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We have reported previously that hearts from type 2 diabetic (db/db) mice show decreased cardiac efficiency due to increased work-independent myocardial [O.sub.2] consumption (unloaded MV[o.sub.2]), indicating higher [O.sub.2] use for nonmechanical processes such as basal metabolism (MV[o.sub.2BM]) and excitationcontraction coupling (MV[o.sub.2ECC]). Although alterations in cardiac metabolism and/or [Ca.sup.2+] handling may contribute to increased energy expenditure in diabetic hearts, direct measurements of the [O.sub.2] cost for these individual processes have not been determined. In this study, we 1) validate a procedure for measuring unloaded MV[o.sub.2] directly (MV[o.sub.2unloaded]) and for determining MV[o.sub.2BM] and MV[o.sub.2ECC] separately in isolated perfused mouse hearts and 2) determine 02 cost for these processes in hearts from db/db mice. Unloaded MV[o.sub.2], extrapolated from the relationship between cardiac work (measured as pressure-volume area, PVA) and MV[o.sub.2], was found to correspond with MV[o.sub.2] measured directly in unloaded retrograde perfused hearts (MV[o.sub.2unloaded]). MV[o.sub.2] in [K.sup.+]-arrested hearts was defined as MV[o.sub.2BM]; the difference between MV[o.sub.2unloaded] and MV[o.sub.2BM] represented MV[o.sub.2ECC]. This procedure was validated by demonstrating that elevations in perfusate fatty acid (FA) and/or [Ca.sup.2+] concentrations resulted in changes in either MV[o.sub.2BM] and/or MV[o.sub.2ECC]. The higher MV[o.sub.2unloaded] in db/db mice was due to both a higher MV[o.sub.2BM] and MV[o.sub.2ECC]. Elevation of glucose and insulin decreased FA oxidation and reduced both MV[o.sub.2unloaded] and MV[o.sub.2BM]. In conclusion, this study provides direct evidence that MV[o.sub.2BM] and MV[o.sub.2ECC] are elevated in diabetes and that acute metabolic interventions can have a therapeutic benefit in diabetic hearts due to a MV[o.sub.2]-lowering effect. cardiac efficiency; pressure-volume area; myocardial oxygen consumption; substrate oxidation

Diabetic (db/ db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult. myocardial metabolism; insulin action; glucose and fatty acid metabolism

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-[alpha], follicle-stimulating hormone (FSH)-[beta] and LH-[beta] mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-Otetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-[alpha]-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activatorinduced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary. pituitary; gene expression; goldfish; gonadotropes; neuroendocrinology; protein kinase C

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the [[alpha].sub.1]-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-[gamma] agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment hut elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the over-expression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes. mice; microvascular function; gene expression; sarcolemmal membrane-associated protein

In the present study, we tested the reliability of measurements of pressure-volume area (PVA) and oxygen consumption (MV[o.sub.2]) in ex vivo mouse hearts, combining the use of a miniaturized conductance catheter and a fiber-optic oxygen sensor. Second, we tested whether we could reproduce the influence of increased myocardial fatty acid (FA) metabolism on cardiac efficiency in the isolated working mouse heart model, which has already been documented in large animal models. The hearts were perfused with crystalloid buffer containing 11 mM glucose and two different concentrations of FA bound to 3% BSA. The initial concentration was 0.3 [+ or -] 0.1 mM, which was subsequently raised to 0.9 [+ or -] 0.1 mM. End-systolic and end-diastolic pressure-volume relationships were assessed by temporarily occluding the preload line. Different steadystate PVA-MV[o.sub.2] relationships were obtained by changing the loading conditions (pre- and afterload) of the heart. There were no apparent changes in baseline cardiac performance or contractile efficiency (slope of the PVA-MV[o.sub.2] regression line) in response to the elevation of the perfusate FA concentration. However, all hearts (n = 8) showed an increase in the y-intercept of the PVA-MV[o.sub.2] regression line after elevation of the palmitate concentration, indicating an FA-induced increase in the unloaded MV[o.sub.2]. Therefore, in the present model, unloaded MV[o.sub.2] is not independent of metabolic substrate. This is, to our knowledge, the first report of a PVA-MV[o.sub.2] relationship in ex vivo perfused murine hearts, using a pressure-volume catheter. The methodology can be an important tool for phenotypic assessment of the relationship among metabolism, contractile performance, and cardiac efficiency in various mouse models. oxygen consumption; pressure-volume area; cardiac metabolism; compliance; steady state

Diabetic dh/db mice exhibit profound insulin resistance in vivo, but the specific degree of cardiac insensitivity to insulin has not been assessed. Therefore, the effect of insulin on cardiomyocytes from db/db hearts was assessed by measuring two metabolic responses (deoxyglucose uptake and fatty acid oxidation) and the phosphorylation of two enzymes in the insulin-signaling cascade [Akt and AMP-activated protein kinase (AMPK)]. Maximal insulin-stimulated deoxyglucose transport was reduced to 58 and 40% of control in cardiomyocytes from db/db mice at two ages (6 and 12 wk). Insulin-stimulated deoxyglucose uptake was also reduced in myocytes from transgenic db/db mice overexpressing the insulin-sensitive glucose transporter (db/db-hGLUT4). Treatment of db/db mice for 1 wk with an insulin-sensitizing peroxisome proliferator-activated receptor-[gamma] agonist (COOH) completely normalized insulin-stimulated deoxyglucose uptake. Insulin had no direct effect on palmitate oxidation by either control or db/db cardiomyocytes, but the combination of insulin and glucose reduced palmitate oxidation, likely an indirect effect secondary to increased glucose uptake. Insulin had no effect on AMPK phosphorylation from either control or db/db cardiomyocytes. Insulin increased the phosphorylation of Akt in all cardiomyocyte preparations (control db/db. COOH-treated db/db) to the same extent. Thus insulin has selective metabolic actions in mouse cardiomyocytes: deoxyglucose uptake and Akt phosphorylation are increased, but fatty acid oxidation and AMPK phosphorylation are unchanged. Insulin resistance in db/db cardiomyocytes is manifested by reduced insulin-stimulated deoxyglucose uptake. cardiac metabolism; glucose uptake; fatty acid oxidation

Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphennxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-[gamma] that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward [K.sup.+] currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization. diabetic cardiomyopathy; cardiac metabolism

Hydrolysis of triacylglycerols (TG) in circulating chylomicrons by endothelium-bound lipoprotein lipase (LPL) provides a source of fatty acids (FA) for cardiac metabolism. The effect of diabetes on the metabolism of chylomicrons by perfused mouse hearts was investigated with db/db (type 2) and streptozotocin (STZ)-treated (type 1) diabetic mice. Endothelium-bound heparin-releasable LPL activity was unchanged in both type 1 and type 2 diabetic hearts. The metabolism of LPL-derived FA was examined by perfusing hearts with chylomicrons containing radiolabeled TG and by measuring [sup.3][H.sub.2]O accumulation in the perfusate (oxidation) and incorporation of radioactivity into tissue TG (esterification). Rates of LPL-derived FA oxidation and esterification were increased 2.3-fold and 1.7-fold in db/db hearts. Similarly, LPL-derived FA oxidation and esterification were increased 3.4-fold and 2.5-fold, respectively, in perfused hearts from STZ-treated mice. The oxidation and esterification of [[sup.3]H]palmitate complexed to albumin were also increased in type 1 and type 2 diabetic hearts. Therefore, diabetes may not influence the supply of LPL-derived FA, but total FA utilization (oxidation and esterification) was enhanced. diabetic cardiomyopathy; lipoprotein lipase; fatty acid metabolism

Control db/+ and diabetic db/db mice at 6 and 12 wk of age were subjected to echocardiography to determine whether contractile function was reduced in vivo and restored in transgenic db/db-human glucose transporter 4 (hGLUT4) mice (12 wk old) in which cardiac metabolism has been normalized. Systolic function was unchanged in 6-wk-old db/db mice, but fractional shortening and velocity of circumferential fiber shortening were reduced in 12-wk-old db/db mice (43.8 [+ or -] 2.1% and 8.3 [+ or -] 0.5 circs/s, respectively) relative to db/+ control mice (59.5 [+ or -] 2.3% and 11.8 [+ or -] 0.4 circs/s, respectively). Doppler flow measurements were unchanged in 6-wk-old db/db mice. The ratio of E and A transmitral flows was reduced from 3.56 [+ or -] 0.29 in db/+ mice to 2.40 [+ or -] 0.20 in 12-wk-old db/db mice, indicating diastolic dysfunction. Thus a diabetic cardiomyopathy with systolic and diastolic dysfunction was evident in 12-wk-old diabetic db/db mice. Cardiac function was normalized in transgenic db/db-hGLUT4 mice, indicating that altered cardiac metabolism can produce contractile dysfunction in diabetic db/db hearts. echocardiography; human glucose transporter 4

Hearts from diabetic db/db mice, a model of Type 2 diabetes, exhibit left ventricular failure and altered metabolism of exogenous substrates. Peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha] ligands reduce plasma lipid and glucose concentrations and improve insulin sensitivity in db/db mice. Consequently, the effect of 4- to 5-wk treatment of db/db mice with a novel PPAR-[alpha] ligand (BM 17.0744; 25-38 mg*[kg.sup.-1]*[day.sup.-1]), commencing at 8 wk of age, on ex vivo cardiac function and metabolism was determined. Elevated plasma concentrations of glucose, fatty acids, and triacylglycerol (34.0 [+ or -] 3.6, 2.0 [+ or -] 0.4, and 0.9 [+ or -] 0.1 mM, respectively) were reduced to normal after treatment with BM 17.0744 (10.8 [+ or -] 0.6, 1.1 [+ or -] 0.1, and 0.6 + 0.1 mM). Plasma insulin was also reduced significantly in treated compared with untreated db/db mice. Chronic treatment of db/db mice with the PPAR-[alpha] agonist resulted in a 50% reduction in rates of fatty acid oxidation, with a concomitant increase in glycolysis (1.7-fold) and glucose oxidation (2.3-fold). Correction of the diabetes-induced abnormalities in systemic and cardiac metabolism after BM 17.0744 treatment did not, however, improve left ventricular contractile function. substrate oxidation; isolated working mouse hearts

A study was conducted on the influence fatty acid and glucose concentrations on lipoprotein lipase activity in cultured cardiomyocytes from adult rat hearts. Results reveal that glucose did not effect either cellular- or heparin-releasable-lipoprotein lipase activities. Results, however, show that fatty acids selectively decrease heparin-releasable lipoprotein lipase activity in cardomyocytes.

Endogenous diacylglycerols (DAG) that are generated in cultured A10 smooth muscle cells (SMCs) by phosphatidylcholine-phospholipase C (PC-PLC) undergo hydrolysis via lipase pathway. Phosphatidylinositol-derived and PC-derived DAGs in the cell membrane of A10 SMCs are hydrolized by DAG lipase to form monoacylglycerols (MG) and fatty acids (FA). Hydrolysis by DAG lipase was detected as the principal fate of DAG while MG and FA are the main products of DAG hydrolysis.

Hydrolysis of phosphatidylinositol biphosphate (PIP2) by a phospholipase C yields the intracellular second messenger diacylglycerol (DAG) that stimulates protein kinase C (PKC). DAG collects in a biphasic manner in vascular smooth muscles, a transient phase resulting from the hydrolysis of PIP2 and a prolonged phase of collection due to phosphatidylcholine degradation. PKC induction through DAG metabolism leads to signal transduction.

Cultured A10 smooth muscle cells metabolized a long-chain diacyclglycerol (DAG), 1-palmitoyl-2(1-14C)oleoyl-sn-glycerol (2-(14C)POG) after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. Control A10 cells, not treated with alpha-toxin, did not reveal much 2-(14C)POG metabolism. Metabolism of cell-permeable DAG analogue was not changed when A10 cells were treated with alpha-toxin. Radioactivity was included in the products of the lipase pathway, monoacylgylcerol and fatty acids caused by the metabolism of 2-(14C)POG due to alpha-toxin treated A10 cells. Involvement of the lipase pathway in the metabolism of a long-chain DAG, 2-(14C)POG and A10 cells is identical to the results of degeneration of (3H)diC8 by A10 cells.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate gene expression of lipoprotein lipase (LPL) in liver and adipose tissue. We examined the direct effect of PPAR-[Alpha], ligands on LPL catalytic activity in cultured cardiomyocytes from adult rat heart. After overnight culture (16 h), 1 [micro]M Wy-14643 and 10 [micro]M BM-17.0744 decreased total cellular LPL activity to ~50% of control with no change in enzyme synthesis or mass; as a consequence, PPAR-[Alpha], activation produced a significant decrease in LPL specific activity (mU/ng LPL protein). Wy-14643 and BM-17.0744 also reduced heparin-releasable LPL activity and mass in the culture medium. Inhibition of LPL activity by Wy-14643 did not reduce the ability of insulin plus dexamethasone to stimulate cellular and heparin-releasable LPL activities. A similar inhibitory effect on cellular and heparin-releasable LPL activity was observed when cardiomyocytes were cultured with 60 [micro]M linoleic acid. In conclusion, two different PPAR-[Alpha], ligands (Wy-14643 and BM-17.0744) inhibited cellular LPL activity in cultured cardiomyocytes by a posttranscriptional and posttranslational mechanism. lipoprotein metabolism; fatty acids; Wy-14643; BM-17.0744

The catabolism of rat chylomicrons, labeled in their triacylglycerol (TG) component, was investigated using perfused working mouse hearts. Perfusion of mouse hearts with heparin increased lipoprotein lipase (LPL) activity in the perfusate. This heparin-releasable LPL pool remained constant over a variety of experimental conditions, including workload and fatty acid concentrations, making the mouse heart a suitable model to study chylomicron catabolism. Endothelium-bound LPL hydrolyzed radiolabeled [sup.3H]-labeled chylomicrons (0.4 mM TG); the fate of LPL-derived [sup.3H]-labeled fatty acids was split evenly between oxidation (production of [[sup.3][H.sub.2]]O) and esterification (incorporation into tissue lipids, mainly TG). In comparison, the oxidation of 0.4 mM [sup.3H]palmitate complexed to albumin was fourfold greater than esterification into tissue lipids. Surprisingly, the addition of unlabeled palmitate (0.4 or 1.2 mM) to perfusions with [sup.3H]-chylomicrons did not affect the fate (either oxidation or esterification) of LPL-derived [sup.3H]-fatty acids. These results suggest that fatty acids produced from lipoprotein hydrolysis by the action of LPL and fatty acids from a fatty acid-albumin complex do not enter a common metabolic pool in the heart. lipoprotein lipase; fatty acid metabolism

Belke, Darrel D., Terje S. Larsen, E. Michael Gibbs, and David L. Severson. Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter. Am J Physiol Endocrinol Metab 280: E420-E427, 2001.--Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m +/ +db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate. glycolysis; glucose oxidation; glycogen; palmitate oxidation; glucose transporter 4 transgenic mice

Belke, Darrell D., Terje S. Larsen, E. Michael Gibbs, and David L. Severson. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice. Am J Physiol Endocrinol Metab 279: E1104-E1113, 2000.--Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-[lepr.sup.db]/[lepr.sup.db] (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-sub.3]H]glucose, [U-sup.14]C]glucose, and [9,[10-sup.3]H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts. diabetic cardiomyopathy

Belke, Darrell D., Terje S. Larsen, Gary D. Lopaschuk, and David L. Severson. Glucose and fatty acid metabolism in the isolated working mouse heart. Am. J. Physiol. 277 (Regulatory Integrative Comp. Physiol. 46): R1210-R1217, 1999.--Although isolated perfused mouse heart models have been developed to study mechanical function, energy substrate metabolism has not been examined despite the expectation that the metabolic rate for a heart from a small mammal should be increased. Consequently, glucose utilization (glycolysis, oxidation) and fatty acid oxidation were measured in isolated working mouse hearts perfused with radio-labeled substrates, 11 mM glucose, and either 0.4 or 1.2 mM palmitate. Heart rate, coronary flow, cardiac output, and cardiac power did not differ significantly between hearts perfused at 0.4 or 1.2 mM palmitate. Although the absolute values obtained for glycolysis and glucose oxidation and fatty acid oxidation are significantly higher than those reported for rat hearts, the pattern of substrate metabolism in mouse hearts is similar to that observed in hearts from larger mammals. The metabolism of mouse hearts can be altered by fatty acid concentration in a manner similar to that observed in larger animals; increasing palmitate concentration altered the balance of substrate metabolism to increase overall energy derived from fatty acids from 64 to 92%. glycolysis; glucose oxidation; fatty acid oxidation