Complex interplay between the lipin 1 and the hepatocyte nuclear factor 4 alpha (HNF4alpha) pathways to regulate liver lipid metabolism

Lipin 1 is a bifunctional protein that serves as a metabolic enzyme in the triglyceride synthesis pathway and regulates gene expression through direct protein-protein interactions with DNA-bound transcription factors in liver. Herein, we demonstrate that lipin 1 is a target gene of the hepatocyte nuclear factor 4α (HNF4α), which induces lipin 1 gene expression in cooperation with peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) through a nuclear receptor response element in the first intron of the lipin 1 gene. The results of a series of gain-of-function and loss-of-function studies demonstrate that lipin 1 coactivates HNF4α to activate the expression of a variety of genes encoding enzymes involved in fatty acid catabolism. In contrast, lipin 1 reduces the ability of HNF4α to induce the expression of genes encoding apoproteins A4 and C3. Although the ability of lipin to diminish HNF4α activity on these promoters required a direct physical interaction between the two proteins, lipin 1 did not occupy the promoters of the repressed genes and enhances the intrinsic activity of HNF4α in a promoter-independent context. Thus, the induction of lipin 1 by HNF4α may serve as a mechanism to affect promoter selection to direct HNF4α to promoters of genes encoding fatty acid oxidation enzymes

Dietary fat and carbohydrates differentially alter insulin sensitivity during caloric restriction

BACKGROUND AND AIMS: We determined the effects of acute and chronic calorie restriction with either a low-fat, high-carbohydrate diet or a low-carbohydrate diet on hepatic and skeletal muscle insulin sensitivity. METHODS: Twenty-two obese subjects (body-mass index, 36.5±0.8kg/m(2)) were randomized to a high-carbohydrate (>180g/d) or low-carbohydrate (<60g/d) energy-deficit diet. A euglycemic–hyperinsulinemic clamp, muscle biopsies, and magnetic resonance spectroscopy were used to determine insulin action, cellular insulin signaling and intrahepatic triglyceride content before, after 48 h, and after ~11 wks (7% weight loss) of diet therapy. RESULTS: At 48 h, intrahepatic triglyceride content decreased more in the low-carbohydrate than the high-carbohydrate diet group (29.6±4.8% vs. 8.9±1.4%; P<0.05), but was similar in both groups after 7% weight loss (low-carbohydrate diet, 38.0±4.5% vs. high-carbohydrate diet, 44.5±13.5%). Basal glucose production rate decreased more in the low-carbohydrate than the high-carbohydrate diet group at 48 h (23.4±2.2% vs. 7.2±1.4%, P<0.05) and after 7% weight loss (20.0±2.4% vs. 7.9±1.2%, P<0.05). Insulin-mediated glucose uptake did not change at 48 h, but increased similarly in both groups after 7% weight loss (48.4±14.3%, P<0.05). In both groups, insulin-stimulated phosphorylation of Jun N-terminal kinase decreased by 29±13% and phosphorylation of Akt and insulin receptor substrate -1 increased by 35±9% and 36±9%, respectively, after 7% weight loss (all p<0.05). CONCLUSION: Moderate calorie restriction causes temporal changes in liver and skeletal muscle metabolism; 48 h of calorie restriction affects the liver (intrahepatic triglyceride content, hepatic insulin sensitivity, and glucose production), whereas moderate weight loss affects muscle (insulin-mediated glucose uptake and insulin signaling)

Complex interplay between the lipin 1 and the hepatocyte nuclear factor 4 α (HNF4α) pathways to regulate liver lipid metabolism.

Lipin 1 is a bifunctional protein that serves as a metabolic enzyme in the triglyceride synthesis pathway and regulates gene expression through direct protein-protein interactions with DNA-bound transcription factors in liver. Herein, we demonstrate that lipin 1 is a target gene of the hepatocyte nuclear factor 4α (HNF4α), which induces lipin 1 gene expression in cooperation with peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) through a nuclear receptor response element in the first intron of the lipin 1 gene. The results of a series of gain-of-function and loss-of-function studies demonstrate that lipin 1 coactivates HNF4α to activate the expression of a variety of genes encoding enzymes involved in fatty acid catabolism. In contrast, lipin 1 reduces the ability of HNF4α to induce the expression of genes encoding apoproteins A4 and C3. Although the ability of lipin to diminish HNF4α activity on these promoters required a direct physical interaction between the two proteins, lipin 1 did not occupy the promoters of the repressed genes and enhances the intrinsic activity of HNF4α in a promoter-independent context. Thus, the induction of lipin 1 by HNF4α may serve as a mechanism to affect promoter selection to direct HNF4α to promoters of genes encoding fatty acid oxidation enzymes

Lipin 1 is a target of HNF4α in HepG2 cells.

<p>[<b>A</b>] The schematic depicts luciferase reporter constructs driven by 2045 bp of 5′ flanking sequence or 2293 bp 3′ from the transcriptional start site of the <i>Lpin1</i> gene. Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with <i>Lpin1.</i>Luc reporter constructs and cotransfected with PGC-1α or PGC-1β expression constructs as indicated. The vector values are normalized ( = 1.0). The results are the mean of 3 independent experiments done in triplicate. *p<0.05 versus pCDNA control. [<b>B and C</b>] Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with +2293.<i>Lpin1.Luc</i> reporter construct and cotransfected expression constructs expressing WT or mL2 PGC-1α. The results are the mean of 3 independent experiments done in triplicate. *p<0.05 versus pCDNA control. **p<0.05 versus pCDNA control and HNF4α or PGC-1α overexpression alone. [<b>D</b>] The images depict the results of chromatin immunoprecipitation studies using chromatin from mouse hepatocytes infected with adenovirus to overexpress HNF4α. Crosslinked proteins were IP’ed with HNF4α antibody or IgG controls. “Input” represents 0.2% of the total chromatin used in the IP reactions. PCR primers were designed to amplify two regions of the <i>Lpin1</i> gene promoter containing NRREs or exon 7 (negative control). [<b>E</b>] Inset images depict results of western blotting analyses for the HNF4α and β-actin in HepG2 cells infected with adenovirus to overexpress PGC-1α or GFP (control) and transfected with siRNA to knockdown HNF4α or scramble control siRNA. Graphs depict the expression of HNF4α or lipin 1 mRNA in HepG2 cells infected with adenovirus to overexpress PGC-1α or GFP (control) and transfected with siRNA to knockdown HNF4α or scramble control siRNA (n = 6). *p<0.05 versus scramble control infected with the same adenovirus. **p<0.05 versus all other groups.</p

Lipin 1 enhances HNF4α-mediated increases in fatty acid oxidation.

<p>[<b>A</b>] The images depict the results of co-immunoprecipitation studies using lysates from HepG2 cells infected with adenovirus driving expression of lipin 1β or lipin 1(LXXFF). HNF4α-containing complexes were immunoprecipitated with an antibody directed against HNF4α or IgG control. Immunoprecipitated proteins were then subjected to immunoblotting with antibody directed against the HA tag of overexpressed lipin 1. Input represents 5% of the total protein used in immunoprecipitation reactions. [<b>B</b>] Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with <i>Acadm</i>.TKLuc or <i>Ppara.</i>Luc and cotransfected with lipin 1 and/or HNF4α expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p<0.05 versus pCDNA control. <b>**</b>p<0.05 versus pcDNA or lipin 1 alone. <b>***</b>p<0.05 versus all other groups. [<b>C and D</b>] Primary hepatocytes were isolated from 6 week old C57BL/6 mice and infected with adenovirus driving expression of GFP or HNF4α in the presence or absence of overexpressed lipin 1β (wild-type or LXXFF). The graphs depict <b>[C]</b> the expression of <i>Ppara</i> and <i>Acadm</i> (n = 5) or [<b>D</b>] mean rates of palmitate oxidation (mean of 3 independent experiments done in triplicate) or *p<0.05 versus GFP control. <b>**</b>p<0.05 versus HNF4α overexpression alone. <b>***</b>p<0.05 versus all other groups.</p

Lipin 1 inhibits <i>Apoc3/Apoa4</i> promoter activity in an HNF4α-dependent manner.

<p>[<b>A</b>] The schematic depicts the luciferase reporter construct under control of the intergenic region between the genes encoding ApoC3 and ApoA4 (<i>Apoc3/Apoa4.Luc</i>). The relative positions of two HNF4α response elements denoted as <i>Apoc3 enhancer</i> and <i>Apoa4 enhancer</i> are indicated. Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with <i>Apoc3/Apoa4.Luc</i> reporter constructs and cotransfected with lipin 1 and/or HNF4α expression constructs as indicated. <i>Apoc3/Apoa4.Luc</i> constructs were either wild-type or contained mutations in the <i>ApoC3 enhancer</i> or <i>ApoA4 enhancer</i> HNF4α response elements. The results are the mean of 3 independent experiments done in triplicate. *p<0.05 versus pCDNA control. <b>**</b>p<0.05 versus vector control or lipin 1 cotransfection. [<b>B</b>] The schematic depicts the heterologous luciferase reporter construct driven by three copies of the <i>Apoc3 enhancer</i> HNF4α response element. Graphs depict results of luciferase assays using lysates from HEK293 cells transfected with <i>Apoc3 enhancer.3X.TKLuc</i> and cotransfected with empty vector (pcDNA and pMT), lipin 1, and/or HNF4α expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p<0.05 versus pCDNA control. <b>**</b>p<0.05 versus vector control or lipin 1 cotransfection.</p