Long-term efficacy and safety of sitagliptin in the treatment of Japanese Type 2 diabetes (ASSET-K1) to a target of HbA1c <7%.

BACKGROUND: Few studies have investigated the factors related to improvement and maintenance of glycemic control with sitagliptin in Type 2 diabetes (T2D) patients. AIM: To identify factors contributing to reaching and maintaining glycated hemoglobin (HbA1c) <7% with sitagliptin in Japanese T2D patients. SUBJECTS AND METHODS: This study included 1327 patients who were: taking sitagliptin as monotherapy; switched to sitagliptin; or taking sitagliptin in combination therapy. At baseline and 1, 3, 6, and 12 months after starting sitagliptin, weight, body mass index (BMI), HbA1c, fasting plasma glucose (FPG), and post-prandial plasma glucose (PPG) were measured. The subjects were divided into a group that achieved HbA1c<7% at 12 months, a poor control group (HbA1c≥8% at 12 months), and a discontinued group. Multiple regression analysis was performed to identify factors contributing to long-term control and maintenance with sitagliptin treatment. RESULTS: HbA1c decreased significantly from 8.0% at baseline to 7.3%, but weight was unchanged. FPG and PPG improved significantly. The HbA1c<7% group had a significantly higher age and a signifi cant ly lower BMI at baseline than the HbA1c≥8% group and the discontinued group. On multivariate regression analysis, baseline HbA1c, baseline BMI, and Δbody weight after 12 months were significantly related to HbA1c reduction. The most common adverse event was hypoglycemia, and the most common adverse event responsible for discontinuation was constipation. CONCLUSIONS: HbA1c<7.0% was achieved in 31% of T2D patients who had poor control with conventional treatment. Weight management is important for maintaining good long-term control with sitagliptin.

Information theoretical approach to the storage capacity of neural networks with binary weights.

The storage capacity of the perceptron with binary weights w(i)in[0,1] is derived by introducing the minimum distance d between input patterns. The approach presented in this paper is based on some results in the information theory, and the obtained storage capacity 0.585 is in good agreement with the well-known value 0.59 by the replica method in statistical physics. A strength of the present information theoretical approach is that it provides an easier and more intuitive understanding for the storage capacity than the replica method, which is believed to be more reliable and informative than the Vapnik-Chervonenkis procedure.

Antibodies to GAD in Japanese diabetic patients: a multicenter study.

We determined the prevalence of antibodies to glutamic acid decarboxylase (anti-GAD) in Japanese diabetic patients. Anti-GAD were detected by RIP Anti-GAD Hoechst, which is a new sensitive radioimmunoassay (RIA) kit using purified pig brain GAD as the antigen. One thousand nine hundred Japanese patients were collected by the Study Group for Antibodies to GAD. The prevalence of anti-GAD in the subjects of this study was: 35.4% (326/921) in all patients with IDDM, 50.3% (96/191) in patients with IDDM less than 1-year duration, 4.3% (29/680) in NIDDM, 37.9% (39/103) in slowly progressive IDDM, 10.5% (4/38) in gestational diabetes mellitus, 0% (0/27) in impaired glucose tolerance, 4.8% (6/124) in the school children with glycosuria, 2.1% (1/47) in the relatives of IDDM and 5.0% (1/20) in neurological diseases without diabetes. The prevalence in normal subjects was 2.2% (7/323). Anti-GAD are frequently detected by the RIA kit in patients with IDDM of short duration and this assay may be useful for population screening for IDDM and for better understanding of its pathogenesis.

A novel microwell indirect immunofluorescence assay to detect antibodies against islet cell surface antigens in insulin-dependent diabetes mellitus.

End-point titers of islet cell surface antibodies (ICSA) were determined in 144 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM), 98 siblings to such patients, and 188 controls using a microwell indirect immunofluorescence assay with an insulin-producing cell line of Syrian hamster origin. The prevalence of ICSA of a titer equal to or greater than 1:4 was 33% (p less than 0.001) in IDDM patients and 15% (p less than 0.01) in siblings, compared to 5% among the controls. In patients under 15 years of age at clinical onset, the prevalence of ICSA was 43% (37/77), which was higher than the 30% (20/67) found in patients older than 15 years at onset (p less than 0.05). The ICSA titer at the time of diagnosis varied between 1:4 and 1:32; however, 43% (23/53) of ICSA positive IDDM patients had a titer equal to or greater than 1:8. Although gender did not affect ICSA prevalence, 56% (15/27) of the female IDDM patients had ICSA titers exceeding 1:8 which was higher than 30% (8/26) among male patients (p less than 0.05). These results confirm that IDDM patients have a high prevalence of ICSA and that this convenient, high capacity immunofluorescence assay allows studies of large numbers of serum samples. The cell-specific reaction following absorption should also allow the islet cell surface antigens to be characterized.

Gene probes to detect cross-culture contamination in hormone producing cell lines.

Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU sequence probe, BLUR, and lacked restriction fragment length polymorphism typical for the human HLA-DQ beta-chain gene. Although a human insulin gene probe showed a weak, nonhuman hybridization pattern, a cDNA probe for the Syrian hamster insulin gene hybridized strongly consistent with a single copy hamster insulin gene. Karyotyping confirmed the absence of human chromosomes in the Clone-16 cells while sizes, centromere indices, and banding patterns were identical to Syrian hamster fibroblasts. We conclude that the insulin-producing Clone-16 cells are of Syrian hamster origin and demonstrate the effective use of gene probes to control the origin of cell cultures.

Detection of islet cell autoantibodies in newly diagnosed diabetic patients using insulin-producing Syrian hamster cells.

An insulin-producing cell line, Clone-16, of hamster origin, was characterized for islet hormone production and for reactivity with islet cell surface (ICSA) and islet cell cytoplasmic (ICA) antibodies in sera from children with newly diagnosed insulin-dependent (Type 1) diabetes mellitus (IDDM). The Clone-16 cells have a doubling time of about 50-60 hr. The cells produced 63 +/- 3 ng (mean +/- SD) immunoreactive insulin and 9.4 +/- 0.3 ng immunoreactive glucagon per day per 10(6) cells, while somatostatin (SRIF) and pancreatic polypeptide (PP) were undetectable. The reactivity with autoantibodies in IDDM sera was assessed by indirect immunofluorescence or 125I-protein A binding assay on intact cells to detect islet cell surface antibodies (ICSA) or on frozen sections of cell pellets to detect islet cell cytoplasmic antibodies (ICCA) by indirect immunofluorescence. Although the proportion of the ICSA-positive Clone-16 cells compared favorably with rat islet cells (r = 0.81; p less than 0.01), we found 5/10 IDDM sera to be positive on rat islet cells but 8/10 on the Clone-16 cells. There was also a good correlation in the 125I-protein A binding assay between mouse islet cells and Clone-16 cells (r = 0.91; p less than 0.01). Frozen sections of Clone-16 cells showed a cytoplasmic immunofluorescence in 8/10 of the IDDM sera and this reaction parallelled the results obtained in the standard indirect immunofluorescence assay with a frozen section of human blood group O pancreas. We conclude that the insulin- and glucagon-producing Clone-16 cells are a useful cell line for detecting islet cell autoantibodies.

Preventive effects of cyclosporin on diabetes in NOD mice.

Non-obese diabetic mice aged 30 to 60 days were treated orally with Cyclosporin at doses of 25, 15 and 2.5 mg/kg every 2 days until 160 days of age. Diabetes developed in 12 out of 18 oil-treated mice (67%), with partial to complete Langerhans' islet destruction associated with lymphocytic infiltration. The non-obese diabetic mice showed a plasma glucose concentration of 6.62 +/- 0.92 mmol/l (mean +/- SD) at 50 days of age. The plasma glucose level of oil-treated non-obese diabetic mice gradually increased after 130 days of age and reached 14.0 to 19.0 mmol/l at 160 days of age, while Cyclosporin-treated non-obese diabetic mice showed neither clear increase of plasma glucose levels nor development of insulitis. The cumulative incidence of diabetes in Cyclosporin-treated mice was significantly lower than that in oil-treated mice (p less than 0.01). Subsequently, Cyclosporin treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cyclosporin was administered every 2 days for 35 days. Cyclosporin appeared to have little therapeutic effect on diabetes in non-obese diabetic mice.

Islet cell antibodies in insulin-dependent (type 1) diabetic children treated with plasmapheresis.

Plasma levels of islet cell cytoplasmic and cytotoxic antibodies were determined in 10 children with insulin-dependent diabetes mellitus (IDDM) treated with plasmapheresis shortly after diagnosis, and in 9 children with IDDM treated by conventional means alone. Islet cell cytoplasmic antibody (ICA) titers were determined by indirect immunofluorescence using unfixed sections of human pancreas, and islet cell cytotoxic antibody levels were determined in a complement-dependent antibody-mediated cytotoxicity (C'AMC) assay using a human fetal cloned insulin-producing cell line (JHPI-1) as target. Before plasmapheresis, ICA was present in 7 out of 10 children and C'AMC was positive in 4. Four successive treatments with plasmapheresis did not consistently decrease plasma levels of ICA or C'AMC. ICA was present in 15 out of the total 19 children at diagnosis, and titers of ICA decreased in 12 out of 15 subjects by at least 1 degree of dilution (1:3) at 18-30 months follow-up, whether or not they had been treated with plasmapheresis; C'AMC was positive in 6 out of the 18 children at diagnosis and decreased in 2 out of 6. Plasma levels of C-peptide did not differ at diagnosis but remained higher in the plasmapheresis treated diabetic children at 3 and 18-30 months follow-up. Neither ICA titers nor C'AMC levels correlated with plasma C-peptide responses at 18-30 months. It is concluded that plasmapheresis decreases ICA and C'AMC but is followed rapidly by a rebound effect, and does not affect the rates at which these islet cell antibodies decrease with increasing duration of IDDM.

[The effect of methyleneblue on insulin and glucagon release stimulated by glucose and arginine in the isolated perfused rat pancreas].

To examine the role of NADPH in the release of insulin and glucagon, isolated rat pancreata were perfused with methyleneblue, which is known to oxidize NADPH. Hormonal release was stimulated by changes in arginine or glucose concentrations as follows. After establishing the basal secretion state during perfusion at various glucose levels for 10 min., pancreata were stimulated by the addition of arginine or a change in glucose concentration of the perfusate for 15 min. Conditions for the stimulation were: (A) addition of 10 mM arginine at constant 4 mM glucose concentration; (B) increase in glucose concentration from 2.8 mM to 11.1 mM, or (C) decrease in glucose concentration from 11.1 mM to 2.8 mM. In some experiments, methyleneblue was added throughout the perfusion period at 1 or 3 micrograms/ml. The effluent from the portal vein was collected over 1 minute intervals: Insulin and glucagon concentrations in the effluent were determined by radioimmunoassay. Insulin release. Stimulation by the addition of arginine and increased glucose concentration produced a typical biphasic insulin response. In both cases, 1 microgram/ml methyleneblue reduced the second phase, and 3 micrograms/ml methyleneblue inhibited both phases almost completely. Glucagon release: Stimulation by arginine and inhibition by increasing glucose concentration were not influenced by methyleneblue; however, glucagon release induced by lowering of glucose concentration was suppressed by 3 micrograms/ml of methyleneblue. Thus, methyleneblue specifically inhibits glucose- and arginine-induced insulin release while it has no effect on arginine-induced glucagon release.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-mediated cytotoxic islet cell surface antibodies to human pancreatic beta cells.

Sera containing islet cell surface antibodies show a complement-dependent cytotoxic reaction against islet cells, but it has not yet been clarified whether islet cell surface antibodies exhibit cell-mediated cytotoxicity to these cells. By 51Cr release assay we investigated whether islet cell surface antibodies showed a cytotoxic reaction to human pancreatic B cells (JHPI-1 clone) in the presence of normal human lymphocytes. The sera from 14 islet cell surface antibody-positive, 16 islet cell surface antibody-negative Type 1 (insulin-dependent) diabetic patients and 18 islet cell surface antibody-negative healthy subjects were studied. Four sera containing islet cell surface antibodies showed specific cytotoxicity above the mean +3SD value of healthy subjects, and the mean specific cytotoxicity of islet cell surface antibody-positive sera differed significantly from that of both islet cell surface antibody-negative groups. These results suggest that this cell-mediated cytotoxic mechanism may play an important role in the pathogenesis of Type 1 diabetes.

[Hyperglycemia and inhibition of glucose-induced insulin release in 6-aminonicotinamide treated rats].

The effects of 6-aminonicotinamide (6-AN) on blood glucose and insulin release were studied in rats. 6-AN at 4mg/100g body weight slowly raised blood glucose concentrations to a significantly higher level than the control values 6 hours after an intraperitoneal injection. At this time severe glucose intolerance and low IRI response were noticed during an intravenous glucose tolerance test. Adrenalectomized rats replaced by hydrocortisone also presented hyperglycemia, glucose intolerance and low IRI response during IVGTT under treatment with 6-AN but to a lesser extent than in the intact rats. In in vitro experiments, decreased insulin release from the perfused pancreata of rats pretreated with 6-AN was found both in the 1st and 2nd phases of response to glucose stimulation. These data indicate that 6-AN-induced hyperglycemia is attributed to the inhibition of insulin release in adrenalectomized rats except for the hypothetical effect of 6-AN which diminishes an action of insulin on cellular glucose transport.

The effect of hypoxia on insulin and glucagon secretion in the perfused pancreas of the rat.

It has been reported that insulin secretion decreases during hypoxia both in vitro and in vitro, while an increase in glucagon secretion is found only in vivo. The effect of acute hypoxia on the secretion of glucagon and insulin was studied in the perfused rat pancreas. Phentolamine, an alpha-adrenergic blocker, was perfused during the period of hypoxia to elucidate the role of alpha-adrenergic stimulation. Sodium ATP and dibutyryl cAMP were also administered to study their effects on insulin and glucagon responses during hypoxia. In the present experiments, insulin secretion was suppressed while glucagon secretion was increased during hypoxia. Phentolamine did not cause any change in insulin of glucagon secretion. When dibutyryl cAMP was added, the increased glucagon secretion was reduced to the basal level, whereas the decreases in insulin secretion were not altered. The addition of sodium ATP reversed the hypoxia-induced decrease in insulin and the increase in glucagon secretion. These results suggest that a decrease in ATP production, which leads to impaired cAMP generation, pays a role in, and that alpha-adrenergic stimulation does not participate in the changes in, insulin and glucagon secretion during hypoxia in vitro.

Human pancreatic islet cell clones secreting insulin, glucagon and somatostatin: immunocytochemical and functional studies.

Human pancreatic A-, B- and D-cell clonal strains, named JHPG-1, JHPI-1 and JHPS-1, were established successfully from adult and fetal pancreata by the single cell plating feeder layer method using a modified Rose's chamber. This is the first time that insulin-, glucagon- and somatostatin-producing clonal strains have been separated into continuous clonal cell lines. The cultured cells are epithelial in nature, free of fibroblast contamination, and can be cloned. Under the phase-contrast microscope, the B-cell clone (JHPI-1) was generally oval or round in shape, the A-cell clone (JHPG-1) was bipolar, and the D-cell clone (JHPS-1) was nerve-like with cytoplasmic processes. By the use of immunocytochemical techniques, insulin-, glucagon- and somatostatin-like immunoreactivities were detected in each clone respectively. By radioimmunoassay it was revealed that each clone produced a single pancreatic hormone. The B-cell clone especially, was found to secrete insulin amply and continuously, for over 150 days. The glucagon release responses by the A-cell clone to insulin and glucose were also studied. The clonal strains obtained in this study provide useful systems for the investigation of the cell-biological aspects of human islet cells in vitro.

Adrenergic modulation of insulin and glucagon secretion from the isolated perfused rat pancreas.

In order to observe the effect of the adrenergic system on pancreatic glucagon secretion in the isolated perfused rat pancreas, phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, were added to the perfused solution. 1.2 microM phenylephrine suppressed glucagon secretion at 2.8 mM glucose, and it also decreased insulin secretion at 11.1 mM glucose. 240 nM isoproterenol enhanced glucagon secretion not only at 2.8 mM glucose, but also at 11.1 mM glucose, as well as insulin secretion at 11.1 mM. In order to study the role of intra-islet noradrenalin, phentolamine, an alpha-adrenergic antagonist, and propranolol, a beta-adrenergic antagonist, were infused with the perfused solution. 10 and 100 microM phentolamine caused an increase in insulin secretion, and 25 microM propranolol decreased insulin secretion, while they did not cause any change in glucagon secretion. From these results, it can be concluded that alpha-stimulation suppresses not only insulin but also glucagon secretion, while beta-stimulation stimulates glucagon secretion, as well as insulin secretion. Intra-islet catecholamine may have some effect on the B cell, whereas it seems to have no influence on the A cell.