Fish oil prevents changes induced by a high-fat diet on metabolism and adipokine secretion in mice subcutaneous and visceral adipocytes

Abstract

Key points: Fish oil (FO), rich in omega-3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities. The effects of FO on adipocytes from different adipose tissue depots in high-fat (HF) diet induced obese mice have not been uninvestigated. This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet-induced obese mice. Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity. HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor-α and interleukin-6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro-inflammatory cytokines) adipose depots. Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet.

Abstract: In the present study, we investigated the effect of fish oil (FO) on metabolism and adipokine production by adipocytes from s.c. (inguinal; ING) and visceral (retroperitoneal; RP) white adipose depots in high-fat (HF) diet-induced obese mice. Mice were divided into CO (control diet), CO+FO, HF and HF+FO groups. The HF group presented higher body weight, glucose intolerance, insulin resistance, higher plasma total and low-density lipoprotein cholesterol levels, and greater weights of ING and RP adipose depots accompanied by hypertrophy of the adipocytes. FO exerted anti-obesogenic effects associated with beneficial effects on dyslipidaemia and insulin resistance in mice fed a HF diet (HF+FO group). HF raised RP adipocyte lipolysis and the production of pro-inflammatory cytokines and reduced de novo synthesis of fatty acids, whereas, in ING adipocytes, it decreased glucose uptake and adiponectin secretion but did not change lipolysis. Therefore, the adipose depots play different roles in HF diet-induced insulin resistance according to their location in the body. Concerning cytokine secretion, adipocytes per se in addition to white adopise tissue infiltrated leukocytes have to be considered in the aetiology of the comorbidities associated with obesity. Evidence is presented showing that previous and concomitant administration of FO can prevent changes in metabolism and the secretion of hormones and cytokines in ING and RP adipocytes induced by HF.

Figure 1. Effects of HF diet and FO administration on BW gain (difference between initial and final BW), adiposity and hypertrophy of adipocytes

Oral supplementation with FO (2 g kg⁻¹ BW, three times a week, by oral gavage) or water, was started 4 weeks before animals received (or not) the HF diet and this was maintained until the end of the experiment as described in the Materials and methods. Animals were fed with either the HF or CO diet for an additional 8 weeks. A, BW gain in the first period (all animals received CO diet; 0–4 weeks). B, BW gain in the second period (animals of the HF and HF+FO groups; 4–12 weeks). C, relative weight (mg g^–1 BW) of ING fat depot. D, relative weight (mg g^–1 BW) of RP depot. E, volume of ING adipocytes. F, volume of RP adipocytes. Mean ± SEM of nine animals. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet. ^& P < 0.05 vs. CO+FO diet.

Figure 2. GTT and ITT in animals fed with either the CO or HF diet associated (or not) with FO administration

A, GTT or glucose concentration vs. time after administration of glucose (2 g kg⁻¹ BW). B, incremental area under the curve from GTT. C, ITT or glucose decay curve vs. time after insulin administration (0.75 mU g^–1 BW). D, glucose decrease rate for ITT (kITT). The plasma glucose levels obtained between 0 and 30 min were used to calculate the kITT. Mean ± SEM of 14 animals. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet. ^& P < 0.05 vs. CO+FO diet.

Figure 3. Effects of HF diet and FO administration on metabolic activities in isolated adipocytes from ING and RP fat depots

The animals were fed with either the CO or HF diet associated (or not) with supplementation with FO as described in the Materials and methods. A, oleate incorporation into TAG in ING adipocytes. B, oleate incorporation into TAG in RP adipocytes. C, acetate incorporation into TAG in ING adipocytes. D, acetate incorporation into TAG in RP adipocytes. E, basal lipolysis in ING adipocytes. F, basal lipolysis in RP adipocytes. G, isoproterenol stimulated lipolysis in ING adipocytes. H, isoproterenol stimulated lipolysis in RP adipocytes. Mean ± SEM of 12 animals. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet. ^& P < 0.05 vs. CO+FO diet.

Figure 4. Effects of HF diet and treatment with FO on glucose uptake and mRNA levels of GLUT‐4 in isolated adipocytes from ING and RP fat depots

The animals were fed with either the CO or HF diet associated (or not) with supplementation with FO as described in the Materials and methods. A, basal 2DG‐glucose uptake by ING adipocytes. B, basal 2DG‐glucose uptake by RP adipocytes. C, insulin stimulated 2DG‐glucose uptake by ING adipocytes. D, insulin stimulated 2DG‐glucose uptake by RP adipocytes. E, mRNA levels of GLUT‐4 in ING adipocytes. F, mRNA levels of GLUT‐4 in RP adipocytes. 36B4 was used as the housekeeping gene. Mean ± SEM of 15 animals. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet.

Figure 5. Effects of HF diet and treatment with FO on mRNA levels of cytokines expressed by isolated adipocytes from ING and RP fat depots

The animals were fed with either the CO or HF diet associated (or not) with supplementation with FO as described in the Materials and methods. A, mRNA levels of TNF‐α in ING adipocytes. B, mRNA levels of TNF‐α in RP adipocytes. C, mRNA levels of IL‐6 in ING adipocytes. D, mRNA levels of IL‐6 in RP adipocytes. E, mRNA levels of resistin in ING adipocytes. F, mRNA levels of resistin in RP adipocytes. G, mRNA levels of adiponectin in ING adipocytes. H, mRNA levels of adiponectin in RP adipocytes. 36B4 was used as the housekeeping gene. Mean ± SEM of 15 animals. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet. ^& P < 0.05 vs. CO+FO diet.

Figure 6. Effects of HF diet and treatment with FO on secretion of cytokines by isolated adipocytes from ING and RP fat depots

Isolated adipocytes were cultured in Dulbecco's modified Eagle's medium/10% FBS for 30 h and adipokine concentrations were determined using specific ELISA kits in culture supernatants. The animals were fed with either the CO or HF diet associated (or not) with supplementation with FO as described in the Materials and methods. A, TNF‐α secretion by ING adipocytes. B, IL‐6 secretion by ING adipocytes. C, IL‐6 secretion by RP adipocytes. D, resistin secretion by ING adipocytes. E, resistin secretion by RP adipocytes. F, adiponectin secretion by ING adipocytes. G, adiponectin secretion by RP adipocytes. Mean ± SEM of 15. ^* P < 0.05 vs. CO diet. ^# P < 0.05 vs. HF diet.

Figure 7. Pearson's correlation between the results obtained in adipocytes isolated from ING and RP fat depots

Results obtained from animals supplemented with FO were not used to calculate the correlations. A, adipocyte sizes vs. lipogenesis in ING adipocytes. B, adipocyte sizes vs. lipogenesis in RP adipocytes. C, adipocyte sizes vs. de novo lipogenesis in ING adipocytes. D, adipocyte sizes vs. de novo lipogenesis in RP adipocytes. E, adipocyte sizes vs. lipolysis in ING adipocytes. F, adipocyte sizes vs. lipolysis in RP adipocytes. G, adipocyte sizes vs. glucose uptake in ING adipocytes. H, adipocyte sizes vs. glucose uptake in RP adipocytes. I, adipocyte sizes vs. IL‐6 secretion in ING adipocytes. J, adipocyte sizes vs. IL‐6 secretion in RP adipocytes. K, adipocyte sizes vs. resistin secretion in ING adipocytes. K, adipocyte sizes vs. resistin secretion in RP adipocytes. M, adipocyte sizes vs. adiponectin secretion in ING adipocytes. N, adipocyte sizes vs. adiponectin secretion in RP adipocytes. O, 2DG‐glucose uptake vs. adiponectin secretion in ING adipocytes. P, 2DG‐glucose uptake vs. adiponectin secretion in RP adipocytes. The values of Pearson's correlation coefficient (r) and significance (when found) are shown to the right.

Figure 8. Changes induced by HF on the whole body and on RP and ING adipose depots and prevention by FO treatment

HF diet led to glucose intolerance and insulin resistance, as well as an increase of BW gain, food ingestion, food efficiency, energetic efficiency, total cholesterol, LDL cholesterol, fasted plasma glucose levels, fasted plasma insulin levels and HOMA‐IR. Additionally, HF diet increased ING and RP depots weight. In ING adipocytes, HF diet caused hypertrophy, increased oleate incorporation into TAG, decreased stimulated 2DG‐glucose uptake and GLUT‐4 mRNA levels, and increased TNF‐α mRNA levels. In RP adipocytes, HF diet caused hypertrophy, increased oleate incorporation into TAG, decreased acetate incorporation into TAG, and increased basal and stimulated lipolysis, TNF‐α mRNA levels and IL‐6 cytokine. Fish oil prevented or reversed all these effects.