Role of Protein Kinase C in the Translational Regulation of Lipoprotein Lipase in Adipocytes

Abstract

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC ␣, ␤, ␦, and ⑀ isoforms, whereas PKC , , , , , and ␥ remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression. Lipoprotein lipase (LPL)1 is a central enzyme in lipid metabolism that is expressed primarily in adipose tissue and muscle (1). The regulation of lipoprotein lipase is complex and regulation may occur at the transcriptional, translational, and posttranslational levels (2). LPL activity is decreased in the adipose tissue of patients with diabetes. After treatment to control hyperglycemia in both type I and type II diabetes, there is an increase in LPL activity (3, 4), along with an increase in LPL synthetic rate with no change in LPL mRNA, suggesting regulation at the level of translation (5). Similar observations have been made in experimental models of insulin-deficient diabetic rats. Whereas short term insulin treatment of insulin-deficient rats increased LPL protein more than LPL mRNA, prolonged insulin treatment increased both LPL protein and LPL mRNA levels (6).Glucose and insulin modulate protein kinase C (PKC) activity in rat adipocytes (7). Hyperglycemia is known to increase cellular diacylglycerol (DAG), which in turn is the natural activator of PKC. Elevated DAG, resulting in PKC activation has been identified in insulin-deficient diabetic adipose tissue (8 -10). The role of hyperinsulinemia and hyperglycemia in the activation of PKC isoforms and involvement in insulin resistance has been studied in various animal models of diabetes (11). PKC is present in the soluble cytoplasmic fraction in cells prior to stimulation, and the treatment of cells with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in activation and translocation of PKC to the membrane. TPA activates PKC by interacting with the DAG binding site (12), although the...

Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress

“…Prolonged exposure of WI-38 cells to TPA at either 10 or 100 nM caused downregulation of cPKC␣, -␥, and nPKC␦, but had only a marginal effect on aPKC levels even at 100 nM ( Fig. 2A), as reported previously (46,47). Phorone at a concentration of 0.25 mM induced HO-1 gene expression in TPA-pretreated WI-38 cells to a level similar to that of TPA-untreated cells, although the level of induction by 1 mM phorone was somewhat suppressed in the PKC downregulated cells (Fig.…”

Regulation of Lipoprotein Lipase by Protein Kinase Cα in 3T3-F442A Adipocytes

“…To determine whether Raf1 kinase is involved in the PKC-mediated regulation of LPL, we measured Raf1 activity and LPL activity in adipocytes where PKC isoforms had been depleted by long term TPA treatment. As demonstrated previously (13), TPA treatment inhibited LPL activity by 70 Ϯ 6%. As shown in Fig.…”

“…We have previously examined the regulation of LPL in adipocytes through modulation of PKC. Depletion of cellular PKC activity, either through prolonged treatment of cells with phorbol ester (11,12) or with drugs that inhibit PKC, resulted in an inhibition of LPL activity through a decrease in translation (13). Thus, PKC is important in the regulation of LPL expression, although the precise mechanism for this effect is not known.…”

“…Western Blot Analysis-The analysis of proteins LPL, PKC␣, or ERK was performed essentially as described earlier (13). The cell monolayer was rinsed in ice-cold phosphate-buffered saline, and the total protein was extracted using the cell lysis buffer described above.…”

“…Involved in LPL Regulation-In previous studies, we observed a decrease in LPL activity following depletion of PKC isoforms ␣, ␤, ␦, and ⑀ after overnight TPA treatment of adipocytes (13). To identify the individual isoforms of PKC involved in LPL regulation, we examined the effect of depleting specific PKC isoforms using antisense oligonucleotide technology.…”