Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse.

AIMS/HYPOTHESIS: T helper type (Th) 17 cells have been shown to play important roles in mouse models of several autoimmune diseases that have been classified as Th1 diseases. In the NOD mouse, the relevance of Th1 and Th17 is controversial, because single-cytokine-deficient NOD mice develop diabetes similarly to wild-type NOD mice. METHODS: We studied the impact of IL-17/IFN-γ receptor double deficiency in NOD mice on the development of insulitis/diabetes compared with IL-17 single-deficient mice and wild-type mice by monitoring diabetes-related phenotypes. The lymphocyte phenotypes were determined by flow cytometric analysis. RESULTS: IL-17 single-deficient NOD mice showed delayed onset of diabetes and reduced severity of insulitis, but the cumulative incidence of longstanding diabetes in the IL-17-deficient mice was similar to that in wild-type mice. The IL-17/IFN-γ receptor double-deficient NOD mice showed an apparent decline in longstanding diabetes onset, but not in insulitis compared with that in the IL-17 single-deficient mice. We also found that double-deficient NOD mice had a severe lymphopenic phenotype and preferential increase in regulatory T cells among CD4(+) T cells compared with the IL-17 single-deficient mice and wild-type NOD mice. An adoptive transfer study with CD4(+)CD25(-) T cells from young non-diabetic IL-17 single-deficient NOD mice, but not those from older mice, showed significantly delayed disease onset in immune-deficient hosts compared with the corresponding wild-type mice. CONCLUSIONS/INTERPRETATION: These results indicate that IL-17/Th17 participates in the development of insulitis and that both IL-17 and IFN-γ signalling may synergistically contribute to the development of diabetes in NOD mice.

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non-obese diabetic mice.

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of ß cells and perforin deficiency effectively reduces diabetes in non-obese diabetic (NOD) mice, it can be deduced that ß cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non-obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB(-/-) mice developed diabetes spontaneously with kinetics similar to those of wild-type NOD (wt-NOD) mice. Adoptive transfer study with regulatory T cell (Treg )-depleted splenocytes (SPCs) into NOD-SCID mice or in-vivo Treg depletion by anti-CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB(-/-) mice and wt-NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre-diabetic NOD.GzmB(-/-) mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide-promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4(+) , CD8(+) and CD4(+) CD25(+) T cells in SPCs from NOD.GzmB(-/-) mice than those from wt-NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for ß cell destruction in NOD mice.

Fuel-mediated teratogenesis. Use of D-mannose to modify organogenesis in the rat embryo in vivo.

The unique embryotoxic properties of D-mannose have been used as the basis for a new technique to secure precise temporal correlations between metabolic perturbations during organogenesis and subsequent dysmorphogenesis. Conscious, pregnant rats were infused with D-mannose or equimolar amounts of D-glucose by "square wave" delivery during the interval in which the neural plate is established and early fusion of neural folds takes place, that is, days 9.5-10.0 of gestation. Infusions of mannose to maternal plasma levels of 150-200 mg/dl did not elicit any toxicity in the mothers: motor activity, eating behavior, and serum components (electrolytes, osmolality, bilirubin) did not differ in glucose- vis-à-vis mannose-infused dams. Embryos were excised by hysterotomy on day 11.6 for evaluation of development. Examination with a dissecting microscope did not disclose developmental abnormalities in any of the 136 embryos from glucose-infused mothers or in 62 additional embryos from mothers that had not received any infusions. By contrast, dysmorphic changes were seen in 17 of 191 embryos (8.9%) from mannose-infused mothers. 14 of the 17 had abnormal brain or neural tube development with incomplete neural tube closure in 9 instances. Abnormal axial rotation was present in 8 of the 191 embryos (4.2%) and lesions of the heart or optic vesicles were seen in 4 (2.1%) and 3 (1.6%), respectively. Embryos from mannose-infused mothers displayed significant retardations in somite number, crown-rump length, and total protein and DNA content. These stigmata of growth retardation were more marked in the 17 dysmorphic embryos. The experiments indicate that D-mannose may be employed in model systems with rodents for precisely timed interruptions of organogenesis in vivo. Initial applications are consistent with our earlier suggestion that multiple dysmorphic changes may supervene after interference with communally observed metabolic dependencies during organogenesis. The studies do not identify the vulnerable site(s) within the conceptus (e.g., investing membranes, embryos, or both). However, the findings suggest that dysmorphic events are manifest most markedly in a general setting of embryo growth retardation.

Islet cell antibodies in patients with autoimmune thyroid disease.

Islet cell antibodies (ICAs) were assayed in 316 patients with autoimmune thyroid disease (AITD; 190 with Graves' disease, 126 with Hashimoto's thyroiditis), 53 patients with insulin-dependent diabetes mellitus (IDDM), and 144 healthy control subjects. ICAs were measured by an immunohistochemical method with peroxidase-labeled protein A and human pancreatic tissues. The prevalence of ICAs in patients with AITD was 7.6% (24 of 316), whereas the prevalence in control subjects was 0.7% (1 of 144). Among 24 ICA+ patients, 20 (83%) had IDDM. In these 20 patients, the duration of diabetes from clinical onset was 5.4 +/- 5.1 yr. ICAs in patients with IDDM alone were positive in 90.9% at 1 yr and 7.7% at 5 yr after the onset of diabetes. These data have shown that most ICA+ patients with AITD have IDDM and that the prevalence of ICAs in patients with AITD in Japanese is as high as that found among whites, whereas the incidence of IDDM in Japanese is approximately one-thirtieth or one-fiftieth of that in whites.

Significance of glutathione depletion and oxidative stress in early embryogenesis in glucose-induced rat embryo culture.

Recent studies have demonstrated the protective effects of supplementing free oxygen radical scavenging enzymes against hyperglycemia-induced embryonic malformations. In this study, the glutathione (GSH)-dependent protection system in hyperglycemia-induced embryopathy was investigated. Rat embryos at the early head-fold stage (day 9.5) cultured in 66.7 mmol/l glucose for 48 h showed significant growth retardation and an increase in the frequency of malformations. The concentration of GSH and activity of the rate-limiting GSH-synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), significantly decreased in embryos exposed to hyperglycemia compared with controls (7.9 +/- 0.6 vs. 12.5 +/- 0.9 nmol/mg protein, P < 0.01 and 13.3 +/- 1.9 vs. 22.6 +/- 1.1 microU/mg protein, P < 0.01, respectively). Decreased activity of gamma-GCS in embryos exposed to hyperglycemia was associated with decreased expression of gamma-GCS mRNA levels. However, the activities of superoxide dismutase and glutathione peroxidase did not significantly change in these embryos. Extracellular and intracellular free oxygen radical formations estimated by Lucigenin-dependent chemoluminescence and flow cytometric analysis using 2',7'-dichlorofluorescein diacetate increased in isolated embryonic cells taken from embryos cultured under hyperglycemia. Supplementation of 2 mmol/l GSH ester into the hyperglycemic culture nearly restored GSH concentration in these embryos (11.9 +/- 0.5 vs. 12.5 +/- 0.9 nmol/mg protein) and reduced the formation of free oxygen radical species leading to almost complete normalization of growth retardation and embryonic dysmorphogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of glutathione-dependent antioxidant system in diabetes-induced embryonic malformations.

Hyperglycemia-induced embryonic malformations may be due to an increase in radical formation and depletion of intracellular glutathione (GSH) in embryonic tissues. In the past, we have investigated the role of the glutathione-dependent antioxidant system and GSH on diabetes-related embryonic malformations. Embryos from streptozotocin-induced diabetic rats on gestational day 11 showed a significantly higher frequency of embryonic malformations (neural lesions 21.5 vs. 2.8%, P<0.001; nonneural lesions 47.4 vs. 6.4%, P<0.001) and growth retardation than those of normal mothers. The formation of intracellular reactive oxygen species (ROS), estimated by flow cytometry, was increased in isolated embryonic cells of diabetic rats on gestational day 11. The concentration of intracellular GSH in embryonic tissues of diabetic pregnant rats on day 11 was significantly lower than that of normal rats. The activity of y-glutamylcysteine synthetase (gamma-GCS), the rate-limiting GSH synthesizing enzyme, in embryos of diabetic rats was significantly low, associated with reduced expression of gamma-GCS mRNA. Administration of buthionine sulfoxamine (BSO), a specific inhibitor of gamma-GCS, to diabetic rats during the period of maximal teratogenic susceptibility (days 6-11 of gestation) reduced GSH by 46.7% and increased the frequency of neural lesions (62.1 vs. 21.5%, P<0.01) and nonneural lesions (79.3 vs. 47.4%, P<0.01). Administration of GSH ester to diabetic rats restored GSH concentration in the embryos and reduced the formation of ROS, leading to normalization of neural lesions (1.9 vs. 21.5%) and improvement in nonneural lesions (26.7 vs. 47.4%) and growth retardation. Administration of insulin in another group of pregnant rats during the same period resulted in complete normalization of neural lesions (4.3 vs. 21.5%), nonneural lesions (4.3 vs. 47.4%), and growth retardation with the restoration of GSH contents. Our results indicate that GSH depletion and impaired responsiveness of GSH-synthesizing enzyme to oxidative stress during organogenesis may have important roles in the development of embryonic malformations in diabetes.

Effects of insulin and myo-inositol on embryo growth and development during early organogenesis in streptozocin-induced diabetic rats.

We have previously shown that myo-inositol depletion in the embryonic tissue at a critical stage of organogenesis has a crucial role in hyperglycemia-induced embryopathy. This study tested whether myo-inositol depletion in early organogenesis contributes to the pathogenesis of streptozocin-induced diabetic embryopathy. Rats were made diabetic by streptozocin administration before conception, and the diabetic rats were treated with diet supplemented by 2% myo-inositol or insulin from 6 to 11 gestational days during the period of maximum teratological susceptibility. In each group on the 11th gestational day, growth retardation and incidence of malformations were recorded, and myo-inositol and sorbitol content in the embryonic and extraembryonic tissues were examined. In diabetic rats, the myo-inositol content of the embryos was decreased by 36% (P less than 0.01) compared with control rats, and there was growth retardation (crown-rump length 3.37 +/- 0.04 vs. 3.87 +/- 0.03 mm, P less than 0.01; somite no. 27.5 +/- 0.2 vs. 29.1 +/- 0.2, P less than 0.01) and a significantly increased incidence of the neural lesions (17.6 vs. 1.9%, P less than 0.01). Insulin treatment resulted in near normalization of maternal serum glucose and complete restoration of myo-inositol content in the embryos with significant improvement of the growth retardation (crown-rump length 3.55 +/- 0.06 vs. 3.37 +/- 0.04 mm, P less than 0.05; somite no. 28.2 +/- 0.13 vs. 27.5 +/- 0.2, P less than 0.05) and a significantly lowered incidence of neural lesions (2.5 vs. 17.6%, P less than 0.01) compared with those of the untreated diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of brief exposure to insulin-induced hypoglycemic serum during organogenesis in rat embryo culture.

We have previously shown that long-term exposure to medium containing insulin-induced hypoglycemic serum during the early phase of organogenesis can adversely affect embryonic development in rat embryo culture and that these effects were mediated through the interruption of glycolytic flux that constituted the principal pathway at this embryonic stage. Further experiments were performed to examine whether brief exposure to the hypoglycemic medium during critical developmental periods would have adverse effects on embryogenesis during embryo culture not only in normal but also in high glucose concentrations. Rat embryos in the early head-fold stage (9.5 days gestation) were grown in vitro for 48 h until neural tube closure occurred; dysmorphogenic lesions were not elicited in either the basal culture medium containing 6.6 mM glucose (control medium) or the hyperglycemic medium supplemented with glucose at a concentration of 33.3 mM. Hypoglycemic mediums (2.2-2.5 mM glucose) were prepared from the serum of rats given insulin intraperitoneally. Postimplantation embryos (in early neural tube formation) were briefly exposed (1 h) to hypoglycemic medium on day 10.3 of gestation during the basal culture. After exposure to the hypoglycemic medium for 1 h during culture in the control medium, embryos showed minor growth retardation and dysmorphogenic lesions (7.1% open neural pores). Exposure to the hypoglycemic medium for 1 h during culture in hyperglycemic medium suplemented with a subteratogenic concentration of glucose (33.3 mM) resulted in greater growth retardation and increased occurrence of dysmorphogenic lesions (17.3% open neural pores).(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to glutamic acid decarboxylase in patients with IDDM and autoimmune thyroid disease.

Autoantibodies to glutamic acid decarboxylase (GAD), previously reported to be the 64,000-M(r) (64K) islet cell protein, were measured by a radioimmunoassay using purified pig brain GAD in 29 insulin-dependent diabetes mellitus (IDDM) patients with autoimmune thyroid disease (AITD) and in 29 sex- and disease duration-matched IDDM patients without AITD. Islet cell antibodies (ICAs) and 64K antibodies were also determined. In IDDM patients with short-duration diabetes (< 1 year), the prevalence and levels of GAD antibodies were 100% (8 of 8) and 609 +/- 166 U (means +/- SE), respectively, in IDDM patients with AITD and 81.8% (9 of 11) and 90 +/- 51 U, respectively, in patients without AITD. In patients with long-standing IDDM (3-22 years), the prevalence and levels of GAD antibodies were 76.2% (16 of 21) and 193 +/- 66 U, respectively, in patients with AITD and 50.0% (9 of 18) and 36 +/- 14 U, respectively, in patients without AITD. For up to 6 years after the onset of IDDM, the levels of GAD antibodies in IDDM patients with AITD were significantly higher than in IDDM patients without AITD. A close and significant correlation was found between GAD antibodies and ICA or 64K antibodies in IDDM patients with AITD. Our results demonstrate that high levels of GAD antibodies were present in IDDM patients with AITD. The observed differences in GAD immunoreactivity between IDDM patients with and without AITD might help evaluate the role of GAD antibodies in IDDM.

Efficacy of troglitazone on body fat distribution in type 2 diabetes.

OBJECTIVE: The insulin-sensitizing action of troglitazone may be mediated through the activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the promotion of preadipocyte differentiation in adipose tissue on which troglitazone has depot-specific effects. We investigated the relationship between efficacy of the drug and body fat distribution. Changes in body fat distribution were also investigated by long-term administration of the drug. RESEARCH DESIGN AND METHODS: Troglitazone was given at a dose of 400 mg/day to 20 patients with type 2 diabetes whose diet and sulfonylurea therapy produced unsatisfactory glycemic control (HbA(1c) >7.8%) and whose insulin secretory capacity was found to be preserved (postprandial C-peptide >3 ng/ml). HbA(1c) values, serum lipid levels, and body weight were measured monthly Body fat distribution was evaluated in subcutaneous (SC) and visceral fat using a computed tomography scan at umbilical levels before and after troglitazone therapy RESULTS: During the 1-year troglitazone treatment, HbA(1c) was significantly decreased (from 9.2 +/- 0.2 to 7.1 +/- 0.2%, P < 0.01), showing lowest values at 4-6 months, whereas body weight was significantly increased (BMI 24.6 +/- 0.6 to 25.7 +/- 0.6 kg/m2, P < 0.01). Reduction of HbA(1c) (deltaHbA(1c)) from the baseline value during treatment was significantly greater in obese patients (BMI >26 kg/m2) than in nonobese patients (-3.2 +/- 0.4 vs. -2.1 +/- 0.3%, P < 0.05) and was more significant in women than in men (-3.2 +/- 0.2 vs. - 1.4 +/- 0.2%, P < 0.01). The level of deltaHbA(1c) during treatment showed a significant negative correlation with SC fat area (r = -0.742, P < 0.01) but not with visceral fat area. Weight gain during troglitazone treatment resulted in increased accumulation of SC fat without a change in visceral fat area and, consequently. in a significant decrease in the visceral-to-SC fat ratio. CONCLUSIONS: Predominant accumulation of SC fat for the visceral fat tissue was an important predictor of the efficacy of troglitazone therapy in patients with type 2 diabetes. Greater efficacy of troglitazone was observed in women who were characterized by more accumulation of SC adipose tissue than men. Long-term administration of the drug resulted in weight gain with increased accumulation of SC adipose tissue, probably because of the activation of PPAR-gamma in the region.

Insulin resistance and arteriosclerosis obliterans in patients with NIDDM.

OBJECTIVE: To investigate the risk factors for arteriosclerosis obliterans (ASO) in NIDDM, we measured insulin sensitivity and other risk factors including lipoprotein(a) [Lp(a)] in NIDDM patients with and without ASO. RESEARCH DESIGN AND METHODS: A case-control study in 100 patients with NIDDM, 35 with and 65 without ASO, was performed. Insulin sensitivity was assessed by the short insulin tolerance test's K index (KITT). Duration of diabetes, a history of smoking, prevalence of hypertension, prevalence of coronary artery disease (CAD), serum C-peptide, 24-h urinary C-peptide, serum lipids, and Lp(a) were compared in the two groups. RESULTS: Age, BMI, HbA1c, and fasting plasma glucose were comparable in the two groups. Patients with ASO were significantly more insulin resistant than patients without ASO (KITT 2.16 +/- 0.16 vs. 3.00 +/- 0.13%/min, P < 0.0001, respectively), had a longer duration of diabetes (10.3 +/- 1.2 vs. 7.5 +/- 0.8 years, P < 0.05), included a greater number of smokers (68.6 vs. 40.0%, P < 0.01), had a higher prevalence of CAD (60.0 vs. 16.9%, P < 0.01), and had a greater percentage of insulin therapy (48.6 vs. 29.2%, P < 0.05). However, urinary and serum C-peptide levels, serum lipids, and Lp(a) levels were comparable in the two groups. Multiple logistic regression analysis indicated that a history of smoking (odds ratio 3.70, P = 0.011), insulin resistance (odds ratio 3.68, P < 0.001), and an elevated Lp(a) level (odds ratio 1.03, P = 0.020) were independently related to ASO. When patients with CAD were removed from the logistic regression analysis, insulin resistance was most strongly related to ASO (odds ratio 20.9, P < 0.001). CONCLUSIONS: Patients with ASO were characterized by a higher prevalence of CAD, a greater percentage of smokers, a greater percentage of insulin therapy, and a higher insulin resistance than were patients without ASO. Insulin resistance, especially, may be the most powerfully related to ASO. Lp(a) may play a minor role in the development of ASO.

Glucose tolerance, insulin secretion, and insulin sensitivity in nonobese and obese Japanese subjects.

OBJECTIVE: To investigate the relative contributions of insulin secretion and insulin resistance to the development of glucose intolerance in Japanese subjects. RESEARCH DESIGN AND METHODS: A cross-sectional study of 756 Japanese subjects (530 nonobese, 226 obese) was performed. A 75-g oral glucose tolerance test (OGTT) was given, and subjects were classified according to the World Health Organization (WHO) criteria (normal glucose tolerance [NGT], impaired glucose tolerance [IGT], and diabetes). Early-phase insulin secretion was assessed by the insulinogenic index (the ratio of the increment of insulin to that of plasma glucose [PG] 30 min after a glucose load [delta IRI0-30 min/delta PG0-30 min]). Total insulin secretion was assessed by mean immunoreactive insulin (IRI) during the OGTT, and insulin resistance was assessed by use of the homeostasis model [HOMA(R)]. RESULTS: Early-phase insulin secretion was significantly decreased in IGT, compared with patients with NGT, in both the nonobese and obese subjects (0.70 +/- 0.05 vs. 0.37 +/- 0.03, P < 0.01 and 1.36 +/- 0.19 vs. 0.73 +/- 0.08, P < 0.01, respectively). However, mean IRI and HOMA(R) in both nonobese and obese subjects with IGT and NGT were not statistically different. Subjects with diabetes showed a significant decline in early-phase and total insulin secretion and a significantly higher level of insulin resistance than did subjects with IGT. When the fasting glucose (FPG) exceeded 100 mg/dl, early-phase insulin decreased progressively. The graphed relationship between FPG and mean IRI did not show an inverted U-shape, and mean IRI decreased progressively when FPG exceeded 100-130 mg/dl. The pattern of changes in insulin secretion and insulin resistance associated with the progression of glucose intolerance was similar in both the nonobese and obese subjects. CONCLUSIONS: The worsening from NGT to IGT in Japanese subjects may be associated with a decrease in early-phase insulin secretion in nonobese as well as in obese subjects. Hyperinsulinemia in IGT is not common. We suggest that impaired early-phase insulin secretion may be the initial abnormality in the development of glucose intolerance in Japanese people. Insulin resistance may be a consequence of hyperglycemia and/or obesity.

Effects of voglibose on glycemic excursions, insulin secretion, and insulin sensitivity in non-insulin-treated NIDDM patients.

OBJECTIVE: To investigate the effects of voglibose, an alpha-glucosidase inhibitor, on daily glycemic excursions, insulin secretion, and insulin sensitivity in non-insulin-treated NIDDM patients. RESEARCH DESIGN AND METHODS: An open prospective study was conducted in 27 NIDDM patients receiving diet therapy alone or treatment with a sulfonylurea drug. Of the study subjects, 14 patients were treated with voglibose; the remaining 13 patients served as the control group. The metabolic parameters were evaluated before treatment and at week 4 of treatment as follows: glycemic excursions by M-value and 1,5-anhydro-D-glucitol (1,5-AG), insulin secretion by area under the curve of daily serum insulin (AUCinsulin), and insulin sensitivity by the K index of the insulin tolerance test (KITT). RESULTS: After the study treatment, HbA1c and plasma glucose in the patients who had received voglibose were comparable to those of patients in the control group. M-value was lower in the patients treated with voglibose than in the control subjects (5.7 +/- 0.9 vs. 9.8 +/- 1.2, P < 0.05). 1,5-AG was higher in the patients treated with voglibose than in the control subjects (12.2 +/- 1.0 vs. 8.2 +/- 0.7 micrograms/ml, P < 0.01). A statistically significant increase in AUCinsulin occurred after treatment with voglibose (2,223.5 +/- 390.6 to 1,546.7 +/- 303.4 pmol.l-1.h, P < 0.05), but no change occurred in the control group (2,364.5 +/- 315.4 to 2,464.2 +/- 269.3 pmol.l-1.h, P = 0.60). Insulin sensitivity (KITT) was improved to a statistically significant level in both the patients treated with voglibose and the patients in the control group. KITT in the patients after voglibose treatment was comparable to that of the control group (3.18 +/- 0.30 vs. 3.21 +/- 0.23%/min, P = 0.94). CONCLUSIONS: The results suggest that voglibose lowers the daily glycemic excursions and inhibits overwork of the pancreatic beta-cells but has little effect on insulin sensitivity in NIDDM patients.

Effects of cyclosporin A and low dosages of steroid on posttransplantation diabetes in kidney transplant recipients.

OBJECTIVE: To investigate the adverse effects of cyclosporin A (CsA) on pancreatic beta-cell function in kidney transplant recipients. RESEARCH DESIGN AND METHODS: The study consisted of 73 patients without a history of diabetes mellitus who had undergone kidney transplantation in our clinic. RESULTS: We experienced a higher incidence of posttransplantation diabetes mellitus (PTDM) in patients receiving CsA and low dosages of methylprednisolone (6/20, 30%, P less than 0.05) than in patients receiving conventional therapy of azathioprine methylprednisolone (4/53, 7.5%) since the introduction of CsA. In all 6 patients in the CsA-treated group, PTDM occurred within 3 mo after transplantation. The CsA level during the initial 3 mo posttransplant was significantly higher in diabetic than nondiabetic subjects, and the highest CsA level was observed shortly (1 mo) before the development of PTDM. After an average of 71 days of insulin therapy, there was complete remission of PTDM in 5 of 6 diabetic patients, with a corresponding decrease in CsA level. For the patients who were in remission for greater than 1 yr, a significant improvement of glucose intolerance was observed in association with a significantly higher insulin response to oral glucose load; however, their glycemic profile still showed a significantly higher plasma glucose concentration and a prolonged continuous elevation without initial peak of the insulin-response curve in contrast to the normal pattern found in nondiabetic subjects in the CsA-treated group. CONCLUSIONS: This study suggests that CsA in combination with low dosages of steroid may have adverse effects on glucose metabolism, which may lead to effects similar to those in non-insulin-dependent diabetes mellitus.

Cellular-tissue localization and regulation of the GLUT-1 protein in both the embryo and the visceral yolk sac from normal and experimental diabetic rats during the early postimplantation period.

We investigated the tissue-specific developmental expression and localization of GLUT-1 protein in the rat embryo and visceral yolk sac (VYS) during the organogenic periods of normal rats. The expression of GLUT-1 protein was then compared to that of experimental diabetic rats to test whether the diabetic state would affect the regulation of the glucose transporter during the early postimplantation periods (9.5-14.5 days), as we have previously demonstrated that GLUT-1 protein in embryo and VYS was down-regulated in culture with hyperglycemic medium. In the embryo, GLUT-1 protein was highly expressed during the early stages of organogenesis (between 9.5-12.5 days) and declined thereafter, whereas in the VYS, its strong expression was observed at the later stages (from 12.5-14.5 days). Immunohistochemical localization of the GLUT-1 protein in the embryo during the main periods of neurulation (9.5-11.5 days) showed that GLUT-1 immunoreactivity was principally observed in the neuroepithelial cells of the neural tube and also noted in the primitive heart, primitive gut, otic, and optic vesicles. At 12.5 days, GLUT-1 protein started to be expressed in the microvessels at the cranial portions of the neural tube, although its expression in the neuroepithelial cells still remained at the caudal (tail) portions of the neural tube. In the later stages (13.5-14.5 days) after completion of neural tube formation, GLUT-1 protein immunoreactivity substantially decreased in the neuroepithelial cells and was found mainly in the microvessels of the brain vesicles and spinal cord, whereas it continued to be expressed in the heart and eyes. In the VYS, its immunoreactivity was noticeably confined to the endodermal layer, which started as a simple layer and developed wave-like folds in the later stages. The levels of GLUT-1 protein in embryo and VYS from diabetic rats, determined by Western blot analysis, were not down-regulated compared to those in control rats at the different gestational days. Likewise, comparison of GLUT-1 protein immunoreactivity of various tissues in embryo and VYS, focusing on the neural tube, also revealed no significant differences between the two groups. We demonstrated that GLUT-1 protein is abundantly expressed in embryonic tissues and VYS during the early periods of organogenesis. The lack of down-regulation and the continuous abundant expression of the GLUT-1 protein despite the diabetic state in embryo and VYS during the early postimplantation periods may increase delivery of glucose from the VYS into various differentiating embryonic cells, leading to diabetes-induced congenital malformations.

Relationships between glucose and mannose during late gestation in normal pregnancy and pregnancy complicated by diabetes mellitus: concurrent concentrations in maternal plasma and amniotic fluid.

Enzymatic assays were modified to permit sensitive and highly reproducible simultaneous measurements of D-mannose and D-glucose in biological fluids during weeks 34-40 of human pregnancy. Plasma mannose and glucose averaged 9.8 +/- 0.4 (+/- SEM) and 790 +/- 16 micrograms/ml, respectively, after an overnight fast in pregnant women (n = 22) with normal carbohydrate metabolism. Significantly higher plasma mannose levels were found, despite only minor increases in plasma glucose, in pregnant women with relatively well controlled diabetes mellitus after an overnight fast (16.9 +/- 0.6 micrograms/ml mannose; 883 +/- 29 micrograms/ml glucose; n = 31) or 3-4 h after breakfast (15.7 +/- 1.2 micrograms/ml mannose; 1159 +/- 101 micrograms/ml glucose; n = 19). Plasma mannose correlated significantly with plasma glucose in the women with diabetes mellitus, particularly after an overnight fast. Samples of amniotic fluid were also obtained from the gravida with diabetes mellitus to provide some index of simultaneous relationships in utero. Amniotic fluid mannose and glucose averaged 5.9 +/- 0.4 and 302 +/- 24 micrograms/ml, respectively, after an overnight fast and 6.7 +/- 1.3 and 459 +/- 84 micrograms/ml 3-4 h after breakfast. In amniotic fluid, as in plasma, the concurrent levels of mannose and glucose conformed to relatively fixed relationships. Thus, both fetus and mother appear to be exposed to readily demonstrable amounts of mannose during late gestation and the absolute as well as relative abundance of mannose may be increased coincident with faulty maternal glucoregulation. However, since mannose did not exceed 3% of the concurrent concentration of glucose in any instance, it does not seem likely that endogenous levels of circulating mannose can modify glucose utilization appreciably by competing with glucose for phosphorylation via hexokinase and subsequent intracellular processing.

High-dose but not low-dose dexamethasone impairs glucose tolerance by inducing compensatory failure of pancreatic beta-cells in normal men.

The diabetogenic effects of glucocorticoids appear to be dose dependent. To determine the effects of different doses of dexamethasone on glucose metabolism, we performed frequently sampled intravenous glucose tolerance tests in 20 healthy young men. Glucose kinetics were analysed by the minimal model. Ten subjects received low-dose dexamethasone (2 mg/day) for 3 days, and the other 10 received high-dose dexamethasone (6 mg/day) for 3 days. The rate of glucose disappearance (KG) did not decrease in the low-dose group (2.46 +/- 0.20 to 2.19 +/- 0.11% min-1, P = 0.35). In contrast, KG in the high-dose group did decrease significantly (2.43 +/- 0.29 to 1.81 +/- 0.11% min-1, P < 0.05). The factor responsible for the decline in KG in the high-dose group was not glucose effectiveness because these values did not change in either group. The insulin sensitivity decreased significantly, by 46% in the low-dose group and 69% in the high-dose group [17.1 +/- 2.7 to 9.2 +/- 1.5 and 18.5 +/- 3.7 to 5.8 +/- 0.9 x 10(-5) min-1 (pmol/L)-1, P < 0.001 and P < 0.01, respectively]. The insulin area (0-20 min) increased significantly, by 104% in the low-dose group and 114% in the high-dose group [3412.6 +/- 609.7 to 6972.7 +/- 1450.1 and 4086.7 +/- 864.5 to 8750.0 +/- 1451.6 (pmol/L) min, P < 0.01 and P < 0.01, respectively]. Insulin sensitivity x insulin area as an estimate of insulin-dependent glucose uptake and insulin's action to suppress hepatic glucose production decreased significantly in the high-dose group (0.588 +/- 0.112 to 0.441 +/- 0.073, P < 0.05), but did not change in the low-dose group (0.436 +/- 0.050 to 0.484 +/- 0.032, P = 0.77). Therefore, the decline in KG in the high-dose group may be associated with the compensatory failure of pancreatic beta-cells against for the insulin resistance.

Troglitazone ameliorates insulin resistance in patients with Werner's syndrome.

Insulin resistance in Werner's syndrome (WS) is probably due to defective signaling distal to the insulin receptor. To analyze the metabolic effects of troglitazone (TRO) in these patients, we performed frequently sampled iv glucose tolerance tests. Glucose kinetics were analyzed by the minimal model. Five patients with WS (mean age, 41.2 yr; body mass index, 17.0 kg/m2) were treated with TRO (400 mg/day) for 4 weeks. Each subject underwent a 75-g OGTT and frequently sampled iv glucose tolerance tests. Treatment reduced the area under the curve of glucose and insulin in the OGTT by 26% and 43%, respectively. Glucose tolerance, as manifested by the glucose disappearance rate improved significantly (1.36 +/- 0.16 to 1.94 +/- 0.30%/min; P < 0.05). Although the first phase insulin secretion was unchanged, insulin sensitivity and glucose effectiveness increased significantly [0.47 +/- 0.11 to 1.38 +/- 0.37 x 10(-4) min/pmol.L (P < 0.05) and 1.72 +/- 0.17 to 2.52 +/- 0.24 x 10(-2) min-1 (P < 0.05), respectively]. However, treatment did not change glucose effectiveness at zero insulin. In patients with WS, TRO ameliorates glucose intolerance mediated by increased insulin sensitivity as well as glucose effectiveness, as assessed by minimal model analysis. TRO may modulate the postreceptor signaling component and be a clinically useful regimen for the treatment of patients with the intracellular insulin signaling defect.

Oxygen-induced embryopathy and the significance of glutathione-dependent antioxidant system in the rat embryo during early organogenesis.

We investigated the effect of glutathione (GSH)-dependent antioxidant system against hydrogen peroxide (H2O2) formation in oxygen-induced embryopathy. Exposure of rat embryos to a high concentration of oxygen (20%) during early neurulation (day 9 to 10) significantly increased the incidence of neural tube defects compared with control embryos (10% vs 0%, p < 0.01) exposed to a low O2 concentration (5%). The concentration of GSH in 20% O2-exposed embryos was significantly reduced compared with that in control embryos (10.68 +/- 0.72 vs 12.34 +/- 0.65 nmol/mg protein, p < 0.001). The activity of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting GSH synthesizing enzyme increased in 20% O2-exposed embryos (24.83 +/- 0.71 vs 21.00 +/- 0.94 microunits/mg protein). Increased activity of gamma-GCS was associated with increased expression of gamma-GCS mRNA. Substantial increases were also observed in the activities of glutathione peroxidase (GPX) and glutathione S-transferase (GST) in 20% O2-exposed embryos. The formation of intracellular H2O2, measured by flow cytometer using 2',7'-dichlorofluorescein diacetate (DCFH-DA), increased in isolated embryonic cells of 20% O2-exposed embryos. The addition of buthionine sulfoxamine (BSO), a specific inhibitor of gamma-GCS, to culture media exposed to 20% O2 produced a marked decrease in the concentration of GSH in association with a further increase in the incidence of embryonic malformations (24.4% vs. 10%, P < 0.01). The addition of 2.0 mM GSH ester to culture media exposed to 20% O2 prevented the development of embryonic malformations through the restoration of normal GSH contents and reduction of H2O2. Our results demonstrated that oxygen-induced embryonic malformations were induced by increased production of H2O2 in the presence of an immature free radical scavenger system. We suggest that impaired responsiveness of the GSH dependent antioxidant system against oxidative stress plays a crucial role in oxygen-induced embryopathy.

The 1986 McCollum award lecture. Fuel-mediated teratogenesis during early organogenesis: the effects of increased concentrations of glucose, ketones, or somatomedin inhibitor during rat embryo culture.

Whole rat embryos were explanted at head-fold, late pre-somite stage (day 9.5 of gestation) and cultured in rat sera varyingly supplemented with glucose (3, 6, 9, or 12 mg/mL), D,L sodium beta-hydroxybutyrate (2, 4, 8, or 16 mM), or both (6 mg/mL D-glucose plus 8 mM beta-hydroxybutyrate). During 48 h culture, increasing glucose alone or beta-hydroxybutyrate alone effected growth retardation and faulty neural and extraneural organogenesis in dose-dependent fashion. Synergistic dysmorphogenic effects occurred when minimally teratogenic concentrations of glucose and beta-hydroxybutyrate were combined. Sera from diabetic animals containing somatomedin inhibitor bioactivity were also able to produce growth retardation and major developmental lesions in presence of amounts of glucose and ketones which of themselves were not teratogenic. Thus, aberrant fuels and fuel-related products can impair growth and organogenesis in early post-implantation embryo. Such fuel-mediated teratogenesis may be multifactorial and include possibilities for synergistic and additive interactions.