Acute Bidirectional Manipulation of Muscle Glucose Uptake by In Vivo Electrotransfer of Constructs Targeting Glucose Transporter Genes

Abstract

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 ؎ 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism. Diabetes 54:2702-2711, 2005

Amelioration of lipid-induced insulin resistance in rat skeletal muscle by overexpression of Pgc-1β involves reductions in long-chain acyl-CoA levels and oxidative stress

“…In both ex vivo assays in isolated EDL strips and in TC and EDL muscles during a hyperinsulinaemic-euglycaemic clamp, Pgc-1β was able to partially ameliorate this diet-induced insulin resistance. The observed improvements in insulin sensitivity are important considering that Pgc-1β was overexpressed only for 1-2 weeks of the 4-week high-fat feeding regime and with the electroporation technique, only ∼50% of muscle fibres within a given muscle are transfected [27]. Animals with whole body overexpression of PGC-1β display increased oxidative capacity in muscle and improved insulin sensitivity compared with wild-type controls [38].…”

“…Animals (5 h fasted) were studied over 2 h in the conscious state (insulin infusion 0.25 units kg -1 h -1 ), with a bolus of 2-deoxy[ 3 H] glucose tracer administered once plasma glucose levels reached steady state [8,27]. At the conclusion of the clamp the TC and EDL muscles were rapidly dissected and freezeclamped for determination of glucose uptake [8,27].…”

“…In vivo electrotransfer Specific details of the electrotransfer procedure have been described previously [8,27]. Animals were electroporated 2.5 weeks after commencement of their respective diets, and were killed 1.5 weeks after electroporation, unless otherwise stated.…”

“…Animals were electroporated 2.5 weeks after commencement of their respective diets, and were killed 1.5 weeks after electroporation, unless otherwise stated. In brief, the murine Pgc-1β coding sequence [28] was cloned into a muscle-specific expression vector [27]. Control and test muscles were pretreated for 2 h with 90 units of hyaluronidase to break down components of the extracellular matrix, thus improving transfection efficiency [29].…”

Overexpression of the orphan receptor Nur77 alters glucose metabolism in rat muscle cells and rat muscle in vivo

“…In vivo electroporation and euglycaemic-hyperinsulinaemic clamp studies with glucose tracers were performed as described previously [18,19]. For the in vivo electroporation experiments, Nur77 was expressed in the right tibialis anterior (TA) and extensor digitorum longus (EDL) muscle and empty vector (pCDNA3.1, Invitrogen) was expressed in the left leg as an internal control.…”