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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.

journal_name

Diabetes

journal_title

Diabetes

authors

Cleasby ME,Davey JR,Reinten TA,Graham MW,James DE,Kraegen EW,Cooney GJ

doi

10.2337/diabetes.54.9.2702

subject

Has Abstract

pub_date

2005-09-01 00:00:00

pages

2702-11

issue

eissn

0012-1797

issn

1939-327X

pii

54/9/2702

journal_volume

pub_type

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Acute bidirectional manipulation of muscle glucose uptake by in vivo electrotransfer of constructs targeting glucose transporter genes.