Liver-Specific Silencing of the Human Gene Encoding Succinyl-CoA: 3-Ketoacid CoA Transferase

Abstract

The human succinyl-CoA: 3-ketoacid CoA transferase (SCOT) gene encodes the ketolytic enzyme that functions in the mitochondrial matrix. The activation of acetoacetate to acetoacetyl-CoA by SCOT is essential for the use of ketone bodies as an energy source. The ketolytic capacity of tissues is proportional to their level of SCOT activity. Normal hepatocytes, the site of ketone body synthesis, have no detectable SCOT protein. The absence of SCOT in hepatocytes is an important element in energy metabolism, suppressing ketolysis in the liver. To study the tissue-specific silencing of SCOT expression, we analyzed the promoter function of SCOT gene in three different human cell lines. Immunoblot analysis showed that SCOT protein was detectable in HeLa cervical cancer cells and Chang liver cells. However, SCOT protein was not detected in HepG2 hepatoma cells and liver tissues, indicating that HepG2 hepatoma cells maintain the characteristics of liver cells in the ketone body metabolism. Luciferase reporter assays in HeLa and Chang liver cells showed that the 361-bp proximal region of the SCOT gene was responsible for the basal promoter activity and contained two GC boxes, each of which was bound in vitro by Sp1, a ubiquitously expressed transcription factor. These results suggest that these GC boxes may be important for SCOT gene expression. Moreover, the region between -2168 and -361 appeared to inhibit the SCOT promoter activity in HepG2 cells. Thus, liverspecific silencing of the SCOT gene expression may be mediated in part by its 5′-flanking sequence.

Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice

“…The ability of AA to stimulate cell proliferation in a SCOT-independent manner was further corroborated by showing that AA also dose-dependently promoted proliferation of HepG2 hepatocellular cells, which is known not to express SCOT protein (Fig. 4b) (65). Previous studies found that SCOT activity was inhibited by AA at concentrations greater than 5 mM (66,67).…”

Metabolite Regulation of Nucleo-cytosolic Trafficking of Carbohydrate Response Element-binding Protein (ChREBP)

“…Ketone bodies cannot be metabolized in liver because the tissue lacks 3-oxoacid CoA transferase (20), and they are therefore exported out of the liver to other tissues where they are used as a source of energy. ␤HB and AcAc increase between 1 and 2 mM in blood and liver of rats when fasting as the liver switches to fatty acid oxidation (21), increasing to as much as 6 -8 mM in liver during prolonged starvation (22).…”

Hepatocellular carcinoma redirects to ketolysis for progression under nutrition deprivation stress

“…A previous report showed that the promoter region of OXCT1 harbors two GC-box sequences which are potential binding sites of transcription factor SP1 [15]; however, the precise contribution of these GC-box sequences to OXCT1 transcription has not been fully explored. Our western blot and qRT-PCR analysis revealed that knockdown of SP1 by shRNAs or blocking SP1 transactivity by mithramycin A (MIT), a specific SP1 inhibitor, significantly decreased OXCT1 expression level in serum-starved HepG2 cells ( Figure 3F and Supplementary information, Figure S3C).…”

“…For the whole organism, ketone bodies provide a fast and efficient way for energy supply during starvation, which links dietary lipids or adipose triglycerides to the TCA cycle and respiratory chain. As a key enzyme of ketolysis, OXCT1 is expressed abundantly in heart, brain and kidney; however, its expression is found to be repressed in adult liver and liver cell lines [14,15], largely considered as a mechanism to prevent a futile cycle of ketone body synthesis and catabolism in the same tissue. Hence, while it is well established that the liver is the major factory for ketone body production, up to date, there is no documentation showing that adult human liver cells could utilize ketone bodies as energy sources, at least to the best of our knowledge.…”