Insulin Regulation of Lipoprotein Lipase Activity in 3T3-L1 Adipocytes is Mediated at Posttranscriptional and Posttranslational Levels

C F Semenkovich,M Wims,L Noe,J Etienne,L Chan

doi:10.1016/s0021-9258(18)81898-1

C F Semenkovich, M Wims + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)81898-1

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Abstract

Insulin is a major regulator of lipoprotein lipase (LPL) activity. The molecular events associated with LPL regulation by insulin in 3T3-L1 adipocytes were studied by determining LPL enzyme activity, mRNA levels, protein synthetic rate, and transcription run-off activity. Adipocytes treated with insulin (10(-6) M for 48 h) had substantially higher LPL activity (mean difference compared to carrier-treated cells 146%) with little difference in LPL mRNA levels (mean level 109% of control). Insulin regulation of LPL activity was dose-dependent but changes in LPL mRNA were not. Within 2 h of hormone addition, LPL activity was higher in insulin-treated versus carrier-treated adipocytes although their LPL mRNA levels were similar. In [35S]methionine pulse-labeled adipocytes, insulin decreased LPL protein synthetic rate measured by immunoprecipitation 42-48%, although increases (75-340%) in heparin-releasable LPL activity were detected in the same cells. In contrast, during differentiation of 3T3-L1 fibroblasts to the adipocyte state, 5-80-fold increases of heparin-releasable LPL activity were closely associated with similar (8-60-fold) increases in LPL mRNA levels. LPL synthetic rate was 16-fold greater, and LPL gene transcription initiation measured by transcriptional run-off was 10-fold higher in adipocytes than in undifferentiated cells. Differentiation of 3T3-L1 fibroblasts increases transcription of the LPL gene leading to increased LPL mRNA, protein synthetic rate, and enzyme activity. Insulin regulation of LPL activity in 3T3-L1 adipocytes, however, is mediated entirely at posttranscriptional and posttranslational levels.

Highlights

From the $Departmentsof Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas77030and the §Departmentof Biochemistry, Centre Hospitalo-Uniuersitaire Saint Antoine-Tenon7,5020Paris, France
Insulin evokes a wide variety of effects frequently classified L1 lipoprotein lipase (LPL) activity in part by transcriptional processes which as “metabolic,” those occurring over a short period of time could be inhibited by actinomycin D or a-amanitin
The in the current study to show that despite substantialincreases effects of insulin on protein and RNA synthesis do noet asily in LPL activity, insulin treatment is not associated with an increased LPL transcriptionrate (Fig. 8) or LPL mRNA levels (Fig. 2 and “Results”)

Summary

Introduction

From the $Departmentsof Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas77030and the §Departmentof Biochemistry, Centre Hospitalo-Uniuersitaire Saint Antoine-Tenon7,5020Paris, France. The morning of the experiment, aliquots were frozen for subsequent assay of media and heparin-releasable LPL enzyme activity; the cells were washed gently with sterile phosphate-buffered saline at 37 "C, and 2 ml of warm methionine-deficient DMEM (containing 4500 mg of Dglucose /liter) plus heat-inactivated 2% FBS (dialyzed overnight to remove methionine and thensterile filtered) were added to each plate.

Results

Conclusion