Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471–E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (∼10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.

Overexpression of carnitine palmitoyltransferase I in skeletal muscle in vivo increases fatty acid oxidation and reduces triacylglycerol esterification

A key regulatory point in the control of fatty acid (FA) oxidation is thought to be transport of FAs across the mitochondrial membrane by carnitine palmitoyltransferase I (CPT I). To investigate the role of CPT I in FA metabolism, we used in vivo electrotransfer (IVE) to locally overexpress CPT I in muscle of rodents. A vector expressing the human muscle isoform of CPT I was electrotransferred into the right lateral muscles of the distal hindlimb [tibialis cranialis (TC) and extensor digitorum longus (EDL)] of rats, and a control vector expressing GFP was electrotransferred into the left muscles. Initial studies showed that CPT I protein expression peaked 7 days after IVE (+104%, P < 0.01). This was associated with an increase in maximal CPT I activity (+30%, P < 0.001) and a similar increase in palmitoyl-CoA oxidation (+24%; P < 0.001) in isolated mitochondria from the TC. Importantly, oxidation of the medium-chain FA octanoyl-CoA and CPT I sensitivity to inhibition by malonyl-CoA were not altered by CPT I overexpression. FA oxidation in isolated EDL muscle strips was increased with CPT I overexpression (+28%, P < 0.01), whereas FA incorporation into the muscle triacylglycerol (TAG) pool was reduced (−17%, P < 0.01). As a result, intramyocellular TAG content was decreased with CPT I overexpression in both the TC (−25%, P < 0.05) and the EDL (−45%, P < 0.05). These studies demonstrate that acute overexpression of CPT I in muscle leads to a repartitioning of FAs away from esterification and toward oxidation and highlight the importance of CPT I in regulating muscle FA metabolism.

Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle

The present study examined the acute effects of metformin on fatty acid (FA) metabolism in oxidative soleus (SOL) and glycolytic epitrochlearis (EPT) rodent muscle. SOL and EPT were incubated for either 30 or 180 min in the absence or presence of 2 mM metformin and with or without insulin (10 mU/ml). Metformin did not alter basal FA metabolism but countered the effects of insulin on FA oxidation and incorporation into triacylglyerol (TAG). Specifically, metformin prevented the insulin-induced suppression of FA oxidation in SOL but did not alter FA incorporation into lipid pools. In contrast, in EPT metformin blunted the incorporation of FA into TAG when insulin was present but did not alter FA oxidation. In SOL, metformin resulted in a 50% increase in AMP-activated protein kinase α2 activity and prevented the insulin-induced increase in malonyl-CoA content. In both fiber types, basal and insulin-stimulated glucose oxidation were not significantly altered by metformin. All effects were similar regardless of whether they were measured after 30 or 180 min. Because increased muscle lipid storage and impaired FA oxidation have been associated with insulin resistance in this tissue, the ability of metformin to reverse these abnormalities in muscle FA metabolism may be a part of the mechanism by which metformin improves glucose clearance and insulin sensitivity. The present data also suggest that increased glucose clearance is not due to its enhanced subsequent oxidation. Additional studies are warranted to determine whether chronic metformin treatment has similar effects on muscle FA metabolism.

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 μM) palmitate oxidation was decreased by 95% (P < 0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P < 0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.