Stimulatory effect of CSE-generated H2S on hepatic mitochondrial biogenesis and the underlying mechanisms

• H2S is vital to maintain hepatic mitochondrial biogenesis in primary hepatocytes.
• H2S augments the protein expression level and activity (by S-sulfhydration) of PPRC and PGC-1α.
• H2S induces hepatic mitochondrial biogenesis via PPRC and PGC-1α signaling pathways.

We previously showed that hydrogen sulfide (H2S) upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α in primary hepatocytes. PGC-1α is a crucial regulator of mitochondrial biogenesis, a process required to maintain cellular energy homeostasis. We investigated the regulation of hepatic mitochondrial biogenesis by cystathionine γ-lyase (CSE)-generated H2S under physiological conditions. Primary hepatocytes isolated from CSE knockout (KO) and wild-type (WT) mice were used in all experiments. Mitochondrial DNA (mtDNA) and mRNA levels were measured via real-time PCR. Protein S-sulfhydration was determined via a modified biotin switch assay. MitoTracker Green was used to quantify mitochondrial content and distribution. CSE-KO hepatocytes produced less mtDNA compared to WT hepatocytes. Mitochondrial content was reduced in CSE-KO hepatocytes compared to WT hepatocytes, which was restored with NaHS (an H2S donor) treatment. CSE-KO hepatocytes exhibited lower levels of mitochondrial transcription factors and the mitochondrial transcription coactivator, peroxisome proliferator-activated receptor-γ coactivator-related protein (PPRC) compared to WT hepatocytes. NaHS administration upregulated PPRC, yet downregulated PGC-1β protein level in mouse hepatocytes. Exogenous H2S induced the S-sulfhydration of PPRC, which was lower in untreated CSE-KO hepatocytes, but not that of PGC-1β. Finally, knockdown of either PGC-1α or PPRC significantly decreased NaHS-stimulated mitochondrial biogenesis in hepatocytes, where knockdown of both genes were required to abolish NaHS-induced mitochondrial biogenesis. Endogenous H2S-induced liver mitochondrial biogenesis is dependent upon PGC-1α and PPRC signaling in primary hepatocytes. This study may offer clues to the regulation of energy homeostasis under physiological conditions as well as mitochondrial dysregulation.