An ANGPTL4-ceramide-protein kinase Cζ axis mediates chronic glucocorticoid exposure-induced hepatic steatosis and hypertriglyceridemia in mice.

Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders

Bile Acid Signal-Induced Phosphorylation of Small Heterodimer Partner by Protein Kinase Cζ is Critical for Epigenomic Regulation of Liver Metabolic Genes

Bile acids (BAs) are recently recognized key signaling molecules that control integrative metabolism and energy expenditure. BAs activate multiple signaling pathways, including those of nuclear receptors, primarily farnesoid X receptor (FXR), membrane BA receptors, and FXR-induced FGF19 to regulate the fed-state metabolism. Small heterodimer partner (SHP) has been implicated as a key mediator of these BA signaling pathways by recruitment of chromatin modifying proteins, but the key question of how SHP transduces BA signaling into repressive histone modifications at liver metabolic genes remains unknown. Here we show that protein kinase Cζ (PKCζ) is activated by BA or FGF19 and phosphorylates SHP at Thr-55 and that Thr-55 phosphorylation is critical for the epigenomic coordinator functions of SHP. PKCζ is coimmunopreciptitated with SHP and both are recruited to SHP target genes after bile acid or FGF19 treatment. Activated phosphorylated PKCζ and phosphorylated SHP are predominantly located in the nucleus after FGF19 treatment. Phosphorylation at Thr-55 is required for subsequent methylation at Arg-57, a naturally occurring mutation site in metabolic syndrome patients. Thr-55 phosphorylation increases interaction of SHP with chromatin modifiers and their occupancy at selective BA-responsive genes. This molecular cascade leads to repressive modifications of histones at metabolic target genes, and consequently, decreased BA pools and hepatic triglyceride levels. Remarkably, mutation of Thr-55 attenuates these SHP-mediated epigenomic and metabolic effects. This study identifies PKCζ as a novel key upstream regulator of BA-regulated SHP function, revealing the role of Thr-55 phosphorylation in epigenomic regulation of liver metabolism

FXR Acetylation is Normally Dynamically Regulated by p300 and SIRT1 but Constitutively Elevated in Metabolic Disease States

The nuclear bile acid receptor FXR is critical for regulation of lipid and glucose metabolism. Here, we report that FXR is a target of SIRT1, a deacetylase that mediates nutritional and hormonal modulation of hepatic metabolism. Lysine 217 of FXR is the major acetylation site targeted by p300 and SIRT1. Acetylation of FXR increases its stability but inhibits heterodimerization with RXRalpha, DNA binding, and transactivation activity. Downregulation of hepatic SIRT1 increased FXR acetylation with deleterious metabolic outcomes. Surprisingly, in mouse models of metabolic disease, FXR interaction with SIRT1 and p300 was dramatically altered, FXR acetylation levels were elevated, and overexpression of SIRT1 or resveratrol treatment reduced acetylated FXR levels. Our data demonstrate that FXR acetylation is normally dynamically regulated by p300 and SIRT1 but is constitutively elevated in metabolic disease states. Small molecules that inhibit FXR acetylation by targeting SIRT1 or p300 may be promising therapeutic agents for metabolic disorders

Modulation of the activity of a key metabolic regulator Small Heterodimer Partner by post-translational modifications

Small Heterodimer Partner (SHP, NR0B2), a member of the nuclear receptor superfamily, is an orphan receptor that lacks a DNA binding domain but contains a putative ligand binding domain. SHP forms non-functional heterodimers with DNA binding transcriptional factors and, thereby, functions as a transcriptional corepressor in diverse biological processes, including cellular metabolism, cell proliferation, apoptosis, and sexual maturation. Of these reported functions of SHP, maintaining cholesterol and bile acid levels by negative feedback regulation of hepatic conversion of cholesterol to bile acids is well established. Cholesterol is essential in many biological activities in mammalian cells. Conversion of hepatic cholesterol into bile acids is a major pathway to eliminate cholesterol from the body. However, excess amounts of cholesterol and bile acids are pathogenic. Therefore, the levels of cholesterol and bile acids need to be tightly regulated. Cholesterol 7??-hydroxylase (CYP7A1), a liver specific P450 enzyme, is the first and rate-limiting enzyme in this process. Increased levels of bile acids repress transcription of CYP7A1 in a feedback manner. In response to elevated bile acid levels, the nuclear bile acid receptor Farnesoid X Receptor (FXR) increases the transcription of SHP. SHP interacts with the hepatic DNA-binding activators, hepatic nuclear factor-4?? (HNF- 4??) or liver receptor homologue-1 (LRH-1) on the CYP7A1 promoter, and represses transcription of the CYP7A1 gene. In addition to regulating cholesterol and bile acid levels, SHP is known to mediate inhibition of fatty acid synthesis, hepatic lipogenesis, and glucose production in response to elevated bile acid levels. Posttranslational modifications profoundly regulate protein stability and activity. Recently, bile acids have been reported to function as signaling molecules that activate kinase pathways. We recently found that SHP stability is increased by bile acid-activated ERK-mediated phosphorylation through inhibition of ubiquitination. We now show that the activity of SHP is increased by post-translational methylation of SHP at Arg-57 by protein arginine methyltransferase 5 in response to bile acids. The overall aim of this study is to delineate the molecular mechanism by which the post-translational modification of SHP regulates SHP functional activity. In recent years, several naturally-occurring mutations in the SHP gene have been reported in human subjects that are associated with mild obesity and diabetes. About 30% of these reported mutations were Arg mutations, including the R57W mutation. Though it is known that the mutations lead to metabolic disorders, the molecular basis underlying the mechanism by which the mutations lead to metabolic disease is unknown. By mass spectrometry, we identified Arg 57 as a site of methylation in SHP catalyzed by Protein Arginine Methyltransferase 5 (PRMT5). Functional activity assays showed that methylation of SHP at Arg-57 by PRMT5 is important for SHP inhibition of LRH1 and HNF-4?? transactivation. Our lab previously showed the molecular mechanism of SHP-mediated repression involving the coordinate recruitment of chromatin modifying repressive cofactors, mSin3A/HDAC1, NCoR1/HDAC3, methyltransferase G9a, and the Swi/Snf-Brm remodeling complex, to the CYP7A1 promoter. Mutation of the Arg-57 site to Trp (R57W is the naturallyoccurring mutant) decreased SHP interaction with corepressors that we had previously identified, and severely impaired inhibition of gene expression by SHP. Overexpression of wild type SHP in mouse liver resulted in decreased lipogenic, bile acid synthetic and gluconeogenic gene expression, and mutation of Arg-57 blocked SHP function, but remarkably in a gene-selective manner. Overexpression of the R57W mutant resulted in elevated levels of triglycerides and bile acids in liver compared to that of wild type SHP. Differential interaction and recruitment of corepressors by SHP in a promoter-specific manner may contribute to gene-selective repression by the R57W mutant. Our studies have shown that SHP is methylated by PRMT5 after bile acid treatment. Tandem mass spectrometry revealed that in addition to methylation at Arg-57, SHP is also phosphorylated at Thr-55 after bile acid treatment. Studies with kinase inhibitors showed that a signaling pathway involving PI3K and PKC ?? is involved in SHP Thr phosphorylation, and also regulates arginine methylation of SHP. The close proximity of the phosphorylation (Thr-55) and methylation (Arg-57) sites suggested a possible interplay between them. Studies with phosphorylation- and methylation-defective mutants demonstrated crosstalk between SHP Thr phosphorylation and Arg methylation. This study demonstrates a critical role for Arg-57 methylation by PRMT5 in SHP function, and suggests a possible mechanism for association of the reported R57W mutation with obesity. This study also reveals Thr-55 phosphorylation of SHP by upstream kinase signaling pathways to be important for SHP functional activity. Targeting post-translational modifications of SHP may be an effective strategy to develop new therapeutic agents to treat SHP-related human diseases, such as metabolic syndrome, cancer, and infertility

Arginine Methylation by PRMT5 at a Naturally Occurring Mutation Site is Critical for Liver Metabolic Regulation by Small Heterodimer Partner

Small Heterodimer Partner (SHP) inhibits numerous transcription factors that are involved in diverse biological processes, including lipid and glucose metabolism. In response to increased hepatic bile acids, SHP gene expression is induced and the SHP protein is stabilized. We now show that the activity of SHP is also increased by posttranslational methylation at Arg-57 by protein arginine methyltransferase 5 (PRMT5). Adenovirus-mediated hepatic depletion of PRMT5 decreased SHP methylation and reversed the suppression of metabolic genes by SHP. Mutation of Arg-57 decreased SHP interaction with its known cofactors, Brm, mSin3A, and histone deacetylase 1 (HDAC1), but not with G9a, and decreased their recruitment to SHP target genes in mice. Hepatic overexpression of SHP inhibited metabolic target genes, decreased bile acid and hepatic triglyceride levels, and increased glucose tolerance. In contrast, mutation of Arg-57 selectively reversed the inhibition of SHP target genes and metabolic outcomes. The importance of Arg-57 methylation for the repression activity of SHP provides a molecular basis for the observation that a natural mutation of Arg-57 in humans is associated with the metabolic syndrome. Targeting posttranslational modifications of SHP may be an effective therapeutic strategy by controlling selected groups of genes to treat SHP-related human diseases, such as metabolic syndrome, cancer, and infertility

The C-terminal fibrinogen-like domain of angiopoietin-like 4 stimulates adipose tissue lipolysis and promotes energy expenditure.

Angptl4 (Angiopoietin-like 4) is a circulating protein secreted by white and brown adipose tissues and the liver. Structurally, Angptl4 contains an N-terminal coiled-coil domain (CCD) connected to a C-terminal fibrinogen-like domain (FLD) via a cleavable linker, and both full-length Angptl4 and its individual domains circulate in the bloodstream. Angptl4 inhibits extracellular lipoprotein lipase (LPL) activity and stimulates the lipolysis of triacylglycerol stored by adipocytes in the white adipose tissue (WAT). The former activity is furnished by the CCD, but the Angptl4 domain responsible for stimulating adipocyte lipolysis is unknown. We show here that the purified FLD of Angptl4 is sufficient to stimulate lipolysis in mouse primary adipocytes and that increasing circulating FLD levels in mice through adenovirus-mediated overexpression (Ad-FLD) not only induces WAT lipolysis in vivo but also reduces diet-induced obesity without affecting LPL activity. Intriguingly, reduced adiposity in Ad-FLD mice was associated with increased oxygen consumption, fat utilization, and the expression of thermogenic genes (Ucp1 and Ppargc1a) in subcutaneous WAT. Moreover, Ad-FLD mice exhibited increased glucose tolerance. Chronically enhancing WAT lipolysis could produce ectopic steatosis because of an overflow of lipids from the WAT to peripheral tissues; however, this did not occur when Ad-FLD mice were fed a high-fat diet. Rather, these mice had reductions in both circulating triacylglycerol levels and the mRNA levels of lipogenic genes in the liver and skeletal muscle. We conclude that separating the FLD from the CCD-mediated LPL-inhibitory activity of full-length Angptl4 reveals lipolytic and thermogenic properties with therapeutic relevance to obesity and diabetes

Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin–proteasomal degradation

Small Heterodimer Partner (SHP) inhibits activities of numerous transcription factors involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin–proteasomal pathway and that bile acids or bile acid-induced intestinal fibroblast growth factor 19 (FGF19) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes

Bile Acid Signal-induced Phosphorylation of Small Heterodimer Partner by Protein Kinase Cζ Is Critical for Epigenomic Regulation of Liver Metabolic Genes

Bile acids (BAs) are recently recognized key signaling molecules that control integrative metabolism and energy expenditure. BAs activate multiple signaling pathways, including those of nuclear receptors, primarily farnesoid X receptor (FXR), membrane BA receptors, and FXR-induced FGF19 to regulate the fed-state metabolism. Small heterodimer partner (SHP) has been implicated as a key mediator of these BA signaling pathways by recruitment of chromatin modifying proteins, but the key question of how SHP transduces BA signaling into repressive histone modifications at liver metabolic genes remains unknown. Here we show that protein kinase Cζ (PKCζ) is activated by BA or FGF19 and phosphorylates SHP at Thr-55 and that Thr-55 phosphorylation is critical for the epigenomic coordinator functions of SHP. PKCζ is coimmunopreciptitated with SHP and both are recruited to SHP target genes after bile acid or FGF19 treatment. Activated phosphorylated PKCζ and phosphorylated SHP are predominantly located in the nucleus after FGF19 treatment. Phosphorylation at Thr-55 is required for subsequent methylation at Arg-57, a naturally occurring mutation site in metabolic syndrome patients. Thr-55 phosphorylation increases interaction of SHP with chromatin modifiers and their occupancy at selective BA-responsive genes. This molecular cascade leads to repressive modifications of histones at metabolic target genes, and consequently, decreased BA pools and hepatic triglyceride levels. Remarkably, mutation of Thr-55 attenuates these SHP-mediated epigenomic and metabolic effects. This study identifies PKCζ as a novel key upstream regulator of BA-regulated SHP function, revealing the role of Thr-55 phosphorylation in epigenomic regulation of liver metabolism