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Obestatin and its fragment analog Nt8U were shown to upregulate glycerolipid metabolism and PPARg signaling and decrease fat accumulation in Swiss albino mice. It was further investigated if these peptides could decrease lipid accumulation under obese conditions. We chose to work on Diet-Induced-Obese (DIO) C57BL/6 mice to study the same. Both obestatin and Nt8U decreased lipid accumulation in DIO-C57BL/6 mice. PPARg was not upregulated in comparison to 60% high-fat diet (HFD) fed control mice, implying there was already enhanced PPARg expression due to HFD consumption. We also wanted to investigate if upregulation of PPARg signaling was a secondary effect of enhanced glycerolipid metabolism. To investigate the same, we administered obestatin pairwise with 2 agonists and 2 antagonists of PPARg. The results revealed obestatin is not a mere agonist of PPARg but can also decrease lipid accumulation brought about by rosiglitazone, a well-studied agonist of PPARg. The antagonists also show a further decrease in lipid accumulation, probably due to inhibition of PPARg activity brought about by HFD and the additive decrease brought about by obestatin in DIO-C57BL/6 mice. This chapter will be structured to briefly introduce obestatin, Nt8U, their effect on gene expression in the adipose tissue, and the effect of PPARg agonists and antagonists on their ability to reduce fat accumulation.
Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India
Uma V. Manjappara*
Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysore, Karnataka, India
*Address all correspondence to: umamanjappara@cftri.res.in
1. Introduction
Obestatin is a regulatory peptide discovered in 2005 by Zhang et al. It was predicted to be a protease cleavage product of preproghrelin. The 23-residue peptide was isolated and characterized for its activity. It was shown to decrease food intake and body weight in rodents. They also continued to show GPR39 an orphan G-protein coupled receptor could be the cognate receptor for obestatin [1]. These claims have been debated upon since [2]. More recent research shows GLP-1 receptor could be mediating its activity [3]. In our laboratory, we synthesized 3 overlapping fragments of obestatin and showed the N-terminal 13 residues mimicked the parent peptide obestatin the best [4]. Subsequently, we synthesized two analogs of the N-terminal peptide with alpha-aminoisobutyric acid (Aib, denoted as U) at position 8 replacing a glycine (Nt8U) and cyclohexyl amino acid (Cha) at position 5 replacing phenylalanine (Nt5Cha). Experiments in Swiss albino mice showed Nt8U to be as active as obestatin in its ability to reduce fat accumulation [5]. Obestatin and its fragment analog Nt8U were shown to upregulate glycerolipid metabolism and PPARg signaling and decrease fat accumulation in Swiss albino mice [6]. It was further investigated if these peptides could decrease lipid accumulation under obese conditions. We chose to work on Diet-Induced-Obese (DIO) C57Bl/6 mice to study the same. Both Obestatin and Nt8U decreased lipid accumulation in DIO-C57BL/6 mice. PPARg was not upregulated in comparison to 60% high-fat diet (HFD) fed control mice, implying there was already enhanced PPARg expression due to HFD consumption [7, 8].
We also wanted to investigate if upregulation of PPARg signaling was a secondary effect of enhanced glycerolipid metabolism. To investigate the same, we administered obestatin pairwise with 2 agonists and 2 antagonists of PPARg. The results revealed obestatin is not a mere agonist of PPARg but can also decrease lipid accumulation brought about by rosiglitazone, a well-studied agonist of PPARg [9]. The antagonists also show a further decrease in lipid accumulation, probably due to inhibition of PPARg activity brought about by HFD and the additive decrease brought about by obestatin in DIO-C57BL/6 mice [5]. The effect of antagonists of PPARg in DIO-C57BL/6 mice will be discussed in this chapter concerning DIO and Rosiglitazone (PPARg agonist) + obestatin administered DIO-C57BL/6 mice.
2. Effect of antagonists of PPARg, GW9662, and T0070907 on DIO-C57BL/6 mice individually and along with obestatin
GW9662 is a potent and selective PPARγ antagonist with an IC50 of 3.3 nM. It has 10 and 600 fold less selectivity towards PPARα and PPARδ respectively. Mass spectrometric analysis revealed Cys285 was covalently modified by GW9662 [10]. T0070907 was identified as a potent and selective PPAR antagonist. It had an apparent binding affinity of 1 nM. It covalently modifies cysteine 313 in helix 3 of human PPAR2. Figure 1 shows the structure of the antagonists [11].
Figure 1.
Structure of the PPARg antagonists GW9662 and T0070907.
2.1 Induction of obesity and mice experiments
Obesity was induced by administering a 60% calorie by high-fat diet (HFD) for a period of 24 weeks to four-week-old male C57BL/6 mice. The gain in weight over the induction period is shown in Figure 2a as a comparison with normal diet-fed male C57BL/6 mice. The HFD fed mice gained an average of 25 g whereas, the normal chow-fed mice gained 5 g of weight over the same period. Subsequently, the mice were grouped into six groups as follows:
Figure 2.
(a) Obesity induction over a period of 24 weeks in comparison to Normal diet fed mice. (b) Change in body weight upon treatment with the respective compounds.
Group 1: HFD Control.
Group 2: Obestatin Control.
Group 3: GW9662 Control.
Group 4: T0070907 Control.
Group 5: Obestatin + GW9662 treatment.
Group 6: Obestatin + T0070907 treatment.
Obestatin was synthesized, purified, and characterized in our laboratory as described previously [4]. GW9662 and T0070907 were purchased from Sigma Aldrich. As per the previous optimization done in our laboratory obestatin was administered at 160 nmol /kg/BW [12]. From the available literature, it was decided GW9662 and T0070907 should be administered at 1 mg/kg/BW to the respective groups [10, 11]. All samples were dissolved in 20% DMSO in 0.9% saline and the same solvent was used as control. All the administrations were intraperitoneal. The mice were administered the respective compound for 8 days after induction of obesity. Food intake was monitored for 5 h on an hourly basis after administration of saline or the respective compounds and gain in body weight was recorded every day. Figure 3 shows the decrease in food intake upon administration of obestatin followed by further decreased food intake by the T0070907 group, Obestatin + T0070907 group, GW9662 group, and Obestatin + GW9662 group, respectively. Figure 2b shows the gain in body weight during 8 days. It can be seen that all the treated groups showed a negative gain in body weight even upon HFD administration. Only the HFD control group showed a steady gain in body weight. After 8 days of experimentation, the mice fasted for 6 hours and blood were drawn through the retro-orbital plexus and the mice were sacrificed according to established protocols. All adipose depots and vital organs were stored in formalin for histopathology studies and part of them were frozen in liquid nitrogen for RNA extraction and profiling.
Figure 3.
Food intake for 5 h after administration of the respective compounds. The mice were administered the respective compounds and fed HFD after 15 min.
2.2 Effect of the treatments on the different fat pad
Epididymal, perirenal, retroperitoneal, inguinal, BAT, gluteal, axillary, and cervical fat pads were collected from each group and weighed and normalized to the respective bodyweight to access the effect of the treatments on fat accumulation in each adipose depot. As not all adipose depots are metabolically the same, a decrease in fat accumulation in certain depots can be more beneficial. Table 1 summarizes the overall effect of the treatment on the fat depots. GW9662 increased epididymal adipose tissue weight in comparison to the HFD and obestatin treated groups and decreased % epididymal fat upon coadministration with obestatin. It significantly decreased % Inguinal fat weight upon coadministration with obestatin by 26.4%. It also decreased total subcutaneous fat by 11.3% but increased % visceral fat by 15% and thereby increased total fat % in the adipose depots by 11.1%. On the other hand, T0070907 increased % epididymal fat by 24% and % visceral fat by 10%. It decreased perirenal fat by 54% and % subcutaneous fat by 10%. Upon coadministration with obestatin, T0070907 decreased % inguinal fat by 27% and % total subcutaneous fat by 15%. And maintaining total fat content is equal to that of the HFD control.
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 % fat pad weight. Data are expressed as the mean ± SEM (N ≧ 8). 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).
2.3 Plasma biochemical analysis and lipid parameters
SGOT, SGPT, alkaline phosphatase (ALP), fasting blood glucose, creatinine, urea, triglyceride, total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were estimated using commercially available kits. Phospholipid estimation was carried out by a colorimetric method [13]. Plasma Leptin and adiponectin concentrations were tested as per the instructions of the manufacturer of commercially available ELISA kits.
Table 2 summarizes the plasma lipid parameters after the 8 days of the experiment. All the treated groups showed a significant decrease in plasma triglyceride. Obestatin + GW9662 and obestatin + T0070907 showed a maximum decrease of 39%. T0070907 and obestatin + T0070907 showed a maximum decrease in plasma total cholesterol by 10% whereas, GW9662 showed a significant decrease in total cholesterol by 4% only upon coadministration with obestatin. Obestatin increased phospholipids by 32%, the coadministered groups show a weak enhancement indicating there is a combined effect and not that of the individual components. Plasma-free fatty acids are decreased in all treated groups. A maximum decrease of 23% is seen in the obestatin + GW9662 treated group, followed by obestatin + T0070907 by 17%, GW9662 by 14%, T0070907, and obestatin by 10%.
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 fasting plasma lipid parameters such as triglycerides, total cholesterol, HDL-cholesterol, and phospholipids level. 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.
Table 3 summarizes the plasma biochemical parameters. Plasma glucose, protein, urea, and creatinine are in the normal range, did not show any significant changes. Marker enzymes SGOT, SGPT, and ALP are in the normal range for all the groups. Adipokine leptin that signals long-term fat reserves to the brain is significantly decreased most by obestatin + GW9662 group by 45% followed by obestatin + T0070907 by 37%, GW9662 by 33%, and T0070907 by 21% indicating a decrease in fat content. There were no significant changes in adiponectin levels.
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 fasting plasma biochemical parameters. 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.
2.4 Adipose tissue lipid parameters
Liver tissue, inguinal, and epididymal fat lipids were extracted by Folch’s method of lipid extraction [14]. Tissue TAG, TC, and phospholipid were estimated by a colorimetric method [13, 15, 16]. Table 4 summarizes the tissue lipid parameters. In epididymal adipose tissue, obestatin + T0070907 decreased triglyceride by 20%, followed by obestatin + GW9662 by 17% and GW9662 by 10%. Phospholipids were increased by about 80% in all the groups. No significant decrease in total cholesterol was observed. In inguinal adipose tissue, a significant decrease by 23% in total cholesterol was observed in both obestatin + GW9662 and obestatin + T0070907 groups followed by T0070907 at 18%. No significant changes were observed in triglyceride or phospholipids levels. In the liver tissue, a significant increase of 44% was seen only with respect to triglyceride in the obestatin treated group compared to that of the HFD control.
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 lipid parameters such as triglycerides, total cholesterol, phospholipid content in epididymal fat and liver tissue. 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 toT0070907.
2.5 mRNA profiling of epididymal and inguinal adipose tissue of lipid metabolism-related genes by quantitative real time-PCR (qPCR)
Total RNA was isolated from the adipocyte cells using TRIzol reagent from Sigma, USA. The quantity and quality of the isolated RNA were assessed using a microspectrophotometer (Eppendorf). Samples having a ratio of A260/280 > 1.8 were used for cDNA synthesis by kit method (Thermo Scientific, Ltd.). Real-time PCR assays were performed using SYBR Green (BioRad CFX96 Touch Real-Time PCR Detection System) and primer sequences of the respective genes are given in SI Table 1. Fold change in gene expression was tabulated by normalizing the values of threshold cycle (CT) of the target gene with the CT value of housekeeping gene GAPDH. Briefly, the fold changes were calculated using the 2−ΔΔCt calculation 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.
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).
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.
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 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.
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).
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.
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).
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.
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, 18, 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, 21, 22]. Decreased levels of FASN, DGAT1, and FABP4 are also indicative of decreased lipid accumulation [23, 24, 25]. Decreases in leptin levels seen in the plasma are also reflected at the mRNA level [26]. SORBS1 is a major regulator of insulin-stimulated 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].
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.
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.
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Written By
Beekanahalli G. Mallikarjuna and Uma V. Manjappara
Submitted: 08 February 2022Reviewed: 14 February 2022Published: 31 March 2022
Open access peer-reviewed chapter
