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Iqbal et al. Vessel Plus 2019;3:40 I http://dx.doi.org/10.20517/2574-1209.2019.28 Page 5 of 13

Figure 2. RORγ gene deletion reduces hepatic lipids and increases plasma triglycerides. Lipids from WT and KO mice livers (n = 3) were
extracted by Bligh and Dyer method to measure cholesterol (A) and triglycerides (B). Total cholesterol (C) and triglycerides (D) were also
measured in the plasma of these mice. Values were plotted as mean ± SD. P values were calculated using two-tailed Student’s t test. **P <
0.01. WT: wild type; KO: knockout; RORγ: retinoic acid-related orphan receptor γ

decrease of 27% in total body weight compared to WT littermate control mice [Figure 1B]. However,
no change was seen in the weight of livers in these mice [Figure 1C]. These results suggest that RORγ is
important in maintaining the body weight in mice and its deficiency leads to reduced body weight gain.

RORγ KO mice decreases hepatic but not plasma lipids
Next, we investigated whether change in body weight after Rorγ gene deletion also affects liver and
plasma lipids. Our data indicate that there were significant decreases of 39% and 56% in the levels of liver
cholesterol [Figure 2A] and triglycerides [Figure 2B], respectively, in Rorγ KO mice compared to WT
littermates. However, we did not see any significant change in plasma cholesterol levels between WT and
Rorγ KO mice [Figure 2C]. Contrary to decreased hepatic lipid levels, we observed an increase of ~37% in
the levels of plasma triglycerides in KO mice compared to WT mice [Figure 2D]. These data suggest that
Rorγ gene deletion affects only liver and not plasma cholesterol. On the other hand, both liver and plasma
triglycerides are altered after the ablation of Rorγ gene.

Ablation of Rorγ activity affects expression of genes involved in lipid metabolism
To gain better understanding of how Rorγ activity regulates body weight and lipids, we looked at the
changes in the expression of lipid metabolism genes after the deletion of Rorγ gene. We started by analyzing
the changes in the genes that regulate cholesterol homeostasis in the liver. Coordinated expression of
several genes regulate cellular cholesterol metabolism [23,24] . We looked at the expression of cholesterol
biosynthesis genes such as hydroxymethylglutaryl-CoA reductase (Hmgr) and hydroxymethylglutaryl-
CoA synthase (Hmgs). The expression of rate-limiting enzyme gene, Hmgr, was not affected by the deletion
of Rorγ in the liver [Figure 3A]. However, we observed a decrease of 63% in the expression of Hmgs in the
livers of Rorγ KO mice compared to WT littermates [Figure 3B]. Next, we looked at the expression of genes