Mitsuyoshi Namba

Glucagonostatic and insulinotropic action of glucagonlike peptide I-(7-36)-amide

We examined the effect of glucagonlike peptides (GLPs), which are cleaved from preproglucagon in the enteroglucagon cells, on rat endocrine pancreas with the isolated perfused system. GLP-I-(7–36)-amide, a truncated form of full-sequence GLP-I-(1–37), showed a potent inhibitory effect on glucagon secretion. This inhibitory effect of GLP-I-(7–36)-amide was demonstrated at concentrations of 0.25, 2.5, and 25 nM in 11.2 and 2.8 mM glucose. In contrast, insulin release was significantly stimulated by GLP-I-(7–36)-amide at its concentration from 0.025 to 25 nM in a high glucose concentration, whereas in a low glucose concentration, the stimulation was seen only at the highest concentration (25 nM). Neither GLP-I-(1–37) nor GLP-II showed any effect on glucagon and insulin release. Although several gastrointestinal hormones have been nominated as incretins, none of them may suppress the glucagon secretion. A truncated form of GLP-I, GLP-I-(7–36)-amide thus seems to be a unique incretin that exerts glucagonostatic action.

Fas and Fas ligand expression in inflamed islets in pancreas sections of patients with recent-onset Type I diabetes mellitus.

Aims/hypothesis. Type I (insulin-dependent) diabetes results mainly from T-cell-mediated autoimmune destruction of pancreatic beta cells. Cytotoxic T lymphocytes destroy target cells via a perforin-based or Fas-based mechanism. Our previous study indicated that the Fas-Fas ligand (FasL) pathway is required for the development of autoimmune diabetes in the NOD mouse. We now investigated whether or not the Fas-FasL system is involved in the beta-cell destruction in human Type I diabetes. Methods. We immunohistochemically analysed pancreas biopsy specimens of 13 recent-onset patients. Results. Pancreatic islets were identified but showed various degrees of reduction in beta-cell volume in all patients. Out of 13 patients 6 had insulitis. In these 6 patients Fas was expressed in both the islets and infiltrating cells but not in either cell type in the 7 other patients without insulitis. Double immunostaining showed that Fas was positive in 92.2 to 97.7 % of beta cells but only in 17.6 to 46.7 % of alpha cells in Fas-positive, insulin-remaining islets. We found FasL was expressed exclusively in islet-infiltrating cells in patients with insulitis. Double immunostaining revealed that the most prevalent phenotype of FasL-positive cells was CD8, which was followed by macrophages and CD4. Conclusion/interpretation. The interaction between Fas on beta cells and FasL on infiltrating cells might trigger selective apoptotic beta-cell death in inflamed islets, leading to immune-mediated Type I diabetes. [Diabetologia (1999) 42: 1332–1340]

Cytokine-induced apoptotic cell death in a mouse pancreatic beta-cell line: inhibition by Bcl-2.

SummaryCytokines are thought to contribute to the induction of pancreatic beta-cell destruction in insulin-dependent diabetes mellitus. The molecular mechanisms that underlie beta-cell death were investigated by studying cytokine-induced cell death in beta-cell lines. A combination of three cytokines (interleukin-1Β, tumour necrosis factor-α, and interferon-γ) induced apoptotic cell death in the mouse pancreatic beta-cell line ΒTC1, as judged from the appearance of cells with hypodiploid nuclei and oligonucleosomal DNA fragmentation. The same treatment also induced apoptosis in the mouse pancreatic alpha-cell line αTC1 and the NOD/Lt mouse beta-cell line NIT-1, although to a lesser extent than in ΒTC1 cells. The abundance of endogenous Bcl-2 in ΒTC1 cells was lower than that in the other two cell lines. Overexpression of human Bcl-2 in ΒTC1 cells partially protected them from cytokine-induced cell death. These results suggest that apoptosis may be responsible, at least in part, for cytokine-induced beta-cell destruction and that Bcl-2 prevents apoptosis in pancreatic islet cells.

Mutation P291fsinsC in the Transcription Factor Hepatocyte Nuclear Factor-1α is Dominant Negative

The type 3 form of maturity-onset diabetes of the young (M0DY3) results from mutations in the gene encoding the transcription factor, hepatocyte nuclear factor-1α (HNF-1α). The mechanism by which mutations in only one allele of the HNF-1α gene impair pancreatic β-cell function is unclear. The functional form of HNF-1α is a dimer—either a homodimer or a heterodimer with the structurally related protein HNF-1β—that binds to and activates transcription of the genes whose expression it regulates. HNF-1α is composed of three functional domains: an amino-terminal dimerization domain (amino acids 1–32), a DNA-binding domain with POU-like and homeodomain-like motifs (amino acids 150–280), and a COOH-terminal transactivation domain (amino acids 281–631). Because the dimerization domain is intact in many of the mutant forms of HNF-1α found in MODY subjects, these mutant proteins may impair pancreatic β-cell function by forming nonproductive dimers with wild-type protein, thereby inhibiting its activity; that is, they are dominant-negative mutations. This hypothesis was tested by comparing the functional properties of the frameshift mutation P291fsinsC, the most common mutation identified to date in MODY3 patients, and wild-type HNF-1α. P291fsinsC-HNF-1α showed no transcriptional transactivation activity in HeLa cells, which lack endogenous HNF-1α. Overexpression of P291fsinsC-HNF-1α in MIN6 cells, a mouse β-cell line, resulted in an ∼40% inhibition of the endogenous HNF1α activity in a dosage-dependent manner. Furthermore, heterodimer formation between wild-type and P291fsinsC mutant proteins were observed by electrophoretic mobility shift assay. These data suggest that the P291fsinsC mutation in HNF-1α functions as a dominant-negative mutation. However, other mutations, such as those in the promoter region and dimerization domain, may represent loss of function mutations. Thus mutations in the HNF-1α gene may lead to β-cell dysfunction by two different mechanisms.

Demonstration of Two Different Processes of β-Cell Regeneration in a New Diabetic Mouse Model Induced by Selective Perfusion of Alloxan

To clarify the regeneration process of pancreatic β-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of β-cells in alloxan-perfused segments, while β-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neoislet formation in the alloxan-perfused segment and the proliferation of spared β-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single β-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for β-cell regeneration in β-cell–depleted segment. In addition to β-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were β-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine–labeling index in β-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet β-cells in the nonperfused segment. In conclusion, the regeneration process of β-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.

Immunological abnormalities in islets at diagnosis paralleled further deterioration of glycaemic control in patients with recent-onset Type I (insulin-dependent) diabetes mellitus

Aims/hypothesis. To determine whether the clinical heterogeneity observed in the development of Type I (insulin-dependent) diabetes mellitus correlates with immunohistochemical differences observed at diagnosis. Methods. Patients (n = 17) with recent-onset diabetes clinically considered to be insulin dependent (Type I), underwent pancreatic biopsy for immunohistological analysis. These patients were divided into two groups based on the presence or absence of islet immunological abnormalities (insulitis or hyperexpression of MHC class I antigens or both). The patients were also HLA typed and tested for islet cell antibodies and antibodies to glutamic acid decarboxylase (GAD-Ab). All patients were followed monthly for 2 years and their fasting plasma glucose, haemoglobin A1C and daily insulin doses were recorded. The clinical course of patients with islet immunological abnormalities was compared with that of patients without those abnormalities. Results. Patients with and without islet immunological abnormalities did not differ with regard to HLA type or islet cell antibodies. Antibodies to glutamic acid decarboxylase correlated with the presence of insulitis and MHC class I hyperexpression. These local immunological abnormalities were also associated with higher haemoglobin A1C values (p < 0.05) and a trend towards greater insulin requirements. Further, patients with the islet abnormalities had higher fasting plasma glucose concentrations 2 years after the biopsy than at the time of the biopsy (p < 0.05). Conclusion/interpretation. The heterogeneous clinical course observed following diagnosis in patients with Type I diabetes correlates with islet immunological abnormalities. Insulitis and hyperexpression of MHC class I correlate with deteriorating glycaemic control. [Diabetologia (1999) 42: 574–578]

Three new mutations in the hepatocyte nuclear factor-1α gene in Japanese subjects with diabetes mellitus: clinical features and functional characterization

Aims/hypothesis. Mutations in the hepatocyte nuclear factor-1α gene are a common cause of the type 3 form of maturity-onset diabetes of the young. We examined the clinical features and molecular basis of hepatocyte nuclear factor-1α (HNF-1α) diabetes. Methods. Thirty-seven Japanese subjects with early onset Type II (non-insulin-dependent) diabetes mellitus and 45 with Type I (insulin-dependent) diabetes mellitus were screened for mutations in this gene. Functional properties of mutant HNF-1α were also investigated. Results. Three new mutations [G415R, R272C and A site of the promoter ( + 102G-to-C)] were found. Insulin secretion was impaired in the three subjects. Insulin and glucagon secretory responses to arginine in the subject with the R272C mutation were also diminished. Molecular biological studies indicated that the G415R mutation generated a protein with about 50 % of the activity of wild-type HNF-1α. The R272C mutation had no transactivating or DNA binding activity and acted in a dominant negative manner. The + 102 G-to-C mutation in the A site of the promoter activity was associated with an increase in promoter activity and it had 42–75 % more activity than the wild-type sequence. Conclusion/interpretation. Mutations in the HNF-1α gene may affect the normal islet function by different molecular mechanisms. [Diabetologia (1999) 42: 621–626]

GLUCAGON-LIKE PEPTIDE-1(7-36) AMIDE ENHANCES INSULIN-STIMULATED GLUCOSE UPTAKE AND DECREASES INTRACELLULAR CAMP CONTENT IN ISOLATED RAT ADIPOCYTES

We investigated the effect of GLPs on glucose uptake in isolated rat adipocytes. GLP-1(7-36)amide significantly enhanced glucose uptake in the presence of 1 nM insulin. GLP-1(7-36)amide at 15 nM increased glucose uptake maximally by 56.4% as compared with 1 nM insulin alone (P < 0.01). In contrast, with less than 1 nM insulin or without insulin GLP-1(7-36)amide showed no effect on glucose uptake. Full-sequence GLP-1(1-37) at 15 nM in the presence of 1 nM insulin increased glucose uptake by 24.6% as compared with 1 nM insulin alone (P < 0.05). GLP-2 showed no effect on glucose uptake. Further, we examined the effect of GLP-1(7-36)amide on cAMP content in isolated rat adipocytes. Insulin at 1 nM caused a significant decrease of cAMP content. The combination of 15 nM GLP-1(7-36)amide and 1 nM insulin caused a further reduction of cAMP content. These data indicate that GLP-1(7-36)amide possesses augmentative effects on insulin action in isolated rat adipocytes. Furthermore, it is suggested that the stimulatory effect of GLP-1(7-36)amide occurs through the reduction of intracellular cAMP content.

Metabolic consequence of long-term exposure of pancreatic β cells to free fatty acid with special reference to glucose insensitivity

Long-term exposure of the pancreatic beta cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta-oxidation. Intracellular content of triglyceride and FFA were elevated, beta-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to beta cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.

Trophic effect of glucagon-(1-21)-peptide on the isolated rat ileal mucosal cells.

The trophic effect of glucagon-(1-21)-peptide on rat ileal epithelial cells was studied in vitro. Glucagon-(1-21)-peptide stimulated [3H]thymidine incorporation of mucosal cells significantly in a dose-dependent manner. Then glucagon-related peptides which have common sequences with glucagon-(1-21)-peptide were also tested. The biological potencies to augment [3H]-thymidine uptake were closely related with their amino-acid residues of N-terminal region. The result suggests that the N-terminal amino-acid sequence of glucagon molecule plays an important role in intestinal cell growth.