco-detection (review).

Detection of variations in blood glucose concentrations by pancreatic beta-cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic beta-cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K(ATP) channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2-/- mice will be described, which indicate that this transporter is essential for glucose sensing by pancreatic beta-cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.     

Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to beta-cells in streptozotocin-treated mice

Betacellulin (BTC) induces differentiation of pancreatic beta-cells and promotes regeneration of beta-cells in experimental diabetes. The present study was conducted to determine if BTC improved glucose metabolism in severe diabetes induced by a high dose of streptozotocin (STZ) in mice. Male ICR mice were injected with 200 microg/g ip STZ, and various doses of BTC were administered daily for 14 days. The plasma glucose concentration increased to a level of >500 mg/dl in STZ-injected mice. BTC (0.2 microg/g) significantly reduced the plasma glucose concentration, but a higher concentration was ineffective. The effect of BTC was marked by day 4 but became smaller on day 6 or later. The plasma insulin concentration and the insulin content were significantly higher in mice treated with 0.1 and 0.2 microg/g BTC. BTC treatment significantly increased the number of beta-cells in each islet as well as the number of insulin-positive islets. Within islets, the numbers of 5-bromo-2-deoxyuridine/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells were significantly increased by BTC. These results indicate that BTC improved hyperglycemia induced by a high dose of STZ by promoting neoformation of beta-cells, mainly from somatostatin-positive islet cells.     

Expression of glucose transporter 4 in the human pancreatic islet of Langerhans

Glucose transporter 4 (GLUT4) is the main insulin-responsive glucose transporter in skeletal muscle and adipose tissue of human and rodent, and is translocated to the plasma membrane in response to insulin. GLUT2 is well known as the main glucose transporter in pancreatic islets and could highly regulate glucose-stimulated insulin secretion by B-cells as a glucose sensor. We confirmed the presence of GLUT4 mRNA and GLUT4 protein in pancreas in the human. Indirect immunohistochemistry showed that the pancreatic islets of human and rat were conspicuously labeled by anti-GLUT4 antibody. The presence of placental leucine aminopeptidase (P-LAP), a homologue of insulin-regulated aminopeptidase (IRAP), was also shown in the human pancreatic islet. IRAP/P-LAP is thought to be involved in glucose metabolism. This study provides the first evidence that GLUT4 is present in human and rat pancreatic islets and may suggest its specific role in glucose homeostasis in conjunction with IRAP/P-LAP.     

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.     

ATP-sensitive potassium channels participate in glucose uptake in skeletal muscle and adipose tissue

ATP-sensitive potassium (K(ATP)) channels are known to be critical in the control of both insulin and glucagon secretion, the major hormones in the maintenance of glucose homeostasis. The involvement of K(ATP) channels in glucose uptake in the target tissues of insulin, however, is not known. We show here that Kir6.2(-/-) mice lacking Kir6.2, the pore-forming subunit of these channels, have no K(ATP) channel activity in their skeletal muscles. A 2-deoxy-[(3)H]glucose uptake experiment in vivo showed that the basal and insulin-stimulated glucose uptake in skeletal muscles and adipose tissues of Kir6.2(-/-) mice is enhanced compared with that in wild-type (WT) mice. In addition, in vitro measurement of glucose uptake indicates that disruption of the channel increases the basal glucose uptake in Kir6.2(-/-) extensor digitorum longus and the insulin-stimulated glucose uptake in Kir6.2(-/-) soleus muscle. In contrast, glucose uptake in adipose tissue, measured in vitro, was similar in Kir6.2(-/-) and WT mice, suggesting that the increase in glucose uptake in Kir6.2(-/-) adipocytes is mediated by altered extracellular hormonal or neuronal signals altered by disruption of the K(ATP) channels.     

WTC rat has unique characteristics such as resistant to streptozotocin

Because we found that WTC rats might be resistant to streptozotocin (STZ), we have elucidated the mechanisms of resistant to the diabetogenic effects of STZ in the WTC rats. Dose response to STZ was evaluated with glucose levels. No significant changes in glucose level to STZ administration were observed in WTC rats. Insulin secretion by suppling glucose was preserved in WTC rats even after STZ administration. Although there was no significant difference in gene expression of both GLUT2 and Kir6.2, which were involved in STZ resistance, between WTC rats and Wistar rats, the expression of metallothionein 2a in pancreas and liver to STZ administration of WTC rats was significantly higher than that of Wistar rats. Moreover, alloxan did not induce diabetes in WTC rats as same as STZ. These results suggest that WTC rats might have powerful antioxidant property to protect β cells in pancreas. Because the STZ-resistant property is very close characteristics to human beings, WTC rats will become a useful animal model in diabetic researches.     

Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the beta-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of beta-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the beta-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 +/- 0.2% (n = 6) of the cells in the pancreas of newborn (P1) and 0.9 +/- 0.1% (n = 5) of 8-wk-old mice were beta-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying beta-cell biology in normal and diabetic animals.     

Molecular target structures in alloxan-induced diabetes in mice

Type 1 diabetes results from irreversible damage of insulin-producing beta-cells. In laboratory animals, diabetes can be induced with alloxan (ALX), a 2,4,5,6-tetraoxopyrimidine. ALX is a potent generator of reactive oxygen species (ROS), which can mediate beta-cell toxicity. However, the initial lesions on essential beta-cell structures are not known. In this study, we report that the glucose transporter 2 (GLUT2) and glucokinase (GK) are target molecules for ALX. Ex vivo, a gradual decrement of both GLUT2 and GK mRNA expression was found in islets isolated from ALX-treated C57BL/6 mice. This reduction was more pronounced for GLUT2 than for GK. The mRNA expression of beta-actin was also slightly affected with time after ALX exposure, the proinsulin mRNA, however, remained unaffected as well as the pancreatic total insulin content. Pretreatment with D-glucose (D-G) protected the mRNA expression of GLUT2 and GK against ALX toxicity and prevented diabetes. Yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. Pretreatment with 5-thio-D-glucose (5-T-G) failed to prevent ALX diabetes, administration of zinc sulfate (Zn(2+))-enriched drinking water, however, reduced ALX-induced hyperglycemia. In conclusion, ALX exerted differential toxicity on beta-cell structures similar to in vitro results reported from this laboratory. Furthermore, the present results differ from those reported for the diabetogen streptozotocin (STZ). Injections of multiple low doses (MLD) of STZ reduced GLUT2 expression only, but failed to affect expression of GK and proinsulin as well as beta-actin as internal control. MLD-STZ diabetes was prevented by pretreatment with both D-G and 5-T-G and administration of Zn(2+)-enriched drinking water. Apparently, ALX and MLD-STZ exert diabetogenicity by different pathways requiring different interventional schedules for prevention.     

Exendin-(9-39) corrects fasting hypoglycemia in SUR-1-/- mice by lowering cAMP in pancreatic beta-cells and inhibiting insulin secretion

Congenital hyperinsulinism is a disorder of pancreatic beta-cell function characterized by failure to suppress insulin secretion in the setting of hypoglycemia, resulting in brain damage or death if untreated. Loss-of-function mutations in the K(ATP) channel (composed of two subunits: Kir6.2 and SUR-1) are responsible for the most common and severe form of congenital hyperinsulinism. Most patients are unresponsive to available medical therapy and require palliative pancreatectomy. Similar to the human condition, the SUR-1(-/-) mouse is hypoglycemic when fasted and hyperglycemic when glucose-loaded. We have previously reported that the glucagon-like peptide-1 receptor antagonist exendin-(9-39) raises fasting blood glucose in normal mice. Here we examine the effect of exendin-(9-39) on fasting blood glucose in SUR-1(-/-) mice. Mice were randomized to receive exendin-(9-39) or vehicle. Fasting blood glucose levels in SUR-1(-/-) mice treated with exendin-(9-39) were significantly higher than in vehicle-treated mice and not different from wild-type littermates. Exendin-(9-39) did not further worsen glucose tolerance and had no effect on body weight and insulin sensitivity. Isolated islet perifusion studies demonstrated that exendin-(9-39) blocked amino acid-stimulated insulin secretion, which is abnormally increased in SUR-1(-/-) islets. Furthermore, cAMP content in SUR-1(-/-) islets was reduced by exendin-(9-39) both basally and when stimulated by amino acids, whereas cytosolic calcium levels were not affected. These findings suggest that cAMP plays a key role in K(ATP)-independent insulin secretion and that the GLP-1 receptor is constitutively active in SUR-1(-/-) beta-cells. Our findings indicate that exendin-(9-39) normalizes fasting hypoglycemia in SUR-1(-/-) mice via a direct effect on insulin secretion, thereby raising exendin-(9-39) as a potential therapeutic agent for K(ATP) hyperinsulinism.     

The carboxyl termini of K(ATP) channels bind nucleotides

ATP-sensitive potassium (K(ATP)) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K(ATP) channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5'-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH(2)- and COOH-terminal cytosolic regions of the three known K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K(+) channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K(ATP) channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K(ATP) channels because this nucleotide did not bind to the NH(2) termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K(ATP) COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K(ATP) COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg(2+) and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K(ATP) tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.     

ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling

We previously found that disruption of Kir6.2-containing ATP-sensitive K+ (KATP) channels increases glucose uptake in skeletal muscle, but the mechanism is not clear. In the present study, we generated knockout mice lacking both Kir6.2 and insulin receptor substrate-1 (IRS-1). Because IRS-1 is the major substrate of insulin receptor kinase, we expected disruption of the IRS-1 gene to reduce glucose uptake in Kir6.2 knockout mice. However, the double-knockout mice do not develop insulin resistance or glucose intolerance. An insulin tolerance test reveals the glucose-lowering effect of exogenous insulin in double-knockout mice and in Kir6.2 knockout mice to be similarly enhanced compared with wild-type mice. The basal 2-deoxyglucose uptake rate in skeletal muscle of double-knockout mice is increased similarly to the rate in Kir6.2 knockout mice. Accordingly, disruption of the IRS-1 gene affects neither systemic insulin sensitivity nor glucose uptake in skeletal muscles of Kir6.2-deficient mice. In addition, no significant changes were observed in phosphatidylinositol 3-kinase (PI3K) activity and its downstream signal in skeletal muscle due to lack of the Kir6.2 gene. Disruption of Kir6.2-containing Katp channels clearly protects against IRS-1-associated insulin resistance by increasing glucose uptake in skeletal muscles by a mechanism separate from the IRS-1/PI3K pathway.