Recovery from exhaustive exercise in the white muscle of rainbow trout (Oncorhynchus mykiss) was used to examine the role of the adenylates in the control of energy metabolism and to assess the validity of equilibrium models of the behaviour of the high energy phosphates. The difficulty of obtaining muscle samples from fish makes detailed analysis of the behaviour of the labile high energy phosphates complex. The use of a new sampling procedure, the infusion of a lethal dose of anaesthetic via an indwelling cannula, minimized this problem. At exhaustion [ATP] and [PCr] were depressed by 75 and 80% respectively relative to the resting values. [ATP] depletion was mirrored by a stoichiometric increase in [IMP]. During recovery [PCr] returned to the resting level within 2 hours, but [ATP] recovery was slow and not complete until 24 hours post exercise. In contrast, energy charge and RATP(the proportion of the free adenylate pool phosphorylated to ATP) were, if anything, higher than the resting values by 2 hours post exercise. Therefore, [ATP] and energy status can be dissociated in tissues like fish white muscle because of the action of the purine nucleotide cycle. At 2 hours post exercise the calculated free ADP concentration dropped to less than one tenth the value at rest. As a result the [ATP] / [ADP] free ratio increased by nearly 6 fold. This condition may be required for glycogen resynthesis from lactate in muscle. Several similar equilibrium models of the behaviour of the adenylates and PCr were applied to the fish white muscle system. In general, the models well describe the relationship between the high energy phosphates. However, the definition of the high energy phosphate pool introduces some complications since this includes the total [ATP]. Because of the action of AMP deaminase the [ATP] concentration can change without measurable changes in the energy status, which is not considered in any of the models. As long as the extent of IMP formation is known the models can be applied, but since the formation of IMP may vary from fish to fish or with exercise regime the models lose much of their predictive power. / Science, Faculty of / Zoology, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.