Heart-type fatty acid-binding protein reciprocally regulates glucose and fatty acid utilization during exercise

The role of heart-type cytosolic fatty acid-binding protein (H-FABP) in mediating whole body and muscle-specific long-chain fatty acid (LCFA) and glucose utilization was examined using exercise as a phenotyping tool. Catheters were chronically implanted in a carotid artery and jugular vein of wild-type (WT, n = 8), heterozygous (H-FABP(+/-), n = 8), and null (H-FABP(-/-), n = 7) chow-fed C57BL/6J mice, and mice were allowed to recover for 7 days. After a 5-h fast, conscious, unrestrained mice were studied during 30 min of treadmill exercise (0.6 mph). A bolus of [(125)I]-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid and 2-deoxy-[(3)H]glucose was administered to obtain rates of whole body metabolic clearance (MCR) and indexes of muscle LCFA (R(f)) and glucose (R(g)) utilization. Fasting, nonesterified fatty acids (mM) were elevated in H-FABP(-/-) mice (2.2 +/- 0.9 vs. 1.3 +/- 0.1 and 1.3 +/- 0.2 for WT and H-FABP(+/-)). During exercise, blood glucose (mM) increased in WT (11.7 +/- 0.8) and H-FABP(+/-) (12.6 +/- 0.9) mice, whereas H-FABP(-/-) mice developed overt hypoglycemia (4.8 +/- 0.8). Examination of tissue-specific and whole body glucose and LCFA utilization demonstrated a dependency on H-FABP with exercise in all tissues examined. Reductions in H-FABP led to decreasing exercise-stimulated R(f) and increasing R(g) with the most pronounced effects in heart and soleus muscle. Similar results were seen for MCR with decreasing LCFA and increasing glucose clearance with declining levels of H-FABP. These results show that, in vivo, H-FABP has reciprocal effects on glucose and LCFA utilization and whole body fuel homeostasis when metabolic demands are elevated by exercise.     

Effect of heterozygous PPARgamma deficiency and TZD treatment on insulin resistance associated with age and high-fat feeding

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is the target receptor for thiazolidinedione (TZD) compounds, which are a class of insulin-sensitizing drugs used in the treatment of type 2 diabetes. Paradoxically, however, mice deficient in PPARgamma (PPARgamma(+/-)) are more insulin sensitive than their wild-type (WT) littermates, not less, as would be predicted. To determine whether PPARgamma deficiency could prevent the development of the insulin resistance associated with increasing age or high-fat (HF) feeding, insulin sensitivity was assessed in PPARgamma(+/-) and WT mice at 2, 4, and 8 mo of age and in animals fed an HF diet. Because TZDs elicit their effect through PPARgamma receptor, we also examined the effect of troglitazone (a TZD) in these mice. Glucose metabolism was assessed by hyperinsulinemic euglycemic clamp and oral glucose tolerance test. Insulin sensitivity declined with age for both groups. However, the decline in the PPARgamma(+/-) animals was substantially less than that of the WT animals, such that, by 8 mo of age, the PPARgamma(+/-) mice were markedly more insulin sensitive than the WT mice. This greater sensitivity in PPARgamma(+/-) mice was lost with TZD treatment. HF feeding led to marked adipocyte hypertrophy and peripheral tissue and hepatic insulin resistance in WT mice but also in PPARgamma(+/-) mice. Treatment of these mice with troglitazone completely prevented the adipocyte hypertrophy and normalized insulin action. In conclusion, PPARgamma deficiency partially protects against age-related insulin resistance but does not protect against HF diet-induced insulin resistance.     

Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Exercise training impacts the myocardial metabolism of older individuals in a gender-specific manner

Aging is associated with decreases in aerobic capacity, cardiac function, and insulin sensitivity as well as alterations in myocardial substrate metabolism. Endurance exercise training (EET) improves cardiac function in a gender-specific manner, and EET has been shown to improve whole body glucose tolerance, but its effects on myocardial metabolism are unclear. Accordingly, we studied the effect of EET on myocardial substrate metabolism in older men and women. Twelve healthy older individuals (age: 60-75 yr; 6 men and 6 women) underwent PET with [(15)O]water, [(11)C]acetate, [(11)C]glucose, and [(11)C]palmitate for the assessment of myocardial blood flow (MBF), myocardial O(2) consumption (MVo(2)), myocardial glucose utilization (MGU), and myocardial fatty acid utilization (MFAU), respectively, at rest and during dobutamine infusion (10 microg.kg(-1).min(-1)). Measurements were repeated after 11 mo of EET. Maximal O(2) uptake (Vo(2max)) increased (P = 0.005) after EET. MBF was unaffected by training, as was resting MVo(2); however, posttraining dobutamine MVo(2) was significantly higher (P = 0.05), as was MGU (P < 0.04). Although overall dobutamine MFAU was unchanged, posttraining dobutamine MFAU increased in women (P = 0.01) but decreased in men (P = 0.03). Thus, EET in older individuals improves the catecholamine response of myocardial glucose metabolism. Moreover, gender differences exist in the myocardial fatty acid metabolic response to training. These findings suggest a role for altered myocardial substrate metabolism in modulating the cardiovascular benefits of EET in older individuals.     

Reduced intestinal fat absorptive capacity but enhanced susceptibility to diet-induced fatty liver in mice heterozygous for ApoB38.9 truncation

Fatty liver is prevalent in apolipoprotein B (apoB)-defective familial hypobetalipoproteinemia (FHBL). Similar to humans, mouse models of FHBL produced by gene targeting (apob(+/38.9)) manifest low plasma cholesterol and increased hepatic triglycerides (TG) even on a chow diet due to impaired hepatic VLDL-TG secretive capacity. Because apoB truncations shorter than apoB48 are expressed in the intestine, we examined whether FHBL mice may have limited capacity for intestinal dietary TG absorption. In addition, we investigated whether FHBL mice are more susceptible to diet-induced hepatic TG accumulation. Fat absorption capacity was impaired in apoB38.9 mice in a gene dose-dependent manner. Relative fractional fat absorption coefficients for apob(+/+), apob(+/38.9), and apob(38.9/38.9) were 1.00, 0.96, and 0.71, respectively. To raise hepatic TG, we fed high-fat (HF) and low-fat (LF) pellets. Hepatic TG level was observed in rank order: HF > LF > chow. On both LF and HF, liver TG level was higher in the apob(+/38.9) than in apob(+/+). Hepatic TG secretion remained impaired in the apob(+/38.9) on the HF diet. Thus the FHBL mice are more susceptible to diet-induced fatty liver despite relatively reduced intestinal TG absorption capacity on a HF diet.     

Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation

When recovering from heart failure (HF), the myocardium displays a marked plasticity and can regain normal gene expression and function; however, recovery of substrate oxidation capacity has not been explored. We tested whether cardiac functional recovery is matched by normalization of energy substrate utilization during post-HF recovery. HF was induced in dogs by pacing the left ventricle (LV) at 210-240 beats/min for 4 wk. Tachycardia was discontinued, and the heart was allowed to recover. An additional group was studied in HF, and healthy dogs served as controls (n = 8/group). Cardiac free fatty acids (FFAs) and glucose oxidation were measured with [3H]oleate and [14C]glucose. At 10 days of recovery, hemodynamic parameters returned to control values; however, the contractile response to dobutamine remained depressed, LV end-diastolic volume was 28% higher than control, and the heart mass-to-body mass ratio was increased (9.8 +/- 0.4 vs. 7.5 +/- 0.2 g/kg, P < 0.05). HF increased glucose oxidation (76.8 +/- 19.7 nmol.min(-1).g(-1)) and decreased FFA oxidation (20.7 +/- 6.4 nmol.min(-1).g(-1)), compared with normal dogs (24.5 +/- 6.3 and 51.7 +/- 9.6 nmol.min(-1).g(-1), respectively), and reversed to normal values at 10 days of recovery (25.4 +/- 6.0 and 46.6 +/- 6.7 nmol.min(-1).g(-1), respectively). However, similar to HF, the recovered dogs failed to increase glucose and fatty acid uptake in response to pacing stress. The activity of myocardial citrate synthase and aconitase was significantly decreased during recovery compared with that in control dogs (58 and 27% lower, respectively, P < 0.05), indicating a persistent reduction in mitochondrial oxidative capacity. In conclusion, cardiac energy substrate utilization is normalized in the early stage of post-HF recovery at baseline, but not under stress conditions.     

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C)ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.     

Effect of modulating cardiac A1 adenosine receptor expression on protection with ischemic preconditioning

Activation of A(1) adenosine receptors (A(1)ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological A(1)AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified A(1)AR expression to define the role of A(1)AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted (A(1)KO(-/-)) or single-copy (A(1)KO(+/-)) A(1)AR, and transgenic mice (A(1)TG) with increased cardiac A(1)AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, A(1)KO(+/-), and A(1)KO(-/-) mice was (in % of risk region) 58 +/- 3, 60 +/- 4, and 61 +/- 2, respectively, and was less in A(1)TG mice (39 +/- 4, P < 0.05). A strong correlation was observed between A(1)AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 +/- 3, P < 0.05 vs. WT), A(1)KO(+/-) + IPC (48 +/- 4, P < 0.05 vs. A(1)KO(+/-)), and A(1)TG + IPC mice (24 +/- 2, P < 0.05 vs. A(1)TG). However, IPC did not decrease IF in A(1)KO(-/-) + IPC mice (63 +/- 2). In addition, A(1)KO(-/-) hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that A(1)ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of A(1)AR gene expression.     

Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance

Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3(-/-) mice exhibit extreme cold sensitivity despite induction of uncoupling and oxidative genes and hydrolysis of brown adipose tissue lipid stores. However, using FABP3 gain- and loss-of-function approaches in brown adipocytes, we detected a correlation between FABP3 levels and the utilization of exogenous fatty acids. Thus, Fabp3(-/-) brown adipocytes fail to oxidize exogenously supplied fatty acids, whereas enhanced Fabp3 expression promotes more efficient oxidation. These results suggest that FABP3 levels are a determinant of fatty acid oxidation efficiency by brown adipose tissue and that FABP3 represents a potential target for modulation of energy dissipation.     

Control of exercise-stimulated muscle glucose uptake by GLUT4 is dependent on glucose phosphorylation capacity in the conscious mouse

Previous work suggests that normal GLUT4 content is sufficient for increases in muscle glucose uptake (MGU) during exercise because GLUT4 overexpression does not increase exercise-stimulated MGU. Instead of glucose transport, glucose phosphorylation is a primary limitation of exercise-stimulated MGU. It was hypothesized that a partial ablation of GLUT4 would not impair exercise-stimulated MGU when glucose phosphorylation capacity is normal but would do so when glucose phosphorylation capacity was increased. Thus, C57BL/6J mice with hexokinase II (HKII) overexpression (HK(Tg)), a GLUT4 partial knock-out (G4(+/-)), or both (HK(Tg) + G4(+/-)) and wild-type (WT) littermates were implanted with carotid artery and jugular vein catheters for sampling and infusions at 4 months of age. After a 7-day recovery, 5-h fasted mice remained sedentary or ran on a treadmill at 0.6 mph for 30 min (n = 9-12 per group) and received a bolus of 2-deoxy[3H]glucose to provide an index of MGU (Rg). Arterial blood glucose and plasma insulin concentrations were similar in WT, G4(+/-), HKTg, and HKTg + G4(+/-) mice. Sedentary Rg values were the same in all genotypes in all muscles studied, confirming that glucose transport is a significant barrier to basal glucose uptake. Gastrocnemius and soleus Rg were greater in exercising compared with sedentary mice in all genotypes. During exercise, G4(+/-) mice had a marked increase in blood glucose that was corrected by the addition of HK II overexpression. Exercise Rg (micromol/100g/min) was not different between WT and G4(+/-) mice in the gastrocnemius (24 +/- 5 versus 21 +/- 2) or the soleus (54 +/- 6 versus 70 +/- 7). In contrast, the enhanced exercise Rg observed in HKTg mice compared with that in WT mice was absent in HKTg + G4(+/-) mice in both the gastrocnemius (39 +/- 7 versus 22 +/- 6) and the soleus (98 +/- 13 versus 65 +/- 13). Thus, glucose transport is not a significant barrier to exercise-stimulated MGU despite a 50% reduction in GLUT4 content when glucose phosphorylation capacity is normal. However, when glucose phosphorylation capacity is increased by HK II overexpression, GLUT4 availability becomes a marked limitation to exercise-stimulated MGU.     

AMPK stimulation increases LCFA but not glucose clearance in cardiac muscle in vivo

AMP-activated protein kinase (AMPK) independently increases glucose and long-chain fatty acid (LCFA) utilization in isolated cardiac muscle preparations. Recent studies indicate this may be due to AMPK-induced phosphorylation and activation of nitric oxide synthase (NOS). Given this, the aim of the present study was to assess the effects of AMPK stimulation by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 10 mg.kg(-1).min(-1)) on glucose and LCFA utilization in cardiac muscle and to determine the NOS dependence of any observed effects. Catheters were chronically implanted in a carotid artery and jugular vein of Sprague-Dawley rats. After 4 days of recovery, conscious, unrestrained rats were given either water or water containing 1 mg/ml nitro-L-arginine methyl ester (L-NAME) for 2.5 days. After an overnight fast, rats underwent one of four protocols: saline, AICAR, AICAR + L-NAME, or AICAR + Intralipid (20%, 0.02 ml.kg(-1).min(-1)). Glucose was clamped at approximately 6.5 mM in all groups, and an intravenous bolus of 2-deoxy-[(3)H]glucose and [(125)I]-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid was administered to obtain indexes of glucose and LCFA uptake and clearance. Despite AMPK activation, as evidenced by acetyl-CoA carboxylase (Ser(221)) and AMPK phosphorylation (Thr(172)), AICAR increased cardiac LCFA but not glucose clearance. L-NAME + AICAR established that this effect was not due to NOS activation, and AICAR + Intralipid showed that increased cardiac LCFA clearance was not LCFA-concentration dependent. These results demonstrate that, in vivo, AMPK stimulation increases LCFA but not glucose clearance by a NOS-independent mechanism.     

Acylation-stimulating protein deficiency and altered adipose tissue in alternative complement pathway knockout mice

Acylation-stimulating protein (C3adesArg/ASP) is an adipokine that acts on its receptor C5L2 to stimulate triglyceride (TG) synthesis in adipose tissue. The present study investigated ASP levels in mouse models of obesity and leanness and the effect of ASP deficiency in C3 knockout (C3KO) mice on adipose tissue morphology. Plasma ASP levels in wild-type (WT) mice correlated positively with plasma nonesterified fatty acids (NEFA) (R = 0.664, P < 0.001) and total cholesterol (R = 0.515, P < 0.001). Plasma ASP was increased by 85% in obese ob/ob leptin-deficient mice and decreased in lean diacylglycerol acyltransferase 1 (DGAT1) KO mice (-54%) and C/EBPalpha(beta/beta) transgenic mice (-70%) compared with WT. Mice lacking alternative complement factor B or adipsin (FBKO or ADKO), required for ASP production, were also ASP deficient. Both FBKO and C3KO mice had delayed postprandial TG and NEFA clearance on low-fat (LF) and high-fat (HF) diets, suggesting that lack of ASP, not C3, drives the metabolic phenotype. Adipocyte size distribution in C3KO mice was polarized (increased number of both small and large cells), with decreased adipsin expression (-33% gonadal HF), DGAT1 expression (-31% to -50%) and DGAT activity (-41%). Overall, a reduction/deficiency in ASP is associated with an antiadipogenic state and ASP may provide a target for controlling fat storage.     

Long-term high-fat feeding induces greater fat storage in mice lacking UCP3

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3's function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3(-/-)) or had a transgenically induced approximately twofold increase in UCP3 levels (UCP3tg). Mice were fed the high-fat (HF) diet for a period of either 4 or 8 mo immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild-type mice (P < 0.05) and were 20% less metabolically efficient than both wild-type and Ucp3(-/-) mice (P < 0.01). Additionally, wild-type mice had 21% lower, whereas UCP3tg mice had 36% lower, levels of adiposity compared with Ucp3(-/-) mice (P < 0.05 and P < 0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3(-/-) mice were more glucose tolerant and insulin sensitive compared with wild-type mice after short-term HF feeding, but this protection was not maintained in the long term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.     

Cardiac metabolism in mice: tracer method developments and in vivo application revealing profound metabolic inflexibility in diabetes

Studies of cardiac fuel metabolism in mice have been almost exclusively conducted ex vivo. The major aim of this study was to assess in vivo plasma FFA and glucose utilization by the hearts of healthy control (db/+) and diabetic (db/db) mice, based on cardiac uptake of (R)-2-[9,10-(3)H]bromopalmitate ([3H]R-BrP) and 2-deoxy-D-[U-14C]glucose tracers. To obtain quantitative information about the evaluation of cardiac FFA utilization with [3H]R-BrP, simultaneous comparisons of [3H]R-BrP and [14C]palmitate ([14C]P) uptake were first made in isolated perfused working hearts from db/+ mice. It was found that [3H]R-BrP uptake was closely correlated with [14C]P oxidation (r2 = 0.94, P < 0.001). Then, methods for in vivo application of [3H]R-BrP and [14C]2-DG previously developed for application in the rat were specially adapted for use in the mouse. The method yields indexes of cardiac FFA utilization (R(f)*) and clearance (K(f)*), as well as glucose utilization (R(g)'). Finally, in the main part of the study, the ability of the heart to switch between FFA and glucose fuels (metabolic flexibility) was investigated by studying anesthetized, 8-h-fasted control and db/db mice in either the basal state or during glucose infusion. In control mice, glucose infusion raised plasma levels of glucose and insulin, raised R(g)' (+58%), and lowered plasma FFA level (-48%), K(f)* (-45%), and R(f)* (-70%). This apparent reciprocal regulation of glucose and FFA utilization by control hearts illustrates metabolic flexibility for substrate use. By contrast, in the db/db mice, glucose infusion raised glucose levels with no apparent influence on cardiac FFA or glucose utilization. In conclusion, tracer methodology for assessing in vivo tissue-specific plasma FFA and glucose utilization has been adapted for use in mice and reveals a profound loss of metabolic flexibility in the diabetic db/db heart, suggesting a fixed level of FFA oxidation in fasted and glucose-infused states.     

Heterozygous knockout of neuregulin-1 gene in mice exacerbates doxorubicin-induced heart failure

Neuregulins and their erbB receptors are essential for cardiac development and postulated to be cardioprotective in the presence of injury in the postnatal heart. We tested the hypothesis that the development of doxorubicin-induced cardiotoxicity in vivo is more severe in mice with heterozygous knockout of the neuregulin-1 gene (NRG-1(+/-)) compared with wild-type mice (WT). Three-month old NRG-1(+/-) and WT mice were injected with a single dose of doxorubicin (20 mg/kg ip). Survival was analyzed by the Kaplan-Meier approach. Left ventricular (LV) function and signaling pathways were analyzed 4 days after treatment. Fifteen days after treatment, survival was significantly lower in doxorubicin-treated NRG-1(+/-) mice (NRG-1(+/-)-Dox) compared with doxorubicin-treated WT mice (WT-Dox) (15% vs. 33%, P < 0.01). LV mass was significantly lower in NRG-1(+/-)-Dox but not in WT-Dox compared with nontreated animals. LV systolic pressure and LV midwall fractional shortening were significantly lower in NRG-1(+/-)-Dox compared with WT-Dox mice. LV protein levels of NRG-1, erbB2, and erbB4 receptors were similar in WT-Dox and NRG-1(+/-)-Dox mice. However, levels of phosphorylated erbB2, Akt, and ERK-1/2 were significantly decreased in NRG-1(+/-)-Dox compared with WT-Dox mice. A significant decrease in phosphorylated P70S6K levels was also observed in NRG-1(+/-)-Dox compared with nontreated NRG-1(+/-) mice. These results demonstrate that heterozygous knockout of the neuregulin-1 gene worsens survival and LV function in the presence of doxorubicin-induced cardiac injury in vivo. This is associated with the depression of activation of the erbB2 receptor as well as Akt, p70S6K, and ERK-1/2 pathways.     

PKCε promotes cardiac mitochondrial and metabolic adaptation to chronic hypobaric hypoxia by GSK3β inhibition

PKCε is central to cardioprotection. Sub-proteome analysis demonstrated co-localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia-inducible factor 1α (HIF-1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF-1α. However, there is no established mechanistic link between PKCε, p-GSK3β and HIF1-α. Here we hypothesized that cardiac-restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF-1α-dependent mechanism. aPKCε and wild-type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O(2)) and cardiac metabolism, functional parameters, p-GSK3β/HIF-1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP-dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia-associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p-GSK3β and HIF1-α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p-GSK3β/HIF-1α-mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia.     

Differential regulation of fatty acid trapping in mouse adipose tissue and muscle by ASP

Acylation-stimulating protein (ASP) is a lipogenic hormone secreted by white adipose tissue (WAT). Male C3 knockout (KO; C3(-/-)) ASP-deficient mice have delayed postprandial triglyceride (TG) clearance and reduced WAT mass. The objective of this study was to examine the mechanism(s) by which ASP deficiency induces differences in postprandial TG clearance and body composition in male KO mice. Except for increased (3)H-labeled nonesterified fatty acid (NEFA) trapping in brown adipose tissue (BAT) of KO mice (P = 0.02), there were no intrinsic tissue differences between wild-type (WT) and KO mice in (3)H-NEFA or [(14)C]glucose oxidation, TG synthesis or lipolysis in WAT, muscle, or liver. There were no differences in WAT or skeletal muscle hydrolysis, uptake, and storage of [(3)H]triolein substrate [in situ lipoprotein lipase (LPL) activity]. ASP, however, increased in situ LPL activity in WAT (+64.8%, P = 0.02) but decreased it in muscle (-35.0%, P = 0.0002). In addition, after prelabeling WAT with [(3)H]oleate and [(14)C]glucose, ASP increased (3)H-lipid retention, [(3)H]TG synthesis, and [(3)H]TG-to-[(14)C]TG ratio, whereas it decreased (3)H-NEFA release, indicating increased NEFA trapping in WAT. Conversely, in muscle, ASP induced effects opposite to those in WAT and increased lipolysis, indicating reduced NEFA trapping within muscle by ASP (P < 0.05 for all parameters). In conclusion, novel data in this study suggest that 1) there is little intrinsic difference between KO and WT tissue in the parameters examined and 2) ASP differentially regulates in situ LPL activity and NEFA trapping in WAT and skeletal muscle, which may promote optimal insulin sensitivity in vivo.     

Fatty acid binding protein in locust and mammalian muscle. Comparison of structure,function and regulation

The flight muscle of adult desert locusts, Schistocerca gregaria, contains a fatty acid binding protein (FABP) that is homologous to mammalian M-FABP (cardiac FABP). In spite of the evolutionary distance between invertebrates and vertebrates, locust muscle FABP is similar to cardiac FABP in its amino acid sequence, structure, and binding behavior. While cardiac FABP is present already in the prenatal period, locust FABP is an adult specific protein; its expression is directly linked to metamorphosis. A correlation seems to exist between fatty acid oxidative capacity and FABP content in both locust and mammals. To accomplish the higher metabolic rate encountered during migratory flight, locust flight muscle cytosol contains more than three times as much FABP as that in mammalian heart. Increased fatty acid utilization by exercise or endurance training apparently induces FABP expression. Similarities and differences between vertebrate and invertebrate M-FABP are discussed in light of the proposed functions of muscle FABP as fatty acid transporter and cytoprotectant.