Stimulation Of Pyruvate Dehydrogenase Activity Research Articles

Stimulating myocardial pyruvate dehydrogenase activity fails to alleviate cardiac abnormalities in a mouse model of human Barth syndrome.

Barth syndrome (BTHS) is a rare genetic disorder due to mutations in the TAFAZZIN gene, leading to impaired maturation of cardiolipin and thereby adversely affecting mitochondrial function and energy metabolism, often resulting in cardiomyopathy. In a murine model of BTHS involving short-hairpin RNA mediated knockdown of Tafazzin (TazKD mice), myocardial glucose oxidation rates were markedly reduced, likely secondary to an impairment in the activity of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. Furthermore, TazKD mice exhibited cardiac hypertrophy with minimal cardiac dysfunction. Because the stimulation of myocardial glucose oxidation has been shown to alleviate diabetic cardiomyopathy and heart failure, we hypothesized that stimulating PDH activity would alleviate the cardiac hypertrophy present in TazKD mice. In order to address our hypothesis, 6-week-old male TazKD mice and their wild-type (WT) littermates were treated with dichloroacetate (DCA; 70 mM in the drinking water), which stimulates PDH activity via inhibiting PDH kinase to prevent inhibitory phosphorylation of PDH. We utilized ultrasound echocardiography to assess cardiac function and left ventricular wall structure in all mice prior to and following 6-weeks of treatment. Consistent with systemic activation of PDH and glucose oxidation, DCA treatment improved glycemia in both TazKD mice and their WT littermates, and decreased PDH phosphorylation equivalently at all 3 of its inhibitory sites (serine 293/300/232). However, DCA treatment had no impact on left ventricular structure, or systolic and diastolic function in TazKD mice. Therefore, it is unlikely that stimulating glucose oxidation is a viable target to improve BTHS-related cardiomyopathy.

Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products.

In Vivo Clinical Measures of Intermediary Metabolism Are Inadequate

The heart derives most of its energy via mitochondrial oxidative metabolism. The heart's fuels or energy substrates include fatty acids, lactate, glucose, and ketones. Substrate concentrations vary during the day, driven mainly by postprandial status. Entry into mitochondrial oxidation requires metabolism to acetyl-coenzyme A (CoA), the fuel of the tricarboxylic acid (TCA) cycle. Acetyl-CoA oxidation generates reduced Nicotinamide adenine dinucleotide (NADH), which drives oxidative phosphorylation, the reduction of O2 to H2O, and, ultimately, the synthesis of ATP, which is essential for myocardial mechanics. The heart uses this highly regulated series of enzyme-catalyzed reactions to convert chemical energy into mechanical energy.1 Metabolism and function in the heart are inextricably linked. Article see p 201 The pathological states of the heart, such as ischemia, hypertrophy, and failure, modify metabolism. Available technologies to measure intermediary metabolism have been inadequate. Magnetic resonance spectroscopy (MRS) can be used to spatially map or at least locally measure metabolism in vivo. Measurement of endogenous triglycerides in patients using 1H MRS has demonstrated that cardiac steatosis precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction.2 In the failing human heart and in preclinical heart failure models, 31P MRS has shown that flux through the creatinine kinase reaction is altered.3,4 Also, ATP and phosphocreatine are reduced after myocardial infarction due to myocyte loss.5 Myocardial energy substrate can be measured with 13C MRS. It has the chemical specificity, but its inherently low sensitivity limits the spatial and temporal resolution necessary for clinical application. In isolated hearts, 13C MRS has to be used with labeled substrate molecules to define TCA cycle kinetics. The metabolites of the TCA cycle have not been measured directly in these experiments. The content of the TCA cycle metabolites is …

BRL37344, but not CGP12177, stimulates fuel oxidation by soleus muscle in vitro

The β 3-adrenoceptor agonist, ( RR+ SS)-(±)-4-[2-)2-)3-chlorophenyl)-2-hydroxyethyl)amino)propyl]phenoxyacetate (BRL37344), stimulated fuel utilisation by isolated mouse soleus muscle at concentrations 10- to 100-fold lower than those required to stimulate lipolysis in brown adipocytes. At 1×10 −10 M BRL37344, uptake and phosphorylation of 2-deoxyglucose was increased (40%), as was glucose-oxidation (50%), palmitate-oxidation (70%) and oxidation of [2- 14C]pyruvate (2-fold), indicating stimulation of tricarboxylic acid cycle reactions. Oxidation of [1- 14C]pyruvate was unaffected, indicating no stimulation of pyruvate dehydrogenase activity. Other β 3-adrenoceptor agonists, disodium( RR)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxazole-2,2-dicarboxylate (CL316,243, 1×10 −7 M) and ( S)-4-{2-[2-hydroxy-3-(4-hydroxyphenoxy)propylamino]ethyl}phenoxymethylcyclohexylphosphiric acid lithium salt (SB226552, 1×10 −9 M), achieved similar stimulation of 2-deoxyglucose uptake and phosphorylation but (±)-4-(3- t-butylamino-2-hydroxypropoxy)benzimidazol-2-one (CGP12177A) had no effect. The inhibitor of protein kinase A, H-89 (isoquinolinesulfonamide), had little effect on the stimulation of pyruvate-oxidation by BRL37344, while the specific inhibitor of protein kinase C, bisindolylmaleimide IX, reduced the stimulated rate to slightly below basal values. We consider that these responses provide evidence of the presence of a novel β-adrenoceptor in skeletal muscle, which we have termed β skel-adrenoceptor.

Effects of dichloroacetate in patients with congestive heart failure.

Conventional approaches to management of congestive heart failure (CHF) rely on drugs that increase myocardial contractility or reduce ventricular afterload. These approaches often improve cardiac symptoms and survival, but may be associated with significant deleterious effects. An alternative approach is to enhance myocardial energy production. Dichloroacetate (DCA) stimulates pyruvate dehydrogenase activity and accelerates aerobic glucose, pyruvate, and lactate metabolism in myocardial cells. These alterations would be expected to improve myocardial function. The purpose of the investigation was to assess the efficacy of DCA in patients with left ventricular systolic dysfunction and to examine the mechanism by which improvement occurs. A total of 25 patients (16 men, 9 women; age range 31-72 years, mean 59) with CHF and ejection fraction < or = 40% received an intravenous infusion of 50 mg/kg DCA over 15 min. Indices of systolic and diastolic function were obtained by two-dimensional and Doppler echocardiography performed at baseline, 30 min, and 60 min following completion of DCA infusion. Baseline ventricular ejection fraction was 27.3 +/- 9.1%; 17 patients (68%) had nonischemic cardiomyopathy. Heart rate increased after DCA infusion from 73.9 +/- 14.5 to 79.2 +/- 14.9 beats/min at 60 min; p = 0.02. Left ventricular diastolic and systolic volumes increased at 30 min compared with baseline (248.7 +/- 98.1 vs. 259.6 +/- 99.6; p = 0.04, and 180.1 +/- 80.4 vs. 192.2 +/- 84.9; p = 0.002, respectively), but stroke volume (49.2 +/- 19.1 vs. 48.9 +/- 18.1; p = 0.9) and ejection fraction (27.3 +/- 9.1 vs. 25.7 +/- 9.8; p = 0.2) were unchanged. Indices of diastolic function were also unchanged. Dichloroacetate infusion in patients with CHF is not associated with improvement in noninvasively assessed left ventricular function.

Insulin stimulates pyruvate dehydrogenase and protects human ventricular cardiomyocytes from simulated ischemia

Impaired myocardial metabolism after cardioplegic arrest results in persistent anaerobic lactate production. Insulin may protect the heart from ischemia and reperfusion by enhancing myocardial metabolic recovery. However, the stimulation of glycolysis during ischemia may be detrimental because of an accumulation of metabolic end-products. We examined the effect of insulin on quiescent human ventricular cardiomyocytes subjected to simulated cardioplegic ischemia and reperfusion. Methods: Primary cardiomyocyte cultures were established from patients undergoing corrective repair of tetralogy of Fallot. Cells were exposed to varying concentrations of glucose and insulin during 30 minutes of stabilization in 10 mL of phosphate-buffered saline solution. Ischemia was simulated by exposing the cells to a low volume (1.5 mL) of deoxygenated phosphate-buffered saline solution for 90 minutes followed by 30 minutes of simulated reperfusion in 10 mL of normoxic phosphate-buffered saline solution. Cell viability was assessed by trypan blue exclusion. The activity of mitochondrial pyruvate dehydrogenase was measured in 3 states: stabilization, ischemia, and reperfusion. In addition intracellular lactate, adenine nucleotides, extracellular lactate, pyruvate, and acid release were measured. Results: Higher ambient glucose concentrations resulted in greater cellular injury although insulin-treated cells displayed less injury after ischemia and reperfusion. Insulin increased the pyruvate dehydrogenase activity by 31% in cardiomyocytes and reduced extracellular lactate production by 40%. Intracellular adenosine triphosphate was improved by 75% in cells exposed to high glucose concentrations in the presence of insulin. Conclusions: Insulin protected human ventricular cardiomyocytes from ischemia and reperfusion. This protection may be due to a stimulation of pyruvate dehydrogenase activity which resulted in improved aerobic metabolism. (J Thorac Cardiovasc Surg 1998;116:485-94)

Improved hemodynamic function and mechanical efficiency in congestive heart failure with sodium dichloroacetate

Objectives. The purpose of this study was to determine whether sodium dichloroacetate improves hemodynmic performance and mechanical efficiency in congestive heart failure.Background. Congestive heart failure is associated with impaired hemodynamic performance and reduced mechanical efficiency. Dichloroacetate stimulates pyruvate dehydrogenase activity by inhibition of pyruvate dehydrogenase kinase, which results in inhibition of free fatty acid metabolism and stimulation of high respiratory quotient glucose and lactate consumption by the heart. Facilitation of glucose and lactate consumption with dichloroacetate should improve mechanical efficiency of the failing ventricle.Methods. Ten patients with New York Heart Association functional class III to IV congestive heart failure were studied. Dichloroacetate (50 mg/kg body weight) was administered intravenously for 30 min, with measurements of hemodynamic variables, coronary sinus blood flow and blood gas, glucose and lactate levels for 2 h. The same patients were also given dobutamine (5 to 12.5 mg/kg per min) for comparison.Results. Therapeutic levels of dichloroacetate were achieved (100 to 160 μg/liter of plasma). Myocardial consumption of lactate was stimulated from 29% to 37.4%. Forward stroke volumes increased (+5.3 ml/beat, p < 0.02), as did left ventricular stroke work (+1.8 g-m/m2per beat, p < 0.02) and left ventricular minute work (from 1.38 to 1.55 kg-m/m2per min, p < 0.01). Myocardial oxygen consumption decreased (from 19.3 to 16.3 ml/min, p = 0.06) as left ventricular minute work increased. Left ventricular mechanical efficiency thus improved from 15.2% to 20.6% (p = 0.03).Dobutamine administration resulted in the opposite trend with respect to myocardial lactate extraction (from 34% to 15.3%, p < 0.02). Stroke volume increased (+7.4 ml/beat, p = NS vs. dichloroacetate), as did left ventricular minute work (from 1.29 to 1,59 g-m/m2per min, p < 0.01 vs. dichloroacetate) and myocardial oxygen consumption (from 18.6 to 21.0 ml/min, p = 0.06 vs. dichloroacetate). Left ventricular mechanical efficiency did not change with dobutamine administration (from 16.4% to 15.8%, p = NS).Conclusions. Dichloroacetate administration stimulates myocardial lactate consumption and improves left ventricular mechanical efficiency. Forward stroke volume and left ventricular minute work increase significantly, with a simultaneous reduction in myocardial oxygen consumption. Dobutamine administration results in similar hemodynamic improvements but with no change in left ventricular mechanical efficiency and with opposite effects on lactate metabolism. The opposing metabolic actions, yet similar hemodynamic responses, of dichloroacetate and dobutamine suggest that these agents may be complementary in the treatment of congestive heart failure.

Testosterone Regulates Pyruvate Dehydrogenase Activity of Prostate Mitochondria

A major and unique function of the prostate is the production of extremely high levels of citrate. Testosterone is an important regulator of this function. This regulation is achieved by hormonal stimulation of citrate synthesis by prostate secretory epithelial cells. Previous studies have established that testosterone stimulates the mitochondrial transamination of aspartate to OAA for citrate synthesis. However, increased citrate synthesis would also require an increased availability of AcCoA which is derived from pyruvate. The present studies were concerned with the in vivo effect of testosterone on pyruvate dehydrogenase (PDH) activity of prostate mitochondria isolated from rat ventral prostate (VP). The results demonstrated that testosterone administration to castrated rats stimulates PDH activity and citrate production from pyruvate in the presence of OAA. Therefore testosterone increases both OAA and acetyl CoA production which results in increased citrate synthesis. The mechanism of testosterone regulation of PDH can now be investigated.

The carboxy terminal 110 amino acid portion of the insulin receptor is important for insulin signalling to pyruvate dehydrogenase

Insulin-like growth factor 1 (IGF-1) had no detectable effect on pyruvate dehydrogenase (PDH) in NIH 3T3 cells that stably overexpressed normal human IGF-1 receptors. Insulin stimulated PDH activity 3–4 — fold in cells that overexpressed normal human insulin receptors, but not in cells expressing TMI receptors, composed of the ligand binding domain of the insulin receptor coupled to the transmembrane and intracellular components of the IGF-1 receptor, or CEX receptors, in which the carboxy terminal 110 amino acid portion of the insulin receptor was exchanged for the corresponding portion of the IGF-1 receptor. In contrast, insulin stimulated glucose uptake in the control cell line and in each of the chimeric receptor-expressing lines with similar dose-response characteristics. These findings suggest the carboxy terminal portion of the IR may play a role in mediating the stimulation of PDH activity.

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.

Metabolic changes induced by maximal exercise in human subjects following L-carnitine administration

In double-blind cross-over experiments, ten moderately trained male subjects were submitted to two bouts of maximal cycle ergometer exercise separated by a 3 day interval. Each subject was randomly given either l-carnitine (2 g) or placebo orally 1 h before the beginning of rach exercise session. At rest l-carnitine supplementation resulted in an increase of plasma-free carnitine without a change in acid-soluble carnitine esters. Treatment with l-carnitine induced a significant post-exercise decrease of plama lactate and pyruvate and a concurrent increase of acetylcarnitine. The determination of the individual carnitine esters in urine collected for 24 h after the placebo exercise trial revealed a decrease of acetyl carnitine and a parallel increase of a C 4 carnitine ester, probably isobutyrylcarnitine. Conversely, acetylcarnitine was strongly increased and C 4 compounds were almost suppressed in the l-carnitine loading trial. These results suggest that l-carnitine administration prior to high-intensity exercise stimulates pyruvate dehydrogenase activity, thus diverting pyruvate from lactate to acetylcarnitine formation.

The effect of amino acids, monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-14C] pyruvate in the presence of 0.05 mM Ca2+ and Mg2+. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(beta-morpholinopropionyl) benzo [b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(beta-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-14C] pyruvate to release 14CO2. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg2+ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg2+. However, at Mg2+ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.

Insulin resistance of glucose metabolism in isolated brown adipocytes of lactating rats. Evidence for a post-receptor defect in insulin action

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.

Role of insulin and glucose oxidation in mediating the protein catabolism of burns and sepsis.

We have investigated the responsiveness of protein kinetics to insulin and the role of glucose oxidation rate as a mediator of the protein catabolic response to burn injury and sepsis by assessing the response of leucine and urea kinetics to a 5-h hyperinsulinemic euglycemic clamp with and without the simultaneous administration of dichloroacetate (DCA) (to further increase glucose oxidation via stimulation of pyruvate dehydrogenase activity) in eight severely burned and eight septic patients. Leucine and urea kinetics were measured by the primed-constant infusions of [1(-13)C]leucine and [15N2]urea. Compared with controls, basal leucine kinetics (flux and oxidation) were significantly elevated (P less than 0.01) in both groups of patients. Hyperinsulinemia elicited significant (P less than 0.05) decreases in leucine kinetics in both groups of patients. Consistent with this observation, hyperinsulinemia caused urea production to decrease significantly (P less than 0.05) in both patient groups. The administration of DCA to patients during hyperinsulinemia elicited a significant increase in glucose oxidation rate compared with the clamp rate (P less than 0.05), and the percent of glucose uptake oxidized increased from 45.5 +/- 5.5 to 53.5 +/- 4.8%; yet the response of leucine and urea kinetics to the clamp plus DCA was not different from the response to the clamp alone. These results suggest that the maximal effectiveness of insulin to suppress protein breakdown is not impaired and that a deficit in glucose oxidation or energy supply is probably not playing a major role in mediating the protein catabolic response to severe burn injury and sepsis.

The insulinomimetic effects of the polar head group of an insulin-sensitive glycophospholipid on pyruvate dehydrogenase in both subcellular and whole cell assays

The polar head group that was released by treating an insulin-sensitive glycophospholipid with a phosphatidylinositol-specific phospholipase C (PI-PLC) stimulated pyruvate dehydrogenase (PDH) in both subcellular and whole cell assays. Stimulation of PDH activity in the subcellular assay was detected after gel filtration chromatography of the polar head group. This stimulation was not due to the presence of contaminating calcium and magnesium. The PDH-stimulating activity was proportional to the amount of polar head group added to the assay. The effect of the polar head group on PDH in the subcellular assay was blocked by sodium fluoride, suggesting that the polar head group activated the PDH phosphatase. In the whole cell assay, the polar head group stimulated PDH activity to an equal or greater extent as a physiological concentration of insulin. The effect of the polar head group was detected at 5 min, peaked at 10 min, and declined thereafter. In contrast, insulin stimulated PDH activity more slowly, but consistently. The PDH-stimulating activity eluted after bacitracin but ahead of ATP during gel filtration chromatography, and it was destroyed by exposure to NH 4OH or alkaline phosphatase and by boiling in water. These data support the proposal that an early step in insulin action is the release of insulinomimetic polar head group from the insulin-sensitive glycophospholipid.

Stimulation of pyruvate dehydrogenase and protein kinase by a peptic fragment from elastase.

A peptic fragment from elastase was separated by affinity chromatography on an immobilized trialanine-agarose column. The fragment lost the enzymatic activity but retained the affinity for trialanyl peptide. It also stimulated pyruvate dehydrogenase activity in rat fat pads and protein kinase activity in the plasma membrane fraction of guinea-pig adipocytes.

Insulin-stimulating protein from human plasma. Chemical characteristics and biological activity.

A protein that potentiates the action of insulin in vitro was purified from human plasma. When reduced with 2-mercaptoethanol and then carboxymethylated, it yielded a single subunit, indicating that it was composed of two identical subunits connected by a single disulfide bond. This modified subunit tended to inhibit rather than stimulate insulin activity. A distinctive feature of the amino acid composition of this protein (H-ISP) was the absence of histidine, arginine, and tryptophan. The molecular mass, subunit composition, the characteristic amino acid composition and the N-terminal amino acid residue of H-ISP are very similar to those of human plasma apolipoprotein A-II (apo A-II). The isoelectric point of H-ISP was estimated to be 4.91, which is identical with that of the major apo A-II isoform. H-ISP did not itself have insulin-like activity in increasing CO2 liberation from labeled glucose and 2-deoxyglucose uptake by isolated rat adipocytes, but it potentiated the action of insulin in these parameters. It had no appreciable affect on the binding or degradation of 125I-labeled insulin by adipocytes. Like H-ISP, apo A-II isolated from human plasma also had no insulin-like activity by itself, but stimulated the effect of insulin on CO2 production from labeled glucose in isolated rat adipocytes. From these results, it is concluded that H-ISP is identical with the major apo A-II isoform. Incubation of isolated adipocytes with H-ISP resulted in marked increase in the activity of pyruvate dehydrogenase in a dose-dependent manner in the absence of added insulin. H-ISP also stimulated pyruvate dehydrogenase activity in a subcellular system consisting of plasma membranes and mitochondria from rat adipocytes. The effect of H-ISP on pyruvate dehydrogenase activity could be produced by treatment of the isolated mitochondrial fraction alone.

Adipocyte glycogen synthase and pyruvate dehydrogenase in obese and type II diabetic subjects.

To determine whether 1) insulin stimulates pyruvate dehydrogenase (PDH) and glycogen synthase (GS) in isolated human adipocytes and 2) adipocytes from subjects with obesity or noninsulin-dependent diabetes mellitus (NIDDM) are resistant to the effects of insulin, PDH and GS were assayed in adipocytes from 11 control, 8 obese, and 9 NIDDM subjects. Basal PDH activities were 123 +/- 20, 129 +/- 21, and 128 +/- 25 pmol pyruvate oxidized/min per 2 X 10(5) adipocytes in these groups. Insulin stimulated PDH activity to a maximum of 223 +/- 38 pmol/min per 2 X 10(5) in adipocytes from control subjects, but did not significantly increase values from obese subjects. Insulin significantly decreased PDH activity in cells from NIDDM subjects (99 +/- 20 pmol/min per 2 X 10(5) cells, P less than 0.05). PDH activity assayed with high magnesium and calcium concentrations was significantly stimulated by insulin in adipocytes from control, but not obese or NIDDM subjects. GS assayed with 1 mM glucose 6-phosphate did not differ significantly among control, obese, or NIDDM subjects (446 +/- 110, 451 +/- 156, and 291 +/- 35 pmol incorporated into glycogen, respectively). Insulin significantly stimulated glycogen synthase in all three groups (827 +/- 179, 764 +/- 177, and 569 +/- 51 pmol incorporated) to a similar extent. Glycogen synthase assayed with 10 mM glucose 6-phosphate was decreased in NIDDM (1,335 +/- 131 pmol incorporated) compared with obese or control subjects (2,512 +/- 451 and 2,239 +/- 230 pmol incorporated, respectively, P less than 0.01).

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase.

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.

Insulin mediator stimulates pyruvate dehydrogenase of intact liver mitochondria.

The effects of putative insulin mediators on the pyruvate dehydrogenase (PDH) activity of intact mitochondria isolated from rat liver were investigated. The mitochondria were judged intact on the basis of electron microscopic examination and demonstrated respiratory control. Only mitochondria having respiratory control ratios of greater than 4, using succinate as a substrate, were used in these studies. Addition of physiologic concentrations of insulin to these mitochondria caused stimulation of PDH activity, attributed to generation of an insulin mediator from plasma membranes contaminating the mitochondrial preparation. Exogenous plasma membranes from rat adipocytes or liver caused further stimulation of PDH activity, which was proportional to the amount of plasma membranes added. Addition of insulin to the mixture of mitochondria and plasma membranes stimulated PDH still further. The stimulation was proportional to the insulin concentration, with maximal effects observed at 50 microU/ml insulin. Partially purified mediators from liver, muscle, H4-II-E hepatoma cells, and IM9 lymphocytes also stimulated PDH activity in intact mitochondria. Mediators prepared from insulin-treated liver, muscle, and cultured hepatoma cells stimulated PDH more than did mediators from the corresponding untreated source. Mediator from insulin-treated IM9 lymphocytes stimulated PDH less than did mediator from untreated IM9 lymphocytes. These findings are consistent with the known effects of insulin on these tissues and with the reported effects of the various mediators on PDH activity in non-intact mitochondria. These observations support the proposal that these mediators are physiologically significant modulators of insulin's effects on PDH activity.