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OBJECTIVE--The fatty acid translocase and scavenger receptor CD36 is important in the recognition and uptake of lipids. Accordingly, we hypothesized that it plays a role in saturated fatty acid-induced macrophage lipid accumulation and proinflanunatory activation. RESEARCH DESIGN AND METHODS--In vitro, the effect of CD36 inhibition and deletion in lipid-induced macrophage inflammation was assessed using the putative CD36 inhibitor, sulfosuccinimidyl oleate (SSO), and bone marrow-derived macrophages from mice with (CD36KO) or without (wild-type) global deletion of CD36. To investigate whether deletion of macrophage CD36 would improve insulin sensitivity in vivo, wild-type mice were transplanted with bone marrow from CD36KO or wild-type mice and then fed a standard or high-fat diet (HFD) for 20 weeks. RESULTS--SSO treatment markedly reduced saturated fatty acid-induced lipid accumulation and inflammation in RAW264.7 macrophages. Mice harboring CD36-specific deletion in hematopoietic-derived cells (HSC CD36KO) fed an HFD displayed improved insulin signaling and reduced macrophage infiltration in adipose tissue compared with wild-type mice, but this did not translate into protection against HFD-induced whole-body insulin resistance. Contrary to our hypothesis and our results using SSO in RAW264.7 macrophages, neither saturated fatty acid-induced lipid accumulation nor inflammation was reduced when comparing CD36KO with wild-type bone marrow-derived macrophages. CONCLUSIONS--Although CD36 does not appear important in saturated fatty acid-induced macrophage lipid accumulation, our study uncovers a novel role for CD36 in the migration of proinflanunatory phagocytes to adipose tissue in obesity, with a concomitant improvement in insulin action. Diabetes 60:1100--1110, 2011, Obesity is a key risk factor for the development of insulin resistance leading to type 2 diabetes and other metabolic pathologies. Obesity induces a state of chronic, low-grade inflammation that [...]

OBJECTIVE--We examined in insulin-resistant muscle if, in contrast to long-standing dogma, mitochondrial fatty acid oxidation is increased and whether this is attributed to an increased nuclear content of peroxisome proliferator--activated receptor (PPAR) γ coactivator (PGC) 1α and the adaptations of specific mitochondrial subpopulations. RESEARCH DESIGN AND METHODS--Skeletal muscles from male control and Zucker diabetic fatty (ZDF) rats were used to determine 1) intramuscular lipid distribution, 2) subsarcolemmal and intermyofibrillar mitochondrial morphology, 3) rates of palmitate oxidation in subsarcolemmal and intermyofibrillar mitochondria, and 4) the subcellular localization of PGC1α. Electotransfection of PGC1α cDNA into lean animals tested the notion that increased nuclear PGC1α preferentially targeted subsarcolemmal mitochondria. RESULTS--Transmission electron microscope analysis revealed that in ZDF animals the number (+50%), width (+69%), and density (+57%) of subsarcolemmal mitochondria were increased (P < 0.05). In contrast, intermyofibrillar mitochondria remained largely unchanged. Rates of palmitate oxidation were ~40% higher (P < 0.05) in ZDF subsarcolemmal and intermyofibrillar mitochondria, potentially as a result of the increased PPAR-targeted proteins, carnitine palmitoyltransferase-I, and fatty acid translocase (FAT)/CD36. PGC1α mRNA and total protein were not altered in ZDF animals; however, a greater (~70%; P < 0.05) amount of PGC1α was located in nuclei. Overexpression of PGC1α only increased subsarcolemmal mitochondrial oxidation rates. CONCLUSIONS--In ZDF animals, intramuscular lipids accumulate in the intermyofibrillar region (increased size and number), and this is primarily associated with increased oxidative capacity in suhsarcolemmal mitochondria (number, size, density, and oxidation rates). These changes may result from an increased nuclear content of PGC1α, as under basal conditions, overexpression of PGC1α appears to target subsarcolemmal mitochondria., Skeletal muscle, due to its mass and high rate of glucose disposal, is an important tissue in the development of insulin resistance. While the etiology of skeletal muscle insulin resistance [...]

A reduction in fatty acid (FA) oxidation has been associated with lipid accumulation and insulin resistance in skeletal muscle of obese individuals. Numerous reports suggest that the reduction in FA oxidation may result from intrinsic mitochondrial defects, although little direct evidence has been offered to support this conclusion. This brief review summarizes recent work from our laboratory that reexamined whether this decrease in skeletal muscle FA oxidation with obesity was attributable to a dysfunction in FA oxidation within mitochondria or simply to a reduction in muscle mitochondrial content. Whole-muscle mitochondrial content and FA oxidation was reduced in the obese, but there was no decrease in the ability of isolated mitochondria to oxidize FA. The mitochondrial content of the transport protein, FA translocase (FAT/CD36), did not differ between lean and obese women but was correlated with mitochondrial FA oxidation. It was concluded that the reduced FA oxidation in obesity is attributable to decreased muscle mitochondrial content and not intrinsic defects in mitochondrial FA oxidation, and that mitochondrial FAT/CD36 is involved in regulating FA oxidation in human skeletal muscle. The reduced skeletal muscle mitochondrial content with obesity may result from impaired mitochondrial biogenesis. However, this did not result from decreased protein contents of various transcription factors, because peroxisome proliferater-activated receptor [gamma] coactivator 1[alpha] (PGC1[alpha]), PGC1[beta], peroxisome proliferator-activated receptor [alpha] (PPAR[alpha]), and mitochondrial transcription factor A (TFAM) were not reduced with obesity. In contrast, it appears that obesity is associated with altered regulation of cofactors (PGC1[alpha] and PGC1[beta]) and their downstream transcription factors (PPAR[alpha], PPAR[delta]/[beta], and TFAM), because relations among these variables were present in muscle from lean individuals but not from obese individuals. These findings imply that obese individuals would benefit from interventions that increase the skeletal muscle mitochondrial content and the potential for oxidizing FAs.

Both pharmacological intervention (i.e., thiazolidinediones [TZDs]) and lifestyle modification (i.e., exercise training) are clinically effective treatments for improving whole-body insulin sensitivity. However, the mechanism(s) by which these therapies reverse lipid-induced [...]

In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/ CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates., Cardiovascular diseases are the most serious complications of obesity (1). A common feature of obesity is insulin resistance, which is characterized by a diminished ability of insulin-sensitive tissues, such as [...]

Contraction of rat cardiac myocytes induces translocation of fatty acid translocase (FAT)/CD36 and GLUT4 from intracellular stores to the sarcolemma, leading to enhanced rates of long-chain fatty acid (FA) and glucose uptake, respectively. Because intracellular AMP/ ATP is elevated in contracting cardiac myocytes, we investigated whether activation of AMP-activated protein kinase (AMP kinase) is involved in contraction-inducible FAT/CD36 translocation. The cell-permeable adenosine analog 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside (AICAR) and the mitochondrial inhibitor oligomycin, similar to 4-Hz electrostimulation, evoked a more than threefold activation of cardiomyocytic AMP kinase. Both AICAR and oligomycin stimulated FA uptake into noncontracting myocytes by 1.4- and 2.0-fold, respectively, but were ineffective in 4 Hz-contracting myocytes. These findings indicate that both agents stimulate FA uptake by a similar mechanism as electrostimulation, involving activation of AMP kinase, as evidenced from phosphorylation of acetyl-CoA carboxylase. Furthermore, the stimulating effects of both AICAR and oligomycin were antagonized by blocking FAT/CD36 with sulfo-N-succinimidylpalmitate, but not by inhibiting phosphatidylinositol 3-kinase with wortmannin, indicating the involvement of FAT/CD36, but excluding a role for insulin signaling. Subcellular fractionation showed that oligomycin was able to mobilize intracellularly stored FAT/CD36 to the sarcolemma. We conclude that AMP kinase regulates cardiac FA use through mobilization of FAT/CD36 from a contraction-inducible intracellular storage compartment., Fatty acid translocase (FAT)/CD36 is increasingly becoming recognized as a physiologically important long-chain fatty acid (FA) transport facilitator within the sarcolemma of muscle tissues (1-3). We have gathered convincing evidence [...]

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle. Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and [beta]-oxidation in skeletal muscle. Design: Fourteen well-trained male cyclists and triathletes with a mean ([+ or -] SE) age of 26.9 [+ or -] 1.7 y, weight of 73.7 [+ or -] 1.7 kg, and peak oxygen uptake of 67.0 [+ or -] 1.3 mL*[kg.sup.-1]* [min.sup.-1] consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70-75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), [beta]-hydroxyacyl-CoA dehydrogenase ([beta]-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm. Results: The gene expression of FAT/CD36 and [beta]-HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet. Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle. KEY WORDS Diet-gene interaction, messenger RNA, high-fat diet, skeletal muscle, metabolism, dietary intervention, fat oxidation, carbohydrate oxidation, gene expression

The existence of an intracellular pool of fatty acid translocase (FAT/CD36), an 88-kDa membrane transporter for long-chain fatty acids (FAs), and the ability of insulin to induce translocation events prompted us to investigate the direct effects of insulin on cellular uptake of FA by the heart. Insulin (0.1 nmol/l and higher) increased FA uptake by isolated rat cardiac myocytes by 1.5-fold. This insulin-induced increase in FA uptake was completely blocked by phloretin, sulfo-N-succinimidylpalmitate (SSP), and wortmannin, indicating the involvement of FAT/CD36 and the dependence on phosphatidylinositol-3 (PI-3) kinase activation. Subcellular fractionation of insulin-stimulated cardiac myocytes demonstrated a 1.5-fold increase in sarcolemmal FAT/CD36 and a 62% decrease in intracellular FAT/ CD36 with parallel changes in subcellular distribution of GLUT4. Induction of cellular contractions upon electrostimulation at 4 Hz enhanced cellular FA uptake 1.6-fold, independent of PI-3 kinase. The addition of insulin to 4 Hz-stimulated cells further stimulated FA uptake to 2.3-fold, indicating that there are at least two functionally independent intracellular FAT/CD36 pools, one recruited by insulin and the other mobilized by contractions. In conclusion, we have demonstrated a novel role of insulin in cardiac FA utilization. Malfunctioning of insulin-induced FAT/CD36 translocation may be involved in the development of type 2 diabetic cardiomyopathies., The heart is among the most sensitive organs to insulin action. This hormone has multiple effects on cardiac intermediary metabolism, one of the best described being the stimulation of glucose [...]

Lactate transporters, or MCT proteins, have been investigated in skeletal and heart muscle. MCT1 was found to be expressed in heart and muscle and to be responsive to exercise training and to increase at a much lower training intensity in heart than in muscle. Whether or not lactate transport changes in exercise still is not known.

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether they do so by inducing the recruitment of GLUT4 transporters from [...]

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether these stimuli mobilize different populations of GLUT4 vesicles. To investigate this [...]

There is doubt whether spontaneously hypertensive rats (SHR; North American strain) are null for fatty acid translocase (FAT/CD36). Therefore, we examined whether FAT/CD36 is expressed in heart, muscle, liver and adipose tissue in SHR. Insulin resistance was present in SHR skeletal muscle. We confirmed that SHR expressed aberrant FAT mRNAs in key metabolic tissues; namely, the major 2.9 kb transcript was not expressed, but 3.8 and 5.4 kb transcripts were present. Despite this, FAT/CD36 protein was expressed in all tissues, although there were tissue-specific reductions in FAT/CD36 protein expression and plasmalemmal content, ranging from 26–85%. Fatty acid transport was reduced in adipose tissue (−50%) and was increased in liver (+47%). Normal rates of fatty acid transport occurred in heart and muscle, possibly due to compensatory upregulation of plasmalemmal fatty acid binding protein (FABPpm) in red (+123%) and white muscle (+110%). In conclusion, SHRs (North American strain) are not a natural FAT/CD36 null model, the North American strain of SHR express FAT/CD36, albeit at reduced levels.

Long chain fatty acids (LCFAs) provide 70-80% of the energy for cardiac contractile activity. LCFAs are also essential for many other cellular functions, such as transcriptional regulation of proteins involved in lipid metabolism, modulation of intracellular signalling pathways, and as substrates for membrane constituents. When LCFA uptake exceeds the capacity for their cardiac utilization, the intracellular lipids accumulate and are thought to contribute to contractile dysfunction, arrhythmias, cardiac myocyte apoptosis and congestive heart failure. Moreover, increased cardiac myocyte triacylglycerol, diacylglycerol and ceramide depots are cardinal features associated with obesity and type 2 diabetes. In recent years considerable evidence has accumulated to suggest that, the rate of entry of long chain fatty acids (LCFAs) into the cardiac myocyte is a key factor contributing to a) regulating cardiac LCFA metabolism and b) lipotoxicity in the obese and diabetic heart. In the present review we i) examine the evidence indicating that LCFA transport into the heart involves a protein-mediated mechanism, ii) discuss the proteins involved in this process, including FAT/CD36, FABPpm and FATP1, iii) discuss the mechanisms involved in regulating LCFA transport by some of these proteins (including signaling pathways), as well as iv) the possible interactions of these proteins in regulating LCFA transport into the heart. In addition, v) we discuss how LCFA transport and transporters are altered in the obese/diabetic heart.

Abstract: Introduction: Long-chain fatty acids and glucose are the predominant substrates for cardiac metabolic energy production. While in the healthy heart there is a distinctive and very finely tuned balance between the utilization of these metabolic substrates, in chronic cardiac disease this balance is upset to the use of primarily glucose (e.g., cardiac hypertrophy and failure) or primarily fatty acids (e.g., diabetic cardiomyopathy). Cardiac substrate preference is regulated not only at the level of mitochondrial oxidation (Randle cycle) but also at the level of sarcolemmal uptake of substrates. Molecular mechanism of cardiac substrate uptake: The latter occurs by translocation of specific substrate transporters, namely fatty acid translocase/CD36 and plasma membrane fatty acid-binding protein (FABPpm) to regulate fatty acid transport, and GLUT4 to regulate glucose transport, from intracellular storage pools to the sarcolemma. Both insulin and cardiac muscle contractions increase the cellular uptake of fatty acids and glucose simultaneously by these mechanisms. Although the signal transduction pathways involved in eliciting substrate transporter trafficking have only partly been disclosed, recent studies indicate the feasibility of selective recruitment of either CD36 or GLUT4 to the sarcolemma, thereby increasing the uptake of a single class of substrates and thus altering the substrate preference of cardiac muscle cells. Concluding remarks: As a result, selective modulation of the sarcolemmal localization of fatty acid- and/or glucose transporters holds promise as a therapeutic tool to rectify a disruption of the cardiac substrate balance occurring in chronic cardiac disease.

Skeletal muscle constitutes 40% of body mass and takes up 80% of a glucose load. Therefore, impaired glucose removal from the circulation, such as that which occurs in obesity and type 2 diabetes, is attributable in large part to the insulin resistance in muscle. Recent research has shown that fatty acids, derived from adipose tissue, can interfere with insulin signalling in muscle. Hence, insulin-stimulated GLUT4 translocation to the cell surface is impaired, and therefore, the rate of glucose removal from the circulation into muscle is delayed. The mechanisms provoking lipid-mediated insulin resistance are not completely understood. In sedentary individuals, excess intramyocellular accumulation of triacylglycerols is only modestly associated with insulin resistance. In contrast, endurance athletes, despite accumulating large amounts of intramyocellular triacylglycerols, are highly insulin sensitive. Thus it appears that lipid metabolites, other than triacylglycerols, interfere with insulin signalling. These metabolites, however, are not expected to accumulate in athletic muscles, as endurance training increases the capacity for fatty acid oxidation by muscle. These observations, and others in severely obese individuals and type 2 diabetes patients, suggest that impaired rates of fatty acid oxidation are associated with insulin resistance. In addition, in obesity and type 2 diabetes, the rates of fatty acid transport into muscle are also increased. Thus, excess intracellular lipid metabolite accumulation, which interferes with insulin signalling, can occur as a result of impaired rates of fatty acid oxidation and/or increased rates of fatty acid transport into muscle. Accumulation of excess intramyocellular lipid can be avoided by exercise, which improves the capacity for fatty acid oxidation.

Abstract: Rates of fatty acid oxidation increase rapidly in both rat heart and skeletal muscle in the early postnatal period. Therefore, we examined in heart and soleus muscle, (a) whether there were rapid changes in fatty acid transporter (FAT/CD36, FABPpm) mRNA and protein expression early in life (days 10 –36) and thereafter (days 84, 160, 365), and (b) whether the rates of fatty acid transport and the plasmalemmal content of FAT/CD36 and FABPpm were altered. Protein expression was altered rapidly from day 10–36 in both heart (FAT/CD36 only, +21%, P < 0.05)) and soleus muscle (FAT/CD36 + 100%, P < 0.05; FABPpm −20%, P < 0.05), with no further changes thereafter (P < 0.05). Rates of fatty acid transport (day 10 vs day 160) were increased in heart (+33%, P < 0.05) and muscle (+85%, P < 0.05), and were associated with concomitant increases in plasmalemmal FABPpm (+44%, P < 0.05) and FAT/CD36 (+16%, P < 0.05) in the heart, and only plasmalemmal FAT/CD36 in muscle (+90%, P < 0.05). Therefore, known changes in the rates of fatty acid oxidation in heart and muscle early in life appear to be accompanied by a concurrent upregulation in the rates of fatty acid transport and the expression of FAT/CD36 in heart and muscle, as well as an increase in plasmalemmal FAT/CD36 and FABPpm in the heart, and only plasmalemmal FAT/CD36 in soleus muscle. We speculate that the rapid upregulation of fatty acid transport rates in heart and muscle are needed to support the increased rates of fatty oxidation that have been previously observed in these tissues.

Abstract: Recently, fatty acid transport across the plasma membrane has been shown to be a key process that contributes to the regulation of fatty acid metabolism in the heart. Since AMP kinase activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) stimulates fatty acid oxidation, as well as the expression of selected proteins involved with energy provision, we examined (a) whether AICAR induced the expression of the fatty acid transporters FABPpm and FAT/CD36 in cardiac myocytes and in perfused hearts and (b) the signaling pathway involved. Incubation of cardiac myocytes with AICAR increased the protein expression of the fatty acid transporter FABPpm after 90 min (+27%, P < 0.05) and this protein remained stably overexpressed until 180 min. Similarly, FAT/CD36 protein expression was increased after 60 min (+38%, P < 0.05) and remained overexpressed thereafter. Protein overexpression, which occurred via transcriptional mechanisms, was dependent on the AICAR concentration, with optimal induction occurring at AICAR concentrations 1–5 mM for FABPpm and at 2–8 mM for FAT/CD36. The AICAR (2 h, 2 mM AICAR) effects on FABPpm and FAT/CD36 protein expression were similar in perfused hearts and in cardiac myocytes. AICAR also induced the plasmalemmal content of FAT/CD36 (+49%) and FABPpm (+42%) (P < 0.05). This was accompanied by a marked increase in the rate of palmitate transport (2.5 fold) into giant sarcolemmal vesicles, as well as by increased rates of palmitate oxidation in cardiac myocytes. When the AICAR-induced AMPK phosphorylation was blocked, neither FAT/CD36 nor FABPpm were overexpressed, nor were palmitate uptake and oxidation increased. This study has revealed that AMPK activation stimulates the protein expression of both fatty acid transporters, FAT/CD36 and FABPpm in (a) time- and (b) dose-dependent manner via (c) the AMPK signaling pathway. AICAR also (d) increased the plasmalemmal content of FAT/CD36 and FABPm, thereby (e) increasing the rates of fatty acid transport. Thus, activation of AMPK is a key mechanism regulating the expression as well as the plasmalemmal localization of fatty acid transporters.

FAT/CD36 is a transmembrane protein that is thought to facilitate cellular long-chain fatty acid uptake. However, surprisingly little is known about the localization of FAT/CD36 in human skeletal muscle. By confocal immunofluorescence microscopy, we demonstrate high FAT/CD36 expression in endothelial cells and weaker but significant FAT/CD36 expression in sarcolemma in human skeletal muscle. No apparent intracellular staining was observed in the muscle cells. There are indications in the literature that caveolae may be involved in the uptake of fatty acids, possibly as regulators of FAT/CD36 or other fatty acid transporters.

Fatty acid translocase (FAT/CD36) is a key fatty acid transporter in skeletal muscle. However, the effects on fatty acid transport by another putative fatty acid transporter, plasma membrane-associated fatty acid binding protein (FABPpm), have not been determined in mammalian tissue. We examined the functional effects of overexpressing FABPpm on the rates of 1) palmitate transport across the sarcolemma and 2) palmitate metabolism in skeletal muscle. One muscle (soleus) was transfected with pTracer containing FABPpm cDNA. The contralateral muscle served as control. After injecting the FABPpm cDNA, muscles were electroporated. FABPpm overexpression was directly related to the quantity of DNA administered. Electrotransfection (200 μg/muscle) rapidly induced FABPpm protein overexpression (day 1, +92%, P< 0.05), which was further increased during the next few days (days 3–7; range +142% to +160%, P< 0.05). Sarcolemmal FABPpm was comparably increased (day 7, +173%, P< 0.05). Neither FAT/CD36 expression nor sarcolemmal FAT/CD36 content was altered. FABPpm overexpression increased the rates of palmitate transport (+79%, P< 0.05). Rates of palmitate incorporation into phospholipids were also increased +36%, as were the rates of palmitate oxidation (+20%). Rates of palmitate incorporation into triacylglycerol depots were not altered. These studies demonstrate that in mammalian tissue FABPpm overexpression increased the rates of palmitate transport across the sarcolemma, an effect that is independent of any changes in FAT/CD36. However, since the overexpression of plasmalemmal FABPpm (+173%) exceeded the effects on the rates of palmitate transport and metabolism, it appears that the overexpression of FABPpm alone is not sufficient to induce completely parallel increments in palmitate transport and metabolism. This suggests that other mechanisms are required to realize the full potential offered by FABPpm overexpression.

Long chain fatty acid uptake across the plasma membrane occurs, in part, via a protein-mediated process involving a number of fatty acid binding proteins known as fatty acid transporters. A critical step in furthering the understandings of fatty acid transport was the discovery that giant vesicles, prepared from tissues such as muscle and heart, provided a suitable system for measuring fatty acid uptake. These vesicles are large (10–15 μm diameter), are oriented fully right side out, and contain cytosolic FABP in the lumen, which acts as a fatty acid sink, while none of the fatty acid taken up is metabolized or associated with the plasma membrane. The key fatty acid transporters FAT/CD36 and FABPpm are expressed in muscle and heart and their plasma membrane content is positively correlated with rates of fatty acid transport. These transporters are regulated acutely (within minutes) and chronically (days). For instance, both muscle contraction and insulin can translocate FAT/CD36 from an intracellular pool to the plasma membrane, thereby increasing fatty acid transport. With obesity, fatty acid transport is increased along with a concomitant increase in plasmalemmal FAT/CD36 (heart, muscle) and FABPpm (heart only), but without change in the expression of these transporters. This latter observation suggests that some of the fatty acid transporters are permanently relocated to the plasma membrane. In other studies it also appears that fatty acid transport rates are altered in a reciprocal manner to glucose transport. Since disorders in lipid metabolism appear to be an important factor contributing to the etiology of a number of common human diseases such as diabetes and obesity, our evidence that protein-mediated fatty acid transport is a key step in lipid metabolism allows the speculation that malfunctioning of the fatty acid transport process could be a common critical factor in the pathogenesis of these diseases.

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial β-oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein. In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.

Skeletal muscle metabolic energy, needed to maintain contractile activity, is mainly obtained from glucose and long-chain fatty acids. Recent studies have revealed a remarkable parallel between the regulation of uptake of glucose and fatty acids by muscle, in that each is mediated by sarcolemmal transporters that are recruited from an intracellular storage site. The focus of this review is to describe newly obtained insights on the recruitment of fatty acid transporters and their malfunctioning in diabetes.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (∼4-fold; p< 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately −2-fold; p< 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (−45%) and white (−84%) skeletal muscle compared with control and pair-fed animals (p< 0.05). Leptin treatment reduced FAT mRNA (red, −70%,p< 0.001; white, −48%, p< 0.01) and FAT/CD36 protein expression (red, −32%; p< 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (−28 and −34%, respectively) and white (−44 and −56%, respectively) muscles (p< 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r= 0.88,p< 0.01) and plasma membrane FABPpm protein (r= 0.94, p< 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Protein-mediated fatty acid uptake and intracellular fatty acid activation are key steps in fatty acid metabolism in muscle.We have examined (a) the abundance of fatty acid translocase (FAT/CD36) mRNA (a fatty acid transporter) and long-chain acyl CoA synthetase (FACS1) mRNA in metabolically heterogeneous muscles (soleus (SOL), red (RG) and white gastrocnemius (WG)), and (b) whether FAT/CD36 and FACS1 mRNAs were coordinately upregulated in red (RTA) and white tibialis muscles (WTA) that had been chronically stimulated for varying periods of time (0.25, 1, 6 and 24 h/day) for 7 days. FAT/CD36 mRNA and FACS1 mRNA abundance were scaled with (a) the oxidative capacity of muscle (SOL > RG > WG) (p < 0.05), (b) the rates of fatty acid oxidation in red and white muscles, and (c) fatty acid uptake by sarcolemmal vesicles, derived from red and white muscles. In chronically stimulated muscles (RTA and WTA), FAT/CD36 mRNA and FACS1 mRNA were up-regulated in relation to the quantity of muscle contractile activity (p < 0.05). FAT/CD36 mRNA and FACS1 mRNA up-regulation was highly correlated (r = 0.98). The coordinated expression of FAT/CD36 and FACS is likely a functional adaptive response to facilitate a greater rate of fatty acid activation in response to a greater rate of fatty acid transport, either among different types of muscles or in muscles in which capacity for fatty acid metabolism has been enhanced.

Mitochondrial myopathies (MM) are characterized by alterations in oxidative phosphorylation. The resultant increase in glycolytic flux produces a variable lactic acidosis. Intracellular acidification can induce both metabolic and, in the case of skeletal muscle, contractile dysfunction. Skeletal muscle lactate transporters have recently been identified which include both monocarboxylate transporter 1 (MCT1) and 4 (MCT4). Lactate import into oxidative skeletal muscle appears to be catalyzed by MCT1, whereas its extrusion from glycolytic fibers may be mediated by MCT4. We describe the expression of these isoforms in a patient with MM as compared to controls (n= 5). MCT4 content was 86% (>3 SD) higher in the patient with MM, whereas MCT1 content was less markedly elevated (47%), as compared to controls. These findings support previous work suggesting that the major role of MCT4 is to defend intracellular pH by extruding lactate and H+to the interstitium. The role of MCT1 in MM is less clear. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 394–398, 2001

We have examined the independent and combined effects of insulin insufficiency (streptozotocin (STZ)-induced diabetes, 85 mg/kg i.p.) and reduced muscle activity (denervation) (7 days) on basal, insulin-stimulated and contraction-stimulated glucose transport in rat muscles (soleus, red and white gastrocnemius). There were four treatments: control, denervated, diabetic, and denervated + diabetic muscles. Contraction-stimulated glucose transport was lowered (~ 50%) (p < 0.05) to the same extent in all experimental groups. In contrast, there was a much smaller reduction insulin-stimulated glucose transport in muscles from diabetic animals (18-24% reduction, p < 0.05) than in denervated muscles (40-60% reduction, p < 0.05) and in denervated + diabetic muscles (40-60% reduction, p < 0.05). GLUT-4 mRNA reduction was greatest in denervated + diabetic muscles (~ -75%, p < 0.05). GLUT-4 protein was decreased (p < 0.05) to a similar extent in all three experimental conditions (~ -30-40%). In conclusion, (1) muscle inactivity (denervation) and STZ-induced diabetes had similar effects on reducing contraction-stimulated glucose transport, but (2) muscle inactivity (denervation), rather than severe diabetes, produced a 2-fold greater impairment in skeletal muscle insulin-stimulated glucose transport.

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABPPM) may be a component of this system. To test the hypothesis that FABPPMis involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABPPM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmaxvalues of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and Kmvalues of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The Vmaxvalues for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABPPM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABPPM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABPPMis a component of this process in muscle.

We used muscle contraction, which increases fatty acid oxidation, as a model to determine whether fatty acid transport is acutely regulated by fatty acid translocase (FAT/CD36). Palmitate uptake by giant vesicles, obtained from skeletal muscle, was increased by muscle contraction. Kinetic studies indicated that muscle contraction increased Vmax, butKmremained unaltered. Sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36, fully blocked the contraction-induced increase in palmitate uptake. In giant vesicles from contracting muscles, plasma membrane FAT/CD36 was also increased in parallel with the increase in long chain fatty acid uptake. Further studies showed that like GLUT-4, FAT/CD36 is located in both the plasma membrane and intracellularly (endosomally). With muscle contraction, FAT/CD36 at the surface of the muscle was increased, while concomitantly, FAT/CD36 in the intracellular pool was reduced. Similar responses were observed for GLUT-4. We conclude that fatty acid uptake is subject to short term regulation by muscle contraction and involves the translocation of FAT/CD36 from intracellular stores to the sarcolemma, analogous to the regulation of glucose uptake by GLUT-4.

Increasing evidence has implicated the membrane protein CD36 (FAT) in binding and transport of long chain fatty acids (FA). To determine the physiological role of CD36, we examined effects of its overexpression in muscle, a tissue that depends on FA for its energy needs and is responsible for clearing a major fraction of circulating FA. Mice with CD36 overexpression in muscle were generated using the promoter of the muscle creatine kinase gene (MCK). Transgenic (MCK-CD36) mice had a slightly lower body weight than control litter mates. This reflected a leaner body mass with less overall adipose tissue, as evidenced by magnetic resonance spectroscopy. Soleus muscles from transgenic animals exhibited a greatly enhanced ability to oxidize fatty acids in response to stimulation/contraction. This increased oxidative ability was not associated with significant alterations in histological appearance of muscle fibers. Transgenic mice had lower blood levels of triglycerides and fatty acids and a reduced triglyceride content of very low density lipoproteins. Blood cholesterol levels were slightly lower, but no significant decrease in the cholesterol content of major lipoprotein fractions was measured. Blood glucose was significantly increased, while insulin levels were similar in the fed state and higher in the fasted state. However, glucose tolerance curves, determined at 20 weeks of age, were similar in control and transgenic mice. In summary, the study documented, in vivo,the role of CD36 to facilitate cellular FA uptake. It also illustrated importance of the uptake process in muscle to overall FA metabolism and glucose utilization.

Recent studies have indicated that lactate traversal of the sarcolemmal membrane of skeletal muscle could be a carrier mediated process. In the present study, the initial rates of L(+)-lactate flux (Jlact) were measured in highly purified rat hindlimb skeletal muscle sarcolemmal vesicles. Fluxes were determined by the vesicle uptake of L(+)-[U-14C] lactate from the extra-vesicular medium. Jlact was saturable with respect to increasing concentrations of L(+)-lactate. Regression of these data to the Michaelis-Menten equation yielded a Km of 12.5 mM. Jlact was inhibited 81% by 10 mM pyruvate and 83% by 5mM alpha-cyano 4 hydroxycinnamate (p<0.05), but not by D-lactate indicating the presence of a stereoselective monocarboxylate transporter in the sarcolemmal membrane. Preincubation of the vesicles with the protein modifier, N-ethylmaleimide (20mM), inhibited Jlact by 86% (p<0.05). An inhibitor of the inorganic anion exchanger, SITS (1mM), had no effect on Jlact. However, Jlact was markedly sensitive to an inwardly directed proton gradient (p<0.05), and the flux was more closely related to the concentration of external ionic L(+)-lactate than to the protonated (HLa) form. These studies suggest that skeletal muscle sarcolemmal membranes possess a specific transport system for L-lactate and other monocarboxylates, which has similar properties to the lactate carrier described for several other tissues.

If lactate is primarily oxidized in skeletal muscle in man, it is expected that lactate uptake would increase linearly with increasing muscle metabolism (VO2). Therefore, lactate removal was investigated (N=9) after 6 min exercise bouts (90% VO2 max), at rest, and during 30 min of recovery exercise, when the relative intensities were constant to equate lactate production while permitting exercise metabolism (VO2) to vary. Recovery exercises were therefore conducted at 26.8% VO2 max for arm exercise, 26.8% VO2 max for leg exercise, and 29% VO2 max for combined arm and leg exercise. These exercise intensities were calculated from VO2 max values established separately for each of the three modes of exercise. Lactate removal was slowest at rest (p<0.05). Removal during leg recovery was faster than during the arm condition (p<0.05), but the leg removal was not different from the combined arm and leg condition (p>0.05). The VO2 cost of the arm (0.73±0.04 l/min), leg (1.04±0.05 l/min) and combined arm and leg exercise (1.23±0.10 l/min) were distinctly different from each other (p<0.05). There was a high correlation (r=0.92) between VO2 cost, and the lactate removal rates of the corresponding recovery conditions. These findings indicate that lactate increases proportionately with the metabolically active muscle mass, providing exercise remains aerobic. Thus in man, it appears that lactate removal from the blood during recovery exercise occurs primarily in skeletal muscle.

Teleost fish, in general, are glucose intolerant; this trait has been attributed to piscine islets secreting insulin primarily in response to amino acid secretogogues rather than glucose. However, pancreatic islets from the teleost fish tilapia, when transplanted into diabetic nude mice, were glucose responsive even though tilapia were severely glucose intolerant. This suggested a strong peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies as well as Northern analysis for mRNA, tilapia tissues were found to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals. The absence of GLUT-4 in peripheral tissues may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant. This suggests that tilapia islets have evolved along mammalian lines to be glucose sensitive while tilapia peripheral tissue have diverged widely. Using the same methods, tilapia were found to have a very limited tissue distribution of the insulin-independent glucose transporter, GLUT-1, which is responsible for basal glucose transport in mammalian cells. It is suggested that tilapia provide a naturally occurring GLUT-4 knockout model.

We compared the incorporation of the blood-borne [14C]-palmitate into selected lipid and phospholipid pools in rat muscles (soleus, red and white gastrocnemius), at rest and during contractions (15 and 60 tetani/min) in one leg (5 min) while the contralateral leg served as a control. [1-14C]-palmitate (20 µCi/rat) was administered into the carotid artery (t = 1 min). [14C]-palmitate deposition was greatest in soleus (100%) and lower in red (82%) and white gastrocnemius muscles (63%), respectively (p < 0.05). [14C] was deposited primarily into the tri-acylglycerol (∼50%) and phospholipid pools (∼30%) of soleus and red gastrocnemius muscles, and into the di-acylglycerol (∼30%), tri-acylglycerol (∼30%) and phospholipid pools (∼30%) in white gastrocnemius muscle. During contraction the concentrations of tri-acylglycerol were not changed. But, contraction increased [14C]-palmitate incorporation into soleus and red gastrocnemius muscles (600-700%) and into white gastrocnemius muscles (200%). Slightly more [14C] was directed from the phospholipids into the tri-acylglycerol pool during contraction. [14C]-palmitate deposition was also increased in the subclasses of phospholipids during contraction in red and white gastrocnemius. In conclusion, the deposition of [14C]palmitate into different lipid and phospholipid pools is quite rapid, and is dependent on contraction and the muscle fiber type. (Mol Cell Biochem 166: 73-83, 1997)

We compared the relationship between predicted VO2 max (ml kg-1min-1) and recreational exercise patterns, using secondary data analysis of a comprehensive national survey (18,293 subjects aged 15-69 years). Exercise participation and predicted VO2 max data were available for about 50% of this sample (4933 females, 4738 males). As expected, VO2 max was significantly lower in the females than in the males at any age (P < 0.0001). Age was the most significant predictor of VO2 max (r = -0.71 for males, r = -0.73 for females). Adjusting the data for the body mass index (BMI) increased this relationship only slightly in the males (R - 0.75) and females (R = 0.79). The simultaneous inclusion of exercise participation data (intensity, duration, energy expenditure) did not increase the predictions meaningfully (R = 0.78 for the males, R = 0.81 for the females). These exercise participation parameters concomitantly accounted for only a very slight amount of the variance of VO2 max in both the females (3.0%) and males (4.5%). To minimize the effects of age, the data were analysed using 5-year intervals. Again, the exercise participation parameters accounted for only a small part of the variance in VO2 max (< 10%), except in the 15- to 19-year-old males (24%). These data suggest that VO2 max is not associated with participation in recreational exercise.

We investigated the effects of 3 wk of moderate- (21 m/min, 8% grade) and highintensity treadmill training (31 m/min, 15% grade) on1) monocarboxylate transporter 1 (MCT-1) content in rat hindlimb muscles and the heart and2) lactate uptake in isolated soleus (Sol) muscles and perfused hearts. In the moderately trained group MCT-1 was not increased in any of the muscles [Sol, extensor digitorum longus (EDL), and red (RG) and white gastrocnemius (WG)] (P> 0.05). Similarly, lactate uptake in Sol strips was also not increased (P> 0.05). In contrast, in the heart, MCT-1 (+36%, P< 0.05) and lactate uptake (+72%, P< 0.05) were increased with moderate training. In the highly trained group, MCT-1 (+70%, P< 0.05) and lactate uptake (+79%, P< 0.05) were increased in Sol. MCT-1 was also increased in RG (+94%,P< 0.05) but not in WG and EDL (P> 0.05). In the highly trained group, heart MCT-1 (+44%, P< 0.05) and lactate uptake (+173%, P< 0.05) were increased. In conclusion, it has been shown that1) in both heart and skeletal muscle lactate uptake is increased only when MCT-1 is increased;2) training-induced increases in MCT-1 occurred at a lower training intensity in the heart than in skeletal muscle; 3) in the heart, lactate uptake was increased much more after high-intensity training than after moderate-intensity training, despite similar increases in heart MCT-1 with these two training intensities; and4) the increases in MCT-1 occurred independently of any changes in the heart’s oxidative capacity (as measured by citrate synthase activity).

The aim of this study was to examine the effects of chronic low frequency stimulation on the lactate transport across the plasma membrane of the tibialis anterior (TA) muscle of the rat. Stimulating electrodes were implanted on either side of the peroneal nerve in one hindlimb. Chronic stimulation (10 Hz, 50 psec bursts, 24 h/day) commenced 7 days after surgery, and were continued for 7 days. Animals were then left for 24 h, and thereafter muscles were obtained. Cytochrome C-oxidase activity was increased 1.9-fold in the stimulated TA compared to the control TA (p < 0.05). Lactate transport (zero-trans) was measured in giant sarcolemmal vesicles obtained from the chronically stimulated TA and the control TA. At each of the concentrations used in these studies a significant increase in lactate transport was observed: 2.8-fold increase at 1 mM lactate p < 0.05); 2-fold increases at both 30 mM and 50 mM lactate p < 0.05). These studies have shown that lactate transport capacity is markedly increased in response to chronic muscle contraction.

The purpose of this study was to compare lactic acid removal rates during three modes of recovery from a standardized exercise bout. Each subject (N6) completed a 1 mile run (92.2 ± 3.7 o2max). Thereafter, lactic acid removal rates were compared in the runners at each of three different modes of recovery a) rest; b) a self-selected, continuous jogging pace (free-jogging); and c) completely uncontrolled recovery (free-intermittent) normally practiced by athletes. Venous blood samples were taken immediately after the mile run and every 5 min thereafter for 20 min. Data were expressed relative to the initial post-exercise blood sample (100). Lactic acid removal was significantly faster during the free-jogging recovery than during the free-intermittent and the resting recoveries (P<0.001). Removal rates during the free-intermittent recovery were significantly faster than during the resting recovery (P<0.001). The results indicated that nearly optimal lactic acid removal rates were attained during the free-jogging recovery.

Certain teleost fish have large anatomically discrete islet organs called Brockmann bodies (BBs). When transplanted into streptozotocin diabetic athymic nude mice, tilapia BBs provide long-term normoglycemia. This has afforded us the opportunity to examine tilapia islet in vivo function in a non-piscine environment and compare this with in vivo function in the donor species. As expected, fasting and non-fasting glycemic levels in long-term murine recipients of tilapia BBs were analogous to corresponding values in donor tilapia, but, surprisingly, tilapia BB grafts provided mammalian-like glucose tolerance profiles. Teleost fish, in general, are severely glucose intolerant. When glucose tolerance tests were performed in tilapia, the mean glucose disappearance rates were very low; however, diabetic nude mice bearing long-term tilapia BB grafts were extremely glucose responsive. This suggested a severe or absolute peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies and then confirmed by Northern analysis, tilapia peripheral tissues appear to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals, but not GLUT-1, the insulin independent glucose transporter. This may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant after pharmacologic glucose-loading. Because tilapia do not tend to consume large quantities of glucose in the wild, it is not surprising that they have evolved without a mechanism to move glucose rapidly from the bloodstream into muscle and fat. Nevertheless, insulin still appears to play an important role in maintaining normoglycemia in tilapia; however, this is mostly likely a result of its effect on glucose uptake in the liver. We also present comparative data on tilapia beta cell function, quantification of islet cell numbers and types, islet products, insulin gene structure and expression, and beta cell sensitivity to the diabetogenic drug streptozotocin.

Objective: Alterations in myocardial metabolism occur early after the onset of diabetes suggesting that they may play a role in the development of cardiac dysfunction. Inhibition of myocardial pyruvate dehydrogenase (PDH), glucose transport and glycolysis have all been reported following diabetes. In vivo lactate is also a potential source of energy for the heart and its oxidation should not be affected by changes in glucose transport and glycolysis. Therefore, the objective of this study, was to test the hypothesis that following diabetes the inhibition of glucose oxidation would be greater than the inhibition of lactate oxidation. Methods: Hearts from control and one-week-old diabetic rats were perfused with [1-13C]glucose (11 mmol/l) alone, [1-13C]glucose plus lactate (0.5 mmol/l) or glucose plus [3-13C]lactate (0.5 or 1.0 mmol/l) as substrates. Glucose and lactate oxidation rates were determined by combining 13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. Results: In diabetic hearts perfused with glucose alone, glucose oxidation was decreased compared to controls (0.31±0.08 vs. 0.71±0.11 µmoles/min/g wet weight; p<0.05). Surprisingly, in hearts perfused with glucose plus 0.5 mmol/l lactate, there was no difference in glucose oxidation between control and diabetic groups (0.20±0.05 vs. 0.16±0.04 µmoles/min/g wet weight respectively). However, under these conditions lactate oxidation was markedly reduced in the diabetic group (0.89±0.18 vs. 0.24±0.05 µmoles/min/g wet weight; p<0.05). At 1.0 mmol/l lactate oxidation was still significantly depressed in the diabetic group. Conclusion: There was a greater decrease in lactate oxidation relative to glucose oxidation in hearts from diabetic animals. These results demonstrate that diabetes leads to a specific inhibition of lactate oxidation independent of its effects on pyruvate dehydrogenase.

Objectives: Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. Methods: Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4–5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. Results: The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p<0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. Conclusion: The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.