Myocardial macronutrient transporter adaptations in the adult pregestational female intrauterine and postnatal growth-restricted offspring

Associations between exponential childhood growth superimposed on low birth weight and adult onset cardiovascular disease with glucose intolerance/type 2 diabetes mellitus exist in epidemiological investigations. To determine the metabolic adaptations that guard against myocardial failure on subsequent exposure to hypoxia, we compared with controls (CON), the effect of intrauterine (IUGR), postnatal (PNGR), and intrauterine and postnatal (IPGR) calorie and growth restriction (n = 6/group) on myocardial macronutrient transporter (fatty acid and glucose) -mediated uptake in pregestational young female adult rat offspring. A higher myocardial FAT/CD36 protein expression in IUGR, PNGR, and IPGR, with higher FATP1 in IUGR, FATP6 in PNGR, FABP-c in PNGR and IPGR, and no change in GLUT4 of all groups was observed. These adaptive macronutrient transporter protein changes were associated with no change in myocardial [(3)H]bromopalmitate accumulation but a diminution in 2-deoxy-[(14)C]glucose uptake. Examination of the sarcolemmal subfraction revealed higher basal concentrations of FAT/CD36 in PNGR and FATP1 and GLUT4 in IUGR, PNGR, and IPGR vs. CON. Exogenous insulin uniformly further enhanced sarcolemmal association of these macronutrient transporter proteins above that of basal, with the exception of insulin resistance of FATP1 and GLUT4 in IUGR and FAT/CD36 in PNGR. The basal sarcolemmal macronutrient transporter adaptations proved protective against subsequent chronic hypoxic exposure (7 days) only in IUGR and PNGR, with notable deterioration in IPGR and CON of the echocardiographic ejection fraction. We conclude that the IUGR and PNGR pregestational adult female offspring displayed a resistance to insulin-induced translocation of FATP1, GLUT4, or FAT/CD36 to the myocardial sarcolemma due to preexistent higher basal concentrations. This basal adaptation of myocardial macronutrient transporters ensured adequate fatty acid uptake, thereby proving protective against chronic hypoxia-induced myocardial compromise.     

Additive effects of insulin and muscle contraction on fatty acid transport and fatty acid transporters, FAT/CD36, FABPpm, FATP1, 4 and 6

Insulin and muscle contraction increase fatty acid transport into muscle by inducing the translocation of FAT/CD36. We examined (a) whether these effects are additive, and (b) whether other fatty acid transporters (FABPpm, FATP1, FATP4, and FATP6) are also induced to translocate. Insulin and muscle contraction increased glucose transport and plasmalemmal GLUT4 independently and additively (positive control). Palmitate transport was also stimulated independently and additively by insulin and by muscle contraction. Insulin and muscle contraction increased plasmalemmal FAT/CD36, FABPpm, FATP1, and FATP4, but not FATP6. Only FAT/CD36 and FATP1 were stimulated in an additive manner by insulin and by muscle contraction.     

脂肪细胞中Munc18c的缺失不影响胰岛素诱导的GLUT4转运(已撤稿2016-01-13)

动物脂肪和肌肉组织中葡萄糖的摄取是通过受胰岛素调控的GLUT4储存囊泡的运输实现的.Sec1p的同源物Munc18c被认为是通过控制SNARE复合物的装配来使GLUT4囊泡锚定到质膜上的重要物质.我们发现Munc18c的缺失没有影响GLUT4的转运上膜,也没有影响Syntaxin4在细胞膜上的定位.在缺少Munc18c和功能性Syntaxin2的时候,GLUT4的转运可能和Munc18b有关.在3T3-L1脂肪细胞中与Syntaxin4具有强烈相互作用的是Munc18c而不是Munc18a和Munc18b.然而,当缺少Munc18c时,Munc18a和Munc18b与Syntaxin4体现出较弱的相互作用.因此,Syntaxin4可能在胰岛素刺激GLUT4转运过程中起到重要的作用,且与SM蛋白的相互作用是有代偿性的.     

Fatty acid transport protein 4 is dispensable for intestinal lipid absorption in mice

FA transport protein 4 (FATP4), one member of a multigene family of FA transporters, was proposed as a major FA transporter in intestinal lipid absorption. Due to the fact that Fatp4(-/-) mice die because of a perinatal skin defect, we rescued the skin phenotype using an FATP4 transgene driven by a keratinocyte-specific promoter (Fatp4(-/-);Ivl-Fatp4(tg/+) mice) to elucidate the role of intestinal FATP4 in dietary lipid absorption. Fatp4(-/-);Ivl-Fatp4(tg/+) mice and wild-type littermates displayed indistinguishable food consumption, growth, and weight gain on either low or high fat (Western) diets, with no differences in intestinal triglyceride (TG) absorption or fecal fat losses. Cholesterol absorption and intestinal TG absorption kinetics were indistinguishable between the genotypes, although Western diet fed Fatp4(-/-);Ivl-Fatp4(tg/+) mice showed a significant increase in enterocyte TG and FA content. There was no compensatory upregulation of other FATP family members or any other FA or cholesterol transporters in Fatp4(-/-);Ivl-Fatp4(tg/+) mice. Furthermore, although serum cholesterol levels were lower in Fatp4(-/-);Ivl-Fatp4(tg/+) mice, there was no difference in hepatic VLDL secretion in-vivo or in hepatic lipid content on either a chow or Western diet. Taken together, our studies find no evidence for a physiological role of intestinal FATP4 in dietary lipid absorption in mice.     

Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis

In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.     

Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia

Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control or HF diet (48 kcal %fat) alone or with Met (500 mg/kg), with treadmill exercise, or with both exercise and Met interventions for 8 wk. HF-fed ZDF rats developed hyperglycemia (mean: 24.4 +/- 2.1 mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance and ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions that included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared with lean rats (P < 0.01) and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity. Thus reduction of skeletal muscle FAT/CD36 and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.     

FIP2 and Rip11 specify Rab11a-mediated cellular distribution of GLUT4 and FAT/CD36 in H9c2-hIR cells

Rab11a has been shown to be involved in different vesicle trafficking processes. To further define the functional role of Rab11a in vesicle movement we knocked down gene expression of Rab11a and two of its effectors, Rip11 and FIP2, in H9c2-hIR cells and measured the cell surface abundance of GLUT4myc and FAT/CD36. We observed that by knocking down Rab11a, both GLUT4myc and FAT/CD36 abundance at the plasma membrane were substantially increased. In the case of GLUT4myc, the in vitro knockdown of FIP2 also increased the cell surface abundance of GLUT4myc. Knockdown of both FIP2 and Rip11 increase the abundance of FAT/CD36 at the plasma membrane. Stimulated translocation of GLUT4myc and FAT/CD36 is not altered after gene knockdown of Rab11a. These data therefore show that (i) Rab11a regulates cell surface abundance of both GLUT4 and FAT/CD36 and that (ii) both Rab11a-dependent processes are differently regulated by Rab11a effector proteins.     

Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles.

Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles.It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain.     

Cardiac substrate uptake and metabolism in obesity and type-2 diabetes: Role of sarcolemmal substrate transporters

Cardiovascular disease is the primary cause of death in obesity and type-2 diabetes mellitus (T2DM). Alterations in substrate metabolism are believed to be involved in the development of both cardiac dysfunction and insulin resistance in these conditions. Under physiological circumstances the heart utilizes predominantly long-chain fatty acids (LCFAs) (60–70%), with the remainder covered by carbohydrates, i.e., glucose (20%) and lactate (10%). The cellular uptake of both LCFA and glucose is regulated by the sarcolemmal amount of specific transport proteins, i.e., fatty acid translocase (FAT)/CD36 and GLUT4, respectively. These transport proteins are not only present at the sarcolemma, but also in intracellular storage compartments. Both an increased workload and the hormone insulin induce translocation of FAT/CD36 and GLUT4 to the sarcolemma. In this review, recent findings on the insulin and contraction signalling pathways involved in substrate uptake and utilization by cardiac myocytes under physiological conditions are discussed. New insights in alterations in substrate uptake and utilization during insulin resistance and its progression towards T2DM suggest a pivotal role for substrate transporters. During the development of obesity towards T2DM alterations in cardiac lipid homeostasis were found to precede alterations in glucose homeostasis. In the early stages of T2DM, relocation of FAT/CD36 to the sarcolemma is associated with the myocardial accumulation of triacylglycerols (TAGs) eventually leading to an impaired insulin-stimulated GLUT4-translocation. These novel insights may result in new strategies for the prevention of development of cardiac dysfunction and insulin resistance in obesity and T2DM.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

Insulin acutely regulates Munc18-c subcellular trafficking: altered response in insulin-resistant 3T3-L1 adipocytes.

Preincubation of 3T3-L1 adipocytes in high glucose or glucosamine decreases acute insulin (100 nm)-stimulated glucose transport provided that insulin (0.6 nm) is included during preincubation. GLUT4 expression is unchanged (Nelson, B. A., Robinson, K. A., and Buse, M. G. (2000) Diabetes 49, 981-991). Munc18-c, a Syntaxin 4-binding protein, is a proposed regulator of the docking/fusion of GLUT4-containing vesicles with the plasma membrane. We examined the subcellular distribution of Munc18-c in response to acute (15-min) insulin (100 nm) stimulation after preincubation in 5 or 25 mm glucose +/- 0.6 nm insulin. Immunoblotting detected Munc18-c mainly in the Triton X-100-soluble plasma membrane (TS-PM) and the Triton X-100-insoluble low density microsomal (TI-LDM) fraction. Under each condition except high glucose + insulin preincubation, acute insulin increased Munc18-c (50-200%) in TS-PM and decreased Munc18-c (60%) in TI-LDM. Munc18-c traffic was time-dependent with a lag time of 3 min compared with GLUT4. Preincubation with high glucose + 0.6 nm insulin significantly impaired acute insulin-stimulated Munc18-c trafficking and decreased basal Munc18-c in the TI-LDM. Preincubation with glucosamine + insulin had similar effects. Total cellular Munc18-c remained unchanged. In conclusion, acute insulin stimulation promotes the translocation of Munc18-c, apparently from a TI-LDM-associated compartment to the TS-PM. Chronically increased glucose flux or exposure to glucosamine disrupts this process, which may negatively impact the fusion of GLUT4-containing vesicles with the plasma membrane.     

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.     

Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase

The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.     

Conditioning Increases the Gain of Contraction-Induced Sarcolemmal Substrate Transport in Ultra-Endurance Racing Sled Dogs

Endurance exercise relies on transsarcolemmal flux of substrates in order to avoid depletion of intramuscular reserves. Previous studies of endurance trained sled dogs have shown a remarkable capacity of these dogs to adapt rapidly to endurance exercise by decreasing the utilization of intramuscular reserves. The current study tested the hypothesis that the dogs'' glycogen-sparing phenotype is due to increased sarcolemmal transport of glucose and fatty acids. Basal and exercise-induced transport of glucose and fatty acids into sarcolemmal vesicles was evaluated in racing sled dogs prior to and after 7 months of exercise conditioning. Sarcolemmal substrate transport capacity was measured using sarcolemmal vesicles and radiolabelled substrates, and transporter abundance was measured using Western blot quantification in whole muscle homogenates and the sarcolemmal vesicle preparations. Conditioning resulted in increased basal and exercise-induced transport of both glucose and palmitate. Neither acute exercise nor conditioning resulted in changes in muscle content of GLUT4 or FAT/CD36, but conditioning did result in decreased abundance of both transporters in the sarcolemmal vesicles used for the basal transport assays, and this decrease was further amplified in the vesicles used for the exercise-induced transport assays. These results demonstrate conditioning-induced increases in sarcolemmal transport of oxidizable substrates, as well as increased gain of exercise-induced sarcolemmal transport of these substrates. These results further indicate that increased sarcolemmal transport of oxidizable substrates may be due to either an increased intrinsic capacity of the existing transporters or to a different population of transporters from those investigated.     

Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.     

Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant

To examine the temporal relationship between pre- and post-docking events, we generated a Munc18c temperature-sensitive mutant (Munc18c/TS) by substitution of arginine 240 with a lysine residue. At the permissive temperature (23 degrees C), overexpression of both the wild type (Munc18c/WT) and the R240K mutant inhibited insulin-stimulated GLUT4/IRAP vesicle translocation. However, at the non-permissive temperature (37 degrees C) only Munc18c/WT inhibited GLUT4/IRAP translocation whereas Munc18c/TS was without effect. Moreover, Munc18c/WT bound to syntaxin 4 at both 23 and 37 degrees C whereas Munc18c/TS bound syntaxin 4 only at 23 degrees C. This was due to a temperature-dependent conformational change in Munc18c/TS, as its ability to bind syntaxin 4 and effects on GLUT4 translocation were rapidly reversible while protein expression levels remained unchanged. Furthermore, insulin stimulation of Munc18c/TS-expressing cells at 23 degrees C followed by temperature shift to 37 degrees C resulted in an increased rate of GLUT4 translocation compared with cells stimulated at 37 degrees C. To date, this is the first demonstration that the rate-limiting step for insulin-stimulated GLUT4 translocation is the trafficking of GLUT4 vesicles and not their fusion with the plasma membrane.     

The fatty acid translocase (FAT)/CD36 and the glucose transporter GLUT4 are localized in different cellular compartments in rat cardiac muscle

The fatty acid translocase (FAT)/CD36 plays an important role in the acute regulation of fatty acid uptake in muscle tissue. We studied the subcellular distribution of FAT/CD36 in rat cardiac muscle after in vivo insulin stimulation by membrane fractionation and immunoisolation of GLUT4- and FAT/CD36-vesicles. FAT/CD36 was equally present in both plasma and microsomal membranes with no effect of insulin on the cellular distribution, whereas GLUT4 increased 2- to 3-fold in the plasma membrane. FAT/CD36 resides in one intracellular pool, whereas GLUT4 is present in two distinct pools. Immunoadsorption of GLUT4-vesicles indicated that FAT/CD36 is undetectable in these vesicles. Likewise, no GLUT4 could be detected in FAT/CD36-vesicles. These vesicles contain a high amount of Rab11 that remained unaffected after insulin stimulation, whereas Rab11 increased about 3-fold in the GLUT4-vesicles in response to insulin. These data show that GLUT4 and FAT/CD36 do not co-localize in cardiac muscle and that FAT/CD36 is not redistributed in response to insulin in the heart. Rab11 may be involved in endosomal recycling of FAT/CD36, however, insulin-associated Rab11 functions appear to be limited to GLUT4-vesicles.     

Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin.

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.     

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.