3.1 Body weight gain
As shown in Fig. 2A, from Week 0 to Week 2, there was significant interaction between the TIME and OLANZAPINE factors (F7, 238=13.23, p<0.001). Olanzapine treatment significantly increased body weight gain compared to vehicle treatment from day 8 (Fig. 2A, p<0.05). Three-way repeated ANOVAs (OLANZAPINE × SIMVASTATIN× TIME as repeated measures) showed significant main effects of TIME (F16, 512=17.78, p<0.001). There was a significant interaction between the OLANZAPINE and SIMVASTATIN factors (F1, 32=12.17, p<0.05) for the last 5 weeks. As shown in Fig. 2A, in the olanzapine-only group, continuous olanzapine treatment significantly increased body weight gain compared to the control throughout the 5 weeks’ treatment (Fig. 2A, p<0.05), whereas the O + S co-treatment group had a lower weight gain compared with the olanzapine-only treatment group after 4 weeks’ co-treatment (Fig. 2A, p<0.05).
3.2 Body temperature
Olanzapine treatment significantly decreased body temperature compared to vehicle from day 12 (Fig. 2B, p<0.05). During the last 5 weeks, there was a significant interaction between the OLANZAPINE factor and TIME factor (F16,512=2.1, p<0.05). Body temperature was still significantly lower in the olanzapine-only compared to the control (Fig. 2B,p<0.05). It was interesting that the O+S co-treatment group increased body temperature at a borderline significance compared with the olanzapine-only group from day 40 (p=0.083).
3.3 Food intake and feeding efficiency
During the first 2 weeks (Day 0-14), there was a significant interaction between the TIME and OLANZAPINE factors (F7, 238=446.87, p<0.001). Compared to the control group from day 12, a significant increase in food intake was observed in the olanzapine group (Fig. 2C, p<0.05). From week 3 to week 7, the two groups were divided into four subgroups. In the olanzapine-only group, a significant increase in food intake was observed (p<0.05). Moreover, feeding efficiency (grams of weight gained/grams of food consumed) was significantly elevated by olanzapine treatment compared with the control group (p<0.05). However, no significant difference in food intake was detected between the O+S co-treatment group and olanzapine-only group (Fig. 2C). Furthermore, O+S co-treatment was not effective in decreasing feeding efficiency (grams of weight gained/grams of food consumed) compared to the olanzapine-only treatment (Fig. 2D).
3.4 Fat deposits
As shown in Fig. 3A, compared with control, BAT weight was significantly higher in olanzapine-treated rats (p<0.05). Histological analysis of BAT revealed significant difference in adipocyte size or number between olanzapine-only group and control (Fig. 3B and 3C, p<0.01). It is important that combination with simvastatin treatment reduced lipid droplet content in BAT (approx. -60%, p<0.05), with a decrease of relative BAT weight as compared to olanzapine-only group (approx. -15%; p=0.093, Fig. 3B and 3C).
3.5 Locomotor activity
There was a significant effect of the OLANZAPINE factor on distance moved (F1, 32=9.02, p<0.05). The olanzapine-only group had significantly less distance moved than the control group (p<0.05). It is important that the rats with simvastatin treatment had a significant increase in the total distance moved over the control group (p<0.05) (Fig. 4A and 4B). There were negative correlations between total distance and body weight gain (r=-0.334, p<0.05). However, no significant difference in locomotor activity was detected between the O+S co-treatment group and olanzapine-only group.
3.6 Serum biochemical parameters
As shown in Table 1, olanzapine led to higher levels of triglycerides, total cholesterol and glucose (all p<0.05) than the control. When the two groups divided into four groups, the chronic olanzapine-only treatment further induced triglycerides, total cholesterol and glucose to remain at higher levels (all p<0.05, Table 2). The simvastatin-only group significantly reduced triglycerides and total cholesterol compared to the control group (all p<0.05, Table 2). Co-treatment of olanzapine and simvastatin reversed the levels of triglyceride, total cholesterol and glucose to normal levels when compared with the olanzapine-only group (all p<0.01, Table 2). There was a positive correlation between body weight gain and triglycerides (r=0.487, p<0.01).
3.7 mRNA expression levels in the liver
As presented in Fig. 5A, Srebp2 (Fig. 5A, 1.71-fold increase, p<0.05) and its target genes hmgcr (Fig. 5B, 2.70-fold increase, p<0.05) and hmgcs (Fig. 5C, 1.94-fold increase, p<0.05) were significantly up-regulated by olanzapine. In addition, there was also an upregulation of hepatic Srebp1 mRNA expression in olanzapine-treated rats compared to controls (Fig. 5D, 2.6-fold, p<0.05). Consistent with the alteration of Srebp1, mRNA expression of Fasn, but not Acc1, was significantly increased by olanzapine treatment (Fig. 5E, 2.1-fold, p<0.05). As an HMG-CoA reductase inhibitor, simvastatin significantly affected mRNA expression of Hmgcr (0.69-fold decrease, p<0.01). Consequently, O+S co-treatment significantly reduced Srebp1, Hmgcr and Fasn transcriptional levels increased by olanzapine. However, an increase of Srebp2 and hmgcs mRNA expression was observed in O+S co-treatment group. Histological analysis of liver revealed that olanzapine-only treatment significantly promoted accumulation of lipid droplets in the liver, whilst the O+S co-treatment decreased lipid droplets (Fig. 5G and 5H, p<0.01). As shown in Fig. 5G and 5H, a significantly lower positive ORO staining was observed in the O+S co-treatment group than the olanzapine-only group (∼45.51% reduction, p< 0.01).
3.8 Protein and mRNA levels of thermogenic gene in brown adipose tissue
Compared to the control, olanzapine treatment dramatically decreased the protein levels of UCP1 (-59%, p<0.01, Fig. 6A and 6E) and PGC-1α (-26%, p<0.05, Fig. 6C and 6E) in the BAT, but not PRDM16. However, there was a significant increase in PPARγ expression in the olanzapine-only treatment group (+42%, p<0.01, Fig. 6B and 6E). The O+S co-treatment significantly increased UCP1 expression compared with olanzapine-only treatment (+48%, p<0.05, Fig. 6A and 6E). The UCP1 protein level was negatively correlated with body weight gain (r=-0.516, p<0.01).
Consistent with changes in protein levels, there was a significant decrease of Ucp1 (-45%, p<0.05, Fig. 6F) and Pgc-1α (-40%, p<0.05, Fig. 6H) mRNA expression in the olanzapine-only group compared with the control. mRNA expression of Pparγ was significantly increased by olanzapine treatment compared with the control group (+40%, p<0.05, Fig. 6G). Compared to the olanzapine-only group, O+S co-treatment upregulated Ucp1 (+88%, p<0.01, Fig. 6F) and Pgc-1α (+43%, p<0.05, Fig. 6H) expression, but not Pparγ and Prdm16 gene (Fig. 6G and 6I).
To further assess whether simvastatin could enhance the expression of UCP1 through the PKA-dependent pathway, we detected the expression of proteins of the cAMP-dependent protein kinase (PKA) and phosphorylated PKA (p-PKA). A decrease in p-PKA was observed in BAT from olanzapine-treated rat (-48%, p<0.05, Fig. 7B and 7D), although we did not detect a significant increase of proteins related to the PKA signaling pathway between the O+S co-treatment and the olanzapine-only groups.