Corn Oil

Fish oil and corn oil induced differential effect on beiging of visceral and subcutaneous white adipose tissue in high fat diet induced obesity

Prerna Sharma, Navneet Agnihotri

PII: S0955-2863(20)30490-3
Reference: JNB 108458

To appear in: The Journal of Nutritional Biochemistry

Received date: 25 October 2019
Revised date: 16 June 2020
Accepted date: 16 June 2020

Please cite this article as: P. Sharma and N. Agnihotri, Fish oil and corn oil induced differential effect on beiging of visceral and subcutaneous white adipose tissue in high fat diet induced obesity, The Journal of Nutritional Biochemistry (2020), 10.1016/j.jnutbio.2020.108458

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© 2020 Published by Elsevier.

Fish oil and corn oil induced differential effect on beiging of visceral and subcutaneous white adipose tissue in high fat diet induced obesity.
Prerna Sharma1, Navneet Agnihotri*1

1. Department of Biochemistry, Panjab University, Chandigarh.
* Corresponding Author

E-mail – [email protected]
1. Introduction
Obesity is a chronic disease and a serious public health concern with an alarming increase in incidence rate worldwide [1]. Global prevalence of overweight and obesity-“globesity” has nearly tripled since 1975. Currently approximately 1/3rd of world’s total population is being classified as either overweight or obese [2]. Another cause of concern is its close association with an array of comorbidities such as dyslipidemia, metabolic syndrome, insulin resistance, diabetes mellitus and cardiovascular disease [3]. Obesity arises due to an imbalance between calories consumed and calories utilised resulting in storage of excess calories as fat in white adipose tissue (WAT). Evidence suggests that WAT undergoes a dysfunctional state characterised by hypertrophy and hyperplasia of adipocytes leading to a subsequent change in pattern of adipokine secretion in obesity [4, 5]. WAT is distributed into two main compartments – centrally located visceral depot of WAT (VWAT) and peripheral subcutaneous WAT (SWAT). Both the compartments of WAT have varied lipid storage and metabolising capabilities. SWAT acts as a protective metabolic sink which avidly absorbs circulating FFAs and triglycerides in hypercaloric conditions. On the other hand, VWAT is more metabolically active and exhibits an increased lipid turnover. Access of VWAT to portal circulation lead to ectopic lipid deposition in liver and resultant metabolic complications [6, 7]. Recently it was shown that SWAT reaches its maximal capacity to store TGs during transition from lean to obese phenotype while VWAT maintains a continuous lipid turnover over a broad range of fat levels and is only reduced in morbidly obese phenotypes [8].
Recent discovery of functional brown adipose tissue (BAT) in humans has opened the possibilities of combatting the metabolic complications associated with obesity by promoting its activity and increasing the energy expenditure [9, 10]. Another intriguing possibility is the observation that white adipocytes can adopt a BAT-like phenotype by trans-differentiation

into beige adipocytes. Beige adipocytes are hypothesised to be formed from white adipocytes and are characterised by an increase in number of mitochondria and transformation of stored fat into small lipid droplets similar to brown adipocytes. Browning is also associated with an increased expression of thermogenic gene, uncoupling protein 1 (UCP1) which uncouples oxidative phosphorylation and leads to enhanced energy dissipation [11, 12]. UCP1 expression, in turn, is regulated by transcription factor PGC-1α known for its ability to induce mitochondrial biogenesis and adaptive thermogenesis. Direct phosphorylation of PGC-1α by AMPK which is the master energy sensor of the cell has been devised as the link between the sensing cellular energetic status and the induction of transcriptional pathways to control energy expenditure [11, 13]. It is therefore postulated that AMPK-PGC1α-UCP1 axis may play a critical role in the process of beiging of WAT.
A plethora of studies from animal models have reported the beneficial effect of fish oil (FO) in obesity and its associated metabolic complications like dyslipidemia, inflammation, insulin resistance and type II diabetes. FO has been reported to alleviate obesity via modulating inflammatory pathways [14], reducing oxidative and endoplasmic reticulum stress [15], improving glucose-insulin axis [16, 17] and inducing UCP2 upregulation in liver [18]. Despite a number of studies reporting beneficial effects of n-3 PUFAs in weight reduction and obesity management in animal models, there still is a lacunae in translating the effects to human populations [19]. As both visceral and subcutaneous depots of adipose tissue have a different metabolic and physiological activities, therefore, it is important to delineate the mechanism of action of any anti-obesity regimen in two structurally and functionally diverse depots, namely VWAT and SWAT. Only a few studies have studied the effect of n-3 PUFAs in both depots of WAT and this needs to be further explored. In addition, this study aims to provide a better understanding of effect of corn oil/n-6 PUFAs in high fat diet induced obesity. It is important in the context that a skewed ratio of n-6/n-3 polyunsaturated fatty acids (PUFAs) appears to be the reason for rising obesity epidemic [20, 21]. Thus the present study was designed to provide deeper insights on differential action of n-3 and n-6 PUFAs in functionally and metabolically diverse depots of white adipose tissue in high fat diet induced obesity. Moreover, a better understanding of effects of n-3 and n-6 PUFAs will have important implications for devising nutritional recommendations and tailoring dietary interventions to correct the metabolic dysregulation and hence reduce obesity.

2. Material & Methods
2.1. Materials
All the chemicals used in the study were of analytical grade. Lard, porcine source (MP Biomedicals, USA) was used as the fat source to induce obese phenotype and it contained approximately 35.9% saturated fatty acids. Fish oil (MAXEPA, Merck) was used as a source of n-3 PUFAs which had 180mg eicosapentaenoic acid (EPA) and 120 mg docosahexaenoic acid (DHA) per 1ml capsule. Corn oil (Sigma) was used as a source of n-6 PUFAs. It contained approximately 39.4-62.0% of n-6 PUFAs particularly, linoleic acid. Bradford reagent and RIPA buffer, polymethylsulfonyl fluoride (PMSF) and RNAlater were procured from Sigma chemical company (St. Louis, MO, USA). Monoclonal antibodies against AMPKα (#5831), phospho-AMPKα (#2535), ACC (#3676), phospho-ACC (#11818) were
purchased from Cell Signalling Technology (Beverly, MA, USA). Monoclonal antibody against GAPDH (AM4300) and Revert aid cDNA synthesis kit was purchased from ThermoFisher Scientific (Waltham, Massachusetts, United States). RNeasy mini kit for RNA isolation was purchased from Qiagen, Germany. Polyvinylidenedifluoride (PVDF) membrane and Clarity™ ECL Western Blotting Substrate was purchased from Bio-Rad (Hercules, CA, USA). Adiponectin and leptin ELISA kits were purchased from Elabscience, (Houston, Texas, USA). Serum triglycerides, cholesterol, HDL-Cholesterol, LDL-cholesterol were purchased from Reckon Diagnostics Pvt. Ltd (Baroda, India).
2.2. Experimental design
Male wistar rats (N=60) weighing 140-170g were procured from Central Animal House, Panjab University, Chandigarh, India. All the experimental protocols were approved via institutional animal ethics committee (PU/IAEC/S/14/43). The animals were housed in polypropylene cages under a 12h light/dark cycle at 25±2ºC. After acclimatization for one week, animals were randomly divided into two main groups.
• Standard low fat diet groups with 10% calories from (C), or an identical diet supplemented with fish oil (FO) or corn oil (CO).
• High fat diet with 60% calories from fat (HFD) or identical diets with 10% calories from fat being replaced with fish oil (HFD+FO) or corn oil (HFD+FO).

Fig 1. Schematic representation of study design

The diets were made according to composition of D12450B (10% calories from fat) and D12492 (60% calories from fat) diets (Research Diets, Inc, USA) and fed ad libitum for a period of 12 weeks as per the previous reports on development of high fat diet induced model of obesity [22, 23]. The 10% replacement of saturated fatty acids in high fat diet with n-3 or n-6 PUFAs was made according to the literature available on high fat diet induced obesity models [24, 25] . The detailed composition of experimental diets and its caloric distribution is shown in Table 1.
To avoid oxidation of unsaturated fatty acids, the diets were prepared afresh daily. The anthropometric measurements were done once a week. At end of 12 weeks, the animals were sacrificed by cervical dislocation after collecting blood via retro-orbital puncture. Epididymal and inguinal adipose tissue was dissected from the experimental animals and rapidly stored at
-80ºC till further use. For histopathology and electron microscopy, the tissue was transferred to 4% neutral buffered formalin and 2.5% glutaraldehyde solution respectively.

Ingredients (g) C HFD FO HFD+FO CO HFD+CO
Casein 200 200 200 200 200 200
L-Cystine 3 3 3 3 3 3
Corn starch 315 0 315 0 315 0
Sucrose 350 68.8 350 68.8 350 68.8
Maltodextrin 35 125 35 125 35 125
Fibre 50 50 50 50 50 50
Soyabean oil 25 25 25 25 25 25
Lard 20 245 0 200 0 200
Fish oil 0 0 20 45 0 0
Corn oil 0 0 0 0 20 45
Vitamin Mix* 10 10 10 10 10 10
Mineral mix** 10 10 10 10 10 10
Potassium citrate 16.5 16.5 16.5 16.5 16.5 16.5
Calcium carbonate 5.5 5.5 5.5 5.5 5.5 5.5
Dicalcium phosphate 13 13 13 13 13 13
Choline bitartarate 2 2 2 2 2 2
Energy (Kcal %)
Protein 20% 20% 20% 20% 20% 20%
Carbohydrate 70% 20% 70% 20% 70% 20%
Total Fat 10% 60% 10% 60% 10% 60%
Saturated fat 10% 60% - 50 % - 50 %
n-3 PUFAs - - 10% 10% - -
n-6 PUFAs - - - - 10% 10%
Table 1. Detailed composition of experimental diets.
* Vitamin Mix contains Vitamin A, Vitamin B1, B2, B3, B5, B6, B12, Folic Acid, Vitamin C, Vitamin D3, Vitamin E along with Chromic Chloride, Cupric Oxide, Manganese Chloride, Sodium Selenate and Zinc Oxide as active ingredients.
** Mineral Mix contains Zinc, Magnesium, Manganese, Iron, Copper, Iodine, Cobalt, Potassium, Sodium, Calcium, Phosphorus, Sulphur.

2.3. Analysis of anthropometric parameters
Body weight, body length, waist circumference were determined in all rats at regular intervals and at the end of feeding period. It was used to calculate body mass index (BMI) and lee index [26].
• Body mass index (BMI) = body weight(kg)/length2(m2)
• Lee index = cube root of body weight(g)/nose to anus length (cm)

2.4. Serum biochemical analysis
For biochemical assays, blood was centrifuged at 1000g for 10 minutes at 4ºC to separate serum. Total lipid in serum was quantified using colorimetric method [27]. Serum triglycerides (TGs), total cholesterol (TC), HDL-Cholesterol (HDL-C), LDL-cholesterol (LDL-C) were estimated using commercial kits (Reckon Diagnostics Pvt. Ltd Baroda, India).

Atherogenic index which is named as a new biomarker of obesity was calculated as log10(TGs/HDL-C) [28].

2.5. Quantification of adipose tissue cholesterol
Adipose tissue cholesterol was quantified using Amplex Red Cholesterol Assay kit according to manufacturer’s protocol. Briefly, adipose tissue was homogenized in RIPA buffer, centrifuged and the supernatant was used for cholesterol quantification. For cholesterol assay, 50 μL of the diluted supernatant (1:25) and 50 μL of working solution of 300 μM Amplex® Red reagent (containing both cholesterol esterase and cholesterol oxidase) was added to the individual wells in a 96-well dark microplate. The plate was incubated at 37°C for 30 minutes following which the fluorescence intensities were measured for excitation and emission spectra at 560±10 and 590±10 nm, respectively. An aliquot of the same supernatant was used for protein quantification using Bradford assay. The results were expressed as μg cholesterol/mg protein.

2.6. Adipokine measurements
Adiponectin and leptin levels were quantified in serum using ELISA Kits (Elabscience, USA). Briefly, 100µL sample was added to each well and incubated for 37ºC for 90 minutes. It was followed by addition of detection antibody and incubation for 1 hour at 37 ºC. After washing, HRP-conjugate was added and incubated for 30 minutes at 37 ºC. Finally the substrate was added at 37 ºC and incubated for 15 minutes after which reaction was stopped using stop solution. The ELISA plate was read at 450 nm. The concentration of adiponectin/leptin was calculated by comparing the optical density (OD) of the sample to OD of standard using a standard curve.

2.7. Histological analysis
For light microscopy, the epididymal and inguinal adipose tissue was removed, washed in cold PBS and was fixed in 10% neutral buffered formalin (n=3 animals per group). 5 μm thick sections were prepared and analysed using standard hematoxylin and eosin staining method. The slides were photomicrographed using Nikon Eclipse 80i microscope and adipocyte area was calculated using ImageJ software (NIH, Bethesda, MD, USA). Transmission electron microscopic analysis was done using the method of Yu et al., 2017 [29]. Briefly, fresh epididymal and inguinal adipose tissue was fragmented into 2 mm3 pieces and immediately transferred to 2.5% glutaraldehyde in phosphate buffer (pH 7.4) for primary

fixation followed by secondary fixation in 1% osmium tetraoxide. The sections are then dehydrated in increasing concentrations of ethanol and embedded in resin. Ultrathin sections were cut and visualised with a Talos transmission electron microscope (Thermo Scientific, Waltham, Massachusetts, United States).
2.8. Immunohistochemical analysis
For immunohistochemical staining of UCP1 and PGC1α, 5µm thick sections were deparaffinised in xylene and rehydrated using a series of alcohol dilutions. Endogenous peroxidase activity was blocked by incubation in 3% H2O2 solution in methanol. Antigen retrieval was carried out using 10mM sodium citrate buffer, pH-6.0 in a hot chamber at 98
°C. Then, the tissue slices (2–3 tissue slices per glass slide) were incubated at 4 °C overnight with anti-UCP1 (sc-6529) and anti-PGC1α (sc-13067) purchased from Santa Cruz Biotechnology, Minneapolis, MN, USA. After rinsing in PBS for two times to remove the excess of antibody, the sections were incubated for 2hr at 37ºC with HRP labelled secondary antibody. The expression was visualised using 3,3′-Diaminobenzidine (DAB) and counterstained with hematoxylin. A minimum of 3 random fields were analysed and the positively stained cells were counted in each of these fields using Nikon Eclipse 80i microscope.
2.9 RNA isolation and conventional reverse transcriptase PCR
Total RNA was isolated from visceral and subcutaneous adipose tissue using RNeasy Mini Kit (Qiagen). Quantity and quality of RNA were estimated on Nanodrop spectrophotometer (NanoDrop™ 2000c, Thermo Scientific, Waltham, MA, USA) and by agarose gel electrophoresis, respectively. cDNA was synthesized from isolated RNA using cDNA synthesis kit as per the manufacturer’s protocol. The cDNA samples were then amplified using PGC1α and GAPDH primers (Table 2). The amplification programme involved: pre- incubation of 3 min at 95 °C and then amplification cycles consisting of the 30s at 95 °C, 45 s at annealing temperature, and 1 min at 72 °C. The PCR products were then incubated for 10 min at 72 °C. The resulting PCR products were subjected to electrophoresis on a 2% agarose gel and stained with ethidium bromide. The band intensity was measured using Alphview software.
Table 2. Primer sequence of the target genes.

Gene Name Forward Primer Reverse Primer Amplicon Size



2.10. Protein expression analysis
Epididymal and inguinal adipose tissue (n=3 per group ) was excised and homogenized in ice cold lysis buffer containing 1mM PMSF and 0.2% EZBlock™ Universal Protease and Phosphatase Inhibitor Cocktail (BioVision, Inc. Milpitas, CA, USA). The isolated protein was quantified using Bradford protein assay. 50 μg protein was electro-transferred to PVDF membranes. After blocking with 3% BSA, the membranes were incubated with primary antibodies for AMPKα (1:3000), Phospho-AMPKα (1:3000), ACC (1:3000), Phospho ACC (1:3000) and GAPDH (1:10000) overnight at 4 °C. The membranes were then incubated with corresponding horse radish peroxidase (HRP)-labelled goat anti-mouse IgG (1:3000) and anti-rabbit-IgG (1:5000) at room temperature for 1 h. The immunoblots were developed with Enhanced Chemiluminescence Detection Kit (Biorad, USA) and visualized on FluorChem M (ProteinSimple, San Jose, USA). Densitometry analysis was performed with AlphaView software provided with the instrument.
2.11. Statistical analysis
Results were expressed as the mean ± standard error mean (SEM). One way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-hoc test was used to analyse the experimental data using GraphPad Prism 5 (GraphPad Software, Inc. La Jolla, CA). Results with p<0.05 were considered as statistically significant.
3. Results
3.1. Effect of fish oil and corn oil on anthropometrical parameters

After 12 weeks of dietary intervention, it was observed that high fat diet feeding led to significantly higher body weight in HFD animals as compared to control groups C, FO and CO (p<0.001 for all comparisons). HFD+FO group, although consuming a hypercaloric diets showed a significantly reduced body weight relative to HFD group (p<0.001). On the other hand, HFD+CO group had a less pronounced effect on body weight as compared to HFD+FO group (p<0.05) (Fig 2A). BMI and waist circumference also showed a similar pattern where HFD group showed a significantly higher BMI as compared to control groups, C, FO and CO (p<0.001 for all comparisons). Supplementation of fish oil in HFD+FO resulted in lowering

the BMI and waist circumference significantly (p<0.001) as compared to HFD group. HFD+CO group also had a significantly lower BMI as compared to HFD group (p<0.05) however, it had no effect on waist circumference of the animals (Fig 2B, 2D). Lee index was significantly higher in HFD with respect to control group (p<0.05) but no significant alteration was observed on fish oil and corn oil supplementation (Fig 2C).

Fig 2. Anthropometrical parameters of experimental animals after 12 weeks (n=10). Final body weight (A), Body mass index (kg/m2),BMI (B), lee index (C) and waist circumference (D). ***p<0.001, **p<0.01, *p<0.05. Results are expressed as mean ± S.E.M.
3.2. Effect of fish oil and corn oil on serum lipid profile

Among the circulating lipid fractions, the total lipid, TG, TC levels were found to be significantly increased in HFD group as compared to C group (p<0.001). Fish oil supplementation led to a significant lowering of total lipid, TG and TC levels in HFD+FO group (p<0.05, p<0.05, p<0.001 respectively). However, corn oil supplementation in HFD+CO group failed to show any difference in these levels as compared to HFD group. HDL-C levels did not show any differences between the experimental groups except FO

group which had significantly higher HDL-C levels as compared to C and HFD group (p<0.001). LDL-C levels on the other hand showed an increase in HFD group as compared to C group (p<0.05). Neither fish oil nor corn oil had any visible effect on the levels of LDL-C in HFD+FO and HFD+CO group as compared to HFD group (Table 3).
Atherogenic index was calculated and it was found that HFD group had a 73.6% higher AI as compared to C group (p<0.001). FO supplementation in HFD+FO group significantly reduced AI by 31.9% relative to HFD group (p<0.05) where as HFD+CO group had no appreciable effect on AI as compared to HFD group (Table 3). Collectively the results indicate that FO supplementation had positively lowered the serum lipid fractions of total lipid, TGs, TC to control levels in HFD+FO group whereas CO failed to show any major alterations.
Table 3. Serum lipid profile of animals fed experimental diets (n=8). Results are expressed as

Serum lipid 288.2±7.6 378.5±21.7*** 294.9±5.2 313.2±6.6# 315.6±15.3 394.3±17.1
Atherogenic 0.412±0.03 0.716±0.05*** 0.276±0.04 0.487±0.05# 0.476±0.04 0.631±0.04
Index (AI)
mean ±SEM. ***p<0.001 and *p<0.05 w.r.t. C; ###p<0.001 and #p<0.05 w.r.t. HFD.

3.3 Effect on adipose tissue cholesterol homeostasis
Changes in adipocyte cholesterol homeostasis are associated with adipose dysfunction and obesity. Thus, total cholesterol levels (free cholesterol + cholesterol esters) were quantified in epididymal and subcutaneous adipose tissue using Amplex red fluorometric assay. There was a significant increase in accumulation of cholesterol in epididymal and inguinal adipose tissue of HFD group as compared to C group (p<0.001 and p<0.01 respectively). Fish oil supplementation led to a significant reduction in cholesterol accumulation in epididymal depot in both FO and HFD+FO group as compared to HFD group (p<0.01 for both

comparisons). In inguinal adipose tissue, fish oil supplementation led to a decrease in cholesterol levels in HFD+FO group as compared to HFD group (p<0.05). However, corn oil supplementation had no significant effect on epididymal and inguinal cholesterol levels as compared to HFD group (Fig 3). The results indicate a protective role of FO supplementation on cholesterol accumulation in adipose tissue.

Fig 3. Total Cholesterol levels in visceral/epididymal adipose tissue (A) and subcutaneous/ inguinal adipose tissue (B); n=6. ***p<0.001, **p<0.01 and *p<0.05. Values are represented as mean ± S.E.M.

3.4. Effect of fish oil and corn oil on adipokine secretion
The HFD feeding led to a dysregulation of adipokine secretion as indicated by decreased adiponectin and elevated leptin levels as compared to control group (p<0.01) (Fig 4). Fish oil supplementation led to a significant increase in adiponectin levels in FO (p<0.01) and HFD+FO (p<0.01) as compared to HFD group. Corn oil however failed to show any significant difference as compared to HFD group. On the other hand, serum leptin levels were raised significantly in HFD group in comparison to control group. Supplementation of fish oil and corn oil led to a significant reduction in the serum leptin levels in HFD+FO and HFD+CO group as compared to HFD group (p<0.001 for both comparisons).

Fig 4. Serum adipokine levels. Adiponectin (A) and Leptin (B). ***p<0.001, **p<0.01 and
*p<0.05. Values are represented as mean ± S.E.M. n=4 per group).

3.5. Analysis of effect of fish oil and corn oil on VWAT and SWAT in HFD induced obesity
3.5.1. Epididymal and inguinal adipose tissue mass

The effect of experimental diets on the adiposity i.e. the accumulation of fat in both epididymal and inguinal depots of adipose tissue was evaluated. It was observed that HFD group showed significantly higher epididymal fat mass as compared to C, FO and CO group (p<0.001). Fish oil supplementation in HFD+FO group led to a significant reduction in epididymal fat pad mass as compared to HFD group (p<0.05). On the other hand, corn oil supplementation in HFD+CO group showed no significant difference in epididymal fat pad weight as compared to HFD group (Fig 5A, 5B). Adiposity in subcutaneous depot of adipose tissue i.e. inguinal fat pad weight was also assessed. A pattern parallel to that of epididymal fat was observed where HFD group had a significantly higher fat mass as compared to C, FO and CO groups (p<0.001 for all comparisons). HFD+FO group a lower fat mass as compared to HFD (-30.6%, p<0.05) whereas HFD+CO group had no significant difference in inguinal fat mass as compared to HFD group (Fig 5C, 5D).

Fig 5. Gross anatomical examination of epididymal adipose tissue (A); epididymal fat pad weight (B); inguinal adipose tissue (C) and inguinal fat pad weight (D). a- Control group, b- HFD group, c- FO group, d-HFD+FO group, e- CO group, f-HFD+CO group; n=8 Black arrows indicate hypertrophied adipose tissue and white arrows indicated normal adipose tissue. ***p<0.001, *p<0.05. Values are represented as mean ± S.E.M.
3.5.2. Epididymal and inguinal adipocyte size

In obesity, the adipose tissue shows expansion by both hypertrophy and hyperplasia. Since hyperplasia is already established by the increase in adipose tissue mass, we wanted to investigate the effect of FO and CO on hypertrophy of adipocytes in HFD fed rats. We observed that there was a significant increase in adipocyte size in HFD fed rats as compared to all the control groups (C, FO and CO; p<0.001 for all comparisons) in both epididymal and inguinal adipose tissue. On FO supplementation, the average adipocyte size of epididymal and inguinal depots was lowered significantly as compared to HFD (p<0.001). HFD+CO group on the other hand showed no reduction in adipocyte size of epididymal or inguinal adipose tissue as compared to HFD group (Fig 6).

Fig 6. Hematoxylin and eosin stained photomicrographs of epididymal adipose tissue (A), inguinal adipose tissue (B). Epididymal adipocyte size (C), Inguinal adipocyte size (D). Magnification 400X. Values are represented as mean ±S.E.M; n=3 animals per group;
***p<0.001 and **p<0.01.

3.5.3. Ultrastructural changes in adipose tissue.

The changes induced by dietary FO and CO supplementation in adipose tissue morphology was analysed by transmission electron microscopic analysis (Fig 6). Analysing VWAT ultramicroscopic images, we observed that HFD induced hypertrophy of adipose tissue as observed by the large size of lipid droplets. The results were in corroboration with histological examination of VWAT. HFD group had larger lipid droplets and showed disruption of basal membrane pointing to lipid overload in cells (Fig 7A- c, d, e). FO and HFD+FO groups had relatively smaller adipocyte size as compared to HFD cells (Fig 7A- f, g, h, i). Corn oil supplementation in CO and HFD+CO group showed presence of macrophages suggesting an inflammatory environment in the adipose tissue. Small degenerative lipid droplets were also observed in these cells (yellow arrows) along with disrupted membrane of adipocytes (black arrows) which again point to degenerative characteristics on corn oil supplementation (Fig 7A, j-n). Subcutaneous adipose tissue from inguinal region was also analysed. C group showed a normal architecture of adipocytes with multilocular lipid droplets and lipid vesicles in cytoplasm (FigB-a, b). Conversely, the tissue from HFD group showed large unilocular lipid droplet with diminished cytoplasm characteristic of WAT suggesting a hypertrophy in adipocytes (Fig 7B c, d). FO and HFD+FO group show the normal architecture of beige adipocytes with multilocular lipid droplets and lipid filled vesicles. Mitochondria were seen in abundance in these cells (Fig 7B, e-j). Corn oil supplemented groups had large unilocular lipid droplets and hypertrophied adipocytes. Membranes show disruption along with macrophagic accumulation depicting inflammatory state (Fig 7B, k-p). CO group showed accumulation of lysosome like vesicles

Fig 7. A-Transmission electron microscopic images of visceral (epididymal) white adipose tissue. a, b-Control group; c, d, e-HFD group; f, g,-FO group; h, i-HFD+FO group; j, k-CO group; l, m, n- HFD+CO group. B- Transmission electron microscopic images of subcutaneous (inguinal) white adipose tissue Subcutaneous/inguinal adipose tissue ultramicroscopic images. a-b: Control; c-d: HFD; e-g: FO; h-j: HFD+FO; k-m: CO; n-p: HFD+CO. N-nucleus, L-lipid droplet. Blue arrows depict the collagen fibres in extracellular matrix. White triangles depict the macrophages as seen in HFD, CO and HFD+CO groups in both VWAT and SWAT. Black arrows indicate the disrupted basal membrane of the adipocytes. Yellow arrows point to the lipid droplets and green arrow depicts lipid filled vesicles.

3.5.4. Beiging of white adipose tissue
Based on the visible observation of browning in inguinal fat pad (Fig 3B), we investigated the effect of different diets on inducing beiging or browning of fat. For this the expression of uncoupling protein 1 (UCP1) and PGC1α was determined using immunohistochemistry in both visceral and subcutaneous adipose tissue. Visceral depot showed no significant changes in UCP1 expression in either of the groups. In subcutaneous depot, we observed that the protein expression of UCP1 and PGC1α was significantly diminished on feeding HFD as compared to control group (p<0.05). FO and HFD+FO group showed a marked increase in the UCP1 and PGC1α expression as seen by an increase in positively stained cells in the fields examined (p<0.01 for both comparisons). On other hand, HFD+CO group had no change in UCP1 expression as compared to that of HFD group. The results corroborate the fact that FO resulted in beiging of inguinal white adipose tissue (Fig 8). Immunohistochemical examinations also showed that UCP1 expressing fat pads contained numerous regions or islands of beige adipose tissue dispersed in the SWAT. On the contrary, fat pads with typical unilocular lipid droplets were negative for UCP1 signals. Therefore, it

can be stated that FO supplementation led to beiging of subcutaneous WAT which implicates their role in mitigation of obesity (Fig 8).

Fig 8. Analysis of beiging in subcutaneous adipose tissue. A- UCP1 immunostained sections from different groups. B- PGC1 α immunostained sections. Arrows indicate the positive stained cells. C- Quantitative analyses of UCP1 stained cells was done by counting cells in atleast 5 different fields and represented as mean ± S.E.M. ; D- Representative image of PGC1α gene expression in SWAT; E- Relative PGC1α mRNA expression in SWAT w.r.t. C group.

3.5.5. Regulation of development of beige phenotype

To investigate the effect of supplementation of fish oil and corn oil on energy expenditure, we analysed the protein expression of master energy sensor of cell, AMPK α in both the epididymal and inguinal adipose tissue. Compared to C group, in HFD group the ratio of p- AMPKα/AMPK α was diminished in both epididymal and subcutaneous depots (p<0.01). FO supplementation in HFD+FO group significantly reversed the HFD effects by increasing p- AMPKα/AMPKα ratio in epididymal (p<0.01) and subcutaneous (p<0.001) adipose tissue. Corn oil supplementation however failed to show any improvements in phosphorylation status of the adipocytes in both depots (Fig 9). In subcutaneous adipose tissue, p- AMPK/AMPKα ratio was significantly reduced by 1.7 fold in HFD as compared to C group (p<0.05). Fish oil was remarkably able to reverse the effects of HFD by increasing phosphorylation of AMPKα by approximately 4 fold in HFD+FO group (p<0.001). HFD+CO group however failed to show any altercation in p-AMPKα levels in SWAT.
A similar pattern was followed by phosphorylated ACC levels which were significantly enhanced by fish oil supplementation in HFD+FO group as compared to HFD group (p<0.05). Corn oil had no effect on the phosphorylation status of ACC. The results point to a preventive role of FO on obesity in HFD induced obesity by increasing the AMPK phosphorylation and accelerating energy expending pathways in adipose tissue.

Fig. 9. Protein expression analysis of AMPK, p-AMPK, ACC, p-ACC in adipose tissue. Representative Images of western blot VWAT adipose tissue (A), densitometric analysis of VWAT pAMPKα/AMPKα (B), and VWAT pACC/ACC (C), western blot SWAT adipose tissue (D), densitometric analysis of SWAT pAMPKα/AMPKα (E) and pACC/ACC (F). The values are represented as mean ± SEM for n=3 animals per group. ***p<0.001, **p<0.01,


Dietary interventions remains the most attractive strategy for combating obesity worldwide. Being the major partakers in onset and progression of obesity, lipids especially long chain PUFAs remain to be the molecules of utmost relevance. An increase in the dietary intake of n-6 PUFAs in comparison to n-3 PUFAs is considered to be an important contributor to spread of global obesity epidemic [21, 30]. Therefore, a thorough understanding of the differential action of n-3 and n-6 PUFAs is required before suggesting any anti-obesity dietary regimen. Various studies have documented the protective effect of fish oil, a known source of n-3 PUFAs in obesity and its associated comorbidities. FO has been reported to have pleiotropic effects by modulating various pathways in obesity including inflammation, oxidative stress, thermogenesis and lipid metabolism [14, 31-33]. However, there still exists a lack of understanding of its effect on the two different WAT depots i.e. VWAT and SWAT which behave differently in genesis of obesity. Though a recent report did study the effect of FO on both epididymal WAT and inguinal WAT metabolism however the dosage used was too high for any pharmacological relevance in humans [34]. On the other hand, the effect of n-6 PUFAs on adipose tissue function has not been given due attention. The current study provides a comparative account of differential action of n-3 and n-6 PUFAs on obesogenic effects of high fat diet in both visceral and subcutaneous WAT in rodent model of obesity.
Development of obesity was validated by an increase in various anthropometrical markers of obesity such as body weight, BMI, lee index and waist circumference as compared to control group. Fish oil led to significant alleviation of these HFD induced changes confirming its beneficial effect in pathogenesis of obesity. The results are consistent with the previous

studies which have reported a similar effect of fish oil supplementation in high fat diet induced obesity [14, 15, 23, 33]. FO dosage used to achieve the anti-obesity effects is the major factor that has to be considered here. Majority of studies have utilised a high dosage of FO ranging from 25%-50% of total calories. In our study, 10% of total calories from FO were supplemented which is a relatively low dosage and is translatable for human benefits. In comparison to fish oil, corn oil supplementation in the present study led to only partial improvements in anthropometrical parameters of obesity. We observed a significant decrease in body weight and BMI whereas there was no change in waist circumference and lee index as compared to HFD group. Though the consensus on anti-obesogenic effect of n-3 PUFAs seems to be emerging, however there are contradictory reports available on n-6 PUFAs. Some of the studies have reported inhibition of adipocyte differentiation and reduction in weight gain and fat accumulation with n-6 PUFAs as compared to diets rich in saturated fats [35, 36]. On the contrary another report demonstrated that 15% caloric supplementation from n-6 PUFAs had a role in increasing adipogenesis and promoting hyperplasia leading to increase in body weight gain [37]. The observations on the decrease of BMI on corn oil supplementation in the present study can be related to the replacement of 10% calories from saturated fat with n-6 PUFAs. This hypothesis is also supported by an earlier report that states that incorporation of any category of PUFAs abrogate the obesogenic effect of HFD, however n-3 PUFAs exhibit a far more remarkable effect as compared to n-6 PUFAs which indeed is the case here also . The authors further explained that n-6 PUFAs can act as either anti or pro- adipogenic depending on the feeding and hormonal status of the adipocytes [38].
The circulating lipid fractions are considered as a potential biomarker for dyslipidemia which is a common hallmark of obesity and its associated comorbidities. Similar observations were made in the current study where HFD fed animals exhibited higher than normal levels of total lipids, TGs, TC and LDL-C suggestive of an altered lipid milieu. Fish oil supplementation abrogated the effects of HFD and reversed the total lipid, TGs and TC to normal levels. Previous studies also report similar effects where fish oil supplementation led to reduced serum TGs and cholesterol levels as compared to high caloric diets [31, 39, 40]. The mechanism for this effect of n-3 PUFAs is by modulation of lipid metabolism by promoting fatty acid oxidation rather than its storage possibly via SREBP1c, a master regulator of lipid homeostasis which prevents hepatic and serum TG accretion [41]. n-3 PUFA supplementation has earlier been reported to reduce the hepatic production of chylomicrons, favor fatty acid oxidation and enhance LDL clearance by increasing lipoprotein lipase

activity [42-44]. On the contrary, corn oil supplementation in present study had no significant effect on serum lipid profile as compared to HFD group. Earlier reports have also demonstrated an increase in plasma TGs and cholesterol by dietary n-6 PUFAs possibly via activation of cellular lipogenic pathways [45]. In addition we also observed a significant increase in adipose tissue cholesterol levels in both visceral and subcutaneous depots in HFD fed animals. In obese phenotype, ~ 50 % of total body cholesterol is stored in adipose tissue and is responsible for metabolic complications like adipocyte hypertrophy and inflammation and dysfunctional adipokine secretion [46-48]. In present study, the accumulation of cholesterol was reversed on FO supplementation in both depots of white adipose tissue as compared to HFD group. The reduction in cholesterol levels on fish oil supplementation may thus be an important mediator of its protective effect on adipocyte hypertrophy, inflammation and adipose tissue function. Corn oil supplementation did not show any reduction in cholesterol accumulation in both the depots of WAT. This may be the possible explanation for no significant alteration in fat mass/adiposity in corn oil supplemented groups. Contradictory to our findings a previous report showed that fish oil supplementation led to an increased accumulation of cholesterol in epididymal adipose tissue and was associated with reduced total and free cholesterol in plasma and in liver [49].
White adipose tissue acts as an important endocrine organ secreting adipokines like adiponectin and leptin involved in maintaining energy homeostasis. Obesity is associated with an increase in circulating leptin with a parallel decrease in adiponectin reflecting obesity-associated alterations in adipokinome leading to a dysfunctional state in adipose tissue. Therefore, adiponectin/leptin ratio has been suggested as a new marker of adipose tissue dysfunction and obesity. Various reports over past years have highlighted that high fat diet induced obesity is associated with high levels of leptin and low levels of adiponectin in serum [14, 40, 50]. In present study also, we observed an increase in serum leptin along with a decrease in adiponectin levels in HFD group. Fish oil supplementation reversed the effects of HFD on adiponectin and leptin levels. Earlier reports have also shown that fish oil supplementation leads to an improved secretory function of adipocytes by regulating the secretion of various adipocytokines. Reduction in leptin and increase in adiponectin levels has been viewed as an important mediator of anti-obesity effects of n-3 PUFAs [51-53]. On the other hand, corn oil supplementation significantly lowered the leptin levels. There was a marked increase in the adiponectin levels but it did not reach any statistical significance as compared to HFD group. Similar to our observation, a previous report using cultured primary

rat adipocytes has also shown a direct inhibitory effect of linoleic acid on leptin levels and to a lower extent on adiponectin levels in presence of insulin [54]. The results of the present study thus reinstate the findings of previous studies where an imbalance in adiponectin and leptin levels is suggested to be a main contributing factor for the increased BMI, adiposity and altered lipid milieu in high fat diet induced obesity as in HFD group in current study [50]. Fish oil supplementation thus protects from the HFD induced adipose tissue dysfunction by regulating both adiponectin and leptin levels whereas corn oil primarily affected leptin levels only.
White adipose tissue is compartmentalised into two main depots- visceral and subcutaneous. Both these depots of WAT are different in their cellularity, vascularization and functionality. Visceral white adipose tissue is situated close to vital organs and directly connects to portal system making visceral adiposity more detrimental to health as compared to subcutaneous adipose tissue which is present beneath the skin. Obesity has been associated with an increased accumulation of fats in adipose tissue in form of TGs leading to increased fat mass/adiposity and enlarged adipocytes. Similar to these observations, HFD group in present study showed an increase in adipose tissue mass along with an increase in adipocyte size in both visceral and subcutaneous adipose tissue depots. On FO supplementation, there was a significant reduction in the fat mass gain and adipocyte size in both the depots of WAT. n-3 PUFAs have been shown to enhance lipolysis and suppress lipogenesis hence decreasing the lipid storage ultimately resulting in reduced size of adipocytes [23]. Conversely, corn oil supplementation showed no change in fat mass and hypertrophy of adipocytes as compared to HFD in both the depots of adipose tissue. A previous report has also showed similar effects as 15% caloric supplementation of corn oil led to increased adipogenesis and epididymal fat pad mass as compared to an isocaloric diet containing a combination of n-6 and n-3 PUFAs [37].
Ultrastructural analysis also reinstated our findings of gross and histological examination as HFD group revealed enlarged adipocytes with unilocular lipid droplets in both VWAT and SWAT. There was also a disruption in membranes pointing towards the lipid overload in HFD adipocytes. In visceral depot, fish oil supplementation was able to reverse the effects of HFD by reducing the size and fat mass of adipocytes whereas in subcutaneous depot there was a brown adipocyte-like phenotype with multilocular lipid droplets (LDs), abundant mitochondria and lesser macrophages. The results of this section indicate a depot specific action of fish oil in reducing lipid load in both visceral and subcutaneous compartment and

selectively promoting white to beige adipocyte conversion in subcutaneous depot of WAT. The results can be attributed to the lipolytic action of n-3 PUFAs ensuing a reduced size of adipocytes and a concomitant shift towards beige phenotype thus enhancing thermogenesis as reported earlier [23, 29]. Corn oil supplementation however did not create any visible difference in adiposity in both depots of WAT as compared to HFD. Ultrastructural examination of the corn oil supplemented group also revealed features similar to HFD group
i.e. presence of large adipocytes with unilocular LDs, disrupted membranes and reduced cytoplasm. There was a visible infiltration of adipose tissue macrophages (ATMs) which have a potential role in orchestrating low-grade chronic adipose tissue inflammation. No other studies to our knowledge have shown ultrastructural changes in adipose tissue depots on n-6 PUFA supplementation. The observations of this section clearly highlight the depot specific and differential action of n-3 and n-6 PUFAs on adipose tissue morphology and physiology in high fat diet induced obesity.
Beige adipocytes are characterised by brown-like phenotype with an increased thermogenic capability that can burn off excess fat to re-establish energy balance and reverse the effects of positive energy balance. The thermogenic capability is envisioned to be a potential energy expenditure system that acts as a promising strategy for obesity management [55, 56]. Mitochondrial uncoupling protein (UCP) is the main driver for thermogenesis in adipocytes. UCP has five different isoforms, UCP1 localised in brown/beige adipocytes, UCP2 ubiquitously in whole body, UCP3 predominantly in BAT and skeletal tissue, UCP4 and UCP5 mainly in brain [57]. UCP1 is main isoform localised in WAT and is the most extensively studied marker of beiging in adipocytes. UCP1 induction by stimuli such as cold, exercise and diet has been known to uncouple respiration from energy production and increase non-shivering thermogenesis (NST), leading to increased energy expenditure and prevention of obesity [58, 59]. Our results from immunohistochemical analysis showed that there was a significantly lower UCP1 expression in HFD group as compared to control group. Previous studies have also reported the similar effect of high fat diet feeding on UCP1 expression [60, 61]. Fish oil had a depot specific action in induction of UCP1 expression where it significantly upregulated UCP-1 expression in subcutaneous depot while there was no significant change observed in visceral depot. Earlier reports have also shown an increase in the UCP-1 expression in interscapular BAT and inguinal adipose tissue on fish oil supplementation at a dose of 119g and 238 g per kg feed [33, 62]. Corn oil supplementation however altogether failed to alter UCP1 expression in both visceral and subcutaneous depots.

Previously n-6 PUFAs have been found to show no effect on UCP-1 expression in obesity [63]. Another key player in acquisition of BAT features is transcription factor, PGC-1α which plays a central role in the regulation of cellular energy metabolism via adaptive thermogenesis and mitochondrial biogenesis. PGC-1α is highly expressed in tissues with high mitochondrial content such as heart, kidney and BAT but is expressed at very low levels in the WAT [64]. In present study we observed a significant increase in PGC1α expression in subcutaneous depot of WAT upon fish oil supplementation. A previous report has also shown an induction in PGC1α expression in BAT and SWAT, though at a very high dose of 25% and 50% of total calories [32, 33]. Corn oil however had no significant change in expression of PGC1α in both visceral and subcutaneous adipose tissue depots. Similar to our findings, earlier report has shown that n-6 PUFAs suppress the activity of PGC1α and UCP1 in primary human adipocytes [65]. Our findings thus re-emphasize the findings of previous section that fish oil selectively promotes white to beige adipocyte conversion in subcutaneous adipocytes and curtail excess fat deposition by enhancing the energy expenditure via non- shivering thermogenesis.
Analyses of fatty acid composition/profile in plasma and selected adipose tissues, as reflected by types of dietary fat consumption, would have strengthened the manuscript, however, ample evidence from existing literature states that supplementation of dietary PUFAs strongly influences the fatty acid profile of plasma and adipose tissue. In 1990, Lands and Libelt showed a linear relationship between tissue triglycerides and dietary PUFAs [66]. In another study by Abbott et al., 2012, it was shown that when n-3 PUFAs are <10% of total PUFAs, the membrane composition completely conforms to the diet [67]. A number of studies in animal models have reported that dietary PUFAs get incorporated into adipose tissue membranes and directly alters the adipocyte metabolism [68-70]. Not only in the animal models, but also in clinical studies it has been shown that dietary supplementation of PUFAs alters the fatty acid profile and membrane composition of adipose tissue [71, 72]. Previous reports from our lab have also shown an increase in n-3 PUFAs content in the colon tissues upon dietary supplementation of fish oil [73, 74]. Therefore, it can be hypothesized that dietary PUFAs make their way into the membrane phospholipids and lipid droplets of adipocytes, where they have the potential to modulate signaling events thereby altering the metabolic activity of adipose tissue. Beiging of adipose tissue as seen in results of previous section is associated with increased energy expenditure via thermogenic pathways to prevent excessive accumulation of fat. AMPK is the master energy sensor of the cell and in adipose

tissue it is localised in adipocyte membranes and the fatty acid changes brought upon by dietary supplementation of n-3 and n-6 PUFAs can have an impact on the downstream signaling events. Activated by phosphorylation at Thr172 residue, AMPK drives the metabolic switch from anabolism to catabolism by regulating downstream target proteins. AMPK is a positive regulator of thermogenic markers, PGC1α and UCP-1, and thus promotes mitochondrial biogenesis and enhanced thermogenesis [11, 75]. AMPK also directly regulates lipid metabolism by phosphorylating its downstream target ACC1 and ACC2 which abrogates fatty acid synthesis and increases fatty acid oxidation [76, 77]. In the present study, HFD feeding led to a marked reduction in AMPK phosphorylation at Thr172 residue in both visceral and subcutaneous adipose tissue depots as compared to control group. The effects are in accordance to reduced PGC1α expression in HFD group as low AMPK phosphorylation fails to activate its downstream targets. We also observed a significant reduction in ACC1/2 phosphorylation in HFD group as compared to C in visceral depot, whereas the effect was not significant in subcutaneous depot of WAT. Fish oil supplementation significantly induced the phosphorylation and activation of AMPK in both depots of adipose tissue and therefore, its downstream targets PGC1α and UCP1 expression (as mentioned earlier). Fish oil supplementation also increased the phosphorylation status of ACC1/2 in HFD+FO animals as compared to HFD group. The effects of fish oil were pronounced in subcutaneous depot as compared to visceral adipose tissue and hence suggest an enhanced thermogenic activity in SWAT. The results clearly suggest that fish oil may lead to beiging of SWAT by modulating PGC-1 and UCP1 via upregulating AMPKα which may be viewed as potential mechanism for its anti-obesity effects. Various reports have also implicated AMPK activation by dietary n-3 PUFAs as the possible mechanism for browning in BAT, regulation of lipid metabolism, insulin sensitivity and inflammation in obesity [32, 78, 79]. On the other hand, corn oil did not show any significant changes in AMPK phosphorylation as compared to HFD group. It also failed to show any significant alteration of phosphorylation status of ACC1/2 as compared to HFD in both depots of adipose tissue. An increase in arachidonic acid series eicosanoids by n-6 PUFAs have been previously implicated to inhibit activation of AMPKα and hence its downstream target proteins [80, 81]. This may also explain negligible effect of corn oil on UCP1 and PGC1α observed in the present study.
Combined together our study highlights that n-3 and n-6 PUFAs act differentially on the adipose tissue dysfunction in high fat diet induced obesity. The study focusses on the depot specific effect of n-3 and n-6 PUFAs in modulation of adipose tissue function and physiology. In visceral depot it led to a reduction in size of adipocytes by AMPK mediated

lipogenic inhibition while in subcutaneous depot along with a reduction in size of adipocytes, n-3 PUFAs drive the metabolic switch towards conversion from white to “beige” phenotype by promoting mitochondrial biogenesis and non-cold induced thermogenesis. On the other hand n-6 PUFAs though did reduce the BMI, however it did not show any remarkable effect on either of the depots. The present study strongly suggests that supplementation of as low as 10% calories with n-3 PUFAs led to a major improvement in adipose tissue function and energy expenditure in obesity. Mechanistically, our data suggests a role of n-3 PUFAs on beiging of adipocytes via the AMPKα-PGC1α-UCP1 axis, however, further studies using appropriate animal models are needed to validate these mechanistic pathways. The depot specific action of n-3 PUFAs as seen in current study can be used to design and lay more effective nutritional and dietary guidelines to improve adipose tissue function and maintain lipid and energy homeostasis to combat obesity.
Author contributions
Prerna Sharma performed the research work, data analysis and wrote the manuscript. Navneet Agnihotri conceived and supervised the project and refined the drafted manuscript. Both authors contributed to discussions concerning the paper. Authors have agreed with the final version of the manuscript and provide their consent for publication.

Declaration of Competing Interest

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The authors acknowledge SAIF-AIIMS, New Delhi, India for transmission electron microscopy of the samples. Prerna Sharma thanks University Grants Commission for providing fellowship. The authors also acknowledge the assistance from University Grants Commission and Department of Science & Technology as the department is supported under UGC – SAP and DST-PURSE program.

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• Dietary fish oil supplementation in high fat diet results in a significant improvement in anthropometrical and serum lipid parameters.
• Fish oil depot specifically alters the structure and function of adipose tissue by reducing adipocyte size and inducing beiging in subcutaneous adipose tissue.
• Fish oil upregulated AMPKα phosphorylation in both visceral and subcutaneous depot which lead to enhanced energy expenditure via ACC1/2 mediated regulation of fatty acid synthesis and oxidation and by PGC1α-UCP1 mediated induction of beiging of white adipose tissue.