Teruaki Yamauchi

Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats

Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor beta subunits (IRbeta) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRbeta in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRbeta- and IRS-2-associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.

Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways

Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation. We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation. Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC). Mannitol activated p38 kinase only at or greater than 22 mM. High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation. However, p38 kinase activation by mannitol or tumor necrosis factor-alpha was not inhibited by GF109203X. Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-beta2 and PKC-delta isoforms were increased. Activities of p38 kinase in PKC-delta- but not PKC-beta1-overexpressed SMC were increased compared with control cells. Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats. Thus, moderate hyperglycemia can activate p38 kinase by a PKC-delta isoform-dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.

Enhanced secretion of endothelin-1 by elevated glucose levels from cultured bovine aortic endothelial cells

We have investigated the effect of glucose on the release of endothelin‐1‐like immunoreactivity (ET‐l‐LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET‐1‐LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose‐induced ET‐1‐LI release, These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET‐1‐LI release at the local site of vascular endothelium. which might be involved in the developments of vascular complications and atherosclerosis.

Antithrombin III stimulates prostacyclin production by cultured aortic endothelial cells

Prostacyclin (PGI2) is a potent vasodilator and an inhibitor of platelet aggregation. We found that antithrombin III (AT III), an anticoagulant present in circulating blood, stimulated PGI2 production by cultured bovine aortic endothelial cells in a dose- and time-dependent manner. The stimulation of PGI2 production by AT III was observed at physiological concentrations and was inhibited by the addition of anti-AT III antiserum and heparin. These results suggest that AT III may stimulate PGI2 production by binding to heparin-like molecules on the endothelial cell membrane.

Identification and characterization of endothelin converting activity in cultured bovine endothelial cells

Using a specific and sensitive radioimmunoassay (RIA) for the carboxyl terminal tail of endothelin (ET) (His16-Trp21), we have confirmed the presence of the converting activity from synthetic human big ET-1 to ET-1 in the homogenate of cultured bovine aortic endothelial cells. The optimal pHs for the converting activities were found at pH 3.0 and pH 7.0. The activity at pH 3.0 was completely inhibited by pepstatin A, whereas the activity at pH 7.0 was not affected by known various protease inhibitors except EDTA and EGTA. When the products from big ET-1 were analyzed on an ODS and a CN columns, only ET-1 was detected at pH 7.0, but various ET-like immunoreactivities other than ET-1 were detected at pH 3.0. These findings strongly suggest that mature ET-1 is formed from big ET-1 in the endothelial cells by a metal-dependent neutral protease.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

Apolipoprotein E polymorphism affects the response to pravastatin on plasma apolipoproteins in diabetic patients.

In the present study, we examined the levels of plasma lipids and apolipoproteins in patients with non-insulin dependent diabetes mellitus (NIDDM) with hypercholesterolemia in different apolipoprotein E (apo E) phenotypes. We also examined the influences of apo E polymorphism on the response to pravastatin. The patients were divided into three groups, E4/E3, E3/E3, and E3/E2. There were no differences in the baseline levels of plasma lipids and apolipoproteins, except that the level of triglycerides in E3/E2 heterozygotes was significantly higher than E3/E3 homozygotes. Three months of pravastatin administration significantly reduced plasma levels of total cholesterol and low-density lipoprotein cholesterol in each group to the same degree. We observed a significant reduction of apo B both in the E4/E3 and E3/E3 groups and apo E in the E3/E3 group. Such reduction was not observed in the E3/E2 group. We conclude that pravastatin is a potent drug to correct lipid abnormalities, particularly in NIDDM patients with apo E4/E3 and E3/E3. In the E3/E2 group, its effectiveness may be diminished.

High Glucose Reduces Specific Binding for D-α-Tocopherol in Cultured Aortic Endothelial Cells

We investigated the effects of glucose on specific D-α-tocopherol binding to cultured bovine aortic endothelial cells. Our results confirmed that cultured bovine aortic endothelial cells have specific binding sites for D-α-tocopherol. These binding sites exhibited time- and temperature-dependent saturation. The specific binding affinity of D-α-tocopherol was significantly lower in endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 7 days compared with cells cultured in a physiological glucose concentration (5.6 mM). No significant reduction occurred in D-α-tocopherol binding when 11.1 mM mannitol was added to cells cultured in 5.6 mM glucose. The addition of an aldose reductase inhibitor (ICI-128436, Statil) did not significantly affect the high-glucose–induced reduction of D-α-tocopherol binding, although it reduced sorbitol levels in the cells compared with those from cells cultured in high concentrations of glucose. Moreover, significantly higher amounts of lipid peroxides were produced in aortic endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for >3 days compared with cells cultured in a physiological concentration of glucose. These results indicate that high concentrations of glucose reduce D-α-tocopherol binding through mechanisms independent of putative osmotic effects of sorbitol accumulation in the cells. Possible mechanisms include glycation of protein or oxidative damage of cells and/or redox and metabolic imbalances associated with increased flux of glucose via the sorbitol pathway. A glucose-mediated reduction in D-α-tocopherol binding could diminish the beneficial effects of D-α-tocopherol to vascular endothelial cells and thereby may increase the vascular toxicity of hyperglycemia in diabetes mellitus.

Genome-wide linkage analysis of type 2 diabetes mellitus reconfirms the susceptibility locus on 11p13–p12 in Japanese

AbstractType 2 diabetes mellitus is a heterogeneous disorder, and the development of type 2 diabetes mellitus is associated with both insulin secretion defect and insulin resistance. The primary metabolic defect leading to type 2 diabetes mellitus has been thought to be varied among populations, especially in Japanese and Caucasians. Here, we have done the genome-wide scan for type 2 diabetes mellitus using 102 affected Japanese sib-pairs to identify the genetic factors predisposing to type 2 diabetes mellitus. Nonparametric linkage analysis showed one suggestive evidence for linkage to 11p13-p12 [D11S905: two-point maximum LOD score (MLS) of 2.89 and multipoint MLS of 2.32] and one nominally significant evidence for linkage to 6q15-q16 (D6S462: two-point MLS of 2.02). Interestingly, the 11p13-p12 region was reported to be a susceptibility locus for Japanese type 2 diabetes mellitus with suggestive evidence of linkage, and D11S905 was within 5 cM to D11S935 with the highest MLS in the previous linkage analysis reported. The only overlapped susceptibility region with suggestive evidence of linkage for Japanese type 2 diabetes mellitus was D11S935-D11S905 among the three reports including this study. These results taken together suggest that a susceptibility gene for type 2 diabetes mellitus in Japanese will reside in 11p13-p12.

A case of insulin allergy: the crystalline human insulin may mask its antigenicity

We report an unusual case of insulin allergy. A 48-year-old man with non-insulin-dependent diabetes mellitus receiving biosynthetic isophane human insulin (Humulin N) developed itchy wheal-and-flare reactions at the sites of injection. When Humulin N was changed to a semi-synthetic crystalline human insulin zinc (Novolin U), the allergic reactions completely disappeared. Evaluation of his serum showed a high level of insulin-specific IgE. Skin testing with all commercially available insulins showed immediate local reactions to all agents tested except for Novolin U. In addition, decrystallized Novolin U prepared by lowering the pH with acetic acid also induced a positive reaction. These observations suggest that the crystallized structure of human insulin may mask its antigenicity for allergic reactions.