Mechanism of adipose tissue iNOS induction in endotoxemia.

The aim of the present study was to investigate the mechanism of adipose tissue inducible nitric oxide synthase (iNOS) induction in endotoxemia. Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) to rats for

Insulin inhibits inducible nitric oxide synthase in skeletal muscle cells.

Recent studies have shown that cytokines and endotoxins impair insulin-stimulated glucose transport by activating the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in skeletal muscle cells. In this study, we investigated whether iNOS induction is modulated by insulin in L6 myocytes. Long term exposure of muscle cells to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) greatly increased iNOS mRNA expression and NO production. Addition of insulin to the cytokine/LPS-treated muscle cells reduced (by approximately 40%) NO production. This inhibition was similar to that observed with the synthetic glucocorticoid dexamethasone, a known inhibitor of iNOS in several cell types. The combination of insulin and dexamethasone was more effective than either agent alone in reducing NO production. Dexamethasone greatly inhibited the effect of cytokines/LPS to induce cellular iNOS mRNA expression. In strong contrast, insulin failed to reduce iNOS mRNA expression under similar conditions. These results show that insulin is a novel inhibitor of iNOS-mediated NO production in skeletal muscle cells. Furthermore, our data indicate that unlike glucocorticoids, insulin does not inhibit NO production by suppression of iNOS gene transcription.

Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.

Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase.

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-alpha, interferon-gamma or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. l-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women.

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.

A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle.

A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse (T) tubule membranes from a rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and T tubules in distinct subcellular fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of both surface domains but is markedly enriched with GLUT-4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT-4. Using this new procedure, we found that acute in vivo insulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by approximately 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (approximately 40%) in GLUT-4 abundance in the intracellular fraction. These results further support the hypothesis that T tubules are involved in the regulation of glucose transport in skeletal muscle. This novel fractionation method will be useful in investigating the regulation of muscle GLUT-4 transporters in other physiological and disease states such as diabetes, where defective translocation of the transporter protein to either one or both cell surface domains is suspected to occur.

DNA methylation associated with repeat-induced point mutation in Neurospora crassa.

Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.

Expression of transcripts encoding steroid UDP-glucuronosyltransferases in human prostate hyperplastic tissue and the LNCaP cell line.

The UDP-glucuronosyltransferase (EC 2.4.1.17) enzymes transform many lipophilic compounds to more water-soluble products via conjugation with glucuronic acid. This conversion is responsible for enhancing the excretion of endogenous aglycones such as steroids. To date, several distinct isoforms of steroid UDP-glucuronosyltransferases (UGTs) have been isolated in the human liver. Among these UGTs, UGT2B7 is specific for estriol and 3,4-catechol estrogens, UGT2B15 glucuronidates 17beta-hydroxy-C19 steroids while UGT2B10 has as yet an undescribed activity. To further demonstrate the presence of UGTs in peripheral tissues we studied the expression of these enzymes in human prostate hyperplastic tissue and the LNCaP cell line. Metabolism studies using intact LNCaP cells in culture indicate the presence of UGT activities involved in the glucuronidation of 3alpha-hydroxysteroids (androsterone) and 17beta-hydroxysteroids (testosterone and dihydrotestosterone). Northern blot analysis of poly(A+) RNA from LNCaP cells and prostate using a UGT2B15 cDNA probe revealed two bands of 2.0 and 2.3 kb. In order to identify more specifically the mRNAs detected in Northern blot analysis we used RNase protection and RT-PCR, although, these approaches did not allow detection of UGT2B7 transcripts. Our studies demonstrate the presence of two UGT activities and at least two types of UGT transcripts in both the human prostate and the LNCaP.

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase.

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K(m) value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K(m) values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V(max) of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.

Isolation and purification of creatine kinase conversion factor from human serum and its identification as carboxypeptidase N.

Creatine kinase conversion factor has been isolated from human serum and purified to electrophoretic and chromatographic homogeneity. The enzyme sequentially converts creatine kinase MM3 to MM2 and MM1 and hydrolyzes lysine and arginine from hippuryl-L-lysine and hippuryl-L-arginine. Data on molecular weight, (316,000 dalton), electrophoretic mobility (alpha-globulin), prevalence in serum (26 mg/L), subunit composition (two subunits, 80,000 and 52,200 dalton) indicate that creatine kinase conversion factor is identical to carboxypeptidase N. The previously reported lower molecular weight of 190,000 dalton of partially purified creatine kinase conversion factor is attributed to proteolytic degradation.