Biomedical subjects

N B Ruderman

Publications and source records attributed to N B Ruderman.

At least 37 records · Page 2Linked to original sources

PubMed1997

Malonyl-CoA regulation in skeletal muscle: its link to cell citrate and the glucose-fatty acid cycle.

Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, the enzyme that controls the oxidation of fatty acids by regulating their transfer into the mitochondria. Despite this, knowledge of how malonyl-CoA levels are regulated in skeletal muscle, the major site of fatty acid oxidation, is limited. Two- to fivefold increases in malonyl-CoA occur in rat soleus muscles incubated with glucose or glucose plus insulin for 20 min [Saha, A. K., T. G. Kurowski, and N. B. Ruderman. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E283-E289, 1995]. In addition, as reported here, acetoacetate in the presence of glucose increases malonyl-CoA levels in the incubated soleus. The increases in malonyl-CoA in all of these situations correlated closely with increases in the concentration of citrate (r2 = 0.64) and to an even greater extent the sum of citrate plus malate (r2 = 0.90), an antiporter for citrate efflux from the mitochondria. Where measured, no increase in the activity of acetyl-CoA carboxylase (ACC) was found. Inhibition of ATP citrate lyase with hydroxycitrate markedly diminished the increases in malonyl-CoA in these muscles, indicating that citrate was the major substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. Studies with enzyme purified by immunoprecipitation indicated that the observed increases in citrate could have also allosterically activated ACC. The results suggest that in the presence of glucose, insulin and acetoacetate acutely increase malonyl-CoA levels in the incubated soleus by increasing the cytosolic concentration of citrate. This novel mechanism could complement the glucose-fatty acid cycle in determining how muscle chooses its fuels. It could also provide a means by which glucose acutely modulates signal transduction in muscle and other cells (e.g., the pancreatic beta-cell) in which its metabolism is determined by substrate availability.

Acetoacetates↗

PubMed1997

Diet-induced muscle insulin resistance in rats is ameliorated by acute dietary lipid withdrawal or a single bout of exercise: parallel relationship between insulin stimulation of glucose uptake and suppression of long-chain fatty acyl-CoA.

Chronic high-fat feeding in rats induces profound whole-body insulin resistance, mainly due to effects in oxidative skeletal muscle. The mechanisms of this reaction remain unclear, but local lipid availability has been implicated. The aim of this study was to examine the influence of three short-term physiological manipulations intended to lower muscle lipid availability on insulin sensitivity in high-fat-fed rats. Adult male Wistar rats fed a high-fat diet for 3 weeks were divided into four groups the day before the study: one group was fed the normal daily high-fat meal (FM); another group was fed an isocaloric low-fat high-glucose meal (GM); a third group was fasted overnight (NM); and a fourth group underwent a single bout of exercise (2-h swim), then were fed the normal high-fat meal (EX). In vivo insulin action was assessed using the hyperinsulinemic glucose clamp (plasma insulin 745 pmol/l, glucose 7.2 mmol/l). Prior exercise, a single low-fat meal, or fasting all significantly increased insulin-stimulated glucose utilization, estimated at either the whole-body level (P < 0.01 vs. FM) or in red quadriceps muscle (EX 18.2, GM 28.1, and NM 19.3 vs. FM 12.6 +/- 1.1 micromol x 100 g(-1) x min(-1); P < 0.05), as well as increased insulin suppressibility of muscle total long-chain fatty acyl-CoA (LC-CoA), the metabolically available form of fatty acid (EX 24.0, GM 15.5, and NM 30.6 vs. FM 45.4 nmol/g; P < 0.05). There was a strong inverse correlation between glucose uptake and LC-CoA in red quadriceps during the clamp (r = -0.7, P = 0.001). Muscle triglyceride was significantly reduced by short-term dietary lipid withdrawal (GM -22 and NM -24% vs. FM; P < 0.01), but not prior exercise. We concluded that muscle insulin resistance induced by high-fat feeding is readily ameliorated by three independent, short-term physiological manipulations. The data suggest that insulin resistance is an important factor in the elevated muscle lipid availability induced by chronic high-fat feeding.

Acyl Coenzyme A↗

PubMed1996

Malonyl coenzyme A and adiposity in the Dahl salt-sensitive rat: effects of pioglitazone.

These studies were designed to assess the effects of pioglitazone, a new oral antidiabetic agent that acts by improving insulin sensitivity, on blood pressure, plasma and tissue lipids, and insulin resistance in the Dahl salt-sensitive (Dahl-S) rat. Reaven et al had reported that male Dahl-S rats are moderately hyperinsulinemic and insulin-resistant. This was of particular interest since these rats are not obese but are hypertriglyceridemic, and on a high-salt diet they become hypertensive. In the current study, male Sprague-Dawley control and Dahl-S rats were compared when fed standard chow of high-fat, high-sucrose (HFHS) diets with or without pioglitazone (20 mg/kg body weight/d) for 3 weeks. On the standard chow diet, Dahl-S rats were hypertriglyceridemic and had high tissue levels of malonyl coenzyme A ([CoA] Dahl-S 5.0 v control 3.3 nmol/g in muscle, and Dahl-S 15.6 v control 10.7 nmol/g in liver); however, they were not hyperinsulinemic. Pioglitazone therapy decreased both malonyl CoA and plasma triglycerides toward control values, but had no effect on plasma insulin levels. On the HFHS diet, both groups became glucose-intolerant and hyperinsulinemic; however, the hyperinsulinemia was greater and more sustained in Dahl-S rats. In addition, the HFHS diet appeared to increase the mass of retroperitoneal fat in the Dahl-S but not in the control group. Treatment with pioglitazone decreased retroperitoneal fat, but as reported previously, it increased the mass of the epididymal fat pad. The results suggest that the hypertriglyceridemia of the Dahl-S rat is associated with an increase in the concentration of malonyl CoA in both liver and muscle. They also show that pioglitazone reverses both of these abnormalities independently of its effect on plasma insulin. Whether these high levels of malonyl CoA predispose the Dahl-S rat to hyperinsulinemia and possibly obesity when placed on a HFHS diet remains to be determined.

Adipose Tissue↗

PubMed1996

Differential stimulation of Na+ pump activity by insulin and nitric oxide in rabbit aorta.

The effect of insulin on Na+ pump activity, measured as ouabain-sensitive (OS) 86Rb uptake, was studied in the rabbit aorta. In the absence of insulin, incubation of endothelium-intact rings for 3 h in a medium containing a high concentration of glucose (44 mM) decreased OS 86Rb uptake by 42% compared with that observed at 5.5 mM glucose. Addition of insulin (0.1-10 microU/ml) increased OS86 86Rb uptake at both glycose concentrations and eliminated the differences between the groups. Insulin also increased OS 86Rb uptake in endothelium-intact and -denuded (ED) rings in the presence of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine. Removal of the endothelium before the incubations did not diminish the insulin-induced increase in OS 86Rb uptake, which was concentration dependent. The NO donor sodium nitroprusside increased OS 86Rb uptake in ED rings, and its effect and that of insulin were additive. Phorbol 12,13-dibutyrate, a direct activator of protein kinase C (PKC), also increased OS 86Rb uptake in ED rings; however, its effect and that of insulin were not additive. The PKC inhibitor bisindolylmaleimide totally inhibited insulin-induced, but not sodium nitroprusside-induced, increases in OS 86Rb uptake. The results suggest that insulin activates the Na+ pump in the aorta and reverses the inhibition of the pump caused by hyperglycemia. This effect of insulin can occur at physiological concentrations, is independent of endothelium-derived NO, and is presumably mediated by an increase in PKC activity, In contrast, activation of the Na+ pump by NO appears to be independent of PKC.

Animals↗

PubMed1996

Vasodilator responses in the forearm skin of patients with insulin-dependent diabetes mellitus.

The integrity of endothelium-dependent vasodilation in the skin of patients with insulin-dependent diabetes mellitus (IDDM) is unclear, especially with respect to the role of nitric oxide. To examine this, forearm skin blood flow by laser Doppler flowmetry and total blood flow by venous occlusion plethysmography was measured in response to brachial artery infusions of an endothelium-dependent (methacholine) and -independent (sodium nitroprusside) vasodilator. Peak hyperemic forearm blood flow, following 5 min of arterial occlusion, was also determined. Responses were compared in 11 control subjects and 16 patients with insulin-dependent diabetes mellitus. In ten normal subjects, co-infusion of NG-monomethyl-L-arginine with methacholine produced a significant reduction in total forearm blood flow response to methacholine (p < 0.002), measured by venous occlusion plethysmography, as well as vascular conductance (p < 0.001), confirming that nitric oxide contributes to this response. In contrast, NG-monomethyl-L-arginine had no significant effect on the methacholine-induced increase in forearm skin blood flow measured by laser Doppler flowmetry indicating that factors other than nitric oxide may be involved. Increases in forearm skin blood flow in response to methacholine, sodium nitroprusside and to an ischemic stimulus were not significantly different between the normal subjects and patients with IDDM. Dose-related increases in total forearm blood flow and vascular conductance were not significantly different between control subjects and diabetic patients during infusions of methacholine. The increases in these parameters during infusions of sodium nitroprusside, however, were significantly less in the diabetic group than in the control group (p < 0.05) as was the peak reactive hyperemic blood flow (p < 0.05). Since skin blood flow was not affected, the reduced vasodilator responses to sodium nitroprusside and an ischemic stimulus in the diabetic group are in forearm skeletal muscle. The reduced muscle blood flow does not reflect a decreased vasodilatory capacity, but rather a functional impairment in response to nitric oxide and ischemia since the methacholine dilation was normal. The normal vasodilator responses in the forearm skin, which is predominantly capillary as opposed to arteriovenous anastomatic blood flow, indicate that the response to nitric oxide and an ischemic stimulus in this vascular bed is intact in patients with IDDM. This is, therefore, an unlikely cause of diabetic skin, complications in these areas.

Adult↗

PubMed1995

A malonyl-CoA fuel-sensing mechanism in muscle: effects of insulin, glucose, and denervation.

Increases in the concentration of malonyl-CoA in skeletal muscle have been observed in the KKAy mouse, an obese rodent with high plasma insulin and glucose levels [Saha et al. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E95-E101, 1994]. To assess whether insulin and glucose directly regulate malonyl-CoA in muscle, soleus muscles from young rats were incubated with insulin and glucose at various concentrations, and their content of malonyl-CoA was determined. In addition, the effect on malonyl-CoA of denervation and electrically induced muscle contractions was assessed. The concentration of malonyl-CoA in the soleus, taken directly from a rat fed ad libitum, was 2.0 +/- 0.2 nmol/g. In muscles incubated for 20 min in a medium devoid of added insulin and glucose, the concentration was decreased to 0.8 +/- 0.2 nmol/g. When the medium contained 0.5, 7.5, or 30 mM glucose, malonyl-CoA levels were 1.3 +/- 0.1, 1.8 +/- 0.1, or 2.4 +/- 0.2 nmol/g, respectively, in the absence of insulin and 1.7 +/- 0.1, 4.6 +/- 0.3, or 5.5 +/- 0.6 nmol/g in its presence (10 mU/ml). Compared with its level in a control muscle, the concentration of malonyl-CoA was increased threefold in the soleus 6-8 h after denervation and remained twofold higher for > or = 48 h. In contrast, muscle contractions induced by sciatic nerve stimulation, in vivo, acutely decreased the concentration of malonyl-CoA by 30-35%. The results indicate that insulin and glucose, and probably contractile activity, regulate the concentration of malonyl-CoA in muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1. Stimulation by insulin and inhibition by Wortmannin.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a heterodimer composed of an 85-kDa subunit that binds tyrosyl-phosphorylated proteins via its SH2 domains and a 110-kDa catalytic subunit. Expression and mutagenesis experiments have shown that the 110-kDa subunit is a dual specificity kinase that possesses both lipid and serine kinase activities. Except for the 85- and 110-kDa subunits of PI 3-kinase, however, no endogenous substrates for the serine kinase have been identified. The results of the present study show that another target of this kinase is the insulin receptor substrate, IRS-1. Serine phosphorylation of IRS-1 as well as the 85-kDa subunit of PI 3-kinase was demonstrated in immunoprecipitates of PI 3-kinase and IRS-1 isolated from rat adipocytes incubated with insulin. In adipocytes incubated in the absence of insulin, only the serine phosphorylation of p85 was observed in immunoprecipitates of PI 3-kinase. Both the serine and lipid kinase activities of PI 3-kinase were abolished by the fungal metabolite Wortmannin. Wortmannin also partially inhibited the ability of insulin to stimulate glucose transport and inhibit lipolysis in fat cells. These data raise the possibility that the serine kinase activity of PI 3-kinase is involved in insulin signaling. They also suggest that inhibition of the lipid or serine kinase activities of PI 3-kinase could explain the effect of Wortmannin to diminish insulin action.

Adipocytes↗

PubMed1994

Insulin increases a biochemically distinct pool of diacylglycerol in the rat soleus muscle.

Insulin stimulates the incorporation of glucose-carbon into diacylglycerol (DAG) in rat skeletal muscle, and its ability to do so is enhanced severalfold after the muscle is denervated (S. J. Heydrick, N. B. Ruderman, T. J. Kurowski, H. A. Adams, and K. S. Chen. Diabetes 40: 1707-1711, 1991). The present studies were carried out to assess the nature of this newly synthesized DAG and to identify factors other than insulin that determine its rate of appearance in the incubated rat soleus muscle. In control muscles, incubated at a medium glucose concentration of 6-7.5 mM, insulin (10 mU/ml) increased DAG content (mass) by 20-25% and increased the incorporation of a 14C label from extracellular [14C]glucose into DAG by 200-300%. The labeling of DAG reached a plateau within 20 min, at which time the labeled DAG comprised a very small percentage of total muscle DAG. Molecular species analysis revealed that DAG species having fatty acids of 18:2/20:4 and 18:2/18:2 each constituted approximately 2% of total DAG content but contained 20 and 15%, respectively, of the glucose-derived label in DAG. In contrast, 16:0/18:1 accounted for > 80% of total DAG content but only 18% of the total label incorporated into DAG. Insulin did not alter this pattern. Denervation also did not alter the molecular species profiles of the labeled DAGs or DAG analyzed by mass. An increased incorporation of glucose-carbon into DAG was observed in muscles incubated with 30 mM glucose in place of the usual 7.5-mM concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Lipid abnormalities in tissues of the KKAy mouse: effects of pioglitazone on malonyl-CoA and diacylglycerol.

Insulin resistance is present in liver and muscle of subjects with type 2 diabetes and obesity. Recent studies suggest that such insulin resistance could be related to abnormalities in lipid-mediated signal transduction; however, the nature of these abnormalities is unclear. To examine this question further, tissue levels of diacylglycerol (DAG), malonyl-CoA, and triglyceride (TG) were determined in liver and soleus muscle of obese insulin-resistant KKAy mice and lean C57 BL control mice. In addition, the effects of treatment with pioglitazone, an antidiabetic agent that acts by increasing insulin sensitivity in muscle, liver, and other tissues, were assessed. The KKAy mice were hyperglycemic (407 vs. 138 mg/dl), hypertriglyceridemic (337 vs. 109 mg/dl), hyperinsulinemic (631 vs. 15 mU/ml), and weighed more (42 vs. 35 g) than the control mice. They also had 1.5- to 2.0-fold higher levels of malonyl-CoA in both liver and muscle, higher DAG (twofold) and TG (1.3-fold) levels in muscle, and higher TG (threefold), but not DAG, levels. Treatment of the KKAy mice with pioglitazone for 4 days decreased plasma glucose, TGs, and insulin by approximately 50% and restored hepatic and muscle malonyl-CoA levels to control values. In contrast, pioglitazone increased hepatic and muscle DAG levels two- or threefold. It has no effect on muscle or hepatic TG content, and it slightly increased hepatic TGs in the control group. The results indicate that abnormalities in tissue lipids occur in both liver and muscle of the KKAy mouse and that they are differentially altered when insulin sensitivity is enhanced by treatment with pioglitazone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Role of endothelium-derived nitric oxide in stimulation of Na(+)-K(+)-ATPase activity by endothelin in rabbit aorta.

An endothelium-derived factor with the properties of nitric oxide (NO) has recently been implicated in the regulation of basal Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in vascular smooth muscle. To determine whether this factor also plays a role in the stimulation of ouabain-sensitive (OS) 86Rb uptake by specific agonists, studies were carried out using rabbit aortic rings. In endothelium-intact rings incubated for 3 h with Krebs-Henseleit solution containing 5.5 mM glucose, endothelin (ET) caused a concentration-dependent increase in OS 86Rb uptake (maximal increase = 205%, with 100 nM ET). Incubation with phenylephrine (Phe; 0.1 and 1 microM) or phorbol 12,13-dibutyrate (PDBu; 0.1 microM), under the same conditions, increased OS 86Rb uptake by 128, 144, and 140%, respectively. Removal of endothelium before incubation decreased the ability of ET to stimulate OS 86Rb uptake by 38-45%, but it did not diminish the stimulation of OS 86Rb uptake by Phe or PDBu. An increase in the concentration of glucose from 5.5 to 44 mM diminished ET-stimulated OS 86Rb uptake by 50% in endothelium-intact rings but had no effect on Phe- or PDBu-induced increases in OS 86Rb uptake. Addition of the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 0.3 mM) to the medium decreased ET-stimulated OS 86Rb uptake by 40%. Guanosine 3',5'-cyclic monophophate (cGMP) formation in endothelium-intact rings was also increased (65%) by ET but not by Phe or PDBu. The increase in cGMP by ET was totally inhibited by L-NMMA or endothelium denudation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Stimulation of vascular Na(+)-K(+)-ATPase activity by nitric oxide: a cGMP-independent effect.

An endothelium-derived factor with the properties of nitric oxide (NO) has been implicated in the regulation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity in vascular smooth muscle. To examine this phenomenon further and to explore its modulation by guanosine 3',5'-cyclic monophosphate (cGMP), studies were carried out in the isolated rabbit aorta. Incubation of endothelium-denuded rings with NO (1 microM) or sodium nitroprusside (SNP, 10 microM) caused a time-dependent increase in ouabain-sensitive (OS) 86Rb uptake with the maximal stimulation (approximately 170%) seen after 20 min. In contrast, increases in cGMP concentration caused by NO and SNP (40- and 20-fold increases, respectively) were transient, with peak values observed after 2 min and significantly lower values by 10 min. The ability of NO or SNP to increase OS Rb uptake in endothelium-denuded rings was not mimicked by incubation with 8-bromo- or dibutyryl-cGMP or increases in cGMP caused by treatment with the phosphodiesterase inhibitor isobutylmethylxanthine. Depletion of intracellular cGMP levels by the guanylate cyclase inhibitor LY83583 also did not alter OS Rb uptake. SNP-stimulated OS Rb uptake was not inhibited by LY83583 in endothelium-denuded rings; however, it was completely prevented by the Na(+)-H+ exchange inhibitors amiloride and ethylisopropylamiloride. The results suggest that NO stimulates Na(+)-K(+)-ATPase activity in rabbit aorta by a mechanism independent of its ability to increase the intracellular cGMP concentration. They also suggest that NO may stimulate Na(+)-K(+)-ATPase activity secondary to increases in Na(+)-H+ exchange.

Amiloride↗

PubMed1993

Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle.

Insulin stimulates the appearance of anti-tyrosine(P)-immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity in adipocytes, predominantly in an intracellular membrane fraction (Kelly, K. L., Ruderman, N. B., and Chen, K. S. (1992) J. Biol. Chem. 267, 3423-3428). Neither the mechanism underlying this activation nor the precise subcellular compartment in which it occurs is known. To address these questions, studies were performed using isolated rat adipocytes and subcellular fractions of these cells. In intact cells, insulin stimulated the rapid appearance of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in 32P-labeled adipocytes without changing the labeling of phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-bisphosphate. This effect was accompanied by the tyrosyl phosphorylation of a 185-kDa protein, tentatively identified as IRS-1, with which PI 3-kinase became associated. The majority of the p85, the regulatory subunit of PI 3-kinase, in untreated adipocytes was present in the cytosol; however, neither the activity of PI 3-kinase nor the total amount of p85 in this fraction was modified in response to insulin. In contrast, insulin increased the association of p85 with IRS-1, the tyrosyl phosphorylation of the IRS-1 associated with p85, and the total activity of PI 3-kinase in the plasma membranes and low density membranes. After insulin treatment, similar amounts of p85 were bound to IRS-1 in the low density and plasma membrane fractions; however, tyrosyl-phosphorylated IRS-1 and PI 3-kinase activity were an order of magnitude greater in the low density membranes. The complex of tyrosyl-phosphorylated IRS-1.p85 that formed in response to insulin was localized to a very low density vesicle subpopulation that could be distinguished from vesicles containing the GLUT-4 glucose transporter and the insulin receptor. These data suggest that the activation of PI 3-kinase by insulin in the adipocyte involves the formation of a complex between IRS-1 and PI 3-kinase in a very low density membrane fraction that is not enriched in GLUT-4 or insulin receptors. They also suggest that PI 3-kinase activation correlates more closely with the extent of tyrosyl phosphorylation of the IRS-1 complexed to PI 3-kinase than it does to the amount of p85 bound to IRS-1.

Adipose Tissue↗

PubMed1993

Regulation of phosphatidylinositol 3-kinase by insulin in rat skeletal muscle.

The presence of phosphatidylinositol 3-kinase (PI 3-kinase) in mammalian skeletal muscle and its response to insulin stimulation were investigated. PI kinase, immunoprecipitated from rat soleus muscle with antibodies directed toward its 85-kDa subunit phosphorylated PI, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5,-bisphosphate [PI(4,5)P2] to yield phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4,-bisphosphate, and phosphatidylinositol trisphosphate in vitro. PI 3-kinase activity was also immunoprecipitated with antiphosphotyrosine [alpha-Tyr(P)] antibodies and with antibodies raised against IRS-1, a substrate of the insulin receptor protein tyrosine kinase that associates with and activates PI 3-kinase. Incubation of the soleus with insulin in vitro, or injection of insulin into rats in vivo, produced three- to fivefold increases in alpha-Tyr(P)- and alpha-IRS-1-immunoprecipitable PI 3-kinase activity. In nonstimulated soleus muscle, PI 3-kinase activity immunoprecipitated with alpha-IRS-1 or with alpha-Tyr(P) antibodies was evenly distributed between particulate (200,000-g pellet) and soluble fractions. Insulin treatment increased immunoprecipitable PI 5-kinase activity in both fractions, but the increase in alpha-Tyr-(P)-precipitable activity was greater in the particulate fraction, whereas the increase in alpha-IRS-1-precipitable activity was greater in the soluble fraction. In intact soleus muscles incubated with 32PO4, insulin increased the labeling of PI(3)P but did not affect the labeling of PI(4)P or PI(4,5)P2. Activation of PI 3-kinase by insulin was unaffected by prior denervation of the muscle, a manipulation that has been shown to cause both insulin resistance and hypersensitivity in muscles, depending on the parameter measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Differential effect of metabolic fuels on the energy state and Na(+)-K(+)-ATPase in isolated cerebral microvessels.

Isolated bovine cerebral microvessels (ICMV) were incubated with different metabolic fuels to determine their ability to support microvessel Na(+)-K(+)-ATPase (quantitated as ouabain-sensitive 86Rb+ uptake) and the ATP/ADP ratio. In comparison with ICMV incubated with glucose, Na(+)-K(+)-ATPase activity was reduced by 55% after a 3-h incubation in fuel-free medium and by 30-40% after incubation with beta-hydroxybutyrate, acetoacetate, or glutamate. However, Na(+)-K(+)-ATPase activity was not significantly decreased in ICMV incubated with pyruvate or oleate plus carnitine. In contrast, only glucose was able to maintain the ATP/ADP ratio. To evaluate the effect of endogenous fatty acid metabolism on these parameters, ICMV were incubated with bromostearate, an inhibitor of fatty acid oxidation. Bromostearate decreased both Na(+)-K(+)-ATPase activity and the ATP/ADP ratio, even in the presence of glucose. These results indicate that the varying effects of different fuels on Na(+)-K(+)-ATPase in ICMV cannot be explained solely by their effects on the ATP/ADP ratio or on glycolytic ATP generation. They suggest that other fuel-modulated factors play a key role in regulating this enzyme.

3-Hydroxybutyric Acid↗

PubMed1993

Reduced Na(+)-K+ pump activity in diabetic rabbit carotid artery: reversal by aldose reductase inhibition.

This study addresses the question of whether a decrease in basal Na+ pump [Na(+)-K(+)-adenosinetriphosphatase (ATPase)] activity occurs in the carotid artery of an alloxan-diabetic rabbit and, if so, whether it is associated with altered 86Rb+ uptake and contractile response to ouabain and K(+)-free solution. Ouabain-sensitive 86Rb+ uptake, an index of Na+ pump activity, was diminished approximately 50% in carotid arteries from diabetic rabbits. Concurrent with this, contractions induced by incubating the carotid arteries in a K(+)-free solution (in the absence of phentolamine) were significantly larger in the diabetic group. Readdition of K+ (1 mM) to arteries contracted with the K(+)-free solution caused relaxations that were slower to occur and of lesser magnitude in diabetic than in control rabbits. In contrast to the contractions caused by the K(+)-free medium, contractions caused by incubation with ouabain (1 mM) in the presence of phentolamine were significantly smaller in the diabetic group. Treatment of diabetic rabbits with an aldose reductase inhibitor, zopolrestat, at both high and low doses restored the alterations in vascular reactivity toward normal. The results indicate that the Na+ pump activity is diminished in the carotid artery of diabetic rabbit, and this is associated with abnormal vascular responsiveness and increased polyol pathway flux.

Aldehyde Reductase↗

PubMed1992

Phosphatidylinositol-3-kinase in isolated rat adipocytes. Activation by insulin and subcellular distribution.

Insulin increases phosphatidylinositol-3-kinase (PI-3-kinase) activity in Chinese hamster ovary cells transfected with human insulin receptor (Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). The subcellular distribution of PI-3-kinase has not been investigated, and it is unclear if insulin has a stimulatory effect on PI-3-kinase in a nonproliferating target tissue, and, if so, whether this effect is subject to counter-regulation. To address these questions, we studied the effect of insulin on PI-3-kinase activity in isolated rat adipocytes. Activity was measured in plasma membranes, intracellular membranes, and cytosol of control and insulin-treated adipocytes, and in anti-Tyr(P) immunoprecipitates prepared from these fractions and from whole cell lysates. Treatment of adipocytes with insulin (200 nM) caused a half-maximal increase in anti-Tyr(P)-immunoprecipitable PI-3-kinase activity in whole cell lysates within 2 min. This effect was concentration-dependent, and it was sensitive to inhibition by norepinephrine. In insulin-stimulated cells, 75% of anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was found in the low density microsomes. This fraction also exhibited the highest specific activity of PI-3-kinase, and insulin caused a further increase in this activity. Anti-Tyr(P)-immunoprecipitable PI-3-kinase activity was also found in the plasma membranes of insulin-treated cells, but this accounted for only a minor portion of the total and anti-Tyr(P)-immunoprecipitable PI-3-kinase activity. The majority of PI-3-kinase activity (90%) in control cells was cytosolic, but this was not increased in response to insulin nor was it anti-Tyr(P)-immunoprecipitable. These data demonstrate that insulin increases the activity of PI-3-kinase in adipocytes and this effect is subject to inhibition by a physiological antagonist of insulin action. The data also indicate that the effect of insulin to increase PI-3-kinase activity is expressed primarily in the low density intracellular membranes and to a lesser extent in the plasma membranes.

Adipose Tissue↗

PubMed1992

Glucose and diabetic vascular disease.

The central therapeutic problem in diabetes mellitus is prevention and treatment of the chronic vascular disease associated with this disorder. Prolonged exposure to hyperglycemia is the primary factor associated with the development of diabetes-specific microvascular disease, and the relationship between deranged glucose metabolism and arterial disease is complicated by many other factors that influence atherogenesis in nondiabetics. Until relatively recently, knowledge about diabetic vascular disease was limited mainly to clinical description. New information about abnormal vascular physiology, ultrastructure, biochemistry, cell biology, and molecular biology now makes it possible to understand in an integrated fashion the major specific mechanisms by which hyperglycemia damages diabetic vessels. Continued progress in this area will further optimize the development of safe and effective drugs for the treatment of diabetic vascular disease.

Animals↗

PubMed1992

Effect of prior exercise and insulin on potential thermogenic systems in rat skeletal muscle.

We previously reported that insulin stimulates oxygen consumption by the perfused rat hindquarter after high-intensity exercise. The purpose of the present study was to examine whether fructose 6-phosphate-fructose 1,6-bisphosphate cycling or an uncoupling of mitochondrial respiration contributes to this phenomenon. Hindquarter skeletal muscle was analyzed after perfusion in the absence or presence of insulin (150-200 microU/ml) for high-energy phosphate content, fructose 6-phosphate-fructose 1,6-bisphosphate cycling of glucose before incorporation into glycogen, and mitochondrial respiratory control. Muscle from exercised rats perfused with insulin did not display greater rates of glucose cycling or mitochondrial uncoupling; in fact, insulin decreased the rate of fructose 6-phosphate cycling and tended to increase respiratory control in skeletal muscle mitochondria. In addition, the concentrations of ATP and creatine phosphate and the calculated free ADP level in muscle of previously exercised rats perfused with insulin were similar to those of control rats. The results do not exclude the possibility that localized subcellular changes in ADP occurred, however. In conclusion, the results suggest that insulin-induced increases in other substrate cycles, ion transport systems, and/or as yet unidentified energy-requiring processes account for the 25-30% increase in hindquarter oxygen consumption after intense exercise.

Animals↗