It is becoming increasingly clear that PGC-1β plays an important role as a regulator of both carbohydrate and lipid metabolism. In the present study we evaluated the metabolic effects of PGC-1β knockdown in rats with fructose-induced insulin resistance. Here we show for the first time that hepatic de novo lipogenesis and hepatic triglyceride synthesis induced by fructose are both decreased by PGC-1 β ASO treatment. Furthermore we show that knockdown of PGC-1β prevents fructose-induced hypertriglyceridemia and hepatic and peripheral insulin resistance.
PGC-1 β has been reported to coactivate its partners through augmentation of their transcriptional activity (Lin et al., 2005
). Though initially described to pair with PPARγ, the number of partners for PGC-1 transcription factors is rapidly growing. One of its partners is SREBP-1c. The transcriptional control of this key regulator of lipogensis is complex. It has been reported that rat and human SREBP-1c promoter contains binding sites for both SREBP itself (Cagen et al., 2005
) and LXR (Dif et al., 2006
) which are both regulated by PGC-1β (Lin et al., 2005
The ChIP assay data showed a marked increase in the association of both SREBP-1 and LXRα/β with the SREBP-1c promoter on a high-fructose diet. The bindings were both decreased by PGC-1β ASO treatment. Undoubtedly, the binding of SREBP-1 to SREBP-1c promoter might be reflected with the amount of SREBP-1 total protein, but the binding of LXR to SREBP-1c promoter was also decreased by PGC-1β ASO treatment. LXR protein was unchanged between all groups implying the decreased binding to the SREBP-1c promoter was due to the decreased coactivation with PGC-1β. Thus, this data suggests that PGC-1β ASO treatment reduces SREBP-1c expression by decreased co-activation of LXR and SREBP.
Consistent with the reduction of SREBP-1 expression, a target lipogenic gene of SREBP-1, FAS was down-regulated in high-fructose fed PGC-1β ASO treated rats. This likely accounted for the observed decreased in hepatic de novo lipogenesis, hepatic triglyceride content and reductions in hypertriglyceridemia in the high-fructose fed PGC-1β ASO treated rats. Supporting these observations, we found that in vitro hepatic triglyceride synthesis was significantly inhibited in primary rat hepatocytes by PGC-1β ASO treatment.
Knockdown of PGC-1β in liver protected rats from fructose-induced hepatic insulin resistance. This protection from fructose-induced hepatic insulin resistance could mostly be attributed to reduction in hepatic lipogenesis resulting in reduced hepatic diacylglycerol content and decreased PKCε activation. These data were also reflected in Akt activity.
In marked contrast to these findings, the effects of PGC-1β ASO on the liver differed in the regular chow and high fructose-fed rats. In regular chow fed rats PGC-1β ASO induced slight, but significant reductions in insulin suppression of hepatic glucose production. These reductions in insulin responsiveness could be attributed to decreased mitochondrial fatty acid oxidation resulting in increased diacylglycerol content and increased PKCε activation resulting in decreased insulin signaling (Samuel, 2007
). PGC-1s have been reported to coactivate both PPAR-α and NRF-1. PPARα plays a key role in the transcriptional control of genes encoding mitochondrial fatty acid oxidation enzymes such as LCAD and MCAD. NRFs regulate expression of mitochondrial transcription factor A (Tfam), a nuclear-encoded transcription factor essential for replication, maintenance, and transcription of mitochondrial DNA. NRFs also control the expression of nuclear genes encoding respiratory chain subunits and other proteins required for mitochondrial function (Scarpulla, 2002
). PGC-1β ASO decreased expression of genes encoding fatty acid oxidation and oxidative phosphorylation as well as mitochondrial copy number, suggesting that PGC-1β is required in liver for normal expression of genes encoding fatty acid oxidation and oxidative phosphorylation. These results are consistent with previous studies in PGC-1 β E3,4-/E3,4-
mice (Vianna et al., 2006
) and LCAD knockout mice, which both develop hepatic steatosis and hepatic insulin resistance due to decreased hepatic fat oxidation resulting in increased hepatic diacylglycerol content and increased PKCε activation (Zhang et al., 2007
PGC-1β ASO also prevented fructose-induced insulin resistance in peripheral tissue. This preservation of insulin responsiveness could be attributed largely to a threefold increase in insulin stimulated WAT glucose uptake. Since the PGC family members are known coactivators of PPARs, we examined the expression of PPAR-γ and its target genes. The expression of PPARγ itself, as well as key target genes were all reduced with PGC-1β ASO treatment and could not explain the observed improvements in insulin action.
We next investigated GLUT4 expression and found that the GLUT4 protein expression in WAT was increased by more than fourfold in the high-fructose fed PGC-1β ASO treated rats. This was compatible with our findings in the glucose uptake, although the mRNA expression of GLUT4 was surprisingly decreased by PGC-1β ASO treatment. A similar dissociation between GLUT4 protein and mRNA expression has been reported by Otani et al. (Otani et al., 2004
). We believe this result may reflect enhanced stability of GLUT4 protein, leading to its accumulation. However, in the present study, we did not find any explanations in the gene expression of GLUT4 trafficking proteins. Further studies are required to explain why the increases of 2DG uptake and GLUT4 protein expression were seen in only high-fructose fed PGC-1β ASO treated rats.
We tested PGC-1β ASO on rats fed a high fat diet to understand if the protection from diet induced insulin resistance was specific for high-fructose diet. PGC-1β ASO failed to ameliorate both hepatic and peripheral insulin resistance in high fat fed rats. The degree of SREBP-1 induction with high-fat diet was less than that with high-fructose diet, and PGC-1β ASO did not affect to the SREBP-1 mRNA expression on high fat diet. Consequently, the unaffected lipogenesis resulted in similar hepatic lipid content between control ASO and PGC-1β ASO group. These findings raise the possibility that fructose can directly induces the transcriptional activity of SREBP-1 via PGC-1β.
Interestingly we also found that PGC-1β ASO treatment increased plasma cholesterol concentrations. We did not observe any increases in the expression of SREBP-2, a key transcriptional regulator of cholesterol biosynthesis or in the SREBP2 responsive genes HMGCoAR or LDL-R following PGC-1β ASO treatment. These data suggest that cholesterol biosynthesis in liver and LDL uptake from plasma by liver were unlikely to be responsible for the observed increases in plasma cholesterol in the PGC-1β ASO treated groups. Consistent with these observations we found that reduction in PGC-1β expression in primary rat hepatocytes had no effect on the incorporation rate of 14
C acetic acid into sterol. We next investigated the effects of PGC-1β knockdown on the expression of LXRα and its target genes. LXRα has been shown to be an important regulator of cholesterol (Kalaany et al., 2005
) and is co-activated by PGC-1s (Lin et al., 2005
). Although we found that LXRα expression was not different in the four groups, hepatic CYP7A1 expression was decreased in both PGC-1β ASO treated groups. Since CYP7A1 is responsible for the rate-controlling step in the bile acid synthesis pathway, it is likely that this reduction in CYP7A1 may explain the increased plasma cholesterol concentrations in the PGC-1β ASO treated rats.
In conclusion these data support an important role for PGC-1 β in the pathogenesis of fructose-induced hypertriglyceridemia and insulin resistance. Furthermore, given recent studies suggesting an important role for increased hepatic de novo
lipogenesis in the pathogenesis of hypertriglyceridemia and NAFLD associated with the metabolic syndrome (Petersen et al., 2007
) these data suggest that PGC-1 β inhibition may be a novel therapeutic target for treatment of this condition.