The increase in skeletal muscle pyruvate dehydrogenase kinase (PDK) activity was measured in skeletal muscle of six healthy males after a eucaloric high-fat/low-carbohydrate (HF/LC; 5% carbohydrate, 73% fat, and 22% protein of total energy intake) diet compared with a standardized prediet (50% carbohdyrate, 30% fat, and 21% protein). Biopsies were obtained from the vastus lateralis muscle after 3 days on the prediet (day 0) and after 1, 2, and 3 days of the HF/LC diet. Intact mitchondria were extracted from fresh muscle and analyzed for PDK activity and Western blotting of PDK2 and PDK4 protein. A second biopsy was taken at each time point and frozen for Northern blot analysis of PDK2 and PDK4 mRNAs. PDK activity increased in a linear fashion over the 3-day HF/LC diet and was significantly higher than control by 1 day. PDK activity was 0.09 +/- 0.03, 0.18 +/- 0.05, 0.30 +/- 0.07, and 0.37 +/- 0.09 min(-1) at 0, 1, 2, and 3 days, respectively. PDK4 protein and mRNA increased maximally by day 1, and PDK2 protein and mRNA were unaffected by the HF/LC diet. Resting respiratory exchange ratios decreased after 1 day of the HF/LC diet (from 0.79 +/- 0.02 to 0.72 +/- 0.02) and remained depressed throughout the 3-day dietary intervention (0.68 +/- 0.01). The immediate shift to fat utilization was accompanied by increased blood glycerol, beta-hydroxybutyrate, and plasma free fatty acid concentrations. These results suggest that the continuing increase in PDK activity over the 3-day HF/LC diet is not due to increasing PDK protein beyond 1 day. This could be due to the contribution of another isoform to the total PDK activity or to a continual increase in PDK4 or PDK2 specific activity.

The variability of the triacylglycerol store in human skeletal muscle (TGm) was examined using the needle biopsy technique. In 13 subjects, three biopsies were sampled from the vastus lateralis muscle of one leg at rest and after 90 min of cycling at 65% of maximal O2 uptake on one or two occasions. Visible fat and blood were removed before the samples were frozen, and remaining blood, connective tissue, and fat were removed from freeze-dried fiber bundles. TGm content was measured in two aliquots of powdered muscle from each biopsy. Within-biopsy variability was low at 6%. Despite precautions, many biopsies from inactive subjects were contaminated with adipose tissue. The TGm between-biopsy coefficient of variation (CV) was 23.5 +/- 14.6% (SD, n = 24) for rest and exercise time points where three noncontaminated biopsies existed. The between-biopsy variability at rest (19.8 +/- 7.9%, n = 10) was not significantly different from that at exercise (26.1 +/- 17.4%, n = 14). The muscle glycogen between-biopsy CV for rest and exercise time points was 10.0 +/- 10.3%. The resting TGm content was 26.3 +/- 4.3 mmol/kg dry muscle, and the net utilization during the 90 min of exercise was less than the between-biopsy variability. It is concluded that the TGm store measured in repeated biopsies of human skeletal muscle is variable, with a CV of 20-26%. Therefore, because of this high variability, only changes greater than approximately 24% of resting TGm content may be considered meaningful.

Evidence indicates that skeletal muscle lipid droplet-associated proteins (PLINs) regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN1 is thought to regulate lipolysis by directly interacting with comparative gene identification-58 (CGI-58), an activator of adipose triglyceride lipase (ATGL). Upon lipolytic stimulation, PLIN1 is phosphorylated, releasing CGI-58 to fully activate ATGL and initiate triglyceride breakdown. The absence of PLIN1 in skeletal muscle leads us to believe that other PLIN family members undertake this role. Our purpose was to examine interactions between PLIN2, PLIN3, and PLIN5, with ATGL and its coactivator CGI-58 at rest and following contraction. Isolated rat solei were incubated for 30 min at rest or during 30 min of intermittent tetanic stimulation [150-ms volleys at 60 Hz with a train rate of 20 tetani/min (25°C)] to maximally stimulate intramuscular lipid breakdown. Results show that the interaction between ATGL and CGI-58 increased 128% following contraction (P ϭ 0.041). Further, ATGL interacts with PLIN2, PLIN3, and PLIN5 at rest and following contraction. The PLIN2-ATGL interaction decreased significantly by 21% following stimulation (P ϭ 0.013). Both PLIN3 and PLIN5 coprecipitated with CGI-58 at rest and following contraction, while there was no detectable interaction between PLIN2 and CGI-58 in either condition. Therefore, our findings indicate that in skeletal muscle, during contraction-induced muscle lipolysis, ATGL and CGI-58 strongly associate and that the PLIN proteins work together to regulate lipolysis, in part, by preventing ATGL and CGI-58 interactions at rest. adipocyte differentiation-related protein; adipophilin; OXPAT; MLDP; TIP47; ABHD5 FATTY ACIDS (FA) RELEASED from intramuscular triglycerides (IMTG) during lipolysis provide an important source of energy during muscle contraction. In skeletal muscle, IMTGs are packaged into lipid droplets that possess a unique coat of proteins associated with the surrounding phospholipid monolayer. This protein coat provides an interface for specific processes, such as transport, lipogenesis, and lipolysis (10, 34). Perilipins (PLINs) are the most recognized family of lipid droplet proteins and are the most likely to be involved in the regulation of lipogenesis and lipolysis in skeletal muscle (31).Our understanding of PLIN proteins in skeletal muscle is limited; however, studies in other tissues and in cell culture indicate that PLIN proteins are key regulators of lipid metabolism, as they appear to be directly involved with how cells and tissues store, mobilize, and utilize fatty acids (8,12,15,34,35,62). The PLIN family consists of five members, PLIN1

To characterize human skeletal muscle enzymatic adaptation to a low-carbohydrate, high-fat, and high-protein diet (LCD), subjects consumed a eucaloric diet consisting of 5% of the total energy intake from carbohydrate, 63% from fat, and 33% from protein for 6 days compared with their normal diet (52% carbohydrate, 33% fat, and 14% protein). Biopsies were taken from the vastus lateralis before and after 3 and 6 days on a LCD. Intact mitochondria were extracted from fresh muscle and analyzed for pyruvate dehydrogenase (PDH) kinase, total PDH, and carnitine palmitoyltransferase I activities and mitochondrial ATP production rate (using carbohydrate and fat substrates). β-Hydroxyacyl CoA dehydrogenase, active PDH (PDHa), and citrate synthase activities were also measured on whole muscle homogenates. PDH kinase (PDHK) was calculated as the absolute value of the apparent first-order rate constant of the inactivation of PDH in the presence of 0.3 mM Mg2+-ATP. PDHK increased dramatically from 0.10 ± 0.02 min−1 to 0.35 ± 0.09 min−1 at 3 days and 0.49 ± 0.06 min−1 after 6 days. Resting PDHa activity decreased from 0.63 ± 0.17 to 0.17 ± 0.04 mmol ⋅ min−1 ⋅ kg−1after 6 days on the diet, whereas total PDH activity did not change. Activities for all other enzymes were unaltered by the LCD. In summary, severe deficiency of dietary carbohydrate combined with a twofold increase in dietary fat and protein caused a rapid three- to fivefold increase in PDHK activity in human skeletal muscle. The increased PDHK activity downregulated the amount of PDH in its active form at rest and decreased carbohydrate metabolism. However, an increase in the activities of enzymes involved in fatty acid oxidation did not occur.