Effects of rebamipide, a novel anti-ulcer agent, on gastric mucosal injury induced by platelet-activating factor in rats

We examined the role of gastric mucosal blood flow, lipid peroxidation, and neutrophil accumulation mediated by platelet-activating factor in the protective effect of rebamipide against gastric mucosal injury in rats. The intravenous injection of platelet-activating factor induced hyperemia and hemorrhagic erosions in rat stomachs. Rebamipide did not affect the decrease in the gastric mucosal blood flow induced by platelet-activating factor. The increase in gastric injury score after platelet-activating factor injection and the increase in thiobarbituric acid-reactive substances were significantly inhibited by the administration of rebamipide. The gastric injury score was closely correlated with the accumulation of lipid peroxides. Tissue-associated myeloperoxidase activity in the gastric mucosa significantly increased after platelet activating factor injection; this increase was not influenced by rebamipide treatment. The protective effect of rebamipide against the platelet-activiting factor-induced gastric mucosal injury may be due to direct inhibition of lipid peroxidation or scavenging of oxygen radicals that initiate lipid peroxidation.     

Immunoglobulin heavy chain junctional diversity in young and aged humans

The important role of hyperglycemia in the genesis of diabetic renal disease has been strengthened by tissue culture studies, experimental animal models, and clinical trials. A mechanistic understanding of the cellular and biochemical processes that link hyperglycemia with the development of diabetic nephropathy is indispensable for directing the most optimal therapeutic interventions. Likely mediators of the effects of high ambient glucose include activation of the polyol pathway, increased protein kinase C activity, nonenzymatic glycation of circulating or matrix proteins, and/or aberrant synthesis or actions of cytokines and vasomodulatory agents. The latter include angiotensin II, thromboxane, platelet-derived growth factor, endothelins, insulin-like growth factor-1, and transforming growth factor-beta. The studies we review here argue strongly in support of the hypothesis that elevated production and/or activity of transforming growth factor-beta in the kidney is a final common mediator of diabetic renal hypertrophy and mesangial matrix expansion.     

Stimulation of prostaglandin biosynthesis mediates gastroprotective effect of rebamipide in rats

The concept that gastroprotection by agents such as mild irritants, antacids, or sucralfate is prostaglandin (PG)-mediated has been challenged recently. These agents do not reproducibly stimulate prostaglandin formation, and indomethacin does not effectively attenuate their protective potency. Rebamipide is a novel antiulcer compound. This study was designed to clarify whether eicosanoids contribute to the gastroprotective activity of the drug. In the rat stomach, rebamipide (100 and 500 mg/kg, intraperitoneally) slightly increased release of PGE2, 6-keto-PGF1 alpha, thromboxane B2, and the metabolite 15-keto-13,14-dihydro-PGE2 from mucosal fragments incubated ex vivo and significantly enhanced secretion of these products into the lumen, resulting in gastric juice eicosanoid levels exceeding those in controls several-fold. Mucosal formation of leukotriene (LT) C4 was not affected by rebamipide. Rebamipide caused substantial protection against gastric damage produced by ethanol, which was antagonized by pretreatment with indomethacin (0.1-5 mg/kg, subcutaneously). The dose-response relationship of indomethacin for inhibition of prostaglandin formation and rebamipide-induced protection correlated well and 5 mg/kg indomethacin completely prevented the protective effect of rebamipide. The results indicate that: (1) in contrast to most other protective agents, protection by rebamipide involves the endogenous prostaglandin system; (2) the increase in prostaglandin formation results from stimulation of biosynthesis, and not inhibition of degradation; (3) gastroprotection by rebamipide occurs despite increased thromboxane formation and is not associated with reduced generation of LTC4; and (4) determinations of gastric juice eicosanoids seem to be particularly useful to evaluate effects of agents increasing formation of cyclooxygenase products in the stomach.     

Effects of glycated protein on the expression of very late antigen 5 (VLA5), a fibronectin receptor, of cultured rat mesangial cells

Advanced glycosylation end products are believed to play a role in the increase in extracellular matrix in diabetic glomerulosclerosis via a pathway involving a mesangial cell fibronectin receptor. In the present study, I assessed cultured rat mesangial cells for the presence of mRNA for VLA5, one of the fibronectin receptors. Using these cells, I also evaluated the effects of glycated proteins on the expression of VLA5 and fibronectin. Mesangial cells of Wistar rats were cultured in RPMI 1640 containing 20% FCS. The cells were incubated for 72 hr in a medium containing 50 mg/ml of nonglycated BSA or in a medium containing 50 mg/ml glycated BSA (AGE-BSA). Immunostaining and Northern blot analyses showed that the cells incubated with AGE-BSA exhibited a decrease in VLA5 protein and related mRNA, but showed an increase in the synthesis of fibronectin and related mRNA. On the other hand, non-glycated BSA did not induce these changes in the expression of VLA5 or fibronectin in the mesanginal cells. Northern blot analysis for VLA5 mRNA was also conducted using mesangial cells cultured in a medium with a high glucose concentration (30 mM). However, the high glucose condition did not have any effect on the expression of VLA5 protein or related mRNA. These results suggest that glycated proteins may regulate fibronectin synthesis in mesangial cells by modifying the expression of fibronectin receptors, such as the inhibition of VLA5 synthesis that acts as negative feedback of fibronectin synthesis.     

High glucose inhibits nitric oxide production in cultured rat mesangial cells

Hyperglycemia directly contributes to the development of diabetic nephropathy. A high-serum glucose concentration alters intraglomerular hemodynamics and promotes deposition of extracellular matrix in the kidney. Nitric oxide (NO) is a short-lived messenger molecule that participates in the regulation of renal blood flow, GFR, and mesangial matrix accumulation. Therefore, in this study it was tested whether high glucose directly modulates NO synthesis by rat mesangial cells in vitro by measuring the accumulation of nitrite, the stable metabolite of NO, in the incubation media. Raising the external glucose concentration to 33.3 mM for 24 to 72 h reduced nitrite levels in cell supernatants in a time-dependent manner to a nadir of 14 +/- 3% of the amount in normal glucose media (5.6 mM) (P < 0.01). The decline in NO synthesis in high glucose media was paralleled by decreased cyclic guanosine monophosphate generation; however, there was no alteration in rat mesangial cell expression of inducible NO synthase protein. The suppressive effect of high glucose on NO production by mesangial cells was not modified by inhibition of protein kinase C (H-7), the addition of antioxidants (vitamin E or superoxide dismutase), or a pan-specific anti-transforming growth factor-beta antibody. An elevated ambient glucose caused a time-dependent reduction in mesangial cell L-arginine content. Addition of L-arginine (10 to 20 mM) to external media partially reversed the inhibitory effect of high glucose on mesangial cell NO production in a dose-dependent manner. The highest dose of L-arginine (20 mM) increased mesangial cell L-arginine content to comparable levels in normal and high glucose media. These results indicate that high glucose causes depletion of L-arginine in mesangial cells and compromises NO synthesis. Limitation in the metabolic precursor and other, as yet unidentified, factors act to reduce NO production by mesangial cells in the presence of an elevated ambient glucose level, a change that may play a role in the development of diabetic glomerulosclerosis.     

Potential role of TGF-beta in diabetic nephropathy

Renal injury in diabetes mellitus is a major cause of morbidity and mortality. Several manifestations of diabetic nephropathy may be a consequence of altered production and/or response to cytokines or growth factors. Transforming growth factor-beta (TGF-beta) is one such factor because it promotes renal cell hypertrophy and regulates the production of extracellular matrix molecules. In addition, high ambient glucose increases TGF-beta1 mRNA and protein level in cultured proximal tubular cells and glomerular epithelial and mesangial cells. Neutralizing anti-TGF-beta antibodies or antisense TGF-beta1 oligodeoxynucleotides prevents the hypertrophic effects of high glucose and the stimulation of matrix synthesis in renal cells. Several reports have described overexpression of TGF-beta in the glomeruli and tubulointerstitium of experimental and human diabetes mellitus. We recently provided evidence that the kidney in diabetic patients displays net renal production of immunoreactive TGF-beta1, whereas there is net renal extraction in nondiabetic subjects. We also demonstrated that short-term treatment of streptozotocin-diabetic mice with neutralizing monoclonal antibody directed against TGF-beta significantly reduces kidney weight and glomerular hypertrophy, and attenuates the increase in extracellular matrix mRNA levels. The factors that mediate increased renal TGF-beta activity involve hyperglycemia per se and the intermediary action of other potent mediators such as angiotensin II, thromboxane, endothelins, and platelet-derived growth factor.     

Diagnostic evaluation of the cervical nystagmus in cervical torsion test

The long-term effects of streptozotocin (30-70 mg/kg) were studied on plasma glucose and insulin levels, islet morphology, and glucose-stimulated insulin secretion in rats. In addition, the protective effect of short-term (7 days) insulin treatment on streptozotocin-induced diabetes was examined. Streptozotocin administration at dose levels exceeding 40 mg/kg resulted in a long-term, stable hyperglycemia with no insulin response to glucose at 3 months and with a marked derangement of islet morphology (few insulin cells, accumulation of glucagon cells). In contrast, at 30 and 40 mg/kg, streptozotocin induced a transient diabetes. Thus, the blood glucose levels, being elevated at days 1-7, returned to normal levels within 10 days after streptozotocin administration and the glucose-induced insulin secretion, being absent at day 1, was normal at 3 months. Furthermore, the islet morphology was also normal in these groups at 3 months. Short-term (7 days) insulin treatment normalized the long-term diabetes in rats given 50 mg/kg streptozotocin, but not in rats given 60 or 70 mg/kg streptozotocin. Thus, after insulin treatment, all rats receiving 50 mg/kg streptozotocin returned to normoglycemia within the following 2 weeks, and the glucose-induced insulin secretion was normal after 3 months, as was islet morphology.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of silibinin and antioxidants on high glucose-induced alterations of fibronectin turnover in human mesangial cell cultures

To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures. An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium. The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control. Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation. This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation. Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail. Silibinin alone had no effects on protein synthesis and culture growth. The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures. The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells.     

Effects of diabetes and hyperglycemia on disaccharidase activities in the rat

BACKGROUND AND METHODS: To elucidate the effect of hyperglycemia on disaccharidase activities, the specific and total activities of the disaccharidases were measured in the intestinal mucosa and kidney cortex of diabetic and hyperglycemic rats. The diabetes was induced with an intraperitoneal injection of streptozotocin (60 mg/kg). The rats were made hyperglycemic with an intravenous instillation of a solution containing 40% dextrose monohydrate at a rate of 1.5 ml/h for 24 h. RESULTS: The blood glucose level was 387+/-45 mg/dl and 382+/-35 mg/dl (mean +/- standard deviation) in diabetic and hyperglycemic rats, respectively. In diabetic rats the intestinal maltase, sucrase, and lactase activities were significantly higher than those in control rats. Similarly, disaccharidase activities in hyperglycemic rats were significantly higher than those in control rats. The renal maltase activity in diabetic rats was significantly lower than that in control rats. The maltase activity in hyperglycemic rats, however, was not significantly different from that in control rats. CONCLUSIONS: These results suggest that 1) hyperglycemia directly increases the activities of intestinal maltase, sucrase, and lactase; 2) hyperglycemia does not influence renal maltase activity; and 3) hyperglycemia is partly responsible for increased activities of intestinal disaccharidases in diabetes mellitus.     

Mechanical strain- and high glucose-induced alterations in mesangial cell collagen metabolism: role of TGF-beta

Cultured mesangial cells (MC) exposed to cyclic mechanical strain or high glucose levels increase their secretion of transforming growth factor-beta1 (TGF-beta1) and collagen, suggesting possible mechanisms for the development of diabetic renal sclerosis resulting from intraglomerular hypertension and/or hyperglycemia. This study examines whether glucose interacts with mechanical strain to influence collagen metabolism and whether this change is mediated by TGF-beta. Accordingly, rat MC were grown on flexible-bottom plates in 8 or 35 mM glucose media, subjected to 2 to 5 d of cyclic stretching, and assayed for TGF-beta1 mRNA, TGF-beta1 secretion, and the incorporation of 14C-proline into free or protein-associated hydroxyproline to assess the dynamics of collagen metabolism. Stretching or high glucose exposure increased TGF-beta1 secretion twofold and TGF-beta1 mRNA levels by 30 and 45%, respectively. However, the combination of these stimuli increased secretion greater than fivefold without further elevating mRNA. In 8 mM glucose medium, stretching significantly increased MC collagen synthesis and breakdown, but did not alter accumulation, whereas those stretched in 35 mM glucose markedly increased collagen accumulation. TGF-beta neutralization significantly reduced baseline collagen synthesis, breakdown, and accumulation in low glucose, but had no significant effect on the changes induced by stretch. In contrast, the same treatment of MC in high glucose medium greatly reduced stretch-induced synthesis and breakdown of collagen and totally abolished the increase in collagen accumulation. These results indicate that TGF-beta plays a positive regulatory role in MC collagen synthesis, breakdown, and accumulation. However, in low glucose there is no stretch-induced collagen accumulation, and the effect of TGF-beta is limited to basal collagen turnover. In high glucose media, TGF-beta is a critical mediator of stretch-induced collagen synthesis and catabolism, and, most importantly, its net accumulation. These data have important implications for the pathogenesis and treatment of diabetic glomerulosclerosis.     

Long-term observation of glomerulonephritis induced by multiple injections with anti-Thy-1 antibody in rats

Multiple injections with a mouse monoclonal anti-rat Thy-1 antibody (five times, at weekly intervals) induced marked glomerular sclerotic lesions which are characterized by adhesion of glomerular capillaries to Bowman's capsule and persistent proteinuria in rats. Abnormal production of type I collagen and increased accumulation of type IV collagen and fibronectin were observed in these glomeruli. The glomerular expression of mRNA for these matrix components and transforming growth factor-beta1 (TGF-beta1) were markedly increased at 4 days after the last injections with anti-Thy-1 antibody, but decreased to below the levels of control rats at 5 weeks. This may be down-regulation of mRNA in mesangial cells. The glomerular sclerotic lesions were not progressive but the process of glomerular healing seemed to be retarded. The proteinuria and the glomerular adhesion were irreversible.     

Ectonucleotidases, purine nucleoside transporter, and function of the bile canalicular plasma membrane of the hepatocyte

Expansion of the glomerular mesangium is a consistent finding of diabetic nephropathy. Negatively charged proteoglycans are an integral part of the mesangium and their synthesis and degradation is disturbed in many forms of glomerulosclerosis. The metabolism of ascorbic acid (AA), which plays an important role in extracellular matrix regulation, is known to be abnormal in diabetes. The action of AA has also been shown to be inhibited by high glucose (HG) concentration. In this study we investigated the effect of AA and HG on proteoglycan (PG) synthesis by examining the incorporation of [35S] sulphate into PG in the cellular, matrix and media components of rat mesangial cell (MC) cultures. MC were grown in 9 or 25 mM glucose for 8 days, with and without the addition of AA. Sulphation of PG was measured by adding 50 microCi of [35S] sulphuric acid to the culture medium and precipitating 35S-labelled PG with cetylpyridinium chloride. In this study AA was shown to have a stimulatory effect on the overall incorporation of [35S] sulphate into cell and matrix PG and this was inhibited by 25 mM glucose. Correcting for protein synthesis and specific activity of [35S] sulphate showed that HG inhibits AA stimulation by decreasing sulphation of the individual PG molecules. These findings may be of particular importance in the pathophysiology of nephropathy in diabetes, a condition where AA concentration is already compromised.     

T-cell specific immunosuppression by prodigiosin isolated from Serratia marcescens

One of the characteristics of obesity-associated diabetes is an elevated fasting plasma insulin concentration with a weak insulin secretory response to subsequent glucose stimulation. Evidence suggests that hyperglycemia and hyperlipidemia may contribute to the initiation and progression of this disordered islet glucose sensing. It has been proposed that reducing hyperglycemia and hyperlipidemia per se may improve islet glucose sensing. Here we studied glucose-dependent insulin release in islets isolated from ob/ob mice treated with dopamine agonists (bromocriptine and SKF38393, BC/SKF) which significantly reduced circulating glucose and lipid levels of ob/ob mice. Islets from BC/SKF-treated mice showed a marked decrease of the elevated basal insulin release to levels similar to lean mice. Such treatment also induced a higher secretory response to glucose stimulation compared with that in ob/ob mice with sustained hyperglycemia and hyperlipidemia. Similarly, when islets from untreated ob/ob mice were cultured for 7 days in 11 mM glucose in the absence of free fatty acid, the basal insulin release was significantly decreased and high glucose stimulated insulin release increased compared with that from islets cultured in medium containing 30 mM glucose and 2 mM oleate. The BC/SKF-induced reduction of elevated basal insulin release was associated with decreased hexokinase activity and basal cyclic AMP content in islet tissue. Our results demonstrate that dopamine agonist treatment improves basal insulin release in ob/ob mice and this effect may be mediated, in part, by a reduction of hyperglycemia and hyperlipidemia.     

Benfluorex normalizes hyperglycemia and reverses hepatic insulin resistance in STZ-induced diabetic rats

We have examined the effect of chronic (20 days) oral administration of benfluorex (35 mg/kg) in a rat model of NIDDM, induced by injection of STZ 5 days after birth and characterized by frank hyperglycemia, hypoinsulinemia, and hepatic and peripheral insulin resistance. We assessed the following: 1) basal blood glucose and insulin levels, 2) glucose tolerance and glucose-induced insulin release in vivo and in vitro, and 3) basal and insulin-stimulated in vivo glucose production and glucose utilization, using the insulin-clamp technique in conjunction with isotopic measurement of glucose turnover. The in vivo insulin response of several individual tissues also was evaluated under the steady-state conditions of the clamp, using the uptake of the glucose analogue 2-deoxy-D-glucose as a relative index of glucose metabolism. In the benfluorex-treated diabetic rats, postabsorptive basal plasma glucose levels were decreased (8.1 +/- 0.2 mM compared with 10.5 +/- 0.5 mM in the pair-fed untreated diabetic rats and 6.1 +/- 0.2 mM in the benfluorex-treated nondiabetic rats), whereas the basal and glucose-stimulated intravenous glucose tolerance test plasma insulin levels were not improved. Such a lack of improvement in the glucose-induced insulin release after benfluorex treatment was confirmed under in vitro conditions (perfused pancreas). In the pair-fed untreated diabetic rats, the basal glucose production and overall glucose utilization were significantly increased, and during hyperinsulinemia both liver and peripheral tissues revealed insulin resistance. In the benfluorex-treated diabetic rats, the basal glucose production and basal overall glucose utilization were normalized. After hyperinsulinemia, glucose production was normally suppressed, whereas overall glucose utilization was not significantly improved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beh?et''s disease with acute inflammatory foci and localized secondary organizing pneumonia]

Both high and low affinity sulfonylurea receptors (SURs) reside on glucose responsive neurons where they influence cell firing and neurotransmitter release via the adenosinetriphosphate (ATP)-sensitive K+ (katp) channel. Here, the effect of diabetes on [3H] glyburide binding to SURs was assessed in male obesity-resistant Sprague-Dawley rats rendered diabetic with streptozotocin (65 mg/kg, i.p.). Additional streptozotocin-treated rats were supplemented with insulin (1.5 U/kg/ day). Streptozotocin reduced plasma insulin to 13% of control associated with hyperglycemia (25.3 +/- 1.7 mmol/l), while insulin lowered plasma glucose (9.56 +/- 1.78 mmol/l) to near control levels (7.65 +/- 0.22 mmol/l). Over 7 days, all streptozotocin-treated rats lost 12% of their initial body wt. while controls gained 1%. Despite equivalent wt. loss, streptozotocin-induced diabetes selectively increased high affinity [3H] glyburide binding in the hypothalamic dorsomedial nuclei (DMN) and ventromedial nuclei (VMN) and lateral area (LH). This was prevented by insulin injections. Low affinity binding was similarly increased in the DMN and VMN, as well as two amygdalar subnuclei but decreased in the substantia nigra, pars compacta. Insulin fully prevented these changes only in the DMN and one amygdalar nucleus and the substantia nigra. Therefore, binding to (SURs) appears to be generally upregulated in the face of hypoinsulinemia with hyperglycemia and this is prevented by insulin treatment. These and other data suggest that this combination of abnormalities in diabetes should have an adverse effect on the glucose sensing capacity of the brain.     

Characterization of cellular defects of insulin action in type 2 (non-insulin-dependent) diabetes mellitus

Seven non-insulin-dependent diabetes mellitus (NIDDM) patients participated in three clamp studies performed with [3-3H]- and [U-14C]glucose and indirect calorimetry: study I, euglycemic (5.2 +/- 0.1 mM) insulin (269 +/- 39 pM) clamp; study II, hyperglycemic (14.9 +/- 1.2 mM) insulin (259 +/- 19 pM) clamp; study III, euglycemic (5.5 +/- 0.3 mM) hyperinsulinemic (1650 +/- 529 pM) clamp. Seven control subjects received a euglycemic (5.1 +/- 0.2 mM) insulin (258 +/- 24 pM) clamp. Glycolysis and glucose oxidation were quantitated from the rate of appearance of 3H2O and 14CO2; glycogen synthesis was calculated as the difference between body glucose disposal and glycolysis. In study I, glucose uptake was decreased by 54% in NIDDM vs. controls. Glycolysis, glycogen synthesis, and glucose oxidation were reduced in NIDDM patients (P < 0.05-0.001). Nonoxidative glycolysis and lipid oxidation were higher. In studies II and III, glucose uptake in NIDDM was equal to controls (40.7 +/- 2.1 and 40.7 +/- 1.7 mumol/min.kg fat-free mass, respectively). In study II, glycolysis, but not glucose oxidation, was normal (P < 0.01 vs. controls). Nonoxidative glycolysis remained higher (P < 0.05). Glycogen deposition increased (P < 0.05 vs. study I), and lipid oxidation remained higher (P < 0.01). In study III, hyperinsulinemia normalized glycogen formation, glycolysis, and lipid oxidation but did not normalize the elevated nonoxidative glycolysis or the decreased glucose oxidation. Lipid oxidation and glycolysis (r = -0.65; P < 0.01), and glucose oxidation (r = -0.75; P < 0.01) were inversely correlated. In conclusion, in NIDDM: (a) insulin resistance involves glycolysis, glycogen synthesis, and glucose oxidation; (b) hyperglycemia and hyperinsulinemia can normalize total body glucose uptake; (c) marked hyperinsulinemia normalizes glycogen synthesis and total flux through glycolysis, but does not restore a normal distribution between oxidation and nonoxidative glycolysis; (d) hyperglycemia cannot overcome the defects in glucose oxidation and nonoxidative glycolysis; (e) lipid oxidation is elevated and is suppressed only with hyperinsulinemia.     

Effect of high glucose on cellular proliferation and lipid peroxidation in cultured Vero cells

Nephropathy is common among the diabetic population. However, the molecular mechanisms by which hyperglycemia can cause alteration in kidney structure and function is not known. In this study, we examined the effect of high glucose levels on cellular growth and membrane lipid peroxidation in Vero cells, an African green monkey kidney cell line. Cell growth was assessed by a tetrazolium salt reduction test (MTT); and lipid peroxidation was measured by thiobarbituric acid reactivity. Our results show that elevated levels of glucose (25, 50 and 100 mM) can cause up to 50% reduction in cell growth in cultured Vero cells as compared with controls (8 mM). Also, we observed a significant increase in the amount of oxidative damage in Vero cells cultured with elevated glucose concentrations. This study demonstrates that hyperglycemia can cause increased lipid peroxidation and affect the cellular proliferation in a monkey kidney cell line.     

Sequential effects of high glucose on mesangial cell transforming growth factor-beta 1 and fibronectin synthesis

BACKGROUND: Transforming growth factor (TGF)-beta is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-beta 1 synthesis and between TGF-beta 1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-beta 1 and fibronectin (FN) mRNA expression and protein synthesis. METHODS: Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. RESULTS: Mesangial cell TGF-beta 1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-beta 1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-beta activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-beta antibody completely abolished HG-induced FN synthesis. CONCLUSIONS: The present finding demonstrate that HG stimulates TGF-beta 1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-beta. This finding is consistent with the view that TGF-beta mediates increased ECM accumulation by MC under high glucose conditions.     

Impaired duodenal bicarbonate secretion in diabetic rats. Salutary effect of nitric oxide synthase inhibitor

We previously reported the impaired HCO3- secretion and the increased mucosal susceptibility to acid in the duodenum of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the salutary effect of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) on these changes and compared it with those of insulin. Animals were injected streptozotocin (STZ: 70 mg/kg, ip) and used after 1, 3-4, and 5-6 weeks of diabetes with blood glucose levels of > 300 mg/dL. Under urethane anesthesia the HCO3- secretion was measured in the proximal duodenal loop using a pH-stat method and by adding 10 mM HCl. L-NAME (20 mg/kg x 2) or insulin (4 units/rat) was administered sc for 4-5 weeks, starting 1 week after STZ treatment. The duodenal HCO3- secretory responses to various stimuli such as mucosal acidification (10 mM HCl for 10 min), 16,16-dimethyl prostaglandin E2 (dmPGE2: 10 micrograms/kg, i.v.), and vagal stimulation (0.5 mA, 2 ms, 3 Hz) were significantly decreased in STZ-treated rats, depending on the duration of diabetes. Repeated administration of L-NAME, starting from 1 week after STZ treatment, significantly reduced blood glucose levels toward normal values and restored the HCO3- responses to various stimuli in STZ rats, the effects being similar to those observed after supplementation of insulin. Diabetic rats developed duodenal lesions after perfusion of the duodenum with 150 mM HCl for 4 h, but this ulcerogenic response was significantly inhibited by the repeated treatment with L-NAME as well as insulin. We conclude that L-NAME is effective in ameliorating hyperglycemic conditions in STZ-diabetic rats, similar to insulin, and restores the impaired HCO3- secretion and the increased mucosal susceptibility to acid in diabetic rat duodenums.