Reactivation of peroxisome proliferator-activated receptor alpha is associated with contractile dysfunction in hypertrophied rat heart.

In pressure overload-induced hypertrophy, the heart increases its reliance on glucose as a fuel while decreasing fatty acid oxidation. A key regulator of this substrate switching in the hypertrophied heart is peroxisome proliferator-activated receptor alpha (PPARalpha). We tested the hypothesis that down-regulation of PPARalpha is an essential component of cardiac hypertrophy at the levels of increased mass, gene expression, and metabolism by pharmacologically reactivating PPARalpha. Pressure overload (induced by constriction of the ascending aorta for 7 days in rats) resulted in cardiac hypertrophy, increased expression of fetal genes (atrial natriuretic factor and skeletal alpha-actin), decreased expression of PPARalpha and PPARalpha-regulated genes (medium chain acyl-CoA dehydrogenase and pyruvate dehydrogenase kinase 4), and caused substrate switching (measured ex vivo in the isolated working heart preparation). Treatment of rats with the specific PPARalpha agonist WY-14,643 (8 days) did not affect the trophic response or atrial natriuretic factor induction to pressure overload. However, PPARalpha activation blocked skeletal alpha-actin induction, reversed the down-regulation of measured PPARalpha-regulated genes in the hypertrophied heart, and prevented substrate switching. This PPARalpha reactivation concomitantly resulted in severe depression of cardiac power and efficiency in the hypertrophied heart (measured ex vivo). Thus, PPARalpha down-regulation is essential for the maintenance of contractile function of the hypertrophied heart.

Load-induced changes in vivo alter substrate fluxes and insulin responsiveness of rat heart in vitro.

It has been observed that opposite changes in cardiac workload result in similar changes in cardiac gene expression. In the current study, the hypothesis that altered gene expression in vivo results in altered substrate fluxes in vitro was tested. Hearts were perfused for 60 minutes with Krebs-Henseleit buffer containing glucose (5 mmol/L) and oleate (0.4 mmol/L). At 30 minutes, either insulin (1 mU/mL) or epinephrine (1 micromol/L) was added. Hearts weighed 35% less after unloading and 25% more after aortic banding. Contractile function in vitro was decreased in transplanted and unchanged in banded hearts. Epinephrine, but not insulin, increased cardiac power. Basal glucose oxidation was initially decreased and then increased by aortic banding. The stimulatory effects of insulin or epinephrine on glucose oxidation were reduced or abolished by unloading, and transiently reduced by banding. Oleate oxidation correlated with cardiac power both before and after stimulation with epinephrine, whereas glucose oxidation correlated only after stimulation. Malonyl-coenzyme A levels did not correlate with rates of fatty acid oxidation. Pyruvate dehydrogenase was not affected by banding or unloading. It was concluded that atrophy and hypertrophy both decrease insulin responsiveness and shift myocardial substrate preference to glucose, consistent with a shift to a fetal pattern of energy consumption; and that the isoform-specific changes that develop in vivo do not change the regulation of key metabolic enzymes when assayed in vitro.

Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids.

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, an important modulator of fatty acid oxidation. We hypothesized that increased fatty acid availability would increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all significantly increased the plasma concentration of nonesterified fatty acids, with a concomitant increase in both rat heart and skeletal muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression returned to basal levels. Fatty acids are known to activate peroxisome proliferator-activated receptor-alpha (PPARalpha). Specific PPARalpha stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPARgamma agonist, decreased MCD expression. The sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPARalpha expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce the expression of MCD in rat heart and skeletal muscle. Additional posttranscriptional mechanisms regulating MCD activity appear to exist.

[5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway.

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.

Improved energy homeostasis of the heart in the metabolic state of exercise.

We postulate that metabolic conditions that develop systemically during exercise (high blood lactate and high nonesterified fatty acids) are favorable for energy homeostasis of the heart during contractile stimulation. We used working rat hearts perfused at physiological workload and levels of the major energy substrates and compared the metabolic and contractile responses to an acute low-to-high work transition under resting versus exercising systemic metabolic conditions (low vs. high lactate and nonesterified fatty acids in the perfusate). Glycogen preservation, resulting from better maintenance of high-energy phosphates, was a consequence of improved energy homeostasis with high fat and lactate. We explained the result by tighter coupling between workload and total beta-oxidation. Total fatty acid oxidation with high fat and lactate reflected increased availability of exogenous and endogenous fats for respiration, as evidenced by increased long-chain fatty acyl-CoA esters (LCFA-CoAs) and by an increased contribution of triglycerides to total beta-oxidation. Triglyceride turnover (synthesis and degradation) also appeared to increase. Elevated LCFA-CoAs caused high total beta-oxidation despite increased malonyl-CoA. The resulting bottleneck at mitochondrial uptake of LCFA-CoAs stimulated triglyceride synthesis. Our results suggest the following. First, both malonyl-CoA and LCFA-CoAs determine total fatty acid oxidation in heart. Second, concomitant stimulation of peripheral glycolysis and lipolysis should improve cardiac energy homeostasis during exercise. We speculate that high lactate contributes to the salutary effect by bypassing the glycolytic block imposed by fatty acids, acting as an anaplerotic substrate necessary for high tricarbocylic acid cycle flux from fatty acid-derived acetyl-CoA.

Regulation of fatty acid oxidation of the heart by MCD and ACC during contractile stimulation.

We tested the hypothesis that the level of malonyl-CoA, as well as the corresponding rate of total fatty acid oxidation of the heart, is regulated by the opposing actions of acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD). We used isolated working rat hearts perfused under physiological conditions. MCD in heart homogenates was measured specifically by (14)CO(2) production from [3-(14)C]malonyl-CoA, and ACC was measured specifically based on the portion of total carboxylase that is citrate sensitive. Increased heart work (1 microM epinephrine + 40% increase in afterload) elicited a 40% increase in total beta-oxidation of exogenous plus endogenous lipids, accompanied by a 33% decrease in malonyl-CoA. The basal activity and citrate sensitivity of ACC (reflecting its phosphorylation state) and citrate content were unchanged. AMP levels were also unchanged. MCD activity, when measured at a subsaturating concentration of malonyl-CoA (50 microM), was increased by 55%. We conclude that physiological increments in AMP during the work transition are insufficient to promote ACC phosphorylation by AMP-stimulated protein kinase. Rather, increased fatty acid oxidation results from increased malonyl-CoA degradation by MCD.

Myocardial glucose uptake measured with fluorodeoxyglucose: a proposed method to account for variable lumped constants.

UNLABELLED: Quantitative assessment of myocardial glucose uptake by the glucose tracer analog 2-deoxy-2-[18F]fluoro-D-glucose (FDG) depends on a correction factor (lumped constant [LC]), which may vary. We propose that this variability is caused by different affinities of FDG and glucose for membrane transport and phosphorylation and can be predicted from the time course of FDG retention. We therefore measured the LC under steady-state metabolic conditions and compared the results with values predicted from the tracer retention alone. METHODS: We measured rates of myocardial glucose uptake by tracer ([2-3H]glucose) and tracer analog methods (FDG) in isolated working Sprague-Dawley rat hearts perfused with Krebs buffer and glucose, or glucose plus insulin or beta-hydroxybutyrate. In separate experiments, we established the theoretical upper and lower limits for the LC (Rt and Rp), which are determined by the relative rates of FDG and glucose membrane transport (Rt, 1.73 +/- 0.22) and the relative rates of FDG and glucose phosphorylation (Rp, 0.15 +/- 0.04). RESULTS: The LC was decreased in the presence of insulin or beta-hydroxybutyrate or both (from 1.14 +/- 0.3 to 0.58 +/- 0.16 [insulin], to 0.75 +/- 0.17 [beta-hydroxybutyrate] or to 0.53 +/- 0.17 [both], P < 0.05). The time-activity curves of FDG retention reflected these changes. Combining the upper and lower limits for the LC with the ratio between unidirectional and steady-state FDG uptake rates allowed the prediction of individual LCs, which agreed well with the actually measured values (r = 0.96, P < 0.001). CONCLUSION: The LC is not a constant but is a predictable quotient. As a result of the fixed relation between tracer and tracee for both membrane transport and phosphorylation, the quotient can be determined from the FDG time-activity curve and true rates of myocardial glucose uptake can be measured.

Insulin improves functional and metabolic recovery of reperfused working rat heart.

BACKGROUND: Glucose, insulin, and potassium solution improves left ventricular function in refractory pump failure. Direct effects of insulin on the heart cannot be determined in vivo. We hypothesized that insulin has a direct positive inotropic effect on the reperfused heart. METHODS: Isolated working rat hearts were perfused with buffer containing glucose (5 mmol/L) plus oleate (1.2 mmol/L). Hearts were subjected to 15 minutes of ischemia and reperfused with or without insulin (100 microU/mL) for 40 minutes. Epinephrine (1 micromol/L) was added for the last 20 minutes. RESULTS: Hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p < 0.05). Whereas oleate oxidation remained unchanged, glucose uptake and oxidation increased during reperfusion with epinephrine (p < 0.01). This increase was significantly greater when hearts were reperfused in the presence of insulin (p < 0.01). Insulin also prevented an epinephrine-induced glycogen breakdown during reperfusion (p < 0.05). CONCLUSIONS: Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine and occurs without delay. Increased rates of glucose oxidation and net glycogen synthesis are more protracted.

Insulin does not change the intracellular distribution of hexokinase in rat heart.

Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.

Regulation of energy metabolism of the heart during acute increase in heart work.

We determined the contribution of all major energy substrates (glucose, glycogen, lactate, oleate, and triglycerides) during an acute increase in heart work (1 microM epinephrine, afterload increased by 40%) and the involvement of key regulatory enzymes, using isolated working rat hearts exhibiting physiologic values for contractile performance and oxygen consumption. We accounted for oxygen consumption quantitatively from the rates of substrate oxidation, measured on a minute-to-minute basis. Total beta-oxidation (but not exogenous oleate oxidation) was increased by the work jump, consistent with a decrease in the level of malonyl-CoA. Glycogen and lactate were important buffers for carbon substrate when heart work was acutely increased. Three mechanisms contributed to high respiration from glycogen: 1) carbohydrate oxidation was increased selectively; 2) stimulation of glucose oxidation was delayed at glucose uptake; and 3) glycogen-derived pyruvate behaved differently from pyruvate derived from extracellular glucose. Despite delayed activation of pyruvate dehydrogenase relative to phosphorylase, glycogen-derived pyruvate was more tightly coupled to oxidation. Also, glycogen-derived lactate plus pyruvate contributed to an increase in the relative efflux of lactate versus pyruvate, thereby regulating the redox. Glycogen synthesis resulted from activation of glycogen synthase late in the protocol but was timed to minimize futile cycling, since phosphorylase a became inhibited by high intracellular glucose.

Energy provision from glycogen, glucose, and fatty acids on adrenergic stimulation of isolated working rat hearts.

We postulated that glycogen is a significant energy substrate compared with fatty acids and glucose in response to adrenergic stimulation of working rat hearts. Oxidation rates were determined at 1-min intervals by release of 3H2O from [9,10-(3)H]oleate (0.4 mM, 1% albumin) and 14CO2 from exogenous [U-14C]glucose (5 mM) or, by a pulse-chase method, from [14C]glycogen. We estimated the 14C enrichment of glycogen metabolized at each time point to determine true rates of glycogen use. Based on the pattern of glycogen enrichment over time, glycogenolysis did not exhibit a high degree of preference for newly synthesized glycogen. Epinephrine (1 microM) increased contractile performance 86% but did not stimulate oleate oxidation. The increased energy demand was supplied by carbohydrates, initially by a burst of glycogenolysis (contributing 35% to total ATP synthesis for 5 min) and followed by delayed increase in the use of exogenous glucose (eventually contributing 29% to ATP synthesis). On the basis of the release of 14CO2 and [14C]lactate specifically from glucose or glycogen, we found that a larger portion of glycogen was oxidized compared with exogenous glucose, augmenting the yield of ATP from glycogen. Thus the heart responds to an acute increase in energy demand by selective oxidation of glycogen.

Effects of insulin on glucose uptake by rat hearts during and after coronary flow reduction.

We tested the hypothesis that low-flow ischemia increases glucose uptake and reduces insulin responsiveness. Working hearts from fasted rats were perfused with buffer containing glucose alone or glucose plus a second substrate (lactate, octanoate, or beta-hydroxybutyrate). Rates of glucose uptake were measured by 3H2O production from [2-3H]glucose. After 15 min of perfusion at a physiological workload, hearts were subjected to low-flow ischemia for 45 min, after which they were returned to control conditions for another 30 min. Insulin (1 mU/ml) was added before, during, or after the ischemic period. Cardiac power decreased by 70% with ischemia and returned to preischemic values on reperfusion in all groups. Low-flow ischemia increased lactate production, but the rate of glucose uptake during ischemia increased only when a second substrate was present. Hearts remained insulin responsive under all conditions. Insulin doubled glucose uptake when added under control conditions, during low-flow ischemia, and at the onset of the postischemic period. Insulin also increased net glycogen synthesis in postischemic hearts perfused with glucose and a second substrate. Thus insulin stimulates glucose uptake in normal and ischemic hearts of fasted rats, whereas ischemia stimulates glucose uptake only in the presence of a cosubstrate. The results are consistent with two separate intracellular signaling pathways for hexose transport, one that is sensitive to the metabolic requirements of the heart and another that is sensitive to insulin.

Substrate metabolism as a determinant for postischemic functional recovery of the heart.

The mammalian myocardium meets its high energy needs through the oxidation of a variety of substrates, chiefly fatty acids. This review examines the hypothesis that efficient energy transfer in the heart occurs through a series of moiety-conserved cycles, which makes the heart an obligatory "omnivore." Ischemia results in a transformation of efficient metabolic cycles to less-efficient linear pathways. Substrate metabolism during reperfusion requires the replenishment of depleted cycles and is a major determinant for the return of contractile function. Although there is growing recognition of the concept that regulation of substrate flux through metabolic pathways is shared by several of the pathway enzymes it is apparent that glucose oxidation and glycogen resynthesis promote the return of normal contractile function in the postischemic heart. This concept is supported by clinical observations on the beneficial effects of a solution containing glucose, insulin, and potassium (GIK) for treatment of refractory left ventricular contractile failure after hypothermic ischemic arrest during cardiac surgery.

Regulation of exogenous and endogenous glucose metabolism by insulin and acetoacetate in the isolated working rat heart. A three tracer study of glycolysis, glycogen metabolism, and glucose oxidation.

Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 microU/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer (3H, 14C, and 13C) technique. Insulin-stimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms.

Preferential oxidation of glycogen in isolated working rat heart.

We tested the hypothesis that glycogen is preferentially oxidized in isolated working rat heart. This was accomplished by measuring the proportion of glycolytic flux (oxidation plus lactate production) specifically from glycogen which is metabolized to lactate, and comparing it to the same proportion determined concurrently from exogenous glucose during stimulation with epinephrine. After prelabeling of glycogen with either 14C or 3H, a dual isotope technique was used to simultaneously trace the disposition of glycogen and exogenous glucose between oxidative and non-oxidative pathways. Immediately after the addition of epinephrine (1 microM), 40-50% of flux from glucose was directed towards lactate. Glycogen, however, did not contribute to lactate, being almost entirely oxidized. Further, glycogen utilization responded promptly to the abrupt increase in contractile performance with epinephrine, during the lag in stimulation of utilization of exogenous glucose, suggesting that glycogen serves as substrate reservoir to buffer rapid increases in demand. Preferential oxidation of glycogen may serve to ensure efficient generation of ATP from a limited supply of endogenous substrate, or as a mechanism to limit lactate accumulation during rapid glycogenolysis.

Metabolic fate of glucose in reversible low-flow ischemia of the isolated working rat heart.

The acute adaptation of myocardial glucose metabolism in response to low-flow ischemia and reperfusion was investigated in isolated working rat hearts perfused with bicarbonate saline containing glucose (10 mM) and insulin (40 microU/ml). Reversible low-flow ischemia was induced by reducing coronary perfusion pressure from 100 to 35 cmH2O. Tritiated glucose was used to assess rates of glucose transport and phosphorylation, flux from glucose to pyruvate, and oxidation of exogenous glucose. Rates of glycogen synthesis and glycolysis were also assessed. With ischemia, cardiac power decreased by more than two-thirds. Rates of glucose uptake and flux from glucose to pyruvate remained unchanged, while glucose oxidation declined by 61%. Rates of lactate release more than doubled, and fractional enrichment of glycogen remained the same. During reperfusion, glucose oxidation returned to the preischemic values. When isoproterenol was added during ischemia, glucose uptake increased, glycogen decreased, and lactate release increased. No effect was seen with pacing. We conclude that during low-flow ischemia and with glucose as the only exogenous substrate, net glucose uptake remains unchanged. There is a reversible redirection between glycolysis and glucose oxidation, while glycogen synthesis continues during ischemia and is enhanced with reperfusion.

Fundamental limitations of [18F]2-deoxy-2-fluoro-D-glucose for assessing myocardial glucose uptake.

BACKGROUND: The glucose tracer analog [18F]2-deoxy-2-fluoro-D-glucose (FDG) is widely used for assessing regional myocardial glucose metabolism in vivo. The reproducibility of this method has recently been questioned because of a discordant affinity of hexokinase for its substrates glucose and 2-deoxyglucose. We therefore compared rates of glucose utilization simultaneously with tissue time-activity curves of FDG uptake before and after changes in the physiological environment of the heart. METHODS AND RESULTS: Isolated working rat hearts were perfused for 60 minutes with recirculating Krebs buffer containing glucose (10 mmol/L), FDG (1 microCi/mL), [2-3H]glucose (0.05 microCi/mL), and [U-14C]2-deoxyglucose (2-DG; 0.025 microCi/mL). Myocardial glucose uptake was measured by tracer ([2-3H]glucose) and tracer analog methods (FDG and 2-DG) before and after the addition of either insulin (1 mU/mL), epinephrine (1 mumol/L), lactate (40 mmol/L), or D,L-beta-hydroxybutyrate (40 mmol/L) at 30 minutes of perfusion and after acute changes in cardiac workload. Under steady-state conditions, myocardial rates of glucose utilization as measured by tritiated water (3H2O) production from metabolism of [2-3H]glucose, FDG uptake, and 2-DG retention were linearly related. The addition of competing substrates decreased glucose utilization immediately. The addition of insulin increased the rate of glucose utilization as measured by the glucose tracer but not as measured by the tracer analogs. The ratio of 3H2O release/myocardial FDG uptake increased by 111% after the addition of insulin, by 428% after the addition of lactate, and by 232% after the addition of beta-hydroxybutyrate. Epinephrine increased rates of glucose utilization and contractile performance, whereas there was no increase in glucose uptake with a comparable increase in workload alone. There was no change in the relation between the glucose tracer and the tracer analog either with epinephrine or with acute changes in workload. CONCLUSIONS: The uptake and retention of FDG in heart muscle is linearly related to glucose utilization only under steady-state conditions. Addition of insulin or of competing substrates changes the relation between uptake of the glucose tracer and FDG. These observations preclude the determination of absolute rates of myocardial glucose uptake by the tracer analog method under non-steady-state conditions.

Glycogen turnover in the isolated working rat heart.

The isolated working rat heart was adapted for simultaneous determination of glycogen synthesis and degradation using a dual isotope technique. After prelabeling of glycogen with [U-14C]glucose, glycogenolysis was determined continuously from the washout of 14CO2 plus [14C]lactate. Glycogen synthesis was determined during the same period from incorporation of [5-3H]glucose. In the absence of added hormones, hearts were predominantly glycogenolytic (1.5 mumol/min/g, dry weight), and there was simultaneous synthesis (11% of the rate of glycogenolysis). The percentage of glucose taken up by the heart that could traverse the glycogen pool as a consequence of glycogen turnover was minor (5%). Insulin (10 milliunits/ml) predictably stimulated glycogen synthesis (3.6-fold) and nearly abolished glycogenolysis. Addition of glucagon (1 microgram/ml) increased contractile performance and initially stimulated glycogenolysis (3.8-fold) until glycogen was largely depleted. Net tritium incorporation was unaffected by glucagon. Both hormones stimulated glycolytic flux from exogenous glucose (3H2O from [5-3H]glucose) as well as total glycolytic flux (3H2O plus glycogenolysis). The initial stimulation in total glycolytic flux with glucagon was largely from glycogen, explaining the lag in stimulation from exogenous glucose. The relationship between the specific radioactivity and amount of glycogen remaining after different degrees of glycogenolysis suggests that the preference of glycogenolysis for newly synthesized glycogen is only partial.

Dietary control and tissue specific expression of branched-chain alpha-ketoacid dehydrogenase kinase.

The branched-chain alpha-ketoacid dehydrogenase complex, catalyst for the rate-limiting step of branched-chain amino acid catabolism, is controlled by a highly specific protein kinase (branched-chain alpha-ketoacid dehydrogenase kinase) that associates tightly with the complex. The activity state (proportion of the enzyme in its active, dephosphorylated state) of the complex varies dramatically in different rat tissues. The activity state of the complex in the liver is greater than that in any other tissue, and liver contains the lowest amount of kinase protein and kinase mRNA. However, protein malnutrition, a condition under which the complex is largely phosphorylated and inactive, resulted in a three- to fourfold increase in hepatic kinase activity with an accompanying increase in amounts of kinase protein and mRNA. Refeeding a 50% protein diet restored the normal activity state and the original levels of kinase protein and mRNA. The amount of kinase protein associated with the complex rather than changes in specific activity of the kinase appears responsible for observed differences in activity states of the complex in several rat tissues tested. Accordingly, the levels of kinase protein and mRNA measured are highest in tissues with greatest kinase activity (heart > kidney > liver), correlating reasonably well inversely with activity state of the branched-chain alpha-ketoacid dehydrogenase complex in the respective tissues. These observations suggest that the amount of kinase protein expressed in various tissues and in response to dietary protein deficiency is an important factor determining the activity state of the complex.