Acupuncture: is it effective for treatment of insulin resistance?

Insulin resistance (IR) is closely associated with obesity, type 2 diabetes mellitus (T2DM), hypertension, polycystic ovary syndrome (PCOS), non-alcohol fatty liver diseases (NAFLD) and metabolic syndrome and is also a risk factor for serious diseases such as cardiovascular diseases. Pharmacological treatments available for IR are limited by drug adverse effects. Because acupuncture has been practiced for thousands of years in China, it has been increasingly used worldwide for IR-related diseases. This review analyses 234 English publications listed on the PubMed database between 1979 and 2009 on the effectiveness of acupuncture as a treatment for IR. These publications provide clinical evidence, although limited, in support of the effectiveness of acupuncture in IR. At this stage, well-designed, evidence-based clinical randomized controlled trial studies are therefore needed to confirm the effects of acupuncture on IR. Numerous experimental studies have demonstrated that acupuncture can correct various metabolic disorders such as hyperglycemia, overweight, hyperphagia, hyperlipidemia, inflammation, altered activity of the sympathetic nervous system and insulin signal defect, all of which contribute to the development of IR. In addition, acupuncture has the potential to improve insulin sensitivity. The evidence has revealed the mechanisms responsible for the beneficial effects of acupuncture, though further investigations are warranted.

Long-term effect of modification of dietary protein intake on the progression of diabetic nephropathy: a randomised controlled trial.

AIMS/HYPOTHESIS: There is currently insufficient evidence to recommend a low-protein diet for type 2 diabetic patients with diabetic nephropathy. We assessed whether a low-protein diet could prevent the progression of diabetic nephropathy. METHODS: This was a multi-site parallel randomised controlled trial for prevention of diabetic nephropathy progression among 112 Japanese type 2 diabetic patients with overt nephropathy. It was conducted in Japan from 1 December 1997 to 30 April 2006. The participants were randomly assigned using a central computer-generated schedule to either low-protein diet (0.8 g kg(-1) day(-1)) and normal-protein diet (1.2 g kg(-1) day(-1)), and were followed for 5 years. The participants and investigators were not blinded to the assignment. The primary outcomes were the annual change in estimated GFR and creatinine clearance, the incidence of doubling of serum creatinine and the time to doubling of baseline serum creatinine. RESULTS: The study was completed by 47 (84%) of 56 participants in the low-protein diet group and 41 (73%) of 56 participants in the normal-diet group. During the study period, the difference in mean annual change in estimated GFR between the low-protein diet and the normal-protein diet groups was -0.3 ml min(-1) 1.73 m(-2) (95% CI -3.9, 4.4; p = 0.93). The difference in mean annual change in creatinine clearance between the low-protein diet and the normal-protein diet groups was -0.006 ml s(-1) 1.73 m(-2) (95% CI -0.089, 0.112; p = 0.80). A doubling of serum creatinine was reached in 16 patients of the low-protein group (34.0%), compared with 15 in the normal-protein group (36.6%), the difference between groups being -2.6% (95% CI -22.6, 17.5; p = 0.80). The time to doubling of serum creatinine was similar in both groups (p = 0.66). CONCLUSIONS/INTERPRETATION: It is extremely difficult to get patients to follow a long-term low-protein diet. Although in the low-protein group overall protein intake was slightly (but not significantly) lower, it did not confer renoprotection. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT00448526. FUNDING: Research grant from the Ministry of Health, Labour and Welfare of Japan.

Impedance detected abnormal bolus transit in patients with normal esophageal manometry. Sensitive indicator of esophageal functional abnormality?

SUMMARY: When used in combination with manometry, multichannel intraluminal impedance better characterizes the established manometric abnormalities into those with and without associated transit defects. However, the significance of the finding of normal manometry and abnormal impedance is not known. The objective of this study is to evaluate the clinical relevance of abnormal impedance associated with normal manometry during esophageal function testing in patients with a variety of esophageal symptoms. All patients referred for esophageal function testing during a 27-month period underwent combined multichannel intraluminal impedance and esophageal manometry studies including 10 liquid and 10 viscous swallows in supine position. From 576 patients with normal esophageal body manometry we identified 158 patients (27%) with abnormal impedance. The primary symptom in these 158 patients was compared to that in 146 consecutive patients with normal manometry and normal impedance selected from the original 576 patients. Abnormal bolus transit was found with viscous, liquid and both type swallows in 60%, 19% and 21% of the patients respectively. Of patients with abnormal bolus transit, 23% presented with dysphagia compared to 10% of normal transit patients (p = 0.0035). In conclusion, abnormal impedance even in patients with normal manometry may be a sensitive indicator of esophageal functional abnormality as represented by the symptom of dysphagia in these patients. Abnormal transit was more frequently identified with viscous than liquid swallows. Prospective studies to further clarify impedance detected transit defects in patients with normal manometry and the role of viscous swallows in diagnostic testing are warranted.

Predictive impact of elevated serum level of IL-18 for early renal dysfunction in type 2 diabetes: an observational follow-up study.

AIMS/HYPOTHESIS: The early identification of type 2 diabetic patients at risk of developing microalbuminuria-an independent risk factor for renal and cardiovascular diseases-is important to improve the patients' outcomes. We investigated whether serum levels of IL-18, a proinflammatory cytokine, were a predictor of early renal dysfunction. MATERIALS AND METHODS: A total of 249 Japanese type 2 diabetic patients without overt proteinuria were enrolled in an observational follow-up study (median follow-up 7 years), and their stage of diabetic nephropathy was classified and their estimated glomerular filtration rate (eGFR) was calculated annually. RESULTS: At baseline, serum levels of IL-18 were higher in subjects with microalbuminuria (n = 76) than in those with normoalbuminuria (n = 173). Elevated serum levels of IL-18 were associated with the progression of nephropathy to a higher stage in normoalbuminuric subjects (118 [interquartile range 91-159] ng/l vs 155 [interquartile range 121-205] ng/l, p = 0.003), but not in microalbuminuric subjects (154 [interquartile range 113-200] ng/l vs 160 [interquartile range 101-190] ng/l, p = 0.50). The adjusted risk for developing microalbuminuria was 3.6 (95% CI 1.2-10.4) in normoalbuminuric subjects with serum IL-18 levels above the median (>/=134.6 ng/l), and was significantly enhanced in those urinary AERs at the upper end of the normal range (7.5 mug/min

Glucocorticoid-induced diabetes mellitus: prevalence and risk factors in primary renal diseases.

BACKGROUND/AIMS: In patients with primary renal diseases the current knowledge of hyperglycemia associated with corticosteroid therapy is limited. We therefore examined the prevalence and risk factors of glucocorticoid-induced diabetes mellitus (DM) in primary renal diseases. METHODS: Patients were recruited with primary renal diseases who were started on corticosteroids between April 2002 and June 2005. In patients with DM, an impaired fasting glucose level and/or positive urinary glucose analyses before corticosteroids therapy were excluded. RESULTS: During corticosteroid therapy (initial dose: prednisolone 0.75 +/- 0.10 mg/kg/day), DM was newly diagnosed in 17 (40.5%) of 42 patients. All of the 17 patients were diagnosed as having DM by postprandial hyperglycemia at 2 h after lunch, although they had normal fasting blood glucose levels. Age (OR 1.40, 95% CI 1.06-1.84) and body mass index (OR 1.87, 95% CI 1.03-3.38) were determined as independent risk factors for glucocorticoid-induced DM. CONCLUSION: Over 40% of patients with primary renal disease developed DM during treatment with corticosteroids. A high age and high body mass index are the independent risk factors for glucocorticoid-induced DM. 24-hour urinary glucose analyses and postprandial plasma glucose are useful for detecting glucocorticoid-induced DM.

Cellular mechanisms in the development and progression of diabetic nephropathy: activation of the DAG-PKC-ERK pathway.

Diabetic nephropathy is characterized functionally by glomerular hyperfiltration and albuminuria and histologically by the expansion of glomerular mesangium. We and others have found that protein kinase C (PKC) is activated through an increase in de novo synthesis of diacylglycerol (DAG) from glucose in glomerular mesangial cells cultured under high glucose conditions and in glomeruli of diabetic rats. The activation of PKC could activate further various intracellular signal transduction systems, such as extracellular regulated kinase (ERK). The activation of the DAG-PKC-ERK pathway is considered to be one of the important molecular mechanisms of the development and progression of diabetic nephropathy. To prove this hypothesis, we examined whether the inhibition of the DAG-PKC-ERK pathway could prevent the development of glomerular dysfunction in diabetic animals. First, we found that thiazolidinedione compounds could inhibit PKC activation by activating DAG kinase. Thiazolidinedione compounds were able to prevent glomerular hyperfiltration, albuminuria, and excessive production of extracellular matrix proteins in glomeruli in streptozotocin-induced diabetic rats, a model for type 1 diabetes. Second, we tried to inhibit PKC directly by oral administration of PKC beta inhibitor. PKC beta inhibitor could prevent albuminuria and mesangial expansion in db/db mice, a model for type 2 diabetes. These results confirmed the importance of the activation of the DAG-PKC-ERK pathway in the development of glomerular dysfunction in diabetes.

Dinucleotide repeat polymorphism of matrix metalloproteinase-9 gene is associated with diabetic nephropathy.

BACKGROUND: Although genetic susceptibility has been proposed as an important factor for the development and progression of diabetic nephropathy, the definitive gene has not been identified. To identify the genetic marker for diabetic nephropathy, we examined the association between the (A-C)n dinucleotide repeat polymorphism upstream of the matrix metalloproteinase-9 (MMP-9) gene and diabetic nephropathy in a group of Japanese patients with type 2 diabetes. METHODS: Patients were divided into three groups based on their urinary albumin excretion rate (AER) and the stage of diabetic retinopathy as follows: uncomplicated group (U), normal albuminuria (AER <20 microg/min) without proliferative retinopathy and with the duration of diabetes more than 20 years (N = 32); microalbuminuria group (M), 20 < or = AER < 200 microg/min (N = 155); overt nephropathy group (O), AER > or = 200 microg/min (N = 63). The region containing the dinucleotide repeat upstream of MMP-9 gene was amplified by polymerase chain reaction (PCR). The amplified products were analyzed with 7% formamide/urea acrylamide gel electrophoresis. The promoter constructs of the MMP-9 gene were transfected with the CMV-beta-galactosidase construct into 293 cells using the liposome method. Twenty-four hours after transfection, cells were harvested, and luciferase and beta-galactosidase activities were measured. RESULTS: Nine alleles of the dinucleotide repeat polymorphism (17 to 25 repeats) were identified, and the frequency of each allele in diabetic subjects was not different from that in nondiabetic controls. The frequency of the allele containing 21 repeats (A21) was most abundant (42.4% in control and 45.6% in diabetic subjects), followed by the allele with 23 repeats (A23; 35.4% in control and 27.6% in diabetic subjects). The A21 allele was less frequent in M and O than U (O, 38.9%; M, 45.5%; U, 59.3%, chi2 = 7.18; P < 0.05, O vs. U), while the frequency of the alleles other than A21 was not different among each group. The calculated odds ratio for nephropathy in the noncarrier, heterozygote, or homozygote of A21 allele was 3.38, 1.97, and 0.2, respectively. Furthermore, the promoter assay for the MMP-9 gene revealed that the A21 allele had a higher promoter activity compared with other alleles. No significant correlation was observed between serum MMP-9 concentrations and the MMP-9 gene polymorphism. CONCLUSION: These results indicate that the patients with A21 allele of the MMP-9 gene may be protected from the development and progression of diabetic nephropathy. Thus, the microsatellite polymorphism upstream of the MMP-9 gene could be a useful genetic marker for diabetic nephropathy.

Enhancement of glomerular heme oxygenase-1 expression in diabetic rats.

An increase in oxidative stress in diabetic subjects is implicated to play a pivotal role in diabetic vascular complications. In response to oxidative stress, antioxidant enzymes are considered to be induced and protect cellular functions to keep in vivo homeostasis. However, it remains to be clarified whether antioxidant enzymes are induced against oxidative stress especially in renal glomeruli at an early stage of diabetes. To answer this question, we examined the gene expression of a variety of antioxidant enzymes in glomeruli isolated from streptozotocin-induced diabetic rats. The mRNA expression of antioxidant enzymes such as catalase, glutathione peroxidase, and CuZn-superoxide dismutase, was unaltered in glomeruli of diabetic rats and was comparable to control rats. In contrast, the mRNA expression of heme oxygenase-1 (HO-1) was enhanced in glomeruli of diabetic rats as compared with control rats. A treatment with insulin as well as with vitamin E (40 mg/kg body weight every other day, intra-peritoneal injection) normalized the mRNA expression of HO-1 in the glomeruli of diabetic rats. Immunohistochemical analysis revealed that the up-regulated expression of HO-1 protein was localized in glomerular cells of diabetic rats. In conclusion, these results provide the first evidence that among antioxidant enzymes HO-1 expression is preferentially increased in diabetic glomeruli.

Evaluation of a new care system provided to diabetic patients in the outpatient clinic.

OBJECTIVE: Evaluation of metabolic states and chronic complications is essential for maintaining a high quality of care for diabetic patients. We have assessed the quality of care in routine outpatient clinics for diabetic subjects in our university hospital, and compared with those in a newly introduced standardized clinic to evaluate the new care system. METHODS: The quality of care was assessed by the chart review in 1995, and compared with those from 1996-1997 in the "Diabetes Follow-up Clinic" which is systematically designed for the standardized care. PATIENTS: The subjects were recruited among 860 patients who visited the outpatient clinic in July and August of 1995 with a diagnosis of diabetes or glucose intolerance. Six hundred seventy-two patients whose follow-up period had been more than 6 months with clinically diagnosed diabetes were used for the analysis. RESULTS: Laboratory tests such as determination of HbA1c, and serum levels of lipids and creatinine were performed in more than 90% of the patients in the routine outpatient clinics. However, ophthalmology referral, 24-hour urine collection for the determination of creatinine clearance and albumin excretion, and electrocardiograms were not well performed and were incompletely documented (40-60% of the patients within a previous year and 70-80% in the last 2 years). In the standardized "Diabetes Follow-up Clinic", only four out of 555 diabetic patients failed to collect their 24-hour urine, and all participants had ankle blood pressure measurements, nerve conduction study, and nylon monofilament tests, etc. Furthermore, more than 95% of the patients had funduscopic examinations by ophthalmologists as well as records of electrocardiogram. CONCLUSION: Introduction of the standardized "Diabetes Follow-up Clinic" may be one of the choices for increasing the quality of outpatient care and for the prevention of chronic diabetic complications.

Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-beta in mesangial cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell proliferation, differentiation, and production of extracellular matrix proteins in various types of cells including mesangial cells. Although TGF-beta has been also known as an important player in the pathogenesis of various fibrotic diseases including glomerulosclerosis, signal-transduction cascades of TGF-beta have remained to be clarified. However, emerging evidence indicates that TGF-beta can activate various signal transduction cascades such as Smad proteins and mitogen-activated protein kinases (MAPKs) in many types of cells. Here, we examine the role of MAPKs in TGF-beta-induced gene expression of extracellular matrix proteins in mesangial cells. TGF-beta increases extracellular signal-regulated kinase (ERK) activity, one of the MAPKs, and the expression of fibronectin mRNA and protein in rat mesangial cells. Furthermore, PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), can inhibit this TGF-beta-induced fibronectin expression. These data suggest that MAPKs play an important role in TGF-beta-mediated extracellular matrix production in mesangial cells.

Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats.

Thiazolidinedione (TZD) compounds are widely used as oral hypoglycemic agents. Herein, we provide evidence showing that troglitazone, one of the TZD compounds, is able to prevent glomerular dysfunction in diabetic rats through a novel mechanism independent of its insulin-sensitizing action. We examined the effect of troglitazone on functional and biochemical parameters of glomeruli in streptozotocin-induced diabetic rats. Troglitazone was able to prevent not only diabetic glomerular hyperfiltration and albuminuria, but an increase in mRNA expression of extracellular matrix proteins and transforming growth factor-beta1 in glomeruli of diabetic rats, without changing blood glucose levels. Biochemically, an increase in diacylglycerol (DAG) contents and the activation of the protein kinase C (PKC)-extracellular signal-regulated kinase (ERK) pathway in glomeruli of diabetic rats were abrogated by troglitazone. The activation of DAG-PKC-ERK pathways in vitro in mesangial cells cultured under high glucose conditions was also inhibited by troglitazone. Troglitazone enhanced the activities of DAG kinase, which could metabolize DAG to phosphatidic acid, in both glomeruli of diabetic rats and mesangial cells cultured under high glucose conditions. Surprisingly, pioglitazone, another TZD compound without alpha-tocopherol moiety in its structure, also prevented the activation of the DAG-PKC pathway and activated DAG kinase in mesangial cells cultured under high glucose conditions. These results may identify the TZDs as possible new therapeutic agents for diabetic nephropathy that prevent glomerular dysfunction through the inhibition of the DAG-PKC-ERK pathway.

Immunohistochemical characterization of glomerular PDGF B-chain and PDGF beta-receptor expression in diabetic rats.

Platelet-derived growth factor (PDGF) was found to contribute to the pathophysiological process in the development and progression of glomerulosclerosis characterized by mesangial cell proliferation and accumulation of extracellular matrix. To examine the role of PDGF in the development of diabetic nephropathy, we conducted immunohistochemical analysis for PDGF B-chain (PDGF-B) and PDGF beta-receptor (PDGFR-beta) in the glomeruli of streptozotocin-induced diabetic rats. At 2, 4, and 12 weeks after the onset of diabetes, the expression of PDGF-B in glomeruli of diabetic rats was increased significantly as compared to control or diabetic rats treated with insulin. Similar changes were observed on PDGFR-beta immunostaining. The immunostaining of mirror sections revealed the existence of PDGF-B or PDGFR-beta not only in mesangial cells but also in visceral epithelial cells. Glomerular volume was significantly increased in diabetes. This early glomerular abnormality was prevented by an inhibition of PDGF system with trapidil as well as by the treatment of insulin. Our results suggest that the activation of the PDGF system in glomerular cells might play an important role in the development of early glomerular lesion in diabetes.

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.

[Kidney disease and insulin resistance--clinical impact of thiazolidinedione compounds for kidney disease].

The thiazolidinedione compounds are well known hypoglycemic agents via increasing insulin-sensitivity. Herein, we provide the possibility that thiazolidinedione compounds could be useful for renal dysfunction through mechanism dependent or independent of its insulin-sensitizing action. In type 2 diabetes, troglitazone could reduce urinary albumin-creatinine ratio compared to metformin. Furthermore, we have shown that troglitazone was able to prevent diabetic glomerular dysfunction through inhibition of diacylglycerol-protein kinase C-extracellular signal-regulated kinase pathway in type 1 diabetic rats. Thus, thiazolidinediones might be effective agents for treating insulin-resistant diabetes as well as diabetes-induced kidney disease.

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP-GlcNAc: Galbeta1-3GalNAcalphaRbeta1-6-N-acetylglucosaminyltransferas e (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc-T with severe increase in cardiac core 2 GlcNAc-T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O-glycan and increases of c-fos gene expression and AP-1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc-T also induced c-fos promoter activation, mitogen activated-protein kinase (MAPK) phosphorylation, Trk receptor glycosylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc-T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.-Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

d-Alpha-tocopherol prevents the hyperglycemia induced activation of diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular smooth muscle cell by an increase of DAG kinase activity.

We have reported that d-alpha-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from alpha-tocopherol's principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of alpha-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of alpha-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of alpha-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-alpha-tocopherol's inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40 +/- 10% (mean +/- S.E.) (P < 0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-alpha-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-alpha-tocopherol was also induced in a time- and dose-dependent manner. DAG kinase activity was increased by 57 +/- 19% (P < 0.05) in human VSMC and 112 +/- 35% (P < 0.05) in rat VSMC after 24 h of incubation with d-alpha-tocopherol (100 microg/ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124 +/- 34%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-alpha-tocopherol treatment. These results provide strong and detailed evidence that d-alpha-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect.

TGF-beta 1 stimulates glucose uptake by enhancing GLUT1 expression in mesangial cells.

BACKGROUND: An increase in the expression of transforming growth factor-beta 1 (TGF-beta 1) has been proposed to play an important role in the excessive production of extracellular matrix (ECM) proteins seen in diabetes. Because the linkage between glucose metabolism and ECM protein production was found in mesangial cells overexpressed with the brain-type glucose transporter (GLUT1), we hypothesized that TGF-beta 1 could affect glucose metabolism. METHODS: To prove this hypothesis, we examined the effect of TGF-beta 1 on glucose uptake, the first step of glucose metabolism, in mesangial cells. 2-Deoxy-D-glucose (2DOG) uptake and the expression of GLUT1 were measured in mesangial cells exposed to various concentrations of TGF-beta 1. The kinetic constants were determined using 2DOG and 3-O-methyl-D-glucose (3OMG). The effect of anti-TGF-beta neutralizing antibody on 2DOG uptake and GLUT1 mRNA was also examined in mesangial cells cultured under high-glucose (22.2 mM) conditions for 72 hours. RESULTS: TGF-beta 1 stimulated 2DOG uptake in mesangial cells by approximately 2.5-fold in a dose- (1.25 ng/ml maximum) and time-dependent manner, with a peak stimulation at nine hours. The increase in 2DOG uptake by TGF-beta 1 was completely abolished by the addition of 1 microgram/ml cycloheximide, and kinetic analysis of 2DOG or 3OMG uptake revealed an increase in Vmax by TGF-beta 1. Furthermore, TGF-beta 1 enhanced the expression of GLUT1 mRNA from one hour, followed by an enhancement of the expression of GLUT1 protein at nine hours. Finally, 2DOG uptake was significantly enhanced in cells cultured under high-glucose (22.2 mM) conditions as compared with that in cells under normal glucose (5.6 mM) conditions, and this increase in 2DOG uptake in cells under high-glucose conditions was inhibited by the addition of anti-TGF-beta neutralizing antibody. CONCLUSIONS: TGF-beta 1 stimulates glucose uptake by enhancing the expression of GLUT1 in mesangial cells, which leads to the acceleration of intracellular metabolic abnormalities in diabetes.

Stretch-induced overproduction of fibronectin in mesangial cells is mediated by the activation of mitogen-activated protein kinase.

An excessive production of extracellular matrix (ECM) proteins in glomerular mesangial cells is considered to be responsible for the development of mesangial expansion seen in diabetic nephropathy. Mechanical stretch due to glomerular hypertension has been proposed as one of the factors leading to an increase in the production of ECM proteins in mesangial cells, but the precise mechanism of stretch-induced overproduction of ECM proteins has not been elucidated. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) may play a key role in the overproduction of fibronectin (FN) in mesangial cells exposed to mechanical stretch. MAPK, also termed extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), was activated by mechanical stretch in time- and intensity-dependent manners. Stretch-induced activation of ERK was inhibited by herbimycin A, a tyrosine kinase inhibitor, but not by GF109203X or calphostin C, the inhibitors of protein kinase C. Mechanical stretch also enhanced DNA-binding activity of AP-1, and this enhancement was inhibited by PD98059, an inhibitor of MAPK or ERK kinase (MEK). Furthermore, mechanical stretch stimulated the expression of FN mRNA followed by a significant increase in its protein accumulation. PD98059 could prevent stretch-induced increase in the expression of FN mRNA and protein. These results indicate that the activation of ERK may mediate the overproduction of ECM proteins in mesangial cells exposed to mechanical stretch, an in vitro model for glomerular hypertension seen in diabetes.