**Table 4.**

### *Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*

method with GAPDH as the internal control gene. The experiment was done in "triplicates of triplicate" three experimental samples were estimated in triplicate to obtain statistical significance.

**Figure 4** shows the mRNA profiling of the epididymal adipose tissue of mice treated with the respective compounds. In the epididymal adipose tissue, transcription factors PPARg, CEBPa, and CHREBP decrease by about 0.5 fold in obestatin, GW9662 and T0070907 treated groups whereas, obestatin + GW9662 and obestatin + T0070907 showed PPARg levels equal to that of the HFD control. CEBPa showed a 0.4 fold increase in both obestatin + GW9662 and obestatin + T0070907 treated groups. Concerning CHREBP, the obestatin treated group showed a decrease of 0.5% whereas, obestatin + GW9662 and obestatin + T0070907 groups showed 1.8 fold and 2 fold increase followed by GW9662 and T0070907 treated groups showing an increase by 0.4 fold above the control group.

Lipolysis was significantly enhanced in the obestatin + GW9662 and obestatin + T0070907 treated groups as seen from upregulation of ATGL and HSL by 0.3 fold and 1.8 fold respectively. GW9662 and T0070907 treated groups showed a 0.4% decrease in ATGL levels and no changes in HSL levels. Obestatin showed a decrease in 0.4 fold concerning ATGL and 0.4 fold decrease in HSL levels. No significant changes were seen to MGL.

No changes in mRNA levels of Perilipin 1 were seen in the GW9662 and obestatin + GW9662 treated groups. Whereas obestatin showed a 0.5 fold decrease in Perilipin levels and T0070907 and obestatin + T0070907 showed a decrease of 0.2 and 0.3 folds respectively.

#### **Figure 4.**

*Effect of obestatin (160 nmol /kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol/kg/ BW + 1 mg/kg/BW) treatment on gene expression in epididymal tissue of obese C57BL/6 mice. β-Actin was used as internal control for gene expression studies by RT-PCR. Data are expressed as the mean ± SEM (N* ≧ *8). P < 0.05 was considered as statistically significant value (\* = significant when compared to control, # = significant when compared to obestatin, \$ = significant when compared to GW9662, @ = significant when compared toT0070907).*

*Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*

With regards to lipid accumulation genes, obestatin + GW9662 showed an increase of LPL by 0.2 fold and obestatin + T0070907 showed no change with respect to the control. The obestatin, GW9662, and T0070907 treated groups showed a significant decrease by 0.7, 0.5, and 0.6 fold respectively. FASN was decreased in all treatment groups 0.8 fold by obestatin + GW9662 followed by obestatin by 0.7 folds, obestatin + T0070907 by 0.5 fold, GW9662 by 0.4 fold, and T0070907 by 0.2 fold. All treated groups showed decreased DGAT1 levels by 0.3–0.5 folds and FABP4 by 0.3–0.6 fold.

The adipokines leptin and adiponectin also showed differential regulation supporting a decrease in total fat content. Leptin levels were decreased by 0.7 folds by obestatin + GW9662 and obestatin + T0070907 groups whereas, obestatin and GW9662 showed leptin levels decreased by 0.5 fold. T0070907 showed 0.3 fold decrease in leptin levels. GW9662 and T0070907 groups showed 0.2 and 0.1 fold increase in adiponectin levels obestatin, obestatin + T0070907 showed a decrease in adiponectin levels by 0.5 fold and obestatin + GW9662 group by 0.3 fold. All treated groups showed a decrease in SORBS1 levels. T0070907 and obestatin + T0070907 at 0.4 fold, GW9962, obestatin + GW9662 and obestatin showed a decrease in SORBS1 levels by 0.3, 0.2 and 0.1 fold respectively.

**Figure 5** shows the effect of the treatments on the inguinal adipose tissue. In inguinal adipose tissue, obestatin showed a 0.4 fold increase in PPARg and CHREBP levels and a 0.3 fold increase in CEBPa levels. All other treated groups showed a decrease in PPARg levels. GW9662 and obestatin + GW9662 showed a 0.4 fold decrease in PPARg levels. T0070907 and obestatin + T0070907 showed a decrease in PPARg levels by 0.9 and 0.6 folds respectively. A very similar trend was seen for

#### **Figure 5.**

*Effect of obestatin (160 nmol /kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol/kg/ BW + 1 mg/kg/BW) treatment on gene expression in inguinal tissue of obese C57BL/6 mice. β-Actin was used as internal control for gene expression studies by RT-PCR. Data are expressed as the mean ± SEM (N* ≧ *8). P < 0.05 was considered as statistically significant value (\* = significant when compared to control, # = significant when compared to obestatin, \$ = significant when compared to GW9662, @ = significant when compared toT0070907).*

all the treated groups except obestatin with respect to CEBPa and CHREBP with obestatin + GW9662 group showing a decrease by 0.2 fold, GW9662 by 0.4 fold, T0070907 by 0.75 fold, and obestatin + T0070907 by 0.7 fold.

With regards to lipolytic genes obestatin, obestatin + GW9662 and obestatin + T0070907 increased ATGL by 0.3 fold and GW9662 and T0070907 decreased by 0.5 fold with respect to the control group. Obestatin + GW9662 and obestatin + T0070907 increased HSL levels by 0.3 and 0.1 fold whereas obestatin, GW9662, and T0070907 decreased HSL levels by 02. 0.6 and 0.25 fold respectively. MGL was decreased by 0.7 fold by T0070907 and obestatin + T0070907, 0.4 fold by GW9662 and obestatin and GW9662, and 0.2 fold by obestatin.

Obestatin + GW9662 and obestatin + T0070907 showed decrease in Perilipin levels by 0.7 fold, T0070907 by 0.25 fold, GW9662 by 0.5 fold, obestatin + GW9662 by 0.8 fold. Whereas obestatin showed an increase in Perilipin levels by 0.5 fold.

LPL was decreased by 0.4 fold in GW9662, T0070907, and obestatin + GW9662 groups. T0070907 decreased LPL levels by 0.5 fold and the obestatin group showed an increase in LPL levels by 0.25 fold.

FASN was increased by obestatin by 0.75 fold whereas, all the other treated groups showed a decrease in FASN levels of 0.3 fold with exception of T0070907 at 0.7 fold. All treated groups showed a decrease in DGAT1. Obestatin, obestatin + GW9662, and T0070907 showed a decrease in DGAT1 levels at 0.5 fold, GW9662 at 0.25 fold, and obestatin + T0070907 at 0.75 fold.

No changes in FABP4 levels were observed in the obestatin and obestatin + GW9662 treated groups with respect to the control. GW9662 and T0070907 showed a decrease in FABP4 by 0.7 fold and Obestatin + T0070907 showed an increase in FABP4 levels by 0.25 fold.

Leptin was increased two-fold in the obestatin treated group, GW9662 and T0070907 showed a decrease in leptin levels by 0.4 fold. Adiponectin was decreased by the T0070907 and obestatin + T0070907 groups by 0.6 fold, GW9662, and obestatin + GW9662 groups by 0.25 fold and obestatin increased adiponectin by 0.2 fold.

SORBS1 levels remained unchanged in T0070907 and obestatin + T0070907 groups, GW9662 and obestatin + GW9662 groups decreased SORBS1 levels by 0.3 fold and obestatin showed no changes.

As seen in **Figure 6**, PPARg was enhanced by obestatin by 2 fold whereas, GW9662 and T0070907 decreased PPARg levels by 0.5 fold. Obestatin + GW9662 and obestatin + T0070907 decreased PPARg by 0.1 and 0.2 fold respectively.

FASN levels were decreased by 0.2 fold by all the treated groups except obestatin + T0070907, which decreased FASN levels by 0.6 fold.

GW9662 and obestatin + GW9662 groups increased SCD1 levels by 0.2 fold whereas, obestatin and obestatin + T0070907 showed no changes. The T0070907 group showed a decrease in SCD1 by 0.2 fold.

DGAT1 was decreased in all treated groups except obestatin. All the groups decreased DGAT1 by 0.5 fold and obestatin increased DGAT1 by 0.3 fold.

In brown adipose tissue, PPARg levels were enhanced by .75 fold by obestatin and obestatin + T0070907 and Obestatin + GW9662 and T0070907 by 1.5 fold. GW9662 enhanced PPARg twofold.

UCP1, responsible for thermogenic lipolysis of fat was increased by obestatin by 2.8 fold, obestatin + GW9662 by 1.6 folds obestatin + T0070907 by 1.5 fold. GW9662 and T0070907 increased UCP-1 by 0.1 and 0.3 fold.

*Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*

**Figure 6.**

*Effect of obestatin (160 nmol/kg/BW), GW9662 (1 mg/kg/BW), T0070907 (1 mg/kg/BW), individually and in combination of obestatin + GW9662 (160 nmol/kg/BW + 1 mg/kg/BW, obestatin + T0070907 (160 nmol/kg/ BW + 1 mg/kg/BW) treatment on gene expression in A. Liver tissue B. BAT of obese C57BL/6 mice. β-Actin was used as internal control for gene expression studies by RT-PCR. Data are expressed as the mean ± SEM (N* ≧ *8). P < 0.05 was considered as statistically significant value (\* = significant when compared to control, # = significant when compared to obestatin, \$ = significant when compared to GW9662, @ = significant when compared toT0070907).*

#### **2.6 Comparison of the effect on lipid parameters observed upon the individual and coadministration of antagonists of PPARg GW9662 and T0070907 with rosiglitazone a well-studied agonist of PPARg**

Administration of GW9662 and T00907 have shown the differential effect on plasma, adipose depots, and liver lipid parameters. All the treatments have shown a significant decrease in gain in body weight when compared to the HFD control. There is a decrease in food intake until 5 h after intraperitoneal administration of the agonists and obestatin, though there is no difference in the food intake after 12 h after administration. Both coadministrations show a decrease in inguinal and subcutaneous normalized fat pad weight. Whereas, triglyceride content is decreased in epididymal adipose tissue and total cholesterol in inguinal adipose tissue. The coadministration has significantly decreased plasma triglyceride and free fatty acids levels. Storage of fat in adipose tissue taking it out of circulation in the plasma is an indication of reduced lipotoxicity. Significantly decreased leptin levels in all the treated groups indicate the long-term effect of these compounds on lipid storage. mRNA profiling of the obestatin + GW9662 and obestatin + T0070907 groups show no change in PPARg levels, the significant increase in CEBPa and CHREBP levels indicating they do play a definite role in glucose and lipid metabolism in the liver and also adipogenesis [17–19]. An increase in ATGL and HSL levels also indicate the breakdown of triglyceride in the tissue and reduced perilipin1 levels indicate decreased storage of fat in the epididymal adipose tissue [20–22]. Decreased levels of FASN, DGAT1, and FABP4 are also indicative of decreased lipid accumulation [23–25]. Decreases in leptin levels seen in the plasma are also reflected at the mRNA level [26]. SORBS1 is a major regulator of insulinstimulated signaling and regulation of glucose uptake, by potentiating insulin-induced phosphorylation and recruitment of CBL to a lipid raft [27]. A decrease in SORBS1 mRNA levels indicates altered glucose uptake in the epididymal adipose tissue.

On the other hand, in the inguinal adipose tissue, a decrease in the levels of transcription factors PPARg, CEBPa, and CHERBP in the antagonists and

coadministration groups indicates decreased triglyceride accumulation. An increase in ATGL, HSL levels, and a decrease in MGL levels indicate decreased fat accumulation also supported by a decrease in Perilipin1 levels. Decreased lipid accumulation is also indicated by a decrease in the mRNA levels of FASN, DGAT1, and leptin. No changes are seen in SERBS1 levels indicating status co with insulin sensitivity.

In comparison, the PPARg agonist rosiglitazone showed an increased gain in body weight. Whereas, obestatin and obestatin + rosiglitazone showed a decrease in gain in body weight. Rosiglitazone showed significant a decrease in plasma triglycerides and free fatty acids by 50.93%, and 24.98% respectively. The same decrease was retained upon coadministration with obestatin. Rosiglitazone showed an increase in gluteal, cervical and subcutaneous fat and total fat content by 60%, 17.8%, 12%, and 20% respectively. Combined administration of obestatin and rosiglitazone reduced all the rosiglitazone increased fat content of gluteal, cervical, subcutaneous, and total fat to the control group levels [9]. In the epididymal adipose tissue, rosiglitazone showed a significant increase in LPL and FASN levels by 4.18 and 4.08 folds respectively. Whereas in the obestatin + rosiglitazone treated groups ATGL, HSL and MGL were upregulated by 3.78, 1.3, and 2.09 folds supporting a decrease in fat accumulation upon coadministration countering the fat accumulation increasing the effect of rosiglitazone. In Inguinal adipose tissue, obestatin + rosiglitazone upregulated ATGL by 2.83 fold. Leptin mRNA levels were upregulated 2.5 fold and 2.7 fold by rosiglitazone and obestatin + rosiglitazone [28].

#### **3. Conclusions**

The coadministration studies, previously with the PPARg agonist rosiglitazone and currently with PPARg antagonists GW9662 and T0070907 indicate that obestatin and PPARg agonists/antagonists show combined beneficial effects on plasma, liver, and adipose tissue lipid parameters. Obestatin reversed the lipid accumulation effect inherent to rosiglitazone. Whereas, the antagonists do not show a tendency towards lipid accumulation and additionally bring about beneficial effects by decreasing plasma and epididymal adipose tissue triglyceride levels. These studies indicate antagonists of PPARg in combination with obestatin could be furthered as lead compounds to counter obesity.

#### **Acknowledgements**

Mallikarjun BG gratefully acknowledges the financial support provided by ICMR for SRF (No.3/1/2/20/Nut./2012).

We acknowledge funding by The Council of Scientific and Industrial Research, India, through the major laboratory project MLP-116 and WELFO under the 12th five-year plan.

#### **Abbreviations**


*Effect of GW9662 and T0070907 Antagonist of PPARg and Their Coadministration Pairwise… DOI: http://dx.doi.org/10.5772/intechopen.103700*


#### **Author details**

Beekanahalli G. Mallikarjuna and Uma V. Manjappara\* Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India

\*Address all correspondence to: umamanjappara@cftri.res.in

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
