Abstract
In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.
Highlights
In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation
We examined the inhibition of protein degradation by insulin and insulin analogues (LysB28,ProB29-insulin (LysPro), AspB10-insulin (B10), and GluB4,GlnB16,PheB17-insulin (EQF)) in H4, HepG2, and L6 cells
H4-II-E Hepatoma cells—Because insulin effects on cellular function are initiated by receptor binding, the binding of [125I]iodoinsulin and the effects of native insulin and various analogues were examined
Summary
Materials—The rat hepatoma cell line, H4-II-E, the human hepatoma cell line, HepG2, and the rat skeletal muscle cell line, L6, were purchased from the ATCC (Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS and 10 g/ml gentamicin and incubated at 37 °C in an atmosphere of 5% CO2/95% air. For L6 cells, on day 9 after plating, the growth medium was removed from the cells and replaced with leucine-free DMEM containing 10% FCS and 10 g/ml gentamicin and 1 Ci/ml [3H]leucine. Cells were incubated for 18 h to allow labeling of cellular proteins with [3H]leucine. Assays in hepatoma cells were carried out as described for L6 cells except the cell labeling medium was leucine-free MEM with 10% FCS, 10 g/ml gentamicin, and 1 Ci/ml leucine. The incubation/chase medium consisted of MEM with 20 mM TES, pH 7.5, 0.1% BSA, and 2 mM leucine.
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