Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation.

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.

Subcutaneous fat modulates insulin sensitivity in mice by regulating TNF-alpha expression in visceral fat.

The distribution of fat in obese persons is related to the risk of developing various metabolic disorders, such as glucose intolerance, dyslipidemia and hypertension, and the combination of these conditions is known as the metabolic syndrome. The aim of this study was to investigate the role of subcutaneous fat in regulating insulin resistance and its influence on TNF-alpha expression in visceral fat, by using mice that were subjected to subcutaneous lipectomy with or without subsequent fat transplantation. After partial subcutaneous lipectomy, mice showed significantly greater accumulation of visceral fat compared with sham-operated control mice. Lipectomy led to higher plasma insulin and lower plasma glucose levels after loading with glucose and insulin, respectively, compared with the levels in control mice. Insulin-induced phosphorylation of IRS-1 was decreased in the skeletal muscles of lipectomized mice. Subcutaneous transplantation of fat pads into lipectomized mice reversed the above-mentioned changes indicating insulin resistance in these animals. The fat storage area of adipocytes and TNF- alpha expression by adipocytes in visceral fat were significantly higher in the lipectomized mice than in controls, while subcutaneous transplantation of fat reduced both the fat storage area and TNF-alpha expression. The insulin resistance of lipectomized mice was also ameliorated by systemic neutralization of TNF-alpha activity using a specific antibody. These findings obtained in mice subjected to subcutaneous lipectomy with/without subsequent fat transplantation indicate that subcutaneous fat regulates systemic insulin sensitivity, possibly through altering fat storage and the expression of TNF-alpha by adipocytes in visceral fat. The balance between accumulation of subcutaneous fat and visceral fat may be important with respect to the occurrence of systemic insulin resistance in the metabolic syndrome.

Insulin resistance and increased pancreatic beta-cell proliferation in mice expressing a mutant insulin receptor (P1195L).

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic beta-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic beta-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR beta-subunit. Homozygous (Ir(P1195L/P1195L)) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir(P1195L/wt)) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir(wt/wt)) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir(P1195L/wt) and Ir(wt/wt) mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir(P1195L/wt) mice. The beta-cell mass increased due to the increased numbers of beta-cells in Ir(P1195L/wt) mice. More proliferative beta-cells were observed in Ir(P1195L/wt) mice, but the number of apoptotic beta-cells was almost the same as that in Ir(wt/wt) mice, even after streptozotocin treatment. These data suggest that, in Ir(P1195L/wt) mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of beta-cells, which in turn was due to increased beta-cell proliferation rather than decreased beta-cell apoptosis.

Aldose reductase inhibition alters nodal Na+ currents and nerve conduction in human diabetics.

BACKGROUND: In diabetic nerves, activation of the polyol pathway via an aldose reductase and the resulting impairment of the Na(+)-K(+) pump would lead to a decreased transaxonal Na+ gradient and thereby reduced nodal Na+ currents. OBJECTIVE: To investigate whether the aldose reductase inhibitor (ARI) epalrestat improves nodal Na+ currents and nerve conduction in human diabetic neuropathy. METHODS: The authors conducted a 6-month, open clinical trial with an ARI, epalrestat, in 30 patients with mild-to-moderate diabetic neuropathy. The latent addition technique and measurements of the strength-duration time constant were used to estimate nodal persistent Na+ currents in median motor axons. Excitability testing and extensive nerve conduction studies including F-wave analyses were performed before and 1 and 6 months after the initiation of treatment with oral epalrestat. RESULTS: Within a month of the start of treatment, there was a significant improvement in nerve conduction, particularly in conduction times across the carpal tunnel and F-wave latencies. The results of latent addition (p < 0.05) and strength-duration time constant (p = 0.06) suggested increased nodal persistent Na+ currents. At 6 months, nerve conduction continued to improve. CONCLUSIONS: Aldose reductase pathway inhibition could rapidly increase nodal Na+ currents and thereby improve the slowing of nerve conduction, presumably because of a restoration of the membranous Na+ gradient.

Differential effects of RAS inhibitors associated with ACE gene polymorphisms in type 2 diabetic nephropathy.

Blood pressure and genetic factors are important factors for diabetic nephropathy. We investigated the relationship between the efficacy of renin angiotensin system (RAS) inhibitors and angiotensin-converting enzyme (ACE) genotypes. Patients with type 2 diabetes without proteinuria, were treated with RAS inhibitors, the first being an ACE inhibitor (ACEI) and the second, an angiotensin II (ATII) receptor blocker (ARB) for 8 weeks each. There was no significant difference (except serum ACE activity) between the two treatments. However, by analysis segregated with ACE gene polymorphism, ARB significantly decreased transforming growth factor-beta1 (TGF-beta) compared to ACEI in patients with the I/I genotype but not in patients with the D/I+D/D genotype. DeltaATII and DeltaTGF-beta have a negative correlation with the I/I genotype and a positive correlation with the D/I+D/D genotypes. These correlation coefficients are significantly different. We suggest that in I/I patients, TGF-beta was reduced by ARB via effects on (ATII) type 2 receptors (AT2). In our experiments, the effect of ARB on TGF-beta reduction was only detected by segregation of ACE genotypes. This indicates that the selection of medicine in light of a patient's genotype is important in treating diabetic nephropathy.

Impaired glucose tolerance is accompanied by decreased insulin sensitivity in tissues of mice implanted with cells that overexpress resistin.

AIM/HYPOTHESIS: Resistin, the expression of which is suppressed by thiazolidinedione treatment in adipocytes, is one of the key molecules for the tight link between adiposity and insulin resistance. Here, we show the in vivo effects of resistin on insulin sensitivity in mature mice using a cell implantation method. METHODS: Resistin cDNA was transfected into 3T3-L1 pre-adipocytes, which were then implanted into subcutaneous areas of nude mice. Metabolic analyses were performed 4 or 6 weeks after implantation. RESULTS: The mice implanted with 3T3-L1 cells overexpressing resistin (R-mice) showed significantly (p<0.05) increased plasma resistin levels. After a glucose load plasma insulin levels were significantly greater in R-mice than in mice implanted with mock-transfected cells (M-mice). The AUC of insulin after glucose loading was positively correlated with circulating resistin levels. Significantly decreased glucose responses after insulin injection were observed in R-mice, compared to M-mice. The insulin-induced phosphorylation level of IRS-1 was significantly lower in muscles of R-mice than M-mice. The expression of TNF-alpha mRNA in intra-peritoneal fat tissues was significantly greater in R-mice than in M-mice, but there was no difference between the two groups with regard to subcutaneous fat tissues. The concentration of TNF-alpha in plasma was positively correlated with resistin levels in R-mice. CONCLUSIONS/INTERPRETATION: Resistin, when actually secreted from cells in mature mice, causes disturbed glucose metabolism, possibly based on decreased insulin sensitivity in muscle. The in vivo effects of resistin on insulin sensitivity might be in part mediated by increased TNF-alpha expression in visceral fat tissues.

Mechanisms involved in the stimulatory effect of advanced glycation end products on growth of rat aortic smooth muscle cells.

Hyperglycemia is an important cause of accelerated atherosclerosis in diabetic patients. We examined the effect of hyperglycemia and advanced glycation end products (AGE) on proliferation of rat aortic smooth muscle cells (SMC) in culture; in vivo, this event is believed to contribute importantly to atherogenesis in diabetes mellitus. Glucose itself dose-dependently inhibited thymidine uptake by SMC, but AGE increased thymidine uptake, suggesting that SMC proliferation is accelerated by AGE. To examine possible mechanisms for this effect, we studied nuclear factor-kappa B (NF-kappaB) activation and the tyrosine phosphorylation pathway; AGE stimulated NF-kappaB activity, but phosphorylation of the platelet-derived growth factor (PDGF) receptor was unchanged. In Chinese hamster ovary (CHO) cells overexpressing galectin-3, an AGE receptor related to atherosclerosis, AGE increased thymidine uptake. This suggests SMC proliferation is enhanced by AGE via galectin-3. As pathways involving AGE-galectin-3 interaction thus may be involved in macroangiopathy, AGE appears to be important to the role of SMC in accelerated atherosclerosis associated with diabetes mellitus.

Pathophysiologic phenotypes of Japanese subjects with varying degrees of glucose tolerance: using the combination of C-peptide secretion rate and minimal model analysis.

We tried to characterize the clinical features associated with glucose metabolism in the development of diabetes. Study subjects were glucose-tolerant subjects without a family history of diabetes (normal glucose tolerance [NGT]1 group, n = 15) and with a first-degree diabetes relative (NGT2, n = 9), 12 subjects with impaired glucose tolerance (IGT), and 13 subjects with type 2 diabetes mellitus (DM). The first phase C-peptide secretion (CS1), insulin sensitivity (Si), and glucose effectiveness (Sg) were assessed by the combination of C-peptide 2-compartment model and minimal model analyses. Using these parameters, each group was characterized: CS1 was decreased in NGT2 and IGT compared with NGT1 and further decreased in DM; Si was not different among NGT1, NGT2, and IGT, whereas Si was decreased in DM; CS1 x Si value was decreased in NGT2 compared with NGT1 and decreased in IGT, DM, progressively; Sg was decreased in IGT and DM compared with NGT1 and NGT2. CS1 x Si and Sg values could segregate each group distinctively, although it had a large variety of phenotypes. CS1 x Si value and Sg are assumed to represent the contributions of insulin-dependent and independent mechanisms to glucose tolerance, respectively, and thus, both mechanisms should play an important role in the characterization of pathophysiologic phenotypes of the subjects with various degrees of glucose tolerance.

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells.

Cytokines induce apoptosis in pancreatic beta cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-alpha) in the apoptosis of beta cells. Using the ribonuclease (RNase) protection assay and the reverse transcriptase-polymerase chain reaction (RT-PCR) method, we confirmed that TNF receptor 1 (TNFR1), TNFR1-associated death domain protein (TRADD), Fas receptor-associated intracellular protein with death domain (FADD), and FADD-like interleukin-1beta-converting enzyme (FLICE) were expressed in the pancreatic beta cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-alpha induced time- and dose-dependent apoptotic nuclear changes in these beta cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-alpha generated new 3'-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-alpha produced characteristic apoptotic patterns of DNA fragments formed by internucleosomal hydrolysis of static chromatin. In addition, C2-ceramides and natural ceramides dispersed in a solvent mixture of ethanol and dodecane induced characteristic features of apoptosis in MIN6 cells, mimicking TNF-induced DNA damage. We also determined endosomal ceramide production after TNF-alpha (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-alpha can cause apoptosis in pancreatic beta cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

Effect of ACE gene on diabetic nephropathy in NIDDM patients with insulin resistance.

We investigated the influence of the angiotensin-converting enzyme (ACE) gene on the onset and/or progression of diabetic nephropathy in 62 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM; type II diabetes). Because a number of factors are believed to be involved in the onset and/or progression of diabetic nephropathy, especially in patients with NIDDM, we selected the patients with well-matched risk factors, duration of disease, glycemic control, blood pressure, and others. All patients had normal renal function and none were receiving ACE inhibitors. Patients were divided into three groups according to albumin excretion rate (AER): group A, patients with an AER less than 15 microg/min (n = 29); group B, patients with an AER between 15 and 70 microg/min (n = 19); and group C, patients with an AER greater than 70 microg/min (n = 14). The glucose disposal rate was estimated using a euglycemic hyperinsulinemic clamp. We determined the mean glucose disposal rate in 132 patients with NIDDM (6.49 mg/kg/min). Patients with a glucose disposal rate less than the mean rate were considered to have a high degree of insulin resistance (n = 36). The presence of an insertion/deletion (I/D) polymorphism of the ACE gene was determined by the polymerase chain reaction method. Among patients with a high degree of insulin resistance, diabetic nephropathy was present in 2 of 11 patients with the II genotype of the ACE gene compared with 19 of 25 patients with the ID or DD genotype (P = 0.0024). The prevalence of diabetic nephropathy was greater in patients with both significant insulin resistance and the D allele (19 of 25) than in the remaining patients (14 of 37; odds ratio, 5.20). These results suggest that the ACE gene influences the onset and/or progression of diabetic nephropathy in patients with NIDDM with significant insulin resistance.

A missense mutation in the CD38 gene, a novel factor for insulin secretion: association with Type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro.

Cyclic adenosine 5'diphosphate-ribose (cADPR) is thought to have a second messenger role in insulin secretion through mobilisation of Ca2+. As human lymphocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activity, it may be important in glucose-induced insulin secretion in islets. Thirty one randomly selected Japanese patients with Type II diabetes mellitus who had first-degree and/or second-degree relative(s) with Type II diabetes mellitus were screened for mutations of this gene using single-stranded conformation polymorphism. Two variant patterns in exon 3 and exon 4 of the CD38 gene were identified. The variant in exon 3 resulted in an amino acid substitution from Arg140 (CGG) to Trp (TGG). The Arg140Trp mutation was observed in 4 of 31 patients, and allele frequencies were significantly different in patients and the control subjects (p = 0.004). One patient with this mutation has two missense mutations on beta cell/liver glucose transporter (GLUT2) gene; her mother, who has impaired glucose tolerance, also has this mutation on the CD38 gene and one missense mutation on the GLUT2 gene. Enzyme activity studies using COS-7 cells expressing the Arg140Trp mutation showed a reduction in ADP-ribosyl cyclase and cADPR hydrolase activity of around 50%. The Arg140Trp mutation on CD38 thus appears to contribute to the development of Type II diabetes mellitus via the impairment of glucose-induced insulin secretion in the presence of other genetic defects.

Molecular cloning of rat Pax4: identification of four isoforms in rat insulinoma cells.

Molecular cloning of rat Pax4 cDNA from a rat insulinoma cell line, RINm5F, library by PCR-based cloning strategy revealed four isoforms of the protein. Analysis of tissue distribution using Northern blotting and RT-PCR showed specific expression of Pax4 mRNA in pancreatic islets and RIN cells. RT-PCR confirmed that the mRNAs of four isoforms are expressed in RIN cells. These Pax4 variants may regulate the transcriptional activity of Pax4 during the development of pancreatic islets.

Expression of human islet amyloid polypeptide/amylin impairs insulin secretion in mouse pancreatic beta cells.

Non-insulin-dependent diabetes mellitus (NIDDM) is associated histopathologically with islet amyloid deposits of which a major component is islet amyloid polypeptide (IAPP)/amylin. We examined whether endogenous IAPP controls insulin secretion via a local effect within pancreatic islets and whether overexpression of this peptide contributes to pancreatic beta-cell dysfunction in this disease. Transgenic mice expressing human IAPP in pancreatic beta cell were used in this study. Human IAPP expression did not influence the mouse proinsulin mRNA level and insulin content. Glucose-induced insulin secretion was decreased in the isolated pancreatic islets of transgenic mice. MIN6, a glucose-responsive pancreatic beta-cell line, was transfected with human IAPP cDNA by a lipofectin method. Human IAPP expression was confirmed by RNA blot and immunohistochemical analysis. In two transfectants expressing the largest amount of human IAPP, insulin secretion was increased in response to glucose stimulation; however, the magnitude of the insulin response in cells transfected with human IAPP was smaller than in control clones. Insulin content was not influenced by the expression. We conclude that endogenous IAPP inhibits insulin secretion via an autocrine effect within pancreatic islets, and that the impaired insulin secretion in this disease may be partly caused by overexpression of IAPP.

Islet amyloid polypeptide/amylin contents in pancreas change with increasing age in genetically obese and diabetic mice.

To search for a possible relationship between islet amyloid polypeptide (IAPP)/amylin and the pathophysiology of non-insulin-dependent (type 2) diabetes mellitus (NIDDM), we examined the changes in IAPP contents in the pancreata of genetically obese and diabetic mice (C57BL/6J ob/ob and C57BL/KsJ db/db mice). In the male ob/ob mice, IAPP and insulin contents began to increase at 16 weeks and continued to increase. In the male db/db mice, IAPP content began to increase at 8 weeks of age and insulin content at 4 weeks. Both contents continued to increase until 16 weeks, but drastically decreased at 24 weeks. Immunohistochemical studies using anti-IAPP8-17 antibody showed the increase of islet cell mass and the heterogeneous immunoreactivity for IAPP in islet cells in the ob/ob mice at 24 weeks of age. In the db/db mice at the same age, the immunoreactivity was heterogeneous and weak in many islet cells. These results suggest that genetic factors that are important in the manifestation of NIDDM influence the capacity of beta-cells to synthesize and secrete IAPP, and that IAPP synthesis and secretion change in the course of the disease.

Formation of islet amyloid fibrils in beta-secretory granules of transgenic mice expressing human islet amyloid polypeptide/amylin.

To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we developed transgenic mice using a human IAPP cDNA connected to an insulin promoter. Ribonucleic acid blotting and immunohistochemistry revealed the expression of the transgene in the pancreatic beta cells. Immunogold electron microscopy showed that beta-secretory granules contained the human C-terminal flanking peptide of the IAPP precursor. Reverse-phase HPLC demonstrated human and mouse IAPP amide in the pancreas. Electron microscopy showed the accumulation of fibril-like material in a considerable number of beta-secretory granules. These results suggest that in transgenic mice, the human IAPP precursor is expressed in beta cells and becomes normally sorted into beta-secretory granules in which normal conversion to mature human IAPP takes place. The human IAPP molecules, because of their amyloidogenesis, aggregate into amyloid fibrils in secretory granules. Glucose tolerance was normal at 7 months old and islet amyloid was not observed. A longer time may be required for islet amyloid deposits and hyperglycemia to develop in mice. Our working hypothesis is that in human NIDDM, IAPP aggregates into amyloid fibrils in beta-secretory granules, and that the fibrils are released into the extracellular space and islet amyloid deposits become substantial with time.

Islet amyloid polypeptide and its N-terminal and C-terminal flanking peptides' immunoreactivity in islet amyloid of diabetic patients.

We determined immunohistochemically whether the islet amyloid polypeptide (IAPP)/amylin precursor is one component of islet amyloid, using polyclonal antibodies specific for human IAPP8-17 and amino (N)-terminal and carboxy (C)-terminal flanking peptides. To enhance immunostaining of the amyloid, we pretreated the pancreatic tissue sections with 100% formic acid. In three non-diabetic subjects, pancreatic islet cells were immunoreactive to anti-IAPP8-17 and anti-N-terminal and C-terminal flanking peptide antibodies and the reactivity was enhanced with formic acid pretreatment. In six type 2 diabetic subjects and a subject with type A insulin resistance, islet amyloid deposits were reactive to anti-IAPP8-17 antibody, but not to anti-N-terminal and C-terminal flanking peptide antibodies. Formic acid pretreatment markedly enhanced the reactivity to anti-IAPP8-17 antibody; however, it failed to show the reactivity to anti-N-terminal and C-terminal flanking peptide antibodies. Formic acid pretreatment of pancreatic tissue sections prepared for immunostaining is useful for visualization of buried epitopes of mature IAPP and its precursor molecules, either in islet amyloid deposits or in the islet cells. We conclude that the IAPP precursor and N-terminal and C-terminal flanking peptides are not constituents of human islet amyloid.

[Construction of transgenic mouse system expressing human islet amyloid polypeptide (IAPP)/amylin].

To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we generated transgenic mice using a human IAPP cDNA connected to an insulin promoter. Analyses by RNA blotting and immunohistochemistry revealed that the transgene was expressed exclusively in the pancreatic Beta-cells. Immunogold electron microscopy showed that the C-terminal flanking peptide of the human IAPP was localized in the Beta-secretory granules. Reverse-phase HPLC demonstrated the presence of mature human IAPP. However, glucose tolerance was normal and amyloid formation was not observed in transgenic mice at 7 months of age. A longer time or other factors may be required for islet amyloid deposits and hyperglycemia to develop in mice.