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

OXPAT/PAT-1 is a PPAR-Induced Lipid Droplet Protein that Promotes Fatty Acid Utilization

Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as OXPAT. Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARα overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARγ agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes

Albiglutide, a Long Lasting Glucagon-Like Peptide-1 Analog, Protects the Rat Heart against Ischemia/Reperfusion Injury: Evidence for Improving Cardiac Metabolic Efficiency

BACKGROUND: The cardioprotective effects of glucagon-like peptide-1 (GLP-1) and analogs have been previously reported. We tested the hypothesis that albiglutide, a novel long half-life analog of GLP-1, may protect the heart against I/R injury by increasing carbohydrate utilization and improving cardiac energetic efficiency. METHODS/PRINCIPAL FINDINGS: Sprague-Dawley rats were treated with albiglutide and subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Left ventricle infarct size, hemodynamics, function and energetics were determined. In addition, cardiac glucose disposal, carbohydrate metabolism and metabolic gene expression were assessed. Albiglutide significantly reduced infarct size and concomitantly improved post-ischemic hemodynamics, cardiac function and energetic parameters. Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake while reducing lactate efflux. Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation which in part was due to an increase in both glucose and lactate oxidation. Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic myocardium by albiglutide. CONCLUSION/SIGNIFICANCE: Albiglutide reduced myocardial infarct size and improved cardiac function and energetics following myocardial I/R injury. The observed benefits were associated with enhanced myocardial glucose uptake and a shift toward a more energetically favorable substrate metabolism by increasing both glucose and lactate oxidation. These findings suggest that albiglutide may have direct therapeutic potential for improving cardiac energetics and function

Change in age at diagnosis of oropharyngeal cancer in the United States, 1975-2016

The emergence of the human papillomavirus (HPV) as the primary etiology of oropharyngeal cancer has changed head and neck cancer (HNC) epidemiology. This study described change in the age at diagnosis of oropharyngeal and non-oropharyngeal HNC in the United States in the last four decades. Using a retrospective cohort analysis, the Surveillance, Epidemiology, and End Results dataset from 1975 to 2016 was queried for eligible adult cases of HNC, grouped as oropharyngeal

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 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

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