Succinate causes α-SMA production through GPR91 activation in hepatic stellate cells

doi:10.1016/j.bbrc.2015.06.023

Biochemical and Biophysical Research Communications

Volume 463, Issue 4, 7 August 2015, Pages 853-858

https://doi.org/10.1016/j.bbrc.2015.06.023 Get rights and content

Highlights

•
Succinate increased expression of GPR91 and α-SMA in hepatic stellate cells.
•
Expression of GPR91 and α-SMA was increased in the isolated hepatic stellate cells of mice fed MCD diet.
•
Plasma succinate concentration was increased in the plasma of MCD diet-fed mice.

Abstract

Succinate acts as an extracellular signaling molecule as well as an intermediate in the citric acid cycle. It binds to and activates its specific G protein-coupled receptor 91 (GPR91). GPR91 is present in hepatic stellate cells (HSCs), but its role in hepatic fibrogenesis remains unclear. Cultured HSCs treated with succinate showed increased protein expression of GPR91 and α-smooth muscle actin (α-SMA), markers of fibrogenic response. Succinate also increased mRNA expression of α-SMA, transforming growth factor β (TGF-β), and collagen type I. Transfection of siRNA against GPR91 abrogated succinate-induced increases in α-SMA expression. Malonate, an inhibitor of succinate dehydrogenase (SDH), increased succinate levels in cultured HSCs and increased GPR91 and α-SMA expression. Feeding mice a methionine- and choline-deficient (MCD) diet is a widely used technique to create an animal model of nonalcoholic steatohepatitis (NASH). HSCs cultured in MCD media showed significantly decreased SDH activity and increased succinate concentration and GPR91 and α-SMA expression. Similarly, palmitate treatment significantly decreased SDH activity and increased GPR91 and α-SMA expression. Finally, C57BL6/J mice fed the MCD diet had elevated succinate levels in their plasma. The MCD diet also decreased SDH activity, increased succinate concentration, and increased GPR91 and α-SMA expression in isolated HSCs. Collectively, our results show that succinate plays an important role in HSC activation through GPR91 induction, and suggest that succinate and GPR91 may represent new therapeutic targets for modulating hepatic fibrosis.

Introduction

Hepatic stellate cells (HSCs) constitute approximately 8–14% of cells in the normal liver, and HSC activation is crucial for the development of liver fibrosis. Following liver injury, HSCs become activated into contractile and highly proliferative myofibroblast-like cells to promote increased extracellular matrix (ECM) production and hepatic fibrosis [1], [2]. This is accompanied by the upregulated expression of cytoskeletal protein such as α-smooth muscle actin (α-SMA) [1]. The molecular signals activated during HSC activation are not completely understood, but transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) are known to play important roles [1], [3], [4], [5].

Succinate is an intermediate in the citric acid cycle (or Krebs cycle). As part of this cycle in the mitochondrial matrix, succinate is produced by the oxidation of succinyl-CoA by the enzyme succinyl-CoA hydrolase and is further converted into fumarate by succinate dehydrogenase (SDH) [6]. In addition, it acts as an extracellular circulating signaling molecule that binds to and activates its specific G protein-coupled receptor (GPCR), G protein-coupled receptor-91 (GPR91) [7].

GPR91 activation triggered by local succinate accumulation increases the release of renin in the glomerular endothelium [8] and in the luminal membrane of the macula densa [9]. GPR91 mRNA is expressed in the polarized cells of the thick ascending limb of Henle's loop and the cortical and inner medullary collecting ducts, and its activation triggers the release of arachidonic acid and prostaglandins in the distal nephron [10].

In the retinal ganglion cells, GPR91 is involved in retinal angiogenesis [11] and modulates the release of vascular endothelial growth factor (VEGF) induced by high levels of glucose [12]. In dendritic cells, succinate triggers GPR91 activation, which is involved in helper T-cell activation and proinflammatory cytokine production [13]. GPR91 has been found in several highly vascularized tissues, including kidney, heart, liver, white adipose tissue, and retina [6], [14], [15].

In the liver, GPR91 protein is expressed in quiescent HSCs [16]; its mRNA is highly expressed in quiescent HSCs but less expressed in LPS-activated HSCs [17]. In a previous study [16], HSCs treated with succinate showed increased HSC activation, suggesting that succinate may be a novel HSC activator.

However, the roles of succinate and its receptor in the development of fibrosis have not been investigated extensively. In the present study, we determined whether succinate, malonate (an SDH inhibitor), MCD media, or palmitate regulate HSC activation and examined plasma levels of succinate and the expression of succinate and GPR91 by isolating HSCs induced in a nonalcoholic fatty liver disease (NAFLD) mouse model.

Section snippets

Materials

Upregulation of α-SMA, a hallmark of myofibroblastic transdifferentiation, was used as a marker for HSC activation [3]. Completely deficient of methionine and choline (MCD medium) and methionine- and choline-supplement (MCS medium, control medium) were purchased from WELGENE (Kyeongsan, Korea). Succinate, malonate, and palmitate were purchased from Sigma (St. Louis, MO, USA).

Cell culture

LX2 cells are immortalized human stellate cells and they were kindly provided by Professor Ja June Jang, Seoul National

Succinate as a GPR91 agonist activates HSCs

The activation of HSCs by succinate was monitored using Western blotting and RT-PCR. To investigate the expression pattern of GPR91 in HSCs, Western blotting was performed using LX2 cells.

LX2 cells treated with succinate for 24 h showed increased protein expression of GPR91, α-SMA, and TIMP-1(Fig. 1A) but not α-SMA mRNA levels (data was not shown). LX2 cells treated with succinate for 8 h demonstrated increased mRNA expression of α-SMA through GPR91 activation (Fig. 1B). LX2 cells treated with

Discussion

Although succinate has been studied extensively for several decades in terms of energy metabolism, recent studies have demonstrated that it is a cellular signaling molecule in many metabolic diseases [7]. The present study provides novel information regarding the key role of succinate and GPR91 in HSC activation as signaling regulators of hepatic fibrosis.

In the present study, we showed that GPR91 is expressed during HSC activation and succinate-GPR91 signaling stimulates HSC activation in vitro

Acknowledgments

This research was funded by research grant NRF-2013R1A1A1058962.

References (29)

S.L. Friedman
Mechanisms of hepatic fibrogenesis
Gastroenterology
(2008)
T. Knittel et al.
Transforming growth factor beta 1-regulated gene expression of Ito cells
Hepatology
(1996)
J.H. Robben et al.
Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells
Kidney Int.
(2009)
J. Hu et al.
Inhibition of high glucose-induced VEGF release in retinal ganglion cells by RNA interference targeting G protein-coupled receptor 91
Exp. Eye Res.
(2013)
J.B. Regard et al.
Anatomical profiling of G protein-coupled receptor expression
Cell
(2008)
P.R. Correa et al.
Succinate is a paracrine signal for liver damage
J. Hepatol.
(2007)
S. De Minicis et al.
Gene expression profiles during hepatic stellate cell activation in culture and in vivo
Gastroenterology
(2007)
J. Muller-Hocker et al.
Defects of the respiratory chain in the normal human liver and in cirrhosis during aging
Hepatology
(1997)
N. Sadagopan et al.
Circulating succinate is elevated in rodent models of hypertension and metabolic disease
Am. J. Hypertens.
(2007)
E. Mills et al.
Succinate: a metabolic signal in inflammation
Trends Cell. Biol.
(2014)

C.J. Aguiar et al.

Succinate modulates Ca(2+) transient and cardiomyocyte viability through PKA-dependent pathway

Cell. Calcium

(2010)

J. Deng et al.

Hypoxia-inducible factor- 1alpha regulates autophagy to activate hepatic stellate cells

Biochem. Biophys. Res. Commun.

(2014)

T. Kisseleva et al.

Hepatic stellate cells and the reversal of fibrosis

J. Gastroenterol. Hepatol.

(2006)

J. George et al.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor beta type II receptor: a potential new therapy for hepatic fibrosis

Proc. Natl. Acad. Sci. U. S. A.

(1999)

Cited by (78)

Succinate as a signaling molecule in the mediation of liver diseases
2024, Biochimica et Biophysica Acta - Molecular Basis of Disease
Succinate, one of the intermediates of the tricarboxylic acid (TCA) cycle, plays an essential role in the metabolism of mitochondria and the production of energy, and is considered as a signaling molecule in metabolism as well as in initiation and progression of hepatic diseases. Of note, succinate activates a downstream signaling pathway through GPR91, and elicits a variety of intracellular responses, such as succinylation, production of reactive oxygen species (ROS), stabilization of hypoxia-inducible factor-1α (HIF-1α), and significant impact in cellular metabolism because of the pivotal role in the TCA cycle. Therefore, it is intriguing to deeply elucidate signaling mechanisms of succinate in hepatic fibrosis, metabolic reprogramming in inflammatory or immune responses, as well as carcinogenesis. This manuscript intends to review current understanding of succinate in mediating metabolism, inflammatory and immunologic reactions in liver diseases in order to establish molecular basis for the development of therapeutic strategies.
Protective effects of the succinate/SUCNR1 axis on damaged hepatocytes in NAFLD
2023, Metabolism: Clinical and Experimental
Succinate and succinate receptor 1 (SUCNR1) are linked to fibrotic remodeling in models of non-alcoholic fatty liver disease (NAFLD), but whether they have roles beyond the activation of hepatic stellate cells remains unexplored. We investigated the succinate/SUCNR1 axis in the context of NAFLD specifically in hepatocytes.
We studied the phenotype of wild-type and Sucnr1^−/− mice fed a choline-deficient high-fat diet to induce non-alcoholic steatohepatitis (NASH), and explored the function of SUCNR1 in murine primary hepatocytes and human HepG2 cells treated with palmitic acid. Lastly, plasma succinate and hepatic SUCNR1 expression were analyzed in four independent cohorts of patients in different NAFLD stages.
Sucnr1 was upregulated in murine liver and primary hepatocytes in response to diet-induced NASH. Sucnr1 deficiency provoked both beneficial (reduced fibrosis and endoplasmic reticulum stress) and detrimental (exacerbated steatosis and inflammation and reduced glycogen content) effects in the liver, and disrupted glucose homeostasis. Studies in vitro revealed that hepatocyte injury increased Sucnr1 expression, which when activated improved lipid and glycogen homeostasis in damaged hepatocytes. In humans, SUCNR1 expression was a good determinant of NAFLD progression to advanced stages. In a population at risk of NAFLD, circulating succinate was elevated in patients with a fatty liver index (FLI) ≥60. Indeed, succinate had good predictive value for steatosis diagnosed by FLI, and improved the prediction of moderate/severe steatosis through biopsy when added to an FLI algorithm.
We identify hepatocytes as target cells of extracellular succinate during NAFLD progression and uncover a hitherto unknown function for SUCNR1 as a regulator of hepatocyte glucose and lipid metabolism. Our clinical data highlight the potential of succinate and hepatic SUCNR1 expression as markers to diagnose fatty liver and NASH, respectively.
Research progress of metformin in the treatment of liver fibrosis
2023, International Immunopharmacology
Liver fibrosis is a disease with significant morbidity and mortality. It is a chronic pathological process characterized by an imbalance of extracellular matrix production and degradation in liver tissue. Metformin is a type of hypoglycemic biguanide drug, which can be used in the treatment of liver fibrosis, but its anti-fibrotic effect and mechanism of action are unclear. The purpose of this article is to review the research progress of metformin in the treatment of liver fibrosis and to provide a theoretical basis for its application in the treatment of liver fibrosis.
Integrating the contributions of mitochondrial oxidative metabolism to lipotoxicity and inflammation in NAFLD pathogenesis
2022, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.
Foresight regarding drug candidates acting on the succinate–GPR91 signalling pathway for non-alcoholic steatohepatitis (NASH) treatment
2021, Biomedicine and Pharmacotherapy
Citation Excerpt :
Their research results also showed that HSCs treated with succinate or a succinate dehydrogenase (SDH) inhibitor (malonic acid, palmitate/choline or methionine-choline) showed not only enhanced expression of GPR91 but also increased expression of α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β) and type I collagen, signifying a fibrotic response (Fig. 2). On the other hand, transfection of these cells with GPR91 siRNA abolishes succinate-induced α-SMA production [70]. These findings indicate that succinate -GPR91 coupling is dependent on HSC activation and fibrogenesis.
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and it is a liver manifestation of metabolic syndrome, with a histological spectrum from simple steatosis to non-alcoholic steatohepatitis (NASH). NASH can evolve into progressive liver fibrosis and eventually lead to liver cirrhosis. The pathological mechanism of NASH is multifactorial, involving a series of metabolic disorders and changes that trigger low-level inflammation in the liver and other organs. In the pathogenesis of NASH, the signal transduction pathway involving succinate and the succinate receptor (G-protein-coupled receptor 91, GPR91) regulates inflammatory cell activation and liver fibrosis. This review describes the mechanism of the succinate–GPR91 signalling pathway in NASH and summarizes the drugs that act on this pathway, with the aim of providing a new approach to NASH treatment.
Pharmaceutical targeting of succinate dehydrogenase in fibroblasts controls bleomycin-induced lung fibrosis
2021, Redox Biology
Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix in the lung with fibroblast-to-myofibroblast transition, leading to chronically compromising lung function and death. However, very little is known about the metabolic alterations of fibroblasts in IPF, and there is still a lack of pharmaceutical agents to target the metabolic dysregulation. Here we show a glycolysis upregulation and fatty acid oxidation (FAO) downregulation in fibroblasts from fibrotic lung, and perturbation of glycolysis and FAO affects fibroblasts transdifferentiation. In addition, there is a significant accumulation of succinate both in fibrotic lung tissues and myofibroblasts, where succinate dehydrogenase (SDH) operates in reverse by reducing fumarate to succinate. Then succinate contributes to glycolysis upregulation and FAO downregulation by stabilizing HIF-1α, which promotes the development of lung fibrosis. In addition, we identify a near-infrared small molecule dye, IR-780, as a targeting agent which stimulates mild inhibition of succinate dehydrogenase subunit A (SDHA) in fibroblasts, and which inhibits TGF-β1 induced SDH and succinate elevation, then to prevent fibrosis formation and respiratory dysfunction. Further, enhanced cell retention of IR-780 is shown to promote severe inhibition of SDHA in myofibroblasts, which may contribute to excessive ROS generation and selectively induces myofibroblasts to apoptosis, and then therapeutically improves established lung fibrosis in vivo. These findings indicate that targeting metabolic dysregulation has significant implications for therapies aimed at lung fibrosis and succinate dehydrogenase is an exciting new therapeutic target to treat IPF.

View all citing articles on Scopus

View full text

Succinate causes α-SMA production through GPR91 activation in hepatic stellate cells

Highlights

Abstract

Introduction

Section snippets

Materials

Cell culture

Succinate as a GPR91 agonist activates HSCs

Discussion

Acknowledgments

Gastroenterology

Hepatology

Kidney Int.

Exp. Eye Res.

Cell

J. Hepatol.