Glucose and octanoate utilization by isolated adult rat heart cells

Rat heart muscle cells continue to beat in the isolated state apparently independent of any innervation. The oxidation of ¹⁴C-glucose to ¹⁴CO₂ was linear for at least 60 minutes of incubation. The rate of glucose oxidation rose rapidly up to a medium glucose concentration of 2.5 mM and then plateaued. Lactate production reached a maximum at 5 mM glucose. Glucose uptake was linearly related to the concentration up to 40 mM. The addition of octanoate reduced, but did not eliminate, glucose oxidation. Octanoate utilization increased with increasing concentration and reached a maximum at 2 mM. The oxidation of octanoate was linearly related to the time of incubation for at least 90 minutes. The presence of glucose, at a concentration of 1.25 mM or higher, increase the oxidation of octanoate by the heart cells. The metabolic parameters measured with the isolated heart cells gave values comparable to those obtained with the perfused rat heart. Decreasing or increasing the concentration of sodium, potassium or magnesium did not effect the oxidation of either glucose or octanoate with the exception that when sodium was increased above 200 mM, a significant increase in glucose oxidation was observed. In contrast, the addition of calcium to a calcium free medium increased glucose oxidation, reaching a maximum at 0.2 mM calcium. The oxidation of octanoate reached a maximum at 0.2 mM and then decreased significantly with increasing calcium concentration. The metabolic activity appears to be independent of the concentration of sodium, potassium or magnesium. In contrast, the isolated heart cell is very sensitive to a change in calcium concentration.     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

Stimulation of polyunsaturated fatty acid oxidation in myocytes by regulating its cellular uptake. On the rate limiting step of polyunsaturated fatty acid oxidation in heart.

In order to investigate the regulation of polyunsaturated fatty acid oxidation in the heart, the effect of the phosphodiesterase inhibitor enoximone on the oxidation of [1-14C] arachidonic acid, and [1-14C] arachidonyl-CoA, were studied in adult rat myocytes, and isolated rat heart mitochondria. Enoximone stimulated arachidonate oxidation by 94%, at a concentration of 0.25 mM. The apparent Vmax value of arachidonate oxidation in the presence of enoximone (6.98 nmol/mg protein/30 min), was approximately 75% higher than the value observed with the control (4.0 nmol/mg protein/30 min) in isolated myocytes. Also, enoximone stimulated arachidonate uptake by 27% at a concentration of 0.25 mM. On the other hand, enoximone had no effect on the oxidation of [1-14C] arachidonyl-CoA in isolated rat heart mitochondria. These results suggest that the oxidation of polyunsaturated fatty acids in myocytes is regulated by the rate of uptake of these acids across sarcolemmal membranes.     

Isolation of Ca2+-tolerant myocytes from adult rat heart

A procedure for the isolation of myocytes from adult rat hearts is described. It is based on successive treatments with Ca2+-free medium, disaggregating enzymes (collagenase and hyaluronidase) and mechanical agitation. Several recent isolation methods were compared and their best features were combined, together with some original modifications. A good yield of high purity myocytes with excellent morphological and functional integrity was obtained. The cells are tolerant to physiological concentrations of Ca2+. Cellular levels of ATP, Na+, and K+ are close to those in intact hearts and glucose oxidation rates and succinate exclusion are also close to normal. These characteristics are maintained for periods over 1 h.     

Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.     

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal andadult cardiomyocytes

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Palmitic and erucic acid metabolism in isolated perfused hearts from weanling pigs

Hearts from 4 week-old weanling pigs were capable of continuous work output when perfused with Krebs-Henseleit buffer containing 11 mM glucose. Perfused hearts metabolized either glucose or fatty acids, but optimum work output was achieved by a combination of glucose plus physiological concentrations (0.1 mM) of either palmitate or erucate. Higher concentrations of free fatty acids increased their rate of oxidation but also resulted in a large accumulation of neutral lipids in the myocardium, as well as a tendency to increased acetylation and acylation of coenzyme A and carnitine. When hearts were perfused with 1 mM fatty acids, the work output declined below control values. Erucic acid is known to be poorly oxidized by isolated rat heart mitochondria and, to a lesser degree, by perfused rat hearts. In addition, it has been reported that erucic acid acts as an uncoupler of oxidative phosphorylation. In isolated perfused pig hearts used in the present study, erucic acid oxidation rates were as high as palmitate oxidation rates. When energy coupling was measured by 31P-NMR, the steady-state levels of ATP and phosphocreatine during erucic acid perfusion did not change noticeably from those during glucose perfusion. It was concluded that the severe decrease in oxidation rates and ATP production resulting from the exposure of isolated pig and heart mitochondria to erucic acid are not replicated in the intact pig heart.     

Increased expression of SERCA in the hearts of transgenic mice results in increased oxidation of glucose

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wild-type and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.     

Fatty acid oxidation in human and rat heart. Comparison of cell-free and cellular systems

Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells.     

Regulation of glucose transport in Ca2+-tolerant myocytes from adult rat heart

Calcium-tolerant cardiac myocytes were isolated from adult rat ventricles and sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog 3-O-methyl-D-glucose in the presence and absence of Ca2+ in the incubation medium. (1) Agents which are known to increase internal Na+ and thus stimulate Ca2+ influx via Na+-Ca2+ exchange stimulated 3-methylglucose transport in the presence of external Ca2+. These include low-Na+ medium, 10(-6) M ouabain and K+-free medium, cyanide and the sodium ionophore, monensin. Hyperosmolarity stimulated transport also in the absence of Ca2+, consistent with release of Ca2+ from internal stores. Transport was decreased in a hypo-osmolar medium and with 10(-9) M ouabain, a concentration which stimulates the Na+ pump. (2) The calcium ionophore A23187 increased basal 3-methylglucose transport but opposed stimulation of transport by insulin. (3) Insulin-stimulated transport was antagonized by palmitate and this effect was reversed by 2-bromostearate, an inhibitor of fatty acid oxidation. These results are identical in all respects to those obtained in intact cardiac and skeletal muscle preparations, confirming that hexose transport in muscle shows Ca2+ dependence and indicating that isolated cardiac myocytes are suitable for the study of this phenomenon.     

Brain α-Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.     

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes

Ginsengs are widely used to improve cardiac health and circulation. Loosely termed as ginsengs, Asian (Panax), Siberian and Ashwagandha (Indian Ginseng) Indian ginsengs are prepared from different plants. We tested the popular belief of cardiotonic effects of ginsengs using both neonatal and adult rat cardiomyocytes, comparing extracts from the three ginsengs. Addition of 10% v/v of extract (100 microl of extract/ml of culture medium) of each of the ginsengs resulted in a rapid (<10 s) cessation of beating in neonatal cardiomyocytes due to calcium overload, while sequential dilutions revealed that treatment with a low dose (0.01% v/v, 0.1 microl/ml of the medium) resulted in constant, regular beats (transients), and a slight elevation of diastolic calcium without overload. Addition of extracts to sparking, calcium-tolerant adult cardiomyocytes resulted in initiation of calcium transients, and adult cells were able to tolerate exposure to high concentrations of extract. Cardiotonic effects in adult cells (cardiotoxicity in neonatal cells) were most profound with Asian ginseng (2.6 times that of Siberian ginseng, 1.6 times that of Indian ginseng) probably due to the active ingredients (ginsenosides in Asian, eleutherosides in Siberian and withanolides in Indian) being structurally different. We conclude that fully developed cardiomyocytes are able to accommodate higher doses of ginseng than neonatal cells, and that the effects of ginseng on newly formed, developing myocytes, could be extremely deleterious to the fetus. However, for adults, ginseng might well be a 'tonic' in its ability to increase beating and intramyocytic calcium levels.     

Impact of lactate in the perfusate on function and metabolic parameters of isolated working rat heart

The goal of this study was to investigate the effect of 1 mM exogenous lactate on cardiac function, and some metabolic parameters, such as glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation, in isolated working rat hearts. Hearts from male Sprague-Dawley rats were isolated and perfused with 5 mM glucose, 1.2 mM palmitate, and 100 μU/ml insulin with or without 1 mM lactate. The rates of glycolysis, glucose, lactate, and fatty acid oxidation were determined by supplementing the buffer with radiolabeled substrates. Cardiac function was similar between lactate+ and lactate− hearts. Glycolysis was not affected by 1 mM lactate. The addition of lactate did not alter glucose oxidation rates. Interestingly, palmitate oxidation rates almost doubled when 1 mM lactate was present in the perfusate. This study suggests that subst rate supply to the heart is crucially important when evaluating the data from metabolic studies.     

The effects of amylin on carbohydrate metabolism in skeletal muscle in vitro and in vivo.

1. The effects of synthetic human amylin on basal and insulin-stimulated (100 and 1000 microunits/ml) rates of lactate formation, glucose oxidation and glycogen synthesis were measured in the isolated rat soleus muscle preparation incubated in the presence of various concentrations of glucose (5, 11 and 22 mM). 2. The rate of glucose utilization was increased by about 2-fold by increasing the glucose concentration from 5 to 22 mM. 3. Synthetic human amylin (10 nM) significantly inhibited (by 46-56%) glycogen synthesis, irrespective of the concentration of insulin or glucose present in the incubation medium. 4. Amylin (10 nM) did not affect insulin-stimulated rates of 2-deoxy[3H]glucose transport and phosphorylation. 5. Intraperitoneal administration of insulin (100 micrograms/kg) to rats in vivo stimulated the rate of [U-14C]glucose incorporation into glycogen in the diaphragm by about 80-fold. This rate was decreased (by 28%) by co-administration of amylin (66 micrograms/kg).     

Structural and functional properties of adult rat heart myocytes lysed with digitonin

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.     

Stimulation of renal gluconeogenesis by angiotensin II.

Renal gluconeogenesis was studied in suspended tubule fragments isolated by collagenase treatment of rat kidney cortices. Angiotensin II increased glucose formation from pyruvate, lactate, and to a lesser extent from oxoglutarate and glutamine, but not from other substrates such as malate, succinate, dihydroxyacetone or fructose. Stimulation was significant with peptide concentration exceeding 1 . 10(-8) M and was also shown with an 8-Sar derivative. Other peptides such as 4-Ala-8-Ile-angiotensin II, hexapeptide and bradykinin had no effect. The stimulatory action of angiotensin II was additive to that of L-lysine, and 3',5'-adenosine cyclic monophosphate, suggesting a different mechanism of action. In the presence of maximally stimulatory concentrations of oleate, phenylephrine and 3',5'-guanosine cyclic monophosphate, however, the stimulatory effect of angiotensin II was absent. Cyclic GMP levels, however, did not increase in tubules after angiotensin II and phenylephrine addition, making a messenger function of this nucleotide unlikely. Omission of Ca2+ from the medium markedly reduced basal gluconeogenesis but did not result in a complete loss of angiotensin II effect. Reduction of medium potassium to 2 mM, however, increased basal gluconeogenesis and blunted the peptide effect. 1 mM ouabain was also able to inhibit the stimulatory effect of angiotensin II. Therefore changes in intracellular potassium levels are discussed as a possible mechanism of angiontensin action, whereas calcium seems not to be specifically linked to this metabolic action of angiotensin on the proximal tubule.     

Diabetic cardiomyocyte dysfunction and myocyte insulin resistance: Role of glucose-induced PKC activity

Increased protein kinase C (PKC) activity has been implicated in the pathogenesis of a number of diabetic complications, and high concentrations of glucose have been shown to increase PKC activity. The present study was designed to examine the role of PKC in diabetes-induced (and glucose-induced) cardiomyocyte dysfunction and insulin resistance (measured by glucose uptake). Adult rat ventricular myocytes were isolated from nondiabetic and type 1 diabetic animals (4-5 days post-streptozotocin treatment), and maintained overnight, with/without the nonspecific PKC inhibitor chelerythrine (CHEL = 1 microM). Myocyte mechanical properties were evaluated using a video edge-detection system. Basal and insulin-stimulated glucose uptake was measured with [3H]-2-deoxyglucose. Blunted insulin-stimulated glucose uptake was apparent in diabetic myocytes, and both mechanical dysfunctions (e.g., slowed shortening/relengthening) and insulin resistance were maintained in culture, and normalized by CHEL. Cardiomyocytes isolated from nondiabetic animals were cultured in a high concentration of glucose (HG = 25.5 mM) medium, with/without CHEL. HG myocytes exhibited slowed shortening/relengthening and impaired insulin-stimulated glucose uptake compared to myocytes cultured in normal glucose (5.5 mM), and both impairments were prevented by culturing cells in CHEL. Our data support the view that PKC activation contributes to both diabetes-induced abnormal cardiomyocyte mechanics and insulin resistance, and that elevated glucose is sufficient to induce these effects.     

Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture

A self-regulatory mechanism of the glucose transport in rat skeletal muscle cells is described. In isolated rat soleus muscles and rat skeletal myocytes and myotubes in culture, pre-exposure to varying glucose concentrations modulated the rate of 2-deoxyglucose uptake. Maximal uptake was observed at glucose concentrations below 3 mM. Between 2.5 and 4.0 mM glucose it was reduced by 25-35%; further elevation of the glucose concentration resulted in a gradual decrease of the transport rate by approximately 2% for each millimolar glucose. The effect of glucose was time-dependent and fully reversible. Insulin rapidly increased the 2-deoxyglucose uptake in the soleus muscle; however, the insulin effect depended on the glucose concentration of the preincubation. Insulin was totally ineffective in muscles pre-exposed to 1.0-3.0 mM glucose, whereas its stimulatory action increased with increasing glucose concentrations above 4 mM. The effect of low glucose and insulin were not additive, and the maximal 2-deoxyglucose uptake rates induced by both conditions were of identical magnitude. It is postulated that glucose may "up- and down-regulate" its transport by affecting the number of active glucose transporters in the plasma membrane, and that insulin exerts its stimulatory effect only when the extracellular glucose reaches a threshold concentration.