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Iqbal et al. Vessel Plus 2019;3:40 I http://dx.doi.org/10.20517/2574-1209.2019.28 Page 9 of 13

with the lower hepatic cholesterol levels [Figure 2A] and decreased expression of downstream genes involved
in cholesterol biosynthesis pathway [Figure 3]. Our results show that ablation of Rorγ activity in the liver leads
to a 68% decrease in the expression of Srebp1c gene [Figure 6B], which is consistent with the reduced hepatic
triglycerides [Figure 2B] and lower expression of the lipid synthesis genes [Figures 4 and 5]. Furthermore, we
also noticed that expression of Srebp1a gene was significantly reduced by 62% in the livers of Rorγ KO mice
compared to WT littermates [Figure 6C]. Next, we looked at the expression of Ppara and Pparg genes in
the livers of Rorγ KO and WT mice. There was an around five-fold increase in the expression of Ppara gene,
which is mainly involved in fatty acid oxidation [Figure 6D]. However, we observed a decrease of 74% in
the expression of Pparg gene [Figure 6E]. These data suggest that expression of transcription factors that
regulate lipid metabolism is modulated by RORγ.

DISCUSSION
Our results identify a critical role of RORγ in lipid metabolism. Here, we show that ablation of Rorγ gene
decreases the body weight and reduces the accumulation of lipids in the liver. Interestingly, we did not
see any significant change in the liver weight between the WT and Rorγ KO mice [Figure 1C]; however,
we noticed a visible decrease in the content of abdominal fat mass in Rorγ KO mice (data not shown).
This decrease in body weight and fat mass may be due to either reduced food intake or increased energy
expenditure. It is known that activation of PPARγ by agonists promote weight gain and fat accumulation
mainly due to increased adipocyte differentiation and lipid storage [29,30] . Our data indicate that Rorγ gene
deletion results in lower expression of Pparg [Figure 6E], which may explain the decrease in body weight
and fat mass. This finding is consistent with the repression of Rorγ activity by inverse agonist that lead to a
[19]
reduction in fat mass and body weight in obese diabetic mice .

A decrease in body weight and fat mass in Rorγ KO mice suggests that these mice do not store enough
lipids in the adipocytes. Inability of adipocytes to store the excess fatty acids coming from the diet suggest
that these fatty acids may be taken up by the liver for either oxidation or synthesis of lipids for storage in
hepatic or extrahepatic tissues such as muscles. A significant increase in the hepatic expression of Cd36
gene responsible for the uptake of long-chain fatty acids suggests that the livers from Rorγ KO mice may be
[17]
taking up more fatty acids from the circulation [Figure 4A]. Takeda et al. also showed that expression of
Cd36 gene was upregulated at all the times during diurnal oscillations in the livers of Rorγ KO mice. Since
both cholesterol and triglycerides levels were reduced in the livers of Rorγ KO mice [Figure 2A and B], we
speculate that most of the fatty acids taken up by these mice are not utilized for lipid biosynthesis and storage.
This was further supported by our observation that the expression of genes involved in lipid biosynthesis
was lower in the livers from Rorγ KO mice [Figure 5B]. It is interesting to note that there was a reduction
in the expression of Acc2 in the livers of Rorγ KO mice. This enzyme is involved in the synthesis of
malonyl-coA that plays an important role in regulating the oxidation of fatty acids in the mitochondria
[31]
by inhibiting the carnitine/palmitoyl shuttle system in contrast to malonyl-coA that is generated by the
[32]
ACC1 and utilized by Fas for the synthesis of fatty acids in the cytosol . Therefore, lower levels of Acc2
gene expression suggests that the oxidation of fatty acids may be enhanced in the livers of Rorγ KO mice,
which is consistent with the inhibition of Rorγ activity by inverse agonist that leads to enhanced fatty acid
[19]
oxidation . Hence, besides reduced lipid biosynthesis, increased fatty acid oxidation may also contribute
to reduced accumulation of triglycerides in the livers of Rorγ KO mice.

Contrary to lower hepatic lipids, we observed an increase in the levels of triglycerides in the plasma of
[17]
Rorγ KO mice. On the other hand, Takeda et al. reported a decrease in the levels of plasma lipids in
Rorγ KO mice. The discrepancy in results may be due to the feeding of high fat diet for six weeks to these
mice, which may affect the metabolism of lipids in the plasma. It is likely that Rorγ gene ablation affects
the uptake or secretion of these lipids from the liver. We did not observe any changes in the expression
of Mttp, a gene responsible for the secretion of lipid rich lipoproteins, suggesting that secretion of lipids