Yoshifumi Suzuki

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the probands lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.

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

Summary 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. [Diabetologia (1998) 41: 1024–1028]

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic β cells

Abstract Cytokines induce apoptosis in pancreatic β cells, but the exact mechanisms and sequence of events are not clear. Here, we investigate a role for tumor necrosis factor alpha (TNF-α) in the apoptosis of β 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-1β—converting enzyme (FLICE) were expressed in the pancreatic β cell line, MIN6 cells. Fluorescent microscopic examination using Hoechst 33342 dye (Sigma, St Louis, MO) demonstrated that TNF-α induced time- and dose-dependent apoptotic nuclear changes in these β cells. In situ end-labeling (ISEL) DNA analysis revealed that 10 nmol/L TNF-α generated new 3′-OH DNA strand breaks. Moreover, qualitative assessment of the induced DNA damage on agarose gels showed that 10 nmol/L TNF-α 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-α (10 nmol/L) treatment in MIN6 cells using the diacylglycerol kinase assay. These results suggest that TNF-α can cause apoptosis in pancreatic β cells through TNFR1-linked apoptotic factors, TRADD, FADD, and FLICE, and TNF-induced ceramide production may be involved in the pathways.

Activation of mouse phosphodiesterase 3B gene promoter by adipocyte differentiation in 3T3-L1 cells.

Activation of phosphodiesterase (PDE) 3B reduces free fatty acid output from adipocytes. Induction of PDE3B gene expression by adipocyte differentiation could improve insulin resistance. To examine whether the PDE3B promoter is activated by this differentiation, the 5′ flanking sequence of the mouse PDE3B gene was isolated. The transcription initiation site was determined to be located 195 bp upstream of the translation start site. No putative binding site for peroxisome proliferator‐activated receptor γ was found within 2 kb upstream of the transcription initiation site. This region had promoter activity, which was further activated on adipocyte differentiation in 3T3‐L1 cells.

Role of Src Homology 2–Containing Tyrosine Phosphatase 2 on Proliferation of Rat Smooth Muscle Cells

Objective—Src homology 2–containing phosphotyrosine phosphatase 2 (SHP2) is ubiquitously expressed and believed to function as part of a positive signaling pathway mediating growth factor–induced protein tyrosine phosphorylation. Proliferation of aortic vascular smooth muscle cells (SMCs) is an important contributor to atherosclerosis. We examined the effect of SHP2 expression on SMC proliferative activity. Methods and Results—SHP2 was abundant in cultured aortic SMCs, and SHP2 staining was markedly increased in the thickened aortic intima in rats with balloon-induced injury. We obtained several SMC clones by using geneticin screening. Endogenous SHP2 expression varied among individual clones. Significant positive relationships were observed between SHP2 expression and bromodeoxyuridine uptake in SMCs stimulated by FBS, platelet-derived growth factor, or insulin-like growth factor-1. In SMCs transiently transfected with SHP2, FBS stimulation significantly increased bromodeoxyuridine uptake beyond the uptake by control SMCs. Conclusions—Increased SHP2 expression in SMCs may accelerate aortic atherosclerosis by increasing cell growth.

Mitogen-activated protein kinase and p70 ribosomal protein S6 kinase are not involved in the insulin-dependent stimulation of cAMP phosphodiesterase kinase in rat adipocytes

To elucidate the mechanism of anti-lipolytic action of insulin in rat epididymal adipocytes, we explored the potential mechanism that might be involved in the hormone-dependent stimulation of cAMP phosphodiesterase (PDE) kinase. PDE kinase was assayed in a cell-free system. Both wortmannin and LY294002, highly specific inhibitors of phosphatidylinositol 3-kinase, almost completely blocked the hormonal effect not only on PDE kinase but also on mitogen-activated protein (MAP) kinase. Neither PD98059, a specific inhibitor of MAP kinase, nor rapamycin, a potent inhibitor of insulin-dependent stimulation of p70 ribosomal protein S6 kinase (p70S6K), had inhibitory effect on that of PDE kinase. These results are consistent with the view that (i) insulin-activated PDE kinase as well as MAP kinase and p70S6K are localized downstream of phosphatidylinositol 3-kinase, (ii) PDE kinase is distinct from either MAP kinase or p70S6K and (iii) PDE kinase does not exist downstream of either MAP kinase or p70S6K. It is suggested that PDE kinase and MAP kinase or p70S6K may be localized in separate branches of the cascade of insulin action. The branching point of the cascade could be either at or below the level of phosphatidylinositol 3-kinase.

Activation of the Smad Pathway in Glomeruli from a Spontaneously Diabetic Rat Model, OLETF Rats

Background/Aims: Transforming growth factor-β (TGF-β) mediates the excess accumulation of extracellular matrix in the diabetic kidney. Smad family proteins have been identified as signal transducers for the TGF-β superfamily. We sought to characterize the role of Smad proteins in mediating TGF-β responses in the development of diabetic nephropathy. Methods: We evaluated the time course of TGF-β1 fibronectin, Smad2 and Smad3 protein expression and Smad3 activation in glomeruli from spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using immunohistochemistry and Western blot analysis. Results: The glomeruli of diabetic OLETF rats showed not only accelerated activation of Smad3, but also enhanced protein expression of Smad2 and Smad3, which occurred in parallel to the increased expression of TGF-β and fibronectin compared with glomeruli of control, Long-Evans Tokushima Otsuka (LETO) rats at 30 weeks of age. No differences were found in TGF-β1 fibronectin, Smad2 and Smad3 protein expression and Smad3 activation in glomeruli between the two strains at 12 weeks of age when OLETF rats were not diabetic. Conclusions: The enhancement of Smad protein expression and activation may be involved in the TGF-β signaling cascade that plays an important role in the development of diabetic nephropathy through progressive expansion of the mesangial matrix.

Defects in insulin binding and receptor kinase in cells from a woman with type A insulin resistance and from her family

SummaryDefects in insulin receptor function lead to impairment of the insulin response. We treated a patient with the typical phenotype of type A syndrome of insulin resistance whose insulin receptor seemed to lack the transmembrane region and cytoplasmic domain. Hyperinsulinaemia and resistance to exogenous insulin were evident, and insulin binding to cells and uptake of 2-deoxyglucose into fibroblasts were greatly decreased. Molecular weight of the α-subunit of the insulin receptor was normal, but autophosphorylation and kinase activity were impaired. In the pedigree analysis, defects in insulin binding were also observed in the mother, maternal grandfather and two maternal aunts, corresponding with the abnormality of the insulin receptor gene and mild insulin resistance. In the mother, much the same kinase defects as were seen in the patient became evident. However, no relatives had clinical symptoms similar to those seen in the patient. In the father there was a mild insulin resistance in the glucose clamp study and a borderline impaired glucose tolerance. Although insulin binding to cells was normal in the father, both autophosphorylation and kinase activity were reduced. Our findings suggest that insulin resistance in the patient may be caused by the defects in insulin receptor kinase activity as well as by a reduction in insulin binding activity.

Analysis of the insulin-sensitive phosphodiesterase 3B gene in type 2 diabetes

We screened for mutations in the gene of insulin-sensitive phosphodiesterase 3B (PDE3B), which regulates antilipolytic actions of insulin via reduction of intracellular cyclic AMP levels, in Japanese patients with type 2 diabetes mellitus and lipoatrophic diabetes mellitus using single-stranded conformation polymorphism analysis and Southern analysis and investigated frequencies of variable number of tandem repeats. A silent polymorphism at the Arg463 codon (AGG-->AGA) in exon 4 was identified after examining all 16 exons and exon-intron splicing junctions of the gene. This polymorphism was found in 53 of 100 subjects with type 2 diabetes mellitus, 2 of 5 lipoatrophic diabetic patients and 24 of 50 control subjects, without any significant difference in allele frequency between groups. An EcoRI restriction fragment length polymorphism was identified in patients with type 2 diabetes mellitus and control subjects, again with no differences in occurrence. The allelic distribution of two polymorphic tandem repeats sequences in introns 5 and 12 of the gene did not differ significantly between patients with type 2 diabetes mellitus and control subjects. In conclusion, alterations in the PDE3B gene are unlikely to contribute importantly to the pathogenesis of type 2 diabetes mellitus or lipoatrophic diabetes mellitus in Japan.