PGC-1α isoforms coordinate to balance hepatic metabolism and apoptosis in inflammatory environments

Abstract

Objective: The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes.

Methods: Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo.

Results: Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines.

Conclusions: PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis.

Keywords: Apoptosis; Inflammation; Liver; Metabolism; PGC-1 isoforms.

Graphical abstract

Figure 1

PGC-1α isoforms differentially regulate inflammatory and metabolic signaling pathways downstream of TNFα. A) Schematic representation of the PPARGC1A gene with known promoters and transcript changes that give rise to different PGC-1α variants. B-E) Gene expression microarrays of mRNA isolated from wild type primary mouse hepatocytes over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection. B) Number of genes changed greater than two-fold 48 h following transduction in the absence or presence of 2 ng/mL TNFα (2 h) (n = 3 biological replicates, adj. p-value <0.01). C) Clustering of genes significantly changed by over-expression of PGC-1α4 in primary hepatocytes in the presence of TNFα. D) Top 10 GO biological processes (adj. p-value <0.05) were identified from each list generated from TNFα-treated samples in B and listed on x-axis. Size of dot represents number of genes identified in each pathway, in comparison to other genotypes. E) GO biological processes (adj. p-value <0.05) associated with 175 genes regulated in the opposite direction. Data sets were generated using biological replicates (n = 3) of each condition from one experiment.

Figure 2

Over-expression of PGC-1α4 attenuates apoptosis induced by inflammatory signals. A) mRNA expression in wild type primary mouse hepatocytes over-expressing PGC-1α1, PGC-1α4, or control vector alone following 2-hour treatment with 2 ng/mL TNFα or vehicle (n = 3). *p < 0.05 effect of TNFα within each genotype compared to untreated cells. ^#p < 0.05 effect of genotype alone compared with control. ^$p < 0.05 TNFα effect compared with control + TNFα. B) Western blot and C) fragmented nucleosomes (n = 4) in wild type primary mouse hepatocytes over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection, treated with or without 20 ng/mL TNFα for 8 h. ***p < 0.001 versus vehicle. D) Western blot of protein from rat INS-1 β-cells over-expressing either PGC-1α1, PGC-1α4, or vector control by adenoviral infection, treated with or without cytokines (TNFα: 50 ng/mL, IFNγ: 50 ng/mL, IL-1β: 10 ng/mL) for 18 h. Western data are biological replicates representative of three independent experiments.

Figure 3

Loss of PGC-1α4 expression enhances apoptosis in response to TNFα. A) Targeting construct for creation of mouse allowing tissue-specific ablation of the alternative Ppargc1a promoter (AltProm^FL/FL). B) Western blot of PGC-1α proteins and C) mRNA of proximal and alternative Pgc-1α transcripts from primary mouse hepatocytes treated with 50 nM glucagon or vehicle. *p < 0.05 versus AltProm^FL/FL Vehicle. #p < 0.05 versus AltPromKO Vehicle, n = 3. D) Western blot and E) fragmented nucleosomes from primary mouse hepatocytes treated with 20 ng/mL TNFα or vehicle for 8 h *p < 0.05 versus AltProm^FL/FL Vehicle, n = 3. Bars are mean ± SEM of biological replicates in one experiment. Data are representative of two independent experiments.

Figure 4

PPARGC1A proximal and alternative transcripts are differentially regulated by TNFα and glucagon. A-C) mRNA levels of transcripts expressing either exon 1a of the proximal promoter, exon 1b or exon 1b′ of the alternative promoter from wild type primary mouse hepatocytes treated with vehicle (PBS), glucagon (50 nM), or TNFα (20 ng/mL) for indicated times. *p < 0.05 effect of treatment compared to individual controls at the same time point, #p < 0.05 effect of time within the same treatments. Data are representative of two independent experiments.

Figure 5

TNFα signaling promotes mobilization of cytoplasmic PGC-1α4 to the nucleus. A) Confocal imaging of H2.35 mouse hepatocytes transfected with plasmids expressing V5-tagged PGC-1α1 or PGC-1α4 treated with 20 ng/mL TNFα or vehicle (PBS) for 3 h. Images are representative of n = 15–20 cells per condition. B) Western blots after cell fractionation of H2.35 mouse hepatocytes transduced with adenovirus expressing control vector, PGC-1α1, or PGC-1α4 and treated with 50 ng/mL TNFα or vehicle (PBS) for 3 h. *non-specific bands. Data are representative of two independent experiments.

Figure 6

PGC-1α1, but not PGC-1α4, represses NF-κB activity and pro-inflammatory gene expression. A) Luciferase activity in wild type primary mouse hepatocytes treated with 2 ng/mL TNFα or vehicle (PBS) 48 h following transfection with a 3x NF-κB reporter and constructs for PGC-1α1 or PGC-1α4 (or vector alone, mean ± SD, n = 3). *p < 0.05 TNFα response compared to vehicle treatment, ^#p < 0.05 genotype effect compared to vector, ^&p < 0.05 genotype effect compared with vector + TNFα. B) mRNA expression of wild type primary mouse hepatocytes over-expressing PGC-1α1, PGC-1α4, or vector control following 2-hour treatment with 2 ng/mL TNFα or vehicle (n = 3). *p < 0.05 effect of TNFα within each genotype compared with untreated cells. ^#p < 0.05 effect of genotype alone compared with control. ^$p < 0.05 TNFα effect compared with control + TNFα. Data are representative of three independent experiments.

Figure 7

PGC-1α4 is necessary and sufficient to prevent LPS-induced hepatocyte apoptosis. A,B) Western blots of liver protein from male wild type littermate control, LKO, or AltPromKO mice (n = 3 mice) 6 h following tail-vein injection of LPS (2 mg/kg) or vehicle (PBS). C) Targeting construct for transgenic mouse allowing tissue-specific over-expression of PGC-1α4. D) mRNA and E) protein from livers of mice (n = 3) following breeding of ^LSLPGC-1α4 mice with Albumin-Cre^Tg mice to drive PGC-1α4 expression only in hepatocytes (PGC-1α4^HepTg). *p < 0.05 versus WT control. F) Western blot of liver protein from male mice 6 h following tail-vein injection of 2 mg/kg LPS (n = 6) or vehicle (PBS) (n = 2). Similar results were obtained in female mice.