Pathways of Acetyl-CoA Metabolism Involved in the Reversal of Palmitate-Induced Glucose Production by Metformin and Salicylate.

The pathways through which fatty acids induce insulin resistance have been the subject of much research. We hypothesise that by focussing on the reversal of insulin resistance, novel insights can be made regarding the mechanisms by which insulin resistance can be overcome. Using global gene and lipid expression profiling, we aimed to identify biological pathways altered during the prevention of palmitate-induced glucose production in hepatocytes using metformin and sodium salicylate. FAO hepatoma cells were treated with palmitate (0.075 mM, 48 h) with or without metformin (0.25 mM) and sodium salicylate (2 mM) in the final 24 h of palmitate treatment, and effects on glucose production were determined. RNA microarray measurements followed by gene set enrichment analysis were performed to investigate pathway regulation. Lipidomic analysis and measurement of secreted bile acids and cholesterol were also performed. Reversal of palmitate-induced glucose production by metformin and sodium salicylate was characterised by co-ordinated down-regulated expression of pathways regulating acetyl-CoA to cholesterol and bile acid biosynthesis. All 20 enzymes that regulate the conversion of acetyl-CoA to cholesterol were reduced following metformin and sodium salicylate. Selected findings were confirmed using primary mouse hepatocytes. Although total intracellular levels of diacylglycerol, triacylglycerol and cholesterol esters increased with palmitate, these were not, however, further altered by metformin and sodium salicylate. 6 individual diacylglycerol, triacylglycerol and cholesterol ester species containing 18:0 and 18:1 side-chains were reduced by metformin and sodium salicylate. These results implicate acetyl-CoA metabolism and C18 lipid species as modulators of hepatic glucose production that could be targeted to improve glucose homeostasis.

Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors.

BACKGROUND AND PURPOSE: The P2X7 receptor is an ATP-gated ion channel predominantly expressed in immune cells and plays a key role in inflammatory processes. Ginseng is a well-known Chinese herb with both pro- and anti-inflammatory properties and many of its actions have been ascribed to constituent ginsenosides. We screened a number of ginsenoside compounds for pharmacological activity at P2X7 receptors, that might contribute to the reported immunomodulatory actions of ginseng. EXPERIMENTAL APPROACH: We used several assays to measure responses of P2X7 receptors, ATP-mediated dye uptake, intracellular calcium measurement and whole-cell patch-clamp recordings. HEK-293 cells stably expressing human P2X7 receptors were used in addition to mouse macrophages endogenously expressing P2X7 receptors. KEY RESULTS: Four ginsenosides of the protopanaxdiol series, Rb1, Rh2, Rd and the metabolite compound K (CK) potentiated the dye uptake responses of P2X7 receptors, whereas other ginsenosides tested were ineffective (1-10 µM). The potentiation was rapid in onset, required a threshold concentration of ATP (>50 µM) and had an EC50 of 1.08 µM. CK markedly enhanced ATP-activated P2X7 currents, probably via an extracellular site of action. One of the consequences of this potentiation effect is a sustained rise in intracellular Ca(2+) that could account for the decrease in cell viability in mouse macrophages after a combination of 500 µM ATP and 10 µM CK that are non-toxic when applied alone. CONCLUSIONS AND IMPLICATIONS: This study identifies selected ginsenosides as novel potent allosteric modulators of P2X7 channels that may account for some of the reported immune modulatory actions of protopanaxdiol ginsenosides in vivo.

Activation of MAP kinase by insulin and vanadate in adipocytes from young and old rats.

Vanadate has insulin-like effects in adipocytes without stimulating insulin receptor kinase activity. However, it activates IRS-1 associated PI 3-kinase, suggesting that it mimics insulin effects by stimulating signaling elements downstream of PI 3-kinase. Here we analysed the stimulation of MAPK by insulin and vanadate and observed that both elicit a rapid 3.5-4 fold activation which is abolished by wortmannin and PD98059. Simultaneous addition of insulin and vanadate does not result in an additive effect neither on MAPK nor in MEK. Whereas insulin action is transient, vanadate stimulation lasts up to 20 min. In insulin-resistant adipocytes from old rats, insulin stimulates poorly MAPK, whereas a normal activation is achieved with vanadate. We conclude that: (a) insulin and vanadate use a common signaling pathway from PI 3-kinase to MEK and MAPK; (b) vanadate but not insulin, elicits a sustained activation of both enzymes; (c) this pathway is functional in old rat adipocytes.

Dolor lumbar e imágenes radiológicas / Lumbar pain and X-ray follow up

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Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism.

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

Ayudas ergogénicas. Beneficios / Ergogenic aids. Benefits

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Effects of chronic treatment with acarbose on glucose and lipid metabolism in obese diabetic Wistar rats.

AIMS: The effect of chronic treatment with acarbose on fasting plasma glucose, insulin, triglyceride, cholesterol and free fatty acid (FFA) concentrations, as well as on the glucose and insulin excursions during oral glucose tolerance test (OGTT), in obese diabetic Wistar (WDF) rats was investigated. METHODS: Forty-five mature male WDF rats were randomly distributed to one of the three treatment groups (no acarbose, 20 mg and 40 mg of acarbose/100 g of chow, respectively). After 3.5, 7.5 and 11.5 months, animals were tested for glucose tolerance by means of an OGTT, and their respective metabolic profiles were determined. Control determinations were done in obese and age-matched lean animals before the start of the trial. RESULTS: The WDF rats exhibit higher body weight and fasting blood glucose, insulin, triglyceride and cholesterol concentrations compared to lean animals. Moreover, they show marked glucose intolerance as indicated by the glucose and insulin excursions during OGTT. Interestingly, in both treated and untreated animals, a reversion of the hyperglycaemic state as well as an improvement of the glucose tolerance is observed. However, whereas in the group receiving no acarbose this is accounted for by dramatic increases in fasting plasma insulin concentrations and insulin secretion during OGTT (as indicated by the DeltaInsulin area), in rats treated with acarbose the reversion of the diabetic state takes place without increments in hormone concentration. In addition, rats treated with acarbose for 3.5 and 7.5 months show lower plasma triglyceride and FFA concentrations, and the same was observed for cholesterol at the highest dosage of the drug. CONCLUSIONS: Chronic treatment with acarbose of WDF rats improves the glycaemic and lipidic control as well as the glucose tolerance, with a lower demand of pancreatic insulin than in untreated rats. This data suggests that the long-term modulation of glucose and insulin excursions after meals improves the insulin sensitivity in this rat strain.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells.

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy- methyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA-AM. While the ionophores or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degrees C, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/calmodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Release of insulin receptor substrate proteins from an intracellular complex coincides with the development of insulin resistance.

Insulin receptor substrate (IRS) proteins are major substrates of the insulin receptor (IR). IRS-1 associates with an insoluble multiprotein complex, possibly the cytoskeleton, in adipocytes. This localization may facilitate interaction with the IR at the cell surface. In the present study, we examined the hypothesis that the release of IRS proteins from this location may be a mechanism for insulin desensitization. We show that a second IRS protein, IRS-2, is associated with a multiprotein complex in adipocytes with similar characteristics to the IRS-1 complex. Insulin treatment (15-60 min) caused the release of IRS-1 and IRS-2 from this complex (high speed pellet; HSP) into the cytosol, whereas the level of tyrosyl-phosphorylated IRS proteins remained constant. Chronic insulin treatment resulted in a dramatic reduction in IRS-1 and IRS-2 in the HSP, eventually (>2 h) leading to IRS protein degradation and decreased levels of tyrosyl-phosphorylated IRS proteins. Okadaic acid, which rapidly induces insulin resistance in adipocytes independently of IR function, caused an almost quantitative release of IRS-1 into the cytosol commensurate with a significant reduction in tyrosyl-phosphorylated IRS proteins. Platelet-derived growth factor, a factor known to compromise insulin signaling, caused a more moderate release of IRS proteins from the HSP. Collectively, these results suggest that the assembly of IRS-1/IRS-2 into a multiprotein complex facilitates coupling to the IR and that the regulated release from this location may represent a novel mechanism of insulin resistance.

Vanadate fully stimulates insulin receptor substrate-1 associated phosphatidyl inositol 3-kinase activity in adipocytes from young and old rats.

Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. We conclude that: (a) vanadate stimulates 2-deoxyglucose uptake using a pathway that converges with that of insulin at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the insulin pathway at steps located both upstream and downstream of PI 3-kinase.

Changes in the kinase activity of the insulin receptor account for an increased insulin sensitivity of mammary gland in late pregnancy.

Mammary gland is an organ that undergoes cycles of growth, differentiation, and function during pregnancy and lactation. Although it is known that the gland enhances its sensitivity to insulin during lactation, it remains to be investigated whether this increased sensitivity develops during pregnancy and which are the molecular mechanisms underlying such a change. To address this issue, virgin and late-pregnant rats were subjected to a continuous infusion with 50% glucose for 72 h to produce a prolonged hyperinsulinemic-euglycemic condition. Insulin sensitivity in mammary gland was determined as the glucose utilization index by using 2-[3H]-deoxyglucose. Furthermore, binding characteristics and kinase activity were studied by means of both [125I]insulin binding and in vitro phosphorylation studies with insulin receptors partially purified from mammary gland. Whereas the glucose utilization index in mammary gland from nonpregnant rats remained unaffected by hyperinsulinemia, glands from pregnant rats displayed a high insulin-dependent glucose uptake. This effect was not paralleled by changes in the binding characteristics of insulin to the high-affinity receptor, suggesting that the high insulin sensitivity of mammary gland in pregnancy is not accounted for by changes at the level of hormone-receptor interaction. Autophosphorylation studies showed that insulin-stimulated kinase activity of insulin receptors from mammary gland was 6- and 20-fold higher in pregnant than in virgin animals under normo- and hyperinsulinemic conditions, respectively. Moreover, insulin dose-response curves revealed that the efficacy of insulin to stimulate kinase activity of the insulin receptor was markedly higher in pregnant than in virgin rats, whereas its potency (ED50 approximately 15 nM) was not changed. These data, therefore, show that mammary glands develop increased insulin sensitivity during late pregnancy, caused by an augmented kinase activity of the insulin receptor.

In vivo insulin-dependent glucose uptake of specific tissues is decreased during aging of mature Wistar rats.

Aging has been associated with peripheral insulin resistance in both humans and rats. However, the specific tissues that become insensitive to insulin before glucose homeostasis is altered remain to be elucidated. In the present work we studied the glucose metabolic index of a number of tissues known to be insulin sensitive in 3- and 24-month-old Wistar rats by measuring 2-deoxy-D-[1-3H]glucose uptake both under euglycemic-hyperinsulinemic conditions and in the basal state. Analysis of the glucose infusion rate to maintain normoglycemia during the clamp confirmed that the old rats show overall insulin resistance at both saturating and subsaturating insulin concentrations. The maximal response of glucose uptake to insulin as well as insulin sensitivity in red and white quadriceps were unaltered in old rats. In contrast, glucose uptake by soleus and diaphragm was poorly stimulated in old animals, and a marked decrease in insulin sensitivity was observed in both tissues. In heart, only the sensitivity to the hormone, not the maximal response, was impaired in old rats. In white adipose tissue, no significant stimulation was detected. We conclude that during aging in Wistar rats and before fasting plasma insulin and glucose levels become altered, specific tissues develop insulin resistance, whereas other remain insulin sensitive. We postulate that fat tissue plays a qualitative important role in eliciting the insulin resistance in old animals. Due to the metabolic characteristics of the aged Wistar rat, the changes reported might reflect what occurs in nonobese elderly humans, nongenetically committed to develop type 2 diabetes.

Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat.

Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.