Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

PGE1 inhibits the expression of PAI-1 mRNA induced by TNF-alpha in human mesangial cells.

We examined the effect of PGE1 on the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA induced by tumor necrosis factor-alpha (TNF-alpha) in human mesangial cells, because PAI-1 is one of major factors for the progression of glomerulosclerosis. The expression of PAI-1 mRNA was increased after stimulation with TNF-alpha, and it was diminished by pre-incubation with PGE1. Next, we examined the effect of PGE1 on the phosphorylation of mitogen activated protein kinase (MAPK) family and Akt. TNF-alpha activated the phosphorylation of p44/42 MAPK, p38 MAPK, SAPK/JNK and Akt in mesangial cells. PGE1 inhibited the TNF-alpha induced phosphorylation of SAPK/JNK and Akt, but not p44/42 MAPK and p38 MAPK. The TNF-alpha induced expression of PAI-1 mRNA was not affected by PD98059, an inhibitor of MEK, SB203580, an inhibitor of p38 MAPK, nor LY294002, an inhibitor of PI3 K. However, DMAP, an inhibitor of SAPK/JNK, inhibited the expression of PAI-1 mRNA, suggesting that the TNF-alpha induced expression of PAI-1 mRNA is regulated by the SAPK/JNK dependent pathway in human mesangial cells. By the incubation with H8, an inhibitor of PKA, the inhibitory effect of PGE1 on the expression of PAI-1 mRNA was abolished, suggesting that PGE1 inhibited the PAI-1 mRNA expression via the PKA pathway. Our results suggest that the inhibition of PAI-1 synthesis by PGE1 in human mesangial cells may have therapeutic implications for glomerulosclerosis such as occurs in diabetic nephropathy.

Infusion of fish oil emulsion: effects on platelet aggregation and fatty acid composition in phospholipids of plasma, platelets, and red blood cell membranes in rabbits.

An emulsion of fish oil was manufactured to contain 10 g of fish oil/100 mL. Of this, 3 g were eicosapentaenoic acid (EPA) and 1 g was docosahexaenoic acid (DHA). We administered 100 mL of the emulsion into six rabbits intravenously on days 1, 4, 7, 10, and 13. Blood samples were taken on days 0 and 16. The EPA content in phospholipids of plasma, platelets, and red blood cell (RBC) membranes increased 16, 4, and 5 times, respectively. The DHA content in phospholipids of plasma and RBC membranes increased two times whereas that in platelet phospholipids did not increase significantly. Platelet aggregation induced by collagen (10 micrograms/mL) and ADP (5 microM) was depressed significantly after infusion of the fish oil emulsion. In control experiments with soybean oil emulsion, there were almost no significant changes. Therefore, fish oil emulsion may be beneficial to patients who cannot take n-3 fatty acids orally but need them.

Comparison of pulse wave velocity of the aorta between inhabitants of fishing and farming villages in Japan.

Pulse wave velocity (PWV) of the aorta was measured in 55 inhabitants of fishing villages and in 49 inhabitants of farming villages, where people normally eat less fish than in the fishing villages. The PWV was significantly slower (indicating less sclerosis) in the fishing villages than in the farming villages (P less than 0.005). This is consistent with a lower incidence of ischemic heart disease in a coastal area, which includes the fishing villages, than in a mountainous area, including the farming villages. It is reported that a long-term fish-diet slows down sclerotic changes of arteries.

Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells.

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.

Pravastatin suppresses the interleukin-8 production induced by thrombin in human aortic endothelial cells cultured with high glucose by inhibiting the p44/42 mitogen activated protein kinase.

1. 3-Hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro-inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL-8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations. 2. Pravastatin significantly decreased the IL-8 synthesis induced by thrombin. 3. Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity. 4. Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin. 5. Pravastatin inhibit the activator protein-1 activity, but did not inhibit the activation of IkappaB-alpha. 6. Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA. 7. Our results suggest that pravastatin inhibits IL-8 synthesis by blocking the ras-MAP (p44/42) kinase pathway rather than nuclear factor-kappaB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non-diabetics.

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse.

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 +/- 36 to 250 +/- 51 mg/dl) and insulin (76 +/- 14 to 41 +/- 14 microU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 +/- 1 vs. 39 +/- 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 microM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

Infusion of emulsified trieicosapentaenoyl-glycerol into rabbits--the effects on platelet aggregation, polymorphonuclear leukocyte adhesion, and fatty acid composition in plasma and platelet phospholipids.

Using 90%-pure free eicosapentaenoic acid, we synthesized 1,2,3-trieicosapentaenoyl-glycerol (EPA-TG) and manufactured an emulsion of EPA-TG with purified phosphatidylcholine from krill as an emulsifier. After two intravenous injections of the EPA-TG emulsion into rabbits, the EPA content in plasma and platelet phospholipids increased markedly. ADP- and collagen-induced platelet aggregation and polymorphonuclear leukocyte adhesion to glass beads were depressed significantly. No significant changes were observed in serum lipids and liver function. In control experiments which were performed in exactly the same manner except that soybean oil emulsion was used instead of the EPA-TG emulsion, there were almost no significant changes. Our results suggest that an EPA-TG emulsion is applicable to those patients who need both intravenous alimentation and preventive care of thrombosis, such as postoperative patients.

Effects of cilostazol on serum lipid concentrations and plasma fatty acid composition in type 2 diabetic patients with peripheral vascular disease.

Cilostazol is an anti-thrombotic and vasodilating agent, reported to have both anti-thrombotic and cerebral vasodilating effects. We investigated the effects of cilostazol on serum lipid concentrations and plasma fatty acid composition in type 2 diabetic patients with peripheral vascular disease. The serum concentrations of total cholesterol, triglycerides, high-density lipoprotein-cholesterol, lipoprotein (a), remnant-like particles-cholesterol, apolipoproteins, and plasma fatty acid composition were measured in 17 diabetic patients with peripheral vascular disease before and 1, 3, and 6 months after administration of cilostazol (200 mg/day). Serum triglyceride concentrations were significantly decreased after cilostazol (from 1.31+/-0.17 mmol/l to 0.86+/-0.07 mmol/l at 6 months, P<0.01). Plasma docosahexaenoic acid levels were significantly increased after cilostazol (4.11+/-0.26% to 4.94+/-0.26% at 6 months, P<0.01). Our findings show that cilostazol can induce some beneficial changes in serum lipid profile and plasma fatty acid composition.

Reduction in microalbuminuria in diabetics by eicosapentaenoic acid ethyl ester.

Eicosapentaenoic acid (EPA) ethyl ester (1.8 g/d) was administered to 16 diabetic patients (5 insulin-dependent and 11 noninsulin-dependent diabetics) for 6 mon. EPA in total plasma fatty acids increased from 4.0 +/- 2.4 mol% (mean +/- SD) to 7.5 +/- 3.1 mol% (p less than 0.001). Albumin excretion, measured with spot urine, was significantly reduced from 65 to 36 mg/g creatinine (geometric means, p less than 0.001). Fasting blood sugar levels, glycohemogloblin, body weight and blood pressure did not change significantly during the study. There were also no significant changes in serum levels of creatinine, urea nitrogen, total cholesterol and triglycerides. Although no overt hemorrhage was observed in the patients, hematocrit was reduced from 42.6 +/- 2.8% to 41.0 +/- 3.9% (p less than 0.02). Ten other similar diabetic patients (4 insulin-dependent and 6 noninsulin-dependent diabetics) were followed as a reference group, not concomitantly, for 6 mon with neither EPA ethyl ester nor placebo. The parameters mentioned above were not changed significantly in this group during 6 mon. EPA administration might retard the appearance of overt diabetic nephropathy.

Injection of tridocosahexaenoyl-glycerol emulsion and fatty acid composition of blood cells.

An injectable emulsion of docosahexaenoic acid (DHA) was prepared. One hundred ml of the emulsion contained 3 g of 93%-pure 1,2,3-tridocosahexaenoylglycerol (DHA-TG), 1.2 g of 93%-pure 2-docosahexaenoyl-phosphatidylcholine as an emulsifier and 2.5 g of glycerol. Thirty ml of the emulsion of DHA-TG was injected into three rabbits on days 1 and 4 of the study. Blood was taken on day 0, on day 4 just before the second injection and on day 7. The percentage of DHA in the total phospholipid fraction of platelets increased from 0.46% (day 0) to 1.88% (day 4, p less than 0.05) and 3.66% (day 7; p less than 0.02 vs day 0). The percentage of eicosapentaenoic acid (EPA) increased from 0.46% (day 0) to 1.03% (day 4, p less than 0.02) and 1.63% (day 7: p less than 0.05 vs day 0). The percentage of arachidonic acid (AA) decreased from 9.45% (day 0) to 4.31% (day 4, p less than 0.05) and 6.68% (day 7; p less than 0.02 vs day 0). The percentage of DHA in the total phospholipid fraction of erythrocyte membranes increased from 0.23% (day 0) to 0.91% (day 4, p less than 0.05) and 1.52% (day 7; p less than 0.005 vs day 0); that of EPA increased from 0.21% (day 0) to 0.34% (day 4, p less than 0.005%) and 0.52% (day 7, p less than 0.01 vs day 0); that of AA was unchanged. Blood lipids were the same before and after the two injections of the emulsion, except that free fatty acids decreased markedly from 0.32 to 0.06 mEq/1 (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Intake of different eicosapentaenoic acid-containing lipids and fatty acid pattern of plasma lipids in the rats.

The ethyl ester of eicosapentaenoic acid (EPA) is the only pure EPA-containing lipid available in bulk for oral administration. However, there is doubt as to whether EPA ethyl ester can efficiently increase the plasma levels of EPA in comparison with the ability of other kinds of EPA-containing lipids to do so. Therefore, two other kinds of EPA-containing lipids were prepared to study the efficiency of oral administration of those lipids for increasing the EPA content in plasma phospholipids and cholesteryl esters. EPA-containing lipids which were investigated were [A] 1,2,3-trieicosapentaenoyl-glycerol, [B] 2-eicosapentaenoyl-phosphatidylcholine and [C] ethyl ester of EPA. An adjusted amount of lipids [A], [B] and [C] was administered to rats through a gastric tube for 4 days (the first experiment) or for 10 days (the second experiment), and the fatty acid composition of plasma phospholipids and cholesteryl esters was determined. In the first experiment, there were no significant differences in the efficiency for increasing EPA levels in either phospholipids or cholesteryl esters among the lipids. In the second experiment, the EPA levels of both plasma phospholipids and cholesteryl esters of rats administered ethyl ester of EPA were significantly higher than those of rats administered 2-eicosapentaenoyl-phosphatidylcholine. The EPA levels of the rats administered 1,2,3-trieicosapentaenoyl-glycerol were between the levels of the two groups mentioned above, but the differences in the EPA levels were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of eicosapentaenoic acid ethyl ester on proteinuria of streptozotocin-induced diabetes mellitus in rats.

Streptozotocin (45 mg/kg) was intravenously administered to 7-week-old Wistar rats through their tail veins. After 11 days, the rats were divided into two groups. One group was fed a lipid-free diet (90%, w/w) plus lard (8%) and safflower oil (2%) for four weeks (Diet 1 group, n = 12). The other group was fed in the same way, except that safflower oil was replaced by 90% pure eicosapentaenoic acid (EPA) ethyl ester (Diet 2 group, n = 13). Twenty-four-hour urine was collected just before the diets started and during the experiment at 7-day intervals. In the second and third week, the levels of proteinuria were significantly lower in the Diet 2 group than they were in the Diet 1 group. There was no significant difference in the levels of creatinine, urea nitrogen, or lipids in plasma or in body weights between the two groups after four weeks on the diets. Because Diet 2 reduced proteinuria of diabetic rats compared to Diet 1, an EPA-rich diet may retard the development of diabetic nephropathy.

Dietary marine lipids suppress continuous expression of interleukin-1 beta gene transcription.

n-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cells ex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1 beta (IL-1 beta) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fat-free balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk. spleen cell preparations were stimulated ex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and proIL-1 beta mRNA (IL-1 beta mRNA) was measured by northern analysis. Levels of IL-1 beta mRNA in both LPS- and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1 beta gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1 beta gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.