This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Type 2 diabetes mellitus (T2DM), obesity, dyslipidemia, hypertension and macrovascular disease have all been linked to impaired insulin action (insulin resistance). Skeletal muscle is the largest insulin responsive organ in the body and as such has been has been identified as the primary site of insulin stimulated glucose uptake and disposal. Studies have confirmed that it is the major locus of insulin resistance. Excess availability of lipid giving rise to obesity, to elevated plasma FFA, triglycerides1 and even excess dietary fat intake have been correlated with insulin resistance. Interestingly, from the public health perspective, the quality as well as the quantity of fat influences the degree of insulin resistance: saturated fats monounsaturates polyunsaturates (especially omega 3 fatty acid oils). In fact, a greater degree of saturated fatty acid acyl chains of muscle membrane phospholipids correlates significantly with the degree of insulin resistance in cross sectional studies. Studies both in animals and humans have confirmed that excess availability of lipid in the form of plasma FFA can acutely bring on a state of insulin resistance indistinguishable from that observed with obesity or T2DM3, suggesting that excess availability of lipid substrate is causal in the induction of insulin resistance. Triglycerides may be stored in muscle, and it is known that this pool is in homeostatic equilibrium with the supply of FFA substrate from the plasma compartment. Muscle cells preferentially oxidize lipid under most conditions and use the intracellular pool for up to 60% of their basal oxidative requirements. It has recently been proposed that the intramyocellular pool of triglycerides, if increased, could also play an etiologic role in the onset or perpetuation of insulin resistance. A number of animal and human biopsy studies have confirmed such an association, however the association has been relatively weak and inconsistent. The PI believes that the results to date have been confounded by problems with the biopsy techniques used: contamination by as little as 1% fat can be predicted to cause a 100-200% error in the triglyceride analysis. In order to resolve this issue, he has recently validated the use of magnetic resonance proton spectroscopy for the measurement of intracellular triglycerides in skeletal muscle. High resolution volume localized proton spectroscopy has allowed Dr. Stein and colleagues to specifically quantify this important metabolic variable in isolation from contaminating adipose fat. Dr. Stein has demonstrated a very strong inverse relationship between intramyocellular triglyceride stores and the degree of insulin resistance. Impressively, after multivariate regression analysis, the relationship remained strong and a better predictor of insulin sensitivity than classical variables such as percent body fat, BMI, age, and regional adiposity. It is not yet known how excess lipid causes insulin resistance. Classically, the effect of an increase in FFA and its oxidation results in an increase in the NADH/NAD and acetyl CoA/CoA ratio which in turn causes a feedback along the glycolytic pathway, an increase in glucose 6-phosphate such that glucose phosphorylation is inhibited limiting glucose entry into the cell (the Randle effect). Studies in human subjects have tended to contradict this dogma, suggesting that FFA may directly inhibit glucose transport initially resulting in decreased glucose 6- phosphate levels, followed by specific effects to inhibit glycogen synthesis directly. Thus in human studies where plasma FFA have been raised with infusion of a lipid emulsion, inhibition in glucose oxidation occurred rapidly (by 60 minutes) and subsequent defects on glucose transport/phosphorylation appeared at ~2 hours and further inhibition of glycogen synthase at ~3-4 hours. It has been suggested that FFA cause their deleterious effects on insulin action via local accumulation of lipid derived mediators such as long chain acyl-CoA's, diacyl glycerol (DAG) or phospholipids. Another cause may be diversion of glucose flux into the hexosamine pathway which may also have independent post receptors effects to antagonize insulin action. These may include alteration in transcription of insulin responsive genes, inhibiting enzyme activity such as glycogen synthase, and altering plasma membrane activation/Glucose transporter (Glut 4) vesicle trafficking. The intramyocellular pool of triglycerides is thought to be in equilibrium with these lipid derived moieties under steady state conditions. How this pool is related to insulin sensitivity in a dynamic setting is unknown. At present, it is currently unknown whether under conditions of acutely imposed hyperlipacidemia, whether the intramyocellular pool of triglyceride tracks with the onset of insulin resistance. Another and related question is whether all types are fat are equal in their ability to induce insulin resistance. The lipid infusion studies in humans used a soybean oil emulsion which is highly enriched in unsaturated fatty acids (18:2 and 18:3), with the balance being mostly 18:1 (oleic) acid. No studies to date have examined the acute effects of various fatty acids on the time course of inducing insulin resistance and whether specific aspects of the insulin response pathway are affected differentially. In fact medium term dietary studies of several weeks duration have been unable to demonstrate the same induction of insulin resistance as seen in high fat feeding models in rodents. Thus what is more important? Is it the total amount of triglyceride within the intramyocellular storage pool, or is it the relative amounts of saturated vs unsaturated triglycerides? By comparing peak heights, either from the proton spectrum or from the 13C spectrum, the degree of saturation/unsaturation of afatty acids can be calculated without invasive biopsy Previous studies utilizing NMR spectroscopy to study the time course of lipid emulsion induced defects in glucose uptake and storage in human subjects have been performed at 2.1 - 4.7T. The present study will be performed at the GCRC MR Core Laboratory using a 4.0T whole body magnet which has high sensitivity for monitoring glycogen. No studies have been performed on intramyocellular lipid metabolism at high field (3.0T), and Dr. Stein's preliminary studies were all performed at 1.5T. This will not only allow increased sensitivity for quantification of lipids, but increased spectral resolution promises to allow specific quantification of fatty species, i.e. the degree of intracellular triglyceride saturation.
SPECIFIC AIMS 1. Whether increases in plasma FFA from saturated dietary fats will decrease whole body and muscle glucose disposal rates faster and to a greater degree compared to unsaturated fat. 2. Whether the time course of fat induced inhibition of glucose disposal is paralleled or preceded by the accumulation of total and specifically saturated long chain intramyocellular triglycerides. 3. Whether increases in plasma FFA from saturated dietary fats will alter total fatty acid oxidation rates compared to unsaturated fat
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