Succinate is a paracrine signal for liver damage

Abstract

Background/aims: A G-protein-coupled succinate receptor has recently been identified in several tissues, including the liver. The objectives of this work were to determine the hepatic cell types that express this receptor and to determine its physiological role.

Methods: Expression and distribution of the succinate receptor was determined by RT-PCR and confocal immunofluorescence. Biochemical assays were used to measure succinate and cAMP. Cytosolic Ca2+ was monitored in single cells by time-lapse imaging. Western blot was used to study the effect of succinate on activation of hepatic stellate cells.

Results: The succinate receptor was expressed in quiescent hepatic stellate cells, and expression decreased with activation. Ischemia induced release of succinate in isolated perfused livers. In contrast to what is observed in cell expression systems, succinate did not inhibit cAMP production or increase cytosolic Ca2+ in primary hepatic stellate cells. However, succinate accelerated stellate cell activation.

Conclusions: Hepatic stellate cells express the succinate receptor. Succinate may behave as a paracrine signal by which ischemic hepatocytes trigger stellate cell activation.

Figure 1

The succinate receptor GPR91 is expressed in quiescent hepatic stellate cells. (a) The 641 base-pair PCR product is found in whole liver samples and hepatic stellate cells, but not in isolated hepatocytes, cholangiocytes, portal fibroblasts, or activated hepatic stellate cells. The positive control, kidney, shows the same product. (b) The PCR product found in quiescent hepatic stellate cells (1d HSC) is not in passaged stellate cells (P3 HSC) or in Kupffer cells or sinusoidal endothelial cells. PCR for GAPDH was used as a loading control in both gels.

Figure 1

The succinate receptor GPR91 is expressed in quiescent hepatic stellate cells. (a) The 641 base-pair PCR product is found in whole liver samples and hepatic stellate cells, but not in isolated hepatocytes, cholangiocytes, portal fibroblasts, or activated hepatic stellate cells. The positive control, kidney, shows the same product. (b) The PCR product found in quiescent hepatic stellate cells (1d HSC) is not in passaged stellate cells (P3 HSC) or in Kupffer cells or sinusoidal endothelial cells. PCR for GAPDH was used as a loading control in both gels.

Figure 2

Visualization of the succinate receptor in hepatic stellate cells. (a) Low-magnification and (b) high-magnification confocal immunofluorescence images of rat liver show that staining for the succinate receptor (green) and the HSC marker desmin (red) co-localize (yellow). The actin label phalloidin (blue) outlines individual hepatocytes for reference. No non-specific staining is seen in liver sections labeled with secondary antibody alone (not shown). (c) Confocal immunofluorescence image of isolated hepatic stellate cells after 24 hr in culture. The cells are stained for the quiescent stellate cell marker GFAP (green, left) and for the succinate receptor (red, center). Note on the merged image (right) that some of the succinate receptor staining is peripheral to GFAP staining. No non-specific staining is seen in stellate cells labeled with secondary antibody alone (not shown).

Figure 2

Visualization of the succinate receptor in hepatic stellate cells. (a) Low-magnification and (b) high-magnification confocal immunofluorescence images of rat liver show that staining for the succinate receptor (green) and the HSC marker desmin (red) co-localize (yellow). The actin label phalloidin (blue) outlines individual hepatocytes for reference. No non-specific staining is seen in liver sections labeled with secondary antibody alone (not shown). (c) Confocal immunofluorescence image of isolated hepatic stellate cells after 24 hr in culture. The cells are stained for the quiescent stellate cell marker GFAP (green, left) and for the succinate receptor (red, center). Note on the merged image (right) that some of the succinate receptor staining is peripheral to GFAP staining. No non-specific staining is seen in stellate cells labeled with secondary antibody alone (not shown).

Figure 3

Succinate in the isolated perfused rat liver. Infusion of succinate (100 μM) into the portal vein does not affect (a) perfusion pressure (n=4) or (b) glucose output (n=3). Values represent mean±SEM. (c) Succinate levels in perfusate outflow rise 14-fold after ischemia (n=3). Values represent mean±SD.

Figure 4

Succinate does not inhibit the production of cAMP induced by forskolin in quiescent hepatic stellate cells. Bar graph representing cAMP production in non-stimulated (control) stellate cells and in cells treated with succinate (1 mM), forskolin (10 μM) and forskolin plus succinate. Values represent mean±SD of quadruplicate measurements.

Figure 5

Succinate does not increase cytosolic Ca²⁺ in hepatic stellate cells. Graphic shows representative tracings of 7 separate quiescent stellate cells serially stimulated with succinate (1 mM), and then the positive control ATP (100 μM). Cells were loaded with the fluorescent Ca²⁺ dye Fluo-4/AM, and then monitored using time-lapse confocal microscopy. Increases in free cytosolic Ca²⁺ were measured and are represented as increases in fluorescence intensity relative to baseline. Results are representative of what was observed in 132 separate stellate cells from 3 separate preparations.

Figure 6

Succinate promotes activation of hepatic stellate cells. (a) Western blot for alpha-smooth muscle actin (α-SMA) in hepatic stellate cells 1, 3, and 5 days after isolation. Three-day HSC were followed either in the absence or presence of succinate (400 μM). (b) Densitometric quantification of western blots for α-SMA, normalized by GAPDH expression. Results of each of three separate experiments at each time point are shown. α-SMA expression is increased 2.3-fold in succinate-treated cells relative to untreated controls (p<0.05).