Activation of cannabinoid receptor type 1 (Cb1r) disrupts hepatic insulin receptor signaling via cyclic AMP-response element-binding protein H (Crebh)-mediated induction of Lipin1 gene

Abstract

Activation of hepatic cannabinoid 1 receptor (Cb1r) signaling has been implicated in the development of phenotypes associated with fatty liver, hypertriglyceridemia, and insulin resistance. In the current study, we have elucidated the critical role of endoplasmic reticulum-bound transcription factor cyclic AMP-response element-binding protein H (Crebh) in mediating activated Cb1r signaling in inducing phosphatidic acid phosphatase Lipin1 gene expression and subsequently deregulating hepatic insulin receptor signaling. Cb1r agonist (2-arachidonoylglycerol (2-AG)) treatment induced Lipin1 gene expression in a Crebh-dependent manner via recruiting CREBH to the endogenous Lipin1 gene promoter. Adenoviral overexpression of Crebh or 2-AG treatment in mice induced Lipin1 gene expression to increase the hepatic diacylglycerol (DAG) level and phosphorylation of protein kinase Cε (PKCε). This in turn inhibited hepatic insulin receptor signaling. Knockdown of Crebh or Cb1r antagonism attenuated 2-AG-mediated induction of Lipin1 gene expression and decreased DAG production in mouse liver and subsequently restored insulin receptor signaling. Similarly, knockdown of Lipin1 attenuated the 2-AG-induced increase in the DAG level and PKCε phosphorylation. Finally, shRNA-mediated knockdown of Crebh partially but significantly blunted Lipin1 expression and the DAG level in db/db mice. These results demonstrate a novel mechanism by which Cb1r signaling induces Lipin1 gene expression and increases DAG production by activating Crebh, thereby deregulating insulin receptor signaling pathway and lipid homeostasis.

FIGURE 1.

Activated Cb1r induces Lipin1 gene expression via Crebh. A, mice (n = 5 per group) were treated with 2-AG at the indicated times or for 12 h, and liver tissues were obtained for semiquantitative PCR (top left) or qPCR (right) analyses and for measuring protein levels (bottom left). *, p < 0.05 versus untreated control. B, mice (n = 5 per group) were treated with 2-AG for 12 h or treated with AM251 for 12 h preceding 2-AG treatment for a further 12 h, and liver tissues were obtained for semiquantitative PCR (top left) or qPCR (right) analyses and for measuring protein levels (bottom left).*, p < 0.05 versus control; **, p < 0.05 versus vehicle (veh) + 2-AG. All data represent mean ± S.E. (error bars).

FIGURE 2.

Crebh is a transcriptional regulator of Lipin1 gene expression. A, primary (1°) rat hepatocytes were infected with adenoviruses encoding GFP, FLAG-ATF6-N, and FLAG-CREBH-N for 48 h. RNA was extracted for semiquantitative PCR (left) and qPCR analyses (right). *, p < 0.05 versus Ad-GFP. B–E, transient transfection assays were performed in AML12 cells with mLipin1-luc reporter constructs along with co-transfection of the indicated expression vectors (B–D) as indicated or treatment with 2-AG for 12 h (B and E) prior to luciferase assays. -Fold activity represents relative luciferase activity:β-gal activity. All transfections were performed in triplicates and are representative of three to four independent experiments. F, untransfected AML12 cells (top) or AML12 cells transfected with wild type or CREBH mutant constructs of mLipin1-Luc (bottom) were treated with vehicle or 2-AG for 12 h, and immunoprecipitation (IP) of AML12 chromatin from cells exposed to vehicle or 2-AG was performed with IgG or CREBH antibody. The percentage of DNA immunoprecipitated with CREBH antibody relative to input chromatin was demonstrated by semiquantitative PCR analysis. *, p < 0.05 versus control. All data represent mean ± S.E. (error bars) of at least three independent experiments. DN, dominant negative.

FIGURE 3.

Crebh-mediated induction of Lipin1 gene expression and increase in hepatic DAG level in vivo. A and B, mice (n = 5) were infected with the indicated adenoviruses for 72 h. Following completion of the experiments, mice were sacrificed, and liver tissues were obtained for semiquantitative PCR (top left) or qPCR (right) analyses and for measuring protein levels (bottom left). The DAG level was measured in Ad-GFP- and Ad-CREBH-N-infected mouse liver samples (B). *, p < 0.05 versus Ad-GFP. C, mice (n = 5) were infected with the indicated adenoviruses, and 96 h postinfection, mice were treated with 2-AG for a further 12 h. Liver tissues were obtained for semiquantitative PCR (top left) or qPCR (right) analyses and for measuring protein (bottom left). *, p < 0.05 versus USi; **, p < 0.05 versus USi + 2-AG. Data represent mean ± S.E. D, mice (n = 5; left) or AML12 cells (right) were infected with the indicated adenoviruses, and 96 h postinfection, mice were treated with 2-AG for a further 12 h. The DAG level was measured in adenovirus-infected mouse liver samples (left) and in AML12 cells (right). *, p < 0.05 versus USi/vehicle (veh); **, p < 0.05 versus USi/vehicle + 2-AG. All data represent mean ± S.E. (error bars).

FIGURE 4.

Crebh-mediated induction of Lipin1 gene expression is linked to perturbation of hepatic insulin receptor signaling in vivo. A–E, experiments were performed as described in Fig. 1 (for A and C) or Fig. 3 (for B, D, and E), and liver tissues (A–D) or AML12 cell lysates (E) were obtained for Western blot analysis with the indicated antibodies. Protein levels were quantified by densitometry analysis (phospho:total form). *, p < 0.05 versus control and Ad-GFP; **, p < 0.05 versus vehicle (veh) + 2-AG and USi + 2-AG. All data represent mean ± S.E. (error bars). IRβ, insulin receptor β.

FIGURE 5.

Knockdown of Crebh reverses perturbation of hepatic insulin receptor signaling in db/db mice. A, semiquantitative PCR (top) or qPCR (bottom) analyses of gene expression in B6 or db/db mice (n = 4–5). *, p < 0.05 versus B6. B–D, semiquantitative PCR (B, left) or qPCR (B, right) analyses, protein levels (C), and DAG levels (D) were measured in db/db mice (n = 5) infected with Ad-USi or Ad-CREBH RNAi. *, p < 0.05 versus USi. All data represent mean ± S.E. (error bars). E, proposed model depicting the effect of endocannabinoid-mediated activation of Cb1r in inducing and activating CREBH to disrupt hepatic insulin signaling via induction of Lipin1 gene expression and by increasing DAG production. IR, insulin receptor.