The glucocorticoid-Angptl4-ceramide axis induces insulin resistance through PP2A and PKCζ

Abstract

Chronic glucocorticoid exposure is associated with the development of insulin resistance. We showed that glucocorticoid-induced insulin resistance was attenuated upon ablation of Angptl4, a glucocorticoid target gene encoding the secreted protein angiopoietin-like 4, which mediates glucocorticoid-induced lipolysis in white adipose tissue. Through metabolomic profiling, we revealed that glucocorticoid treatment increased hepatic ceramide concentrations by inducing enzymes in the ceramide synthetic pathway in an Angptl4-dependent manner. Angptl4 was also required for glucocorticoids to stimulate the activities of the downstream effectors of ceramide, protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ). We further showed that knockdown of PP2A or inhibition of PKCζ or ceramide synthesis prevented glucocorticoid-induced glucose intolerance in wild-type mice. Moreover, the inhibition of PKCζ or ceramide synthesis did not further improve glucose tolerance in Angptl4^-/- mice, suggesting that these molecules were major downstream effectors of Angptl4. Overall, our study demonstrates the key role of Angptl4 in glucocorticoid-augmented hepatic ceramide production that induces whole-body insulin resistance.

Fig. 1.. Dexamethasone-induced glucose, insulin, and pyruvate intolerance were improved in Angptl4^−/− mice.

Wild-type (WT) and Angptl4^−/− mice were treated with phosphate-buffered saline (PBS) or dexamethasone (Dex). (A) GTT was performed after 16 hours of fasting. (B) Relative area under curve (AUC) for GTT results (relative to PBS-treated WT mice) is shown. Error bars represent SEM. n = 5 to 7 mice per group. *P < 0.05. (C) Plasma insulin concentrations were measured before glucose injection (0 time point), and 15 and 30 min after glucose injection. Error bars represent SEM. n = 5 to 7 mice per group. * indicates significant (P < 0.05) effect of dexamethasone (compared to PBS) at the given time point, ** indicates significant difference (P < 0.05) between WT dexamethasone and Angptl4^−/− dexamethasone at the given time point, and *** indicates significant (P < 0.05) difference between time points. (D) ITT was performed, and results are displayed as percentage of initial plasma glucose concentrations at different time points. (E) Relative AUC for ITT results (relative to PBS-treated WT mice) is shown. Error bars repre sent SEM. n = 5 to 7 mice per group. *P < 0.05. (F) Pyruvate tolerance test (PTT) was performed. (G) Relative AUC for PTT results (relative to PBS-treated WT mice) is shown. Error bars represent SEM. n = 3 to 5 mice per group. *P < 0.05.

Fig. 2.. Dexamethasone-induced insulin resistance in the liver and gastrocnemius muscle was improved in Angptl4^−/− mice.

WT and Angptl4^−/− mice were treated with PBS or dexamethasone and then vehicle or insulin (Ins). (A to C) The amounts of total Akt and phosphorylated Akt (p-Akt) were measured in the liver (A), gastrocnemius muscle (B), and eWAT (C). Gapdh was used as an internal control. The intensity of bands was normalized to those for Gapdh. The relative ratio of p-Akt to Akt represents the Akt activity. Error bars represent SEM. n = 3 to 4 mice per group. *P < 0.05.

Fig. 3.. Metabolomics analysis in the liver and gastrocnemius muscle of control and dexamethasone-treated WT and Angptl4^−/− mice.

WT and Angptl4^−/− mice were treated with PBS or dexamethasone for 7 days. (A) Metabolomics analysis was performed on hepatic lipids. The heat map shows metabolites that are significantly altered in content (P < 0.05) upon dexamethasone treatment in the livers of WT mice. The relative content was displayed in the heat map compared to the WT PBS group. Red shading on the heat map indicates higher relative amounts, and green shading represents lower relative amounts. (B) Six lipid metabolites that were significantly increased in abundance in dexamethasone-treated WT mice liver but not in dexamethasone-treated Angptl4^−/− mice are shown. Error bars represent SEM. n = 4 mice per group. *P < 0.05.

Fig. 4.. Dexamethasone-activated hepatic ceramide synthetic program was attenuated in Angptl4^−/− mice.

WT and Angptl4^−/− mice were treated with PBS or dexamethasone for 7 days. (A to C) The concentrations of 16 different ceramide species in the liver (A), 5 different free fatty acids (FFAs) in plasma (B), and 4 ceramide species in plasma (C) of these mice were measured. n = 4 mice per group. (D) The expression of genes encoding enzymes involved in ceramide production was monitored using quantitative polymerase chain reaction (qPCR). The heat map shows the relative expression compared to that in the WT PBS group. Red shading on the heat map indicates higher expression, and green shading represents lower expression. The changes in fold induction are shown in fig. S1. n = 16 mice per group. (E) Schematic representation of ceramide synthesis pathways. The genes and metabolites that were induced by dexamethasone in WT mice liver are shown in red. The metabolites that were reduced by dexamethasone are shown in green. (F) The abundance of Cers5 and Cers6 proteins was monitored using Western blot. The bar graph represents the average intensity of bands normalized to those for Gapdh. Error bars represent SEM. n = 3 to 4 mice per group. *P < 0.05.

Fig. 5.. The roleof PKCζ andPP2A indexamethasone-induced glucose tolerance in mice.

WT and Angptl4^−/− mice were treated with PBS or dexamethasone for 7 days. (A) Hepatic PP2A activity was measured. The bar graph shows relative PP2A activity (to PBS-treated WT mice). (B) The amounts of total PKCζ and p-PKCζ in the liver were monitored by Western blots. The bar graph shows the average intensity of bands normalized to those for β-actin. The ratio of p-PKCζ to PKCζ is an indicator of PKCζ activity. For both (A) and (B), error bars represent SEM. n = 3 to 4 mice per group. *P < 0.05. (C) GTT was performed after a 6-hour fast on WT and Angptl4^−/− mice infected with adenovirus expressing scrambled (Scr) shRNA or shRNA targeting Ppp2ca and treated with dexamethasone for 7 days. Relative AUC for GTT results (relative to PBS-treated WT mice) is shown. Error bars represent SEM. n = 4 to 6 mice per group. *P <0.05. (D) WT and Angptl4^−/− mice were treated with dexamethasone. ACPD was injected into mice starting on day 4. GTT was performed at day 7 after a 6-hour fast. Relative AUC for GTT results (relative to PBS-treated WT mice) is shown. Error bars represent SEM. n = 3 to 4 mice per group. *P < 0.05.

Fig. 6.. Myriocin improved glucose tolerance in dexamethasone-treated WT but not in Angptl4^−/− mice.

(A) WT and Angptl4^−/− mice were treated with dexamethasone. Myriocin was injected intraperitoneally into mice starting on day 4. GTT was then performed after 6 hours of fasting. (B) Western blots were performed to monitor the amounts of total PKCζ and p-PKCζ in the liver of mice treated with or without myriocin. The bar graph shows the average intensity of bands normalized to those for b-actin. The ratio of p-PKCζ to PKCζ is an indicator of PKCζ activity. Error bars represent SEM. n = 3 to 4 mice per group for (A) and (B). *P < 0.05.

Fig. 7.. The proposed model for the role of Angptl4 in glucocorticoid-induced hepatic insulin resistance.

Glucocorticoid activates the expression of Angptl4, which promotes lipolysis in WAT. Fatty acids (FAs) generated from lipolysis of triglycerides (TGs) serve as both precursors for ceramide synthesis (such as palmitate) and also signals to act with glucocorticoids to increase the expression of Cers3, Cers4, Cers5, and Cers6. Spt1 and Spt2 expression is also induced by glucocorticoids, though this induction does not require Angptl4. Ceramides subsequently activate PKCζ and PP2A, which inhibit Akt and result in insulin resistance.