Free fatty acid-induced PP2A hyperactivity selectively impairs hepatic insulin action on glucose metabolism

Abstract

In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is "selective", in the sense that the insulin-mediated decrease of glucose production is blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity.

Figure 1. Palmitate does not affect insulin-receptor phosphorylation, but impairs Akt activation.

□ Control; ▪ 0.5 mM Palmitate. Palmitate has no effect on insulin receptor phosphorylation, but Akt activation is impaired in cultured primary rat hepatocytes treated for 20 h with palmitate. (a) The insulin-stimulated insulin receptor phosphorylation to total insulin receptor ratio is not altered in hepatocytes treated with palmitate. (b) However, insulin-stimulated phosphorylation of Akt Ser473 is significantly inhibited by palmitate. This effect is conferred onto the Akt substrates FoxO1 (Ser256) (c) and Gsk3α (Ser21) (d) which both show decreased insulin-stimulated phosphorylation. Fold change is relative to no insulin. Phosphorylation levels of individual proteins were normalized to the total amounts of the respective proteins. Data are averages of western blot quantifications +/− std. error of the mean, n = 8. * indicates p<0.05 between groups. Representative western blots are shown.

Figure 2. Palmitate-induced insulin resistance results in impaired G6pc suppression and continued action on aPKC regulated Srebp1c.

□ Control; ▪ 0.5 mM Palmitate. (a) Insulin-suppression of G6pc mRNA was significantly inhibited in hepatocytes treated for 20 h with palmitate in vitro. This suppression was not affected by preincubation with 20 µM of a pseudosubstrate inhibitor of the aPKCs (Inh). (b) On the other hand, insulin-stimulated induction of Srebp1c mRNA was maintained in palmitate-treated cells. Induction could, however, be inhibited by addition of the aPKC inhibitor (Inh). Relative expression is relative to no insulin. Data are averages of real-time PCR results +/− std. error of the mean, n = 10. * indicates p<0.05 between groups.

Figure 3. Palmitate-induced insulin resistance reflects an impairment of insulin action on Akt-dependent processes.

□ Control; ▪ 0.5 mM Palmitate. However, insulin action on TG accumulation is maintained. (a) Insulin-stimulated suppression of gluconeogenesis (from medium containing 2 mM pyruvate), (b) incorporation of glucose into glycogen (from 5.5 mM glucose) and (c) glucose disposal (from 15.5 mM glucose) were significantly inhibited in hepatocytes treated for 20 h with palmitate in vitro. Meanwhile, insulin-stimulated TG accumulation was maintained in palmitate-treated hepatocytes (d). Results are expressed as differences from the rates in cells given no insulin. Data are averages of 8–10 independent experiments +/− std. error of the mean. * indicates p<0.05 between groups.

Figure 4. PP2A activity is increased in hepatocytes treated with palmitate.

□ Control; ▪ 0.5 mM Palmitate. The protein level of a PP2A structural subunit (PP2Aα) was upregulated in hepatocytes treated with palmitate (a). Meanwhile, PP2A activity was increased by approximately 30 per cent in insulin-resistant palmitate-treated hepatocytes (b, c). PP2A-activity was estimated by the degree of methylation of the PP2A catalytic subunit (b) or a direct phosphatase activity assay (c). Protein levels were normalized to β–actin. Data are averages of western blot quantifications or PP2A activity measurements +/− std. error of the mean, n = 10. * indicates p<0.05. Representative western blots are shown for a and b.

Figure 5. Inhibition of PP2A activity results in an increased insulin-stimulated activation of Akt.

Cantharidin (Can) (a) or Okadaic acid (OA) (b) inhibition of PP2A resulted in an increased Ser473 phosphorylation of Akt in palmitate-treated hepatocytes stimulated with 10 nM insulin. Fold change is relative to no Cantharidin or Okadaic acid. Akt phosphorylation levels of were normalized to the total amounts of Akt. Data are averages of western blot quantifications +/− std. error of the mean, n = 8. * indicates p<0.05 vs. no Cantharidin or Okadaic acid. Representative western blots are shown.

Figure 6. The Akt-inactivating phosphatase PP2A is hyperactive in insulin-resistant ZDF rats.

PP2A levels were upregulated in all major insulin target tissues while PP2A activity was upregulated in liver and muscle in severely insulin-resistant ZDF rats. (a) Hepatic, muscle and fat mRNA levels of the catalytic subunit of PP2A (Ppp2ca) and (b) hepatic and muscle PP2A activity were found to be significantly elevated in obese fa/fa ZDF rats compared to their lean Fa/Fa counterparts. Relative expression and fold change is relative to Fa/Fa expression or activity levels. Data are averages of real-time PCR results or PP2A activity measurements +/− std. error of the mean. n = 8 for fa/fa rats and n = 6 for Fa/Fa rats. * indicates p<0.05.

Figure 7. Several PP2A subunits are upregulated in ZDF rats.

The PP2A subunits PP2Aα (Ppp2r1a), B56β (Ppp2r5b), B55α (Ppp2r2a) and Ptpa (Ppp2r4) were significantly upregulated in both liver and muscle of fa/fa rats compared to their lean Fa/Fa counterparts. Relative expression is relative to Fa/Fa expression levels. Data are averages of real-time PCR results +/− std. error of the mean. n = 8 for fa/fa rats and n = 6 for Fa/Fa rats. * indicates p<0.05.

Figure 8. Proposed model for selective insulin resistance.

Black arrows designate physiological conditions, whereas red arrows designate altered conditions in insulin resistance. For further explanation, see text.