Enhanced insulin gene expression by reduced intracellular glutathione level in insulin secreting cells MIN6.

Glutathione (GSH) participates in deoxidization and elimination of hydrogen peroxide and other reactive oxygen species, and plays an important part in the antioxidant system. To investigate the effect of GSH content on insulin gene expression, we utilized a stable transfectant, designated as ribo-MIN6 cells, which were stably transfected with the ribozyme of gamma-glutamylcysteine synthetase (gamma-GCS), exhibiting approximately 50% reduction of intracellular GSH content. We transiently transfected a luciferase expression vector driven by human preproinsulin gene promoter spanning from -1998 to +237 (pINS-1998/luc) and several deletion constructs into ribo-MIN6. Furthermore, transient transfection with ribozyme vector and pINS-1998/luc into wild-type MIN6 cells was also carried out. Luciferase activity was about 9-fold higher in ribo-MIN6 cells as compared to wild-type MIN6 cells. In the transient transfection of pINS-1998/luc with gamma-GCS ribozyme vector into wild-type MIN6 cells, the luciferase activity was increased in proportion to the added amounts of ribozyme vector. In transfection with deletion constructs, two major sites were found to be critical for insulin promoter activity. For the wild-type MIN6 cells, regions important for the promoter activity were also located at regions similar to those of ribo-MIN6 cells. Our results suggest that the suppression of intracellular GSH level might, in part, regulate the insulin gene expression.

Acute effects of ethanol on feeding behavior and leptin-induced STAT3 phosphorylation in rat hypothalamus.

OBJECTIVE: Drinking ethanol stimulates the appetite, producing a positive energy balance. The mechanism by which ethanol regulates the appetite in the central nervous system, however, has not been fully understood. The aim of this study is to investigate the interaction of ethanol with the satiety effect of leptin, a hormone which suppresses the appetite in the hypothalamic region. DESIGN: : Leptin (7.5 micro g) or the same dose of phosphate buffer saline (PBS) was administered into the third ventricle (i.c.v.), 30 min after an intraperitoneal injection (i.p.) of ethanol (0.5 g/kg body weight) or the same dose of PBS. MATERIALS: Adult male Sprague-Dawley rats weighing 290-320 g were used. MEASUREMENTS: Food intake was measured 2, 12 and 24 h after leptin i.c.v. infusion. The tyrosine phosphorylation of signal transducer and activator transcription factor 3 (STAT3) in the hypothalamus was analyzed by Western blotting. RESULTS: The cumulative food intakes in the saline/leptin group (saline i.p.+leptin i.c.v.) were markedly reduced to about 45% of the saline/PBS group (saline i.p.+PBS i.c.v.) at 2, 12 and 24 h time points (P<0.05, 0.001, and 0.005, respectively). As compared with the saline/leptin group, those of the ethanol/leptin group (ethanol i.p.+leptin i.c.v.) were significantly increased to the level seen in the saline/PBS group at 12 and 24 h time points (P<0.05, and P<0.005 vs the saline/leptin group, respectively). Ethanol administration resulted in about a 50% reduction of the leptin-induced STAT3 tyrosine phosphorylation seen in the hypothalamic protein as compared to that of the saline/leptin group. CONCLUSION: These findings suggest that ethanol-induced enhancement of the appetite may, in part, result from leptin resistance transiently caused by ethanol to attenuate the leptin signal transduction.

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization.

To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. (125)I-insulin binding was inhibited by 93% after preincubation with B-IgG at 37 degrees C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4 degrees C, the insulin binding completely recovered the control value by acid washing. (125)I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37 degrees C, but never at 4 degrees C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHO-K1018R at 37 degrees C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient.

CTLA4 gene polymorphism contributes to the mode of onset of diabetes with antiglutamic acid decarboxylase antibody in Japanese patients: genetic analysis of diabetic patients with antiglutamic acid decarboxylase antibody.

AIM: The mode of onset is occasionally similar in Type 1 and Type 2 diabetes mellitus, and some patients with Type 2 diabetes are positive for antiglutamic acid decarboxylase antibody (GAD Ab). We investigated the contribution of Type 1 diabetes susceptibility genes to the progression of the insulin-deficient state and mode of onset of Type 2 diabetes in GAD Ab-positive (GAD-Ab+) patients. We examined the variable number of tandem repeats in the promoter region of the insulin gene (INS-VNTR, insulin-dependent diabetes mellitus (IDDM) 2) and cytotoxic T lymphocyte antigen 4 (CTLA4, IDDM12) as representative of Type 1 diabetes susceptibility genes. METHODS: Patients with Type 2 diabetes who were GAD-Ab+ (n = 51) were selected for this study. In INS-VNTR, the class I allele was classified according to length (1S, 25-38 repeat units; 1M, 39-41 repeat units; 1L, 42-44 repeat units) and the exact class I allele length was analysed by specific polymerase chain reaction (PCR) amplifications. Analyses of classes II and III were performed by Southern blot. CTLA4 gene polymorphism (exon 1 position 49, G/A) was analysed by PCR-restriction fragment length polymorphism. RESULTS: The distribution of INS-VNTR was no different between Type 1 diabetes and Type 2 diabetes with GAD Ab. The allele frequencies of CTLA4 gene polymorphism G and A in Type 2 diabetes/GAD-Ab+ were significantly different from those of Type 1 diabetes/GAD-Ab+ (G: 53%, A: 47% vs. G: 84%, A: 16%; P < 0.0001). CONCLUSIONS: Our data showed that GAD-Ab+ Japanese patients presenting with Type 2 diabetes have shifted A allele while patients with abrupt onset have shifted G allele of CTLA4 gene polymorphism. Our results suggest that immunological function and polymorphism of the CTLA4 gene may contribute to the pathogenesis and progression of Type 1 diabetes.

Hepatocyte nuclear factor-1alpha inhibits insulin promoter factor 1-dependent transactivation of the human insulin gene.

To investigate the regulational interaction of hepatocyte nuclear factor-1alpha (HNF-1alpha) and insulin promoter factor 1 (IPF1) on insulin gene expression, either or both of the expression vectors carrying each transcription factor were transiently transfected into HeLa cells, RINm5F cells and MIN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (-1998 to +237) designated as, pINS-1998/luc. IPF1-transfection into HeLa cells strongly stimulated the luciferase activity to 725 fold that of the basal level. In contrast, HNF-1alpha-transfection resulted in only a 6.7 fold increase. In co-transfection experiments, increasing the amount of HNF-1alpha resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respectively. Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-185/luc were transfected into RINm5F cells to determine the role of the A3 element and its 5' flanking sequence in the inhibitory effect of HNF-1alpha. The results showed that the inhibiting effects of HNF-1alpha with pINS-213/luc and pINS-185/luc were significantly smaller than those with both pINS-1998/luc and pINS-248/luc. Transfection into MN6 cells with pINS-1998/luc in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1alpha. The present data indicate that IPF1 rather than HNF-1alpha predominantly transactivates the insulin gene, and that HNF-1alpha inhibits IPF1-dependent insulin gene transactivation mediated through the 5' flanking sequence of the A3 element. It is suggested that HNF-1alpha may be involved in insulin gene expression as a negative regulator.

Association between IA-2 autoantibody epitope specificities and age of onset in Japanese patients with autoimmune diabetes.

The IA-2 is a major autoantigen of type 1 diabetes belonging to the protein tyrosine phosphatase family. We report on the humoral autoimmunity to an alternatively-spliced variant of IA-2 (IA-2 variant) and autoimmune-mediated diabetes age of onset association with IA-2 autoantibody epitope specificities, in 144 recent-onset patients with type 1 diabetes and 54 GAD autoantibody-positive patients with type 2 diabetes. The cytoplasmic domain of IA-2 (IA-2ic) detected a somewhat greater proportion of patients expressing autoantibodies than IA-2 variant (56%vs. 52% of patients with type 1 diabetes and 17%vs. 9% of GAD autoantibody-positive patients with type 2 diabetes). Conversely, only 1% of IA-2 variant autoantibody-positive patients failed to react to IA-2ic construct. Among 80 patients with type 1 diabetes who were positive for autoantibodies to IA-2ic, 8% recognized the juxtamembrane region (JM, representing amino acids 601-629) only, 64% bound the protein tyrosine phosphatase (PTP)-like domain of IA-2 only, and 29% bound both JM and PTP epitopes. Autoantibodies to the PTP-like domain were prevalent in children and adolescents with type 1 diabetes. The age of disease onset in patients with IA-2JM autoantibodies only, was significantly higher than those in patients reacted with the PTP-like domain of IA-2 (P< 0.02). Among GAD autoantibody-positive patients with type 2 diabetes reacted with IA-2ic, 44% bound the JM region only, and 33% bound epitopes in the PTP-like domain only; 22% had autoantibodies to both regions. The frequency of GAD autoantibody-positive patients with type 2 diabetes positive for autoantibodies to the JM region only, was significantly higher than that in patients with type 1 diabetes (P< 0.01). IA-2PTP autoantibodies were significantly associated with HLA-DR4, while the additional reactivity to IA-2JM was associated with HLA-DR9 allele. These results suggest that autoantibody recognition of IA-2 epitopes in autoimmune diabetes is associated with age of disease onset, which may reflect the intensity of the beta-cell destruction process.

Evaluation of insulin response in glucose tolerance test in a patient with Werner's syndrome: a 16-year follow-up study.

To clarify the effect of Werner's syndrome (WS) on beta-islet cell function, the oral glucose tolerance test (OGTT) was repeatedly performed over a period of 16 years in one patient with WS. The data obtained on insulin secretion were assessed in this study. The patient was a 50-yr-old woman of consanguineous parentage. She presented with gray hair, cataracts, a beak-shaped nose and high-pitched voice. She was diagnosed as WS on the basis of her characteristic appearance. OGTT was performed 14 times during 9 admissions to our hospital. After ingestion of glucose, plasma glucose (PG) levels and immuno-reactive insulin (IRI) at 0, 30, 60, 90, 120 and 180 min were determined. PG levels during OGTT gradually increased during dietary therapy and, at the age of 48, insulin treatment was started [PG level at 120 min during OGTT at 46 yr (before treatment) was 1.5 times that at 34 yr]. Insulin secretion had also gradually decreased during the follow-up period (sum of IRI at 34 yr during OGTT post-treatment; 550.8 IU/ml, sum of IRI at 50 yr during OGTT post-treatment; 244.5 IU/ml). However, the insulinogenic indices were maintained at almost the same level value. Our results indicate that insufficient insulin secretion, which could not overcome insulin resistance, might play a crucial role in the pathophysiology and progression of diabetes in WS along with insulin resistance due to a post-receptor defect.

In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin.

To determine whether leptin signal transduction is exerted directly upon insulin-sensitive tissues in vivo, we examined the ability of iv leptin to acutely stimulate phosphorylation of STAT3, STAT1, and MAPK, and activities of PI 3-kinase and Akt, in insulin-sensitive tissues of normal rats. Both leptin (1 mg/kg iv x 3 min) and insulin (10 U/kg iv x 3 min) stimulated tyrosine phosphorylation of STAT3 5.6- to 6.0-fold and of STAT1 4.0-fold in adipose tissue. Leptin tended to increase STAT3 phosphorylation in liver and muscle. Both hormones also increased MAPK phosphorylation: leptin increased it 3.2- to 3.8-fold in adipose tissue and liver, whereas insulin stimulated MAPK phosphorylation 5.0-fold in adipose tissue, 6.8-fold in liver, and 2.5-fold in muscle. Leptin was much less effective than insulin at stimulating IRS pathways. Leptin increased IRS-1-associated PI 3-kinase activity in adipose tissue only 2.0-fold (P < 0.01) compared with the 10-fold effect of insulin. IRS-2-associated PI 3-kinase activity was increased 1.7-fold (P < 0.01) by leptin in liver and 6-fold by insulin. Akt phosphorylation and activity were not changed by leptin but increased with insulin. Lower concentrations of leptin (10 and 50 microg/kg) also stimulated STAT3 phosphorylation in fat. These effects appear to be direct because 3 min after leptin intracerebroventricular injection, phosphorylation of STAT3, STAT1, and MAPK were not stimulated in hypothalamus or adipose tissue. Furthermore, leptin activated STAT3 and MAPK in adipose tissue explants ex vivo and in 3T3-L1 adipocytes. Leptin did not activate STAT3 or MAPK in adipose tissue of db/db mice. Thus, leptin rapidly activates signaling pathways directly at the level of insulin sensitive tissues through the long-form leptin receptor, and these pathways overlap with, but are distinct from, those engaged by insulin.

Identification and functional analysis of mutations in the hepatocyte nuclear factor-1alpha gene in anti-islet autoantibody-negative Japanese patients with type 1 diabetes.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene are the cause of maturity-onset diabetes of the young type 3 (MODY 3), which is characterized by a severe impairment of insulin secretion and early onset of the disease. Although the majority of patients with type 1 diabetes have type 1A, immune-mediated diabetes, there is a significant percentage of the patients who have no evidence of an autoimmune disorder at the onset of disease. The aim of this study was to estimate the prevalence of MODY 3 in antiislet autoantibody negative patients with type 1 diabetes. From a large population-based sample of unrelated Japanese patients with type 1 diabetes, 28 patients who lacked autoantibodies to glutamic acid decarboxylase, islet cell antigen 512/insulinoma-associated antigen-2, phogrin (phosphate homolog of granules of insulinoma)/insulinoma-associated antigen-2beta, and insulin at the onset of type 1 diabetes were examined by PCR-based direct sequencing of the 10 exons, flanking introns, and the promoter region of the HNF-1alpha gene. Two (7.1%) of 28 autoantibody-negative patients with type 1 diabetes were identified as carrying mutations in the HNF-1alpha gene. One patient carried a frameshift mutation (Pro379fsdelCT) in exon 6, and another patient carried a novel 2-bp substitution at nucleotides +45 (G to A) and +46 (C to A) from the transcriptional site of the promoter region. These mutations were identified in heterozygous form and were not identified in 64 unrelated healthy control subjects or 54 unrelated islet autoantibody-positive patients with type 1 diabetes. Functional analysis of the mutant HNF-1alpha gene indicated that the Pro379fsdelCT mutation had no transcriptional trans-activation activity and acted in a dominant negative manner. The +45/46 GC to AA mutation in the promoter region showed reduced promoter activity by 10-20% compared to the wild-type sequence. In conclusion, about 7% of Japanese diabetic patients lacking antiislet autoantibodies initially classified as having type 1 diabetes could have diabetes caused by mutations in the HNF-1alpha gene.

Anti-insulin receptor autoantibodies in a patient with type B insulin resistance and fasting hypoglycemia.

We studied a patient with systemic lupus erythematosus and type B insulin resistance who showed almost complete normalization of postprandial plasma glucose in 3 months and a transient occurrence of fasting hypoglycemia from day 35 (i.e. the 35th day of hospitalization) to day 77. To determine the clinical relevance of the biological ability of anti-insulin receptor antibodies (anti-IRAb), we made multiple preparations of the patient's dialyzed serum and IgG. Dialyzed serum prepared on day 1 showed 95% inhibition of insulin binding. The binding inhibition was, however, decreased parallel to the normalization of insulin sensitivity. For 2DG uptake, 6.2 microM IgG purified on 3 different days (days 7, 35 and 78, designated IgG-NOV, -JAN, and -FEB, respectively) stimulated 2DG uptake into CHO-hIR at 3.4-, 3.1-, and 1.5-fold, respectively. Phosphotyrosine immunoblotting revealed that apparent insulin receptor autophosphorylation was visible only with IgG-NOV, not with the IgG-JAN or -FEB. Mutation of tyrosine-960 or lysine-1018 of the insulin receptor failed to transduce the IgG's stimulatory effect. IgG-NOV was not able to stimulate the autophosphorylation of the human IGF-I receptor. In the present study, the insulin binding inhibitory activities of the dialyzed sera prepared at different time points were shown to be altered parallel to insulin sensitivity in vivo. Stimulatory activities of the patient's IgG were, however, discordant for the occurrence of fasting hypoglycemia observed in vivo. Other pathogenic factors or mechanisms in addition to the insulin-like action of the anti-IRAb may be also required to fully understand the development of fasting hypoglycemia in type B insulin resistance.

Functional properties of leptin receptor isoforms: internalization and degradation of leptin and ligand-induced receptor downregulation.

Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play essential roles in mediating leptin signaling and the transport and degradation of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no information on the ability of individual receptor species to mediate leptin internalization and degradation or to undergo ligand-induced downregulation. We therefore studied these parameters in Chinese hamster ovary (CHO) cells stably expressing either ObRa or ObRb isoforms of the leptin receptor. We determined that both ObRa and ObRb mediated internalization of 125I-labeled leptin by a temperature- and coated pit-dependent mechanism. Both ObRa and ObRb also mediated degradation of 125I-leptin by a lysosomal mechanism, and this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the conserved Box 1 motif. By studying deletion mutants of ObRa, we found that efficient internalization was dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressing ObRa or ObRb to unlabeled leptin for 90 min at 37 degrees C produced downregulation of available surface receptors, and this effect was of greater magnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after exposure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effect on expression of ObRa or ObRb, and DEX reduced binding to cells expressing ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, mediate leptin internalization by a coated pit-dependent mechanism, leptin degradation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the different roles of the receptor isoforms in the biology of leptin.

Functional properties of leptin receptor isoforms containing the gln-->pro extracellular domain mutation of the fatty rat.

Mutations of the leptin receptor have been found to cause obesity in rodents. The fa mutation that is responsible for obesity in Zucker rats is a missense mutation (269 gln-->pro) in the extracellular domain of the leptin receptor. We have characterized the effects of this mutation on the two major isoforms of the leptin receptor, Ob-Rb and Ob-Ra, by studying cell-surface expression, leptin binding affinity, signaling capacity, and receptor-mediated internalization and degradation of leptin in transfected mammalian cell lines. Both Ob-Rb(269 gln-->pro) and Ob-Ra(269 gln-->pro) have decreased cell-surface expression and decreased leptin binding affinity. Ob-Rb(269 gln-->pro) was shown to have defective signaling to the JAK-STAT pathway and markedly diminished ability to activate transcription of the egr-1 promoter. Constitutive ligand-independent activation of Ob-Rb(269 gln-->pro) was observed for activation of egr-1-luc but only under conditions when JAK2 was coexpressed with Ob-Rb(269 gln-->pro), Finally, Ob-Ra(269 gln-->pro) has an increased ability to internalize leptin but is less efficient at degrading leptin, as compared with Ob-Ra. In conclusion, both Ob-Ra(269 gln-->pro) and Ob-Rb(269 gln-->pro) have multiple functional defects.

Divergent signaling capacities of the long and short isoforms of the leptin receptor.

Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo.

Adenosine 3',5'-cyclic monophosphate mimics the inhibitory effect of high glucose on MAP kinase phosphorylation in rat mesangial cells.

Alteration in mesangial cell function induced by high glucose levels is implicated in the development of diabetic nephropathy. The aim of this study was to investigate the mechanism by which high glucose attenuates mesangial cell proliferation. Thymidine incorporation in cultured mesangial cells decreased in the presence of high glucose concentrations in a dose dependent manner, with the maximum decrease of 25% occurring at a glucose concentration of 55.5 mM. Phosphorylation of mitogen-activated protein (MAP) kinase was abolished when the cells were treated with 55.5 mM glucose compared with 11.1 mM glucose. The concentration of intracellular cAMP doubled in the presence of 55.5 mM glucose. The addition of 8Br-cAMP (1.0 mM) to the culture media containing 11.1 mM glucose decreased thymidine incorporation by 34%, similar to the effect of high glucose. In order to clarify the contribution of protein kinase A (PKA) to the MAP kinase cascade, we used PKA inhibitor (H-8). The addition of H-8(10 microM) recovered MAP kinase phosphorylation in the presence of 55.5 mM glucose. Our data indicated that the inhibition of this mitogenic pathway mediated by activation of PKA, which is probably induced by high glucose levels, may play an important role in the perturbation of mesangial cells.

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.

TNF-alpha stimulates glucose uptake in L6 myoblasts.

The mechanism of TNF-alpha to regulate glucose metabolism remains unclear. To further delineate the TNF-alpha signal transduction pathway mediating glucose metabolism, we utilized L6 rat myoblasts which contain the receptors for the insulin-like growth factor-I (IGF-I) and TNF-alpha, and the ability of both ligands to stimulate glucose uptake was compared. IGF-I (6.5 nM) maximally stimulated glucose uptake 7-fold after 24 h incubation, while 23 nM TNF-alpha maximally stimulated glucose uptake 3-fold only after 48 h incubation. IGF-I receptor beta-subunit, insulin receptor substrate-1 (IRS-1), and mitogen-activated protein (MAP) kinase were all phosphorylated in response to 6.5 nM IGF-I after 10 min incubation. In contrast, the treatment with 23 nM TNF-alpha failed to phosphorylate either IGF-I receptor beta-subunit or IRS-1 but did phosphorylate MAP kinase as much as IGF-I did. Despite a similar extent to which TNF-alpha induced MAP kinase phosphorylation as IGF-I did, TNF-alpha stimulated glucose uptake less compared to IGF-I. The results indicate that MAP kinase phosphorylation is not sufficient for glucose uptake in L6 myoblasts. TNF-alpha-elicited signal transduction to glucose uptake may utilize a different pathway from that seen with IGF-I.

Autoantibodies to glutamic acid decarboxylase in patients with autoimmune thyroid disease: relation to competitive insulin autoantibodies.

We evaluated the presence of autoantibodies to glutamic acid decarboxylase (GAD), competitive insulin autoantibodies (IAA) and islet cell antibodies (ICA) in sera from 288 non-diabetic patients with autoimmune thyroid disease (AITD), including 212 patients with Graves' disease and 76 with Hashimoto's thyroiditis, and in 235 age- and sex-matched healthy control subjects. GAD antibodies and IAA were assayed using radioimmunoassay with 125I-labelled purified pig brain GAD and human insulin, respectively. Titers of greater than 4.7 units for GAD antibodies and 50 nU/ml for IAA, respectively, the mean + 3SD of 235 age- and sex-matched healthy individuals, were defined as positive. The mean titers of GAD antibodies in patients with Graves' disease and in patients with Hashimoto's thyroiditis were 3.6 +/- 4.6 (mean +/- SD, range 0.6-52.0) units and 3.2 +/- 1.4 (range 0.6-10.0) units, respectively. Titer of GAD antibodies in patients with AITD was significantly higher than in healthy controls (P < 0.0005). Thirteen of 212 (6.1%) patients with Graves' disease and 6 of 76 (7.9%) patients with Hashimoto's thyroiditis had positive GAD antibody titers, whereas titers in healthy control sera were < 4.7 units in all but two individuals (P < 0.005). In competition analysis with purified unlabelled GAD, binding tracer was inhibited in all of 13 GAD antibody-positive Graves' sera and 5 of 6 GAD antibody-positive sera from patients with Hashimoto's thyroiditis. Eight of 212 (3.8%) patients with Graves' disease and 3 of 76 (3.9%) patients with Hashimoto's thyroiditis, but none of healthy controls had IAA levels exceeding the range for normal controls (P < 0.005). Positive IAA levels ranged between 50 and 2383 nU/ml. Strikingly, all of 19 GAD antibody-positive sera were negative for IAA. ICA were not detected in any of the patients or healthy controls. These data demonstrate that GAD antibodies in sera of AITD patients are of low titer but significantly elevated compared to healthy controls, and are independent of the appearance of IAA. They also indicate that, in patients with AITD, an autoimmune response to GAD may occur with no relationship to production of IAA.

Insulin response after treatment depends on fasting plasma glucose level in NIDDM.

We investigated the relationship between the improvement in insulin secretion and glycemic control in non-insulin-dependent diabetes mellitus (NIDDM). Fifty-two patients were classified into three groups according to their pretreatment fasting plasma glucose (FPG) level: Group A, FPG < 7.8 mM, n = 20; Group B, 7.8 mM < or = FPG < 11.1 mM, n = 17; and Group C, 11.1 mM < or = FPG, n = 15. A 75-g oral glucose tolerance test (OGTT) and a glucagon loading test were performed to evaluate insulin secretion before and after treatment. Plasma glucose levels during a 75-g OGTT were decreased significantly after treatment in all groups (P < 0.01). In Group A, there was no significant change in insulin secretion before and after treatment (1466 +/- 213 pM to 1565 +/- 191 pM, P = 0.35). In contrast, in Groups B and C, insulin secretion was poor and suppressed initially, but increased significantly when good glycemic control was obtained after treatment (respectively, 587 +/- 70 pM to 863 +/- 79 pM, P < 0.01, and 621 +/- 94 pM to 1236 +/- 232 pM, P < 0.01). The degree of improvement in insulin secretion in 75-g OGTT correlated positively with the degree of improvement in FPG level after treatment (r = 0.5, P < 0.001). However, the C-peptide response to glucagon did not change before and after treatment. In conclusion, impaired insulin secretion recovered by the good glycemic control in NIDDM with FPG levels above 7.8 mM. Therefore, strict glycemic control (FPG below 7.8 mM) seems important for maintaining good insulin secretion.

Molecular analysis of insulin receptor gene in Werner's syndrome.

Werner's syndrome is characterized by premature aging and frequent impaired glucose tolerance or overt diabetes. Insulin resistance may play an important role and may be caused by a post-receptor defect or dysfunctional insulin receptor. The present study was undertaken to investigate the insulin receptor gene mutation in Werner's syndrome. The genomic DNAs were obtained from four patients with Werner's syndrome. Exons 2-22 of the insulin receptor gene except exon 1 were amplified from genomic DNA by the polymerase chain reaction and screened for nucleotide variation by examining for single-stranded conformational polymorphisms. There were no nucleotide variations in exons 2, 4-->7, 9 and 12-->22. Variants were thus found in exons 3, 8, 10 and 11 and each were sequenced. The variant in exon 8 was due to a silent polymorphism (GAT-->GAC/T, Asp519) and other variants in exons 3, 10 and 11 were caused by nucleotide substitutions in introns. These results suggest that the patients with Werner's syndrome express normal insulin receptors and that the primary genetic lesion for insulin resistance is not in the insulin receptor gene. Insulin resistance in Werner's syndrome is thus likely by a post-receptor defect.