Koji Yamamoto

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]

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.

Expression of heparin-binding epidermal growth factor-like growth factor during pancreas development. A potential role of PDX-1 in transcriptional activation.

The development of the pancreas appears to be regulated by various growth factors. We report here the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in the developing pancreas. Immunostaining of fetal and neonatal rat pancreata, in which endocrine cells are visible as cell clusters often associated with primitive ducts or ductular cells, revealed that most of the cluster-forming cells and primitive ducts or ductular cells express HB-EGF protein. In contrast, the exocrine pancreas lacked HB-EGF expression. Based on findings that the expression pattern was similar to that of the homeodomain-containing transcription factor PDX-1 (IDX-1/STF-1/IPF1) and that the regulatory region of the HB-EGF gene contained sequences similar to the PDX-1-binding A element, we examined whether PDX-1 could be a potential activator of HB-EGF gene expression. The results of reporter gene analyses suggested that the HB-EGF gene promoter is PDX-1-responsive and that the activity of the promoter in pancreatic beta cell-derived βTC1 cells depends on the PDX-1 binding site-like sequences. Gel-mobility shift analyses using an anti-PDX-1 antibody indicated that PDX-1 is a specific and dominant binding factor for an A element-like sequence in the HB-EGF gene. These observations suggest the possible involvement of HB-EGF in pancreas development. While PDX-1 is essential for pancreas development, HB-EGF may function as a mediator of PDX-1 and thus be involved in the development of the endocrine 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]

Day-to-day changes in oxygen uptake kinetics at the onset of exercise during strenuous endurance training

SummaryThe aim of this study was to assess the effect of strenuous endurance training on day-to-day changes in oxygen uptake (VO2) on-kinetics (time constant) at the onset.of exercise. Four healthy men participated in strenuous training, for 30 min·day−1, 6 days·week−1 for 3 weeks. The VO2 was measured breath-by-breath every day except Sunday at exercise intensities corresponding to the lactate threshold (LT) and the onset of blood lactate accumulation (OBLA) which were obtained before training. Furthermore, an incremental exercise test was performed to determine LT, OBLA and maximal oxygen uptake (VO2max) before and after the training period and every weekend. The 30-min heavy endurance training was performed on a cycle ergometer 5 days·week−1 for 3 weeks. Another six men served as the control group. After training, significant reductions of the VO2 time constant for exercise at the pretraining LT exercise intensity (P<0.05) and at OBLA exercise intensity (P<0.01) were observed, whereas the VO2 time constants in the control group did not change significantly. A high correlation between the decrease in the VO2 time constant and training day was observed in exercise at the pretraining LT exercise intensity (r=−0.76; P<0.001) as well as in the OBLA exercise intensity (r= −0.91; P<0.001). A significant reduction in the blood lactate concentration during submaximal exercise and in the heart rate on-kinetics was observed in the training group. Furthermore, VO2 at LT, VO2 at OBLA and VO2max increased significantly after training (P<0.05) but such was not the case in the control group. These findings indicated that within a few weeks of training a rapidly improved VO2 on-kinetics may be observed. This may be explained. by some effect of blood lactate during exercise on VO2 on-kinetics, together with significantly improved cardiovascular kinetics at the onset of exercise.

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]

Oxidant-free direct coupling of internal alkynes and 2-alkylpyridine via double C-H activations by alkylhafnium complexes.

We have developed a novel oxidant-free direct cross-coupling reaction of 2,6-lutidine and internal alkynes leading to five-membered carbocyclic compounds mediated by nonmetallocene cationic hafnium alkyl complexes. Mechanistic studies of the coupling reaction showed that the reaction begins with C(sp(3))-H bond activation via σ-bond metathesis, after which the coordinatively unsaturated hafnium center mediates further insertion, migration, and β-H elimination reactions to give five-membered carbocycles from readily available substrates.

Analysis of expression profiles of islet-associated transcription and growth factors during β-cell neogenesis from duct cells in partially duct-ligated mice

Introduction &bgr;-cell neogenesis from pancreatic duct cells has been reported to occur in duct-ligated rat. Nevertheless, detailed process of this phenomenon has not been clarified. Aims and Methodology To clarify the mechanism of &bgr;-cell neogenesis, a partial pancreatic duct ligation mouse model was created. Proliferation of duct cells, &bgr;-cell neogenesis, and expression of transcription factors and differentiation/growth factors were studied by immunohistochemistry, cDNA array, and RT-PCR methods. Results In the duct-ligated portion of the pancreas, newly formed islet-like cell clusters (ICCs) were observed arising from the ducts on day 7 and afterward. Transcription factors, such as pancreatic and duodenal homeobox gene-1 (PDX-1), paired box factor 6 (Pax6), islet1 and Nkx2.2-positive cells, and protein gene product 9.5 (PGP9.5) were also induced in duct lining cells. By cDNA microarray analysis, expression of insulin-like growth factor-1 (IGF-1) and transforming growth factor &bgr;1 (TGF-&bgr;1) were above control levels on day 5, and RT-PCR showed an increase from day 5 to day 28. IGF-1 and activin A–positive cells were detected in ducts. In addition, expression of betacellulin (BTC), heparin-binding epidermal growth factor–like growth factor (HB-EGF), and TGF-&agr; were also increased from day 3 or 5. Conclusion These findings suggest that &bgr;-cell or endocrine precursors are localized among duct lining cells. Induction of several islet cell–associated transcription factors and differentiation and/or growth factors may play important roles during &bgr;-cell neogenesis in this model.

Relationship between cardiac output and oxygen uptake at the onset of exercise

SummaryThe purpose of the present study was to assess the relationship between the rapidity of increased gas exchange (i.e. oxygen uptake