Superoxide anion can modulate vascular smooth muscle tone and potentially affect the growth response in vascular disease. The present studies were undertaken to characterize the source of superoxide in rabbit aorta. Rings of aorta (5 mm) were incubated in physiological salt solution (PSS) for 30 min at 37 degrees C in the presence of 10 mM diethyldithiocarbamate (DDC) with or without inhibitors of superoxide-generating systems. Rings were then placed in PSS containing 250 microM lucigenin at 37 degrees C in the presence or absence of inhibitors, and changes in amounts of superoxide were determined by measuring chemiluminescence (units). The inhibitors of xanthine oxidase, oxypurinol (300 microM), and of mitochondrial NADH dehydrogenase, rotenone (50 microM), had no significant effect on superoxide levels. An inhibitor of NADPH oxidase, iodonium thiophen, caused a concentration-dependent inhibition of superoxide anion (12.49 +/- 1.48 vs 5.27 +/- 1.81 and 2.30 +/- 0.36 units, control vs 7 microM and 70 microM iodonium thiopen, respectively). A structurally related iodonium compound, diphenyleneiodonium (20 microM), caused a 78% reduction in basal and DDC-evoked superoxide levels. In the presence or absence of DDC, exogenous administration of NADPH (10 microM-1 mM), but not NADP (1 mM), elicited a concentration-dependent rise in superoxide levels that was inhibited by iodonium thiophen. Particulate fractions of whole aortic tissue exhibited NADPH-dependent superoxide production that was inhibited by 1 microM diphenyleneiodonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract-Glycolysis increases in hypertrophied hearts but the mechanisms are unknown. We studied the regulation of glycolysis in hearts with pressure-overload LV hypertrophy (LVH), a model that showed marked increases in the rates of glycolysis (by 2-fold) and insulin-independent glucose uptake (by 3-fold). Although the V max of the key glycolytic enzymes was unchanged in this model, concentrations of free ADP, free AMP, inorganic phosphate (P i ), and fructose-2,6-bisphosphate (F-2,6-P 2 ), all activators of the rate-limiting enzyme phosphofructokinase (PFK), were increased (up to 10-fold). Concentrations of the inhibitors of PFK, ATP, citrate, and H ϩ were unaltered in LVH. Thus, our findings show that increased glucose entry and activation of the rate-limiting enzyme PFK both contribute to increased flux through the glycolytic pathway in hypertrophied hearts. Moreover, our results also suggest that these changes can be explained by increased intracellular free [ADP] and [AMP], due to decreased energy reserve in LVH, activating the AMP-activated protein kinase cascade. This, in turn, results in enhanced synthesis of F-2,6-P 2 and increased sarcolemma localization of glucose transporters, leading to coordinated increases in glucose transport and activation of PFK. Key Words: cardiac function Ⅲ hypertrophy Ⅲ protein kinases Ⅲ cardiac metabolism Ⅲ cyclic AMP G lucose utilization is increased in hypertrophied and failing hearts, 1-4 but the underlying mechanisms are poorly understood. Increased glycolytic flux in the hypertrophied myocardium is important because ATP synthesis via glucose utilization may compensate for decreased capacity for ATP synthesis via other pathways. 5,6 In hearts with chronic pressure overload hypertrophy, it was recently reported that chronic depletion of the energy reserve compound PCr coupled with large changes in the ratio of PCr to free creatine led to activation of AMP-activated protein kinase (AMPK) by elevated AMP concentrations. 7 AMPK acts as a low-on-fuel sensor and, when the cytosolic AMP concentration increases, AMPK activates enzymes in pathways that synthesize ATP and inhibits enzymes in pathways that use ATP. 8,9 Among the many consequences of activated AMPK is increased localization of glucose transporters in the sarcolemma and hence increased glucose uptake by an insulinindependent mechanism. 10,11 In addition, in a study of acute myocardial ischemia, AMPK was found to phosphorylate and thereby activate heart phosphofructokinase-2 (PFK-2), leading to increased production of fructose-2,6-bisphosphate (F-2,6-P 2 ), a potent activator of the rate-limiting glycolytic enzyme phosphofructokinase (PFK). 12 In the present study, we tested the hypothesis that increased glycolysis in hypertrophied hearts occurs as a consequence of chronic decreases in the energy reserve and activation of AMPK.Using a model of pressure overload left ventricular hypertrophy (LVH) of the rat heart, in which reduced energy reserve, increased AMPK activity, and increased insulin-independent glucose upt...

Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta-cells. Abundant evidence favors a role for cytosolic long-chain acyl-CoA (LC-CoA), including the rapid rise in malonyl CoA, the inhibitory effect of hydroxycitrate or acetyl CoA carboxylase knockout, both of which prevent malonyl CoA formation, and the stimulatory effect of exogenous FFA. On the other hand, some evidence opposes the concept, including the fall in total LC-CoA levels in response to glucose, the stimulatory effect of LC-CoA on K(ATP) channels and the lack of inhibition of glucose-stimulated secretion either by overexpression of malonyl CoA decarboxylase, which markedly lowers malonyl CoA levels, or by triacsin C, which blocks FFA conversion to LC-CoA. Alternative explanations for these data are presented. A revised model of nutrient-stimulated secretion involving two arms of signal transduction that occur simultaneously is proposed. One arm depends on modulation of the K(ATP) channel evoked by changes in the ATP/ADP ratio. The other arm depends upon anaplerotic input into the tricarboxylic acid cycle, generation of excess citrate, and increases in cytosolic malonyl-CoA. Input from this arm is increased LC-CoA. Signaling through both arms would be required for normal secretion. LC-CoA esters and products formed from them are potent regulators of enzymes and channels. It is hypothesized that their elevations directly modulate the activity of enzymes, genes and various beta-cell functions or modify the acylation state of key proteins involved in regulation of ion channels and exocytosis.

The role of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, in regulating the oxidation of fatty acids in rat skeletal (1, 2) and cardiac (3, 4) muscle has been intensively investigated. Recent studies have demonstrated that its concentration in rat muscle is governed, at least in part, by changes in the activity of the muscle isoform of acetyl-CoA carboxylase (ACC ␤ ) 1 (5), the enzyme that catalyzes malonylCoA synthesis. Thus, in keeping with their observed effects on malonyl-CoA concentration and fatty acid oxidation, insulin and glucose appear to activate ACC ␤ in muscle by increasing the cytosolic concentration of citrate, an allosteric activator of ACC ␤ and a precursor of its substrate, cytosolic acetyl-CoA. Conversely, decreases in malonyl-CoA concentration and increases in fatty acid oxidation in muscle during exercise (contraction) have been linked to decreases in ACC ␤ activity, attributable to its phosphorylation and inhibition by the ␣ 2 isoform of AMP-activated protein kinase (AMPK) (5). AMPK can also be activated and the concentration of malonyl-CoA decreased by exposing resting muscle to 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is taken into the muscle and phosphorylated to form the 5Ј-AMP analogue ZMP (6).Whether a change in malonyl-CoA turnover contributes to the alterations in its concentration in muscle during exercise and other conditions is not known. In a lipogenic tissue such as liver, the de novo synthesis of fatty acids is thought to be the major mechanism by which malonyl-CoA is utilized. In contrast, in skeletal muscle fatty acid synthesis occurs at a very low rate, if at all (7), and attention has been focused on malonyl-CoA decarboxylase (MCD) for removal of malonyl-CoA (1). Evidence has been presented that MCD is present in both cardiac (8, 9) and skeletal (1, 10, 11) muscle. In skeletal muscle, its activity is similar to that of ACC (1). In heart, in which MCD activity is substantially greater than in skeletal muscle, a decrease in the K m of MCD for malonyl-CoA has been reported following an increase in its work load (9). On the other hand, no change in activity has been observed following ischemia-reperfusion of the heart, a situation in which AMPK is activated (8). The question of whether MCD is acutely regulated in skeletal muscle and, if so, how has not been studied previously.In this study, we describe the characteristics of purified MCD from rat skeletal muscle and contraction-induced changes in its maximal activity and affinity for malonyl-CoA. In addition, the effects of the AMPK activator 5-aminoimida-

Normal insulin secretion is oscillatory in vivo and in vitro, with a period of approximately 5-10 min. The mechanism of generating these oscillations is not yet established, but a metabolic basis seems most likely for glucose-stimulated secretion. The rationale is that 1) spontaneous oscillatory operation of glycolysis is a well-established phenomenon; 2) oscillatory behavior of glycolysis involves oscillations in the ATP/ADP ratio, which can cause alternating opening and closing of ATP-sensitive K+ channels, leading to the observed oscillations in membrane potential and Ca2+ influx in pancreatic beta-cells, and may also have downstream effects on exocytosis; 3) spontaneous Ca2+ oscillations are an unlikely basis in this case, since intracellular stores are not of primary importance in the stimulus-secretion coupling, and furthermore, insulin oscillations occur under conditions when intracellular Ca2+ levels are not changing; 4) a neural basis cannot account for insulin oscillations from perifused islets and clonal beta-cells or from transplanted islets or pancreas in vivo; 5) observed oscillations in metabolite levels and fluxes further support a metabolic basis, as does the presence in beta-cells of the oscillatory isoform of phosphofructokinase (PFK-M). The fact that normal oscillatory secretion is impaired in patients with NIDDM and in their near relatives suggests that such derangement may be involved in the development of the disease; furthermore, this probably reflects an early defect in the regulation and operation of the fuel metabolizing/sensing pathways of the pancreatic beta-cell.

Abstract-It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of 3 H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with 3 H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

The knowledge of the mechanism whereby glucose and other fuel stimuli promote the release of insulin by the pancreatic beta cell remains fragmentary. The closure of metabolically sensitive K+ channels and a rise in cytosolic free Ca2+ are key features of beta-cell metabolic signal transduction. However, these two signalling events do not account for the dose dependence of glucose-induced insulin secretion. In fact, recent evidence indicates that there are KATP channel and Ca2+ independent pathway(s) of beta-cell activation which remain to be defined. In this review, we have limited our attention to the recent developments in our understanding of the mode of action of nutrient secretagogues. A particular emphasis is placed in summarising the evidence in support of two new concepts: 1) oscillations in the glycolytic pathway and beta-cell metabolism contribute to the oscillatory nature of beta-cell ionic events and insulin secretion; 2) malonyl-CoA and long chain acyl-CoA esters may act as metabolic coupling factors in beta-cell signalling. Finally, we propose that the altered expression of genes encoding enzymes in the pathway of malonyl-CoA formation and fatty acid oxidation contributes to the beta-cell insensitivity to glucose in some patients with non-insulin-dependent diabetes mellitus.

Glucose transport into the brain is depressed in chronically hyperglycemic (diabetic) rats. To determine whether hypoglycemia has the opposite effect, brain transport of hexoses and other substrates was examined in chronically and acutely hypoglycemic rats. We produced chronic hypoglycemia by implanting insulin-secreting tumors or insulin-releasing osmotic mini-pumps or by repeated injection of protamine zinc insulin (PZI) and acute hypoglycemia by intravascular injection of regular insulin. Blood-brain barrier (BBB) transport was measured using the brain uptake index (BUI) method. In the three models of chronic hypoglycemia, brain glucose extraction was increased compared with controls. The extraction of deoxyglucose and several other hexoses was also increased by chronic hypoglycemia. Acute hypoglycemia had no effect on brain transport. The transport of other substrates was either not affected or depressed, suggesting increased brain hexose transport is specific. Studies of freeze-blown brain in insulinoma-engrafted rats showed that brain glucose levels were depressed while creatine phosphate, ATP, and glucose 6-phosphate were maintained. Tumor removal led to a reversion of brain glucose transport to control rates but only after 5-25 days. These findings support the view that glucose transport across the BBB is modulated by chronic alterations in the ambient glucose concentration. They also may explain why some patients with chronic hypoglycemia tolerate low blood glucose concentrations.