Obesity, inflammation, insulin resistance and cardiovascular disease (CVD) risk are inter-related. Both weight-loss and long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) are independently known to reduce metabolic risk, but the combined effects are unclear.
This study examines whether addition of LC n-3 PUFA to a low fat/high carbohydrate weight-loss programme results in greater improvements in inflammation, insulin sensitivity and CVD risk, than weight-loss alone.
One hundred and sixteen overweight insulin-resistant women entered a 24-week randomised intervention study. Thirty-nine women were randomised to a weight-loss programme, with LC n-3 PUFA (WLFO), 38 to a weight-loss programme with placebo oil (WLPO), and 39 to receive placebo oil, with no weight-loss programme (control).
Ninety-three women completed the study (35 WLFO, 32 WLPO and 26 control), with significant weight-loss in WLFO (10.8±1.0%) and WLPO (12.4±1.0%) compared to the control group (P<0.0001). The WLFO, but not WLPO or control group, showed significant increases in adipose tissue LC n-3 PUFA (0.34±0.20 vs 0.17±0.10 and 0.16±0.10 %DHA, P<0.0001). Weight-loss showed significant improvements in insulin sensitivity (P<0.001), lipid profile (triglycerides P<0.05) and inflammation (sialic acid P<0.05). Time*group effects showed significant decreases in triglycerides (P<0.05) and increases in adiponectin (P<0.01) with LC n-3 PUFA, in the WLFO vs WLPO groups.
Weight-loss improved risk factors associated with CVD, with some additional benefits of LC n-3 PUFA on triglycerides and adiponectin. Given the current low dietary intake of LC n-3 PUFA, greater attention should be given to increase these fatty acids in the treatment of obesity.
Obesity is associated with an increased risk of premature death1 and a substantial increase in morbidity resulting from a wide range of comorbid diseases.2 In particular, risk for diabetes, dyslipidaemia, hypertension and cardiovascular disease (CVD) increase with increasing body weight.3, 4, 5, 6 Modest weight-loss of 5–10% body weight is associated with improvements in cholesterol, blood pressure and insulin sensitivity,7 known risk factors for CVD and type 2 diabetes. Indeed a weight-loss of 6% as part of a lifestyle modification programme has been shown to reduce the rate of progression from impaired glucose tolerance to type 2 diabetes by almost 60% in two separate large intervention programmes.8, 9
Background or habitual inflammatory status is increasingly recognised as an additional obesity-related disease risk factor. A number of markers of inflammation are positively related to measures of adiposity including BMI, waist circumference and abdominal obesity.10, 11, 12 However, in addition, inflammation is associated with CVD and type 2 diabetes risk factors, independent of body mass index (BMI).13, 14, 15 Prospective studies have also shown that inflammatory markers are independent predictors of both CVD16, 17 and type 2 diabetes.18, 19 Reducing background inflammation may provide a new approach to dietary strategies for risk reduction.
Dietary or supplemental long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) have well-documented potent triglyceride lowering effects,20 which may offset any rise in triglyceride which often accompanies weight-loss programmes based on low-fat/high-carbohydrate dietary strategies.21 Additionally, animal studies suggest that LC n-3 PUFA may protect against weight gain, raising possibility that LC n-3 PUFA could facilitate greater weight-loss or differential changes in body composition when incorporated into a weight-loss programme.22 Furthermore, LC n-3 PUFA have an anti-inflammatory effect. Long-chain fatty acids of the n-3 and n-6 classes are precursors for inflammatory eicosanoids. The LC n-3 PUFA-derived eicosanoids have a comparatively lesser inflammatory profile than the equivalent n-6-derived eicosanoids.23 LC n-3 PUFA are natural ligands for PPARγ, the target of the insulin sensitising drugs the thiazolidinediones.24 Thus increases in LC n-3 PUFA intake may have direct insulin sensitising effects by activation of PPARγ. Accordingly, increased intake of LC n-3 PUFA may provide additional benefit over weight-loss alone for metabolic and CVD risk reduction.
This study examines the hypothesis that increases in LC n-3 PUFA intake, combined with a reduction in energy intake, may have synergistic effects on weight-loss, insulin sensitivity and CVD risk factors, promoting benefits over and above weight-loss alone. It also explores the possibility that the metabolic effects of weight-loss or LC n-3 PUFA may be mediated by changes in inflammation.
Subjects and methods
Overweight female subjects were recruited from the community to participate in a 24-week randomised controlled intervention study at MRC Human Nutrition Research. The study was approved by the Local Research Ethics Committee, and all subjects gave written informed consent. Subjects were overweight or obese (BMI>27 kg/m2) and hyperinsulinaemic at a screening appointment (fasting plasma insulin >70 pmol/l). Subjects were excluded if they were pregnant, had symptoms of intercurrent infection, known diabetes, a chronic inflammatory condition, treated dyslipidaemia, liver disease or malignancy. Smokers, subjects with excessive alcohol consumption (>25 U/week) and those taking NSAIDS, steroids or oil supplements were excluded.
Subjects were randomly assigned to either a control group (no weight-loss and placebo oil) or one of two weight-loss intervention groups with either supplemental LC n-3 PUFA (WLFO) or placebo oil (WLPO). The weight-loss programme was designed to achieve a 10% weight-loss in 12 weeks, followed by a 12-weeks weight maintenance phase. In a double blind manner, the WLFO group received five 1 g oil capsules per day with predominantly LC n-3 PUFA, totalling 1.3 g eicosapentaenoic acid (EPA) and 2.9 g docosahexaenoic acid (DHA) per day (EPAX 2050TG, Pronova, Norway). The WLPO and control group received five 1 g oil capsules per day, containing 2.8 g linoleic acid and 1.4 g oleic acid per day (Pronova, Norway). This composition was chosen to most closely reflect the composition of the UK diet, and have minimal impact on subject's fatty acid profile. Measurements were made at baseline, and after 12 weeks, and 24 weeks. Investigations included weight and body composition measurements, fasting blood collection and a standard 5-point, 75 g oral glucose tolerance test (OGTT). Abdominal adipose tissue samples were collected in subjects who separately consented to this additional procedure.
Of the 116 women randomised into the study, 12 women (11 control, one WLPO) dropped out of the study for personal reasons before baseline investigations. A further 11 women (four WLFO, five WLPO, two control) dropped out within a few weeks of commencing the study (seven personal reasons, three unable to manage the weight-loss programme, one medical reasons). Ninety-three women completed the study. Seventy-eight women underwent adipose tissue biopsies to assess incorporation of fatty acids into adipose tissue. The flow of subjects through the study is shown in Figure 1.
Subjects in the two weight-loss groups (WLPO and WLFO) attended fortnightly group sessions where they received detailed dietary advice regarding composition and total energy intake. These subjects also received standard advice to increase their physical activity. Subjects were advised to consume an energy-restricted diet of approximately 3.3–3.8 MJ/day (800–900 kcals/day), predominantly of semi-skimmed milk for the first 5 weeks to facilitate acute weight-loss. This was followed by a staged re-introduction of meals so that by the end of 12 weeks subjects were prescribed a diet providing sufficient energy to match their maintenance energy requirements (approximately 2500 kcal/day) and a prescribed composition of 15% protein, 35% fat and 50% carbohydrate. The control group received no dietary advice, and attended only for measurement appointments at baseline, 12 weeks and 24 weeks (although they were offered the opportunity to take part in a weight loss programme at a later date, after completion of this study).
Weight was measured to the nearest 10 g using a digital scale. Height was measured to the nearest 0.5 cm using a wall-mounted stadiometer. Waist circumference was taken as the smallest circumference between the lower costal margin and the pelvic brim measured to the nearest 0.5 cm. Body fat mass was measured using dual X-ray absorptiometry (DXA) on a Hologic QDR-100 W scanner (Hologic Inc., 590 Lincoln Street, Waltham, MA 02154 USA). A measurement of abdominal fat was calculated by isolating the section between the L2 and L4 lumbar vertebrae. Blood pressure was measured using an automated Dinamap Pro 200 machine (Critikon Ltd, Basingstoke, Hampshire, UK) with the patient rested for 5 min in the seated position, arm supported and using an appropriate sized cuff. Systolic and diastolic values were taken as the average of two readings.
Fasting plasma samples were used to measure glucose insulin, and leptin concentrations and fatty acid profiles. Serum samples were collected for lipids (HDL, total cholesterol and triglycerides), high-sensitivity C-reactive protein (CRP), Interleukin-6 (IL-6), sialic acid, tumour necrosis factor-α (TNFα) and adiponectin concentrations. Insulin and glucose concentrations were also measured following a 75 g glucose load, at 0, 30, 60, 90 and 120 min and used to calculate area under the curve (AUC) values for both insulin and glucose.
Plasma insulin concentration was analysed on the Access Immunoassay System (Sanofi Pasteur Diagnostics). Cross reactivity with intact pro-insulin is <0.2% at 400 pmol/l, 32–33 split pro-insulin <1% at 400 pmol/l. Between-run coefficients of variation are 3.4% at 33.3 pmol/l and 3.1% at 81.0 pmol/l. Plasma glucose concentrations were analysed on the Dade Behring Nephelometer II (Germany). Glycated haemoglobin (HbA1c) was measured using a high-performance liquid chromatography (HPLC) on a TOSOH A1c 2.2 analyser. An index of insulin sensitivity was estimated by the homeostasis model assessment method (HOMA) using the standard formula25 and also by the area under the insulin curve over five 30-min increments following the 75 g oral glucose challenge.
Serum CRP was measured using a high-sensitivity assay (Dade-Behring, Walton, UK). Serum cytokines (IL-6, TNFα) were measured using a high-sensitivity assay (Quantikine kit, R&D Systems, Abingdon, UK). Fasting serum lipids were measured on a Dade Behring Nephelometer II (Germany). LDL cholesterol was calculated using the Friedewald formula.26 Serum adiponectin was measured using a radioimmunoassay (LINCO Research Inc., St Charles, MO, USA). Plasma leptin concentration was measured using an ELISA method (R & D Systems, Quantikine Human Leptin kit).
On completion of the OGTT, a sample of subcutaneous abdominal adipose tissue was collected from consenting subjects using a modified mini-liposuction technique performed using sterile technique, under local anaesthetic, without adrenaline.27 The adipose sample (approximately 15 g) was cleaned to remove small blood clots. Once the sample was clean a small piece of adipose was transferred into an aliquot tube and stored at −80°C until analysed for fatty acid composition.
Total plasma fatty acids were extracted using a modified Folch extraction.28 Both plasma and adipose fatty acids were measured using capillary gas chromatography. Individual fatty acids were expressed as a percentage of total fatty acids. The coefficient of variation was 5.5% for plasma EPA, 3.9% for plasma DHA, 4.0% for adipose EPA and 3.7% for adipose DHA.
Data was analysed using SPSS (SPSS Inc., IL, USA) and SAS (SAS Institute Inc., NC, USA) and is expressed as mean and standard deviation (s.d.) for normally distributed data, or as geometric mean and s.d. (calculated before transformation) for loge transformed data. Random effects analysis of the change in outcome variable, adjusted for baseline values, menopausal status and hormone use, was used to determine the effects of the intervention. Where there were significant time*group effects, data was explored to determine where these significances occurred. The model makes adjustment for multiple comparisons.
The baseline characteristics of subjects who completed the study are shown in Table 1. All women were overweight, with a weight in the range 67.9–129.1 kg (mean 92.7±15.3 kg), aged between 21 and 69 years (mean 44.7±13.2 years). Whereas at screening all subjects had fasting insulin concentrations >70 pmol/l (mean and range), at baseline fasting insulin ranged from 22.6 to 231.0 pmol/l (mean 85.0±41 pmol/l), highlighting within subject variability in fasting insulin values. Whereas subjects with known diabetes were excluded at screening, eight subjects were retrospectively found to have diabetes at baseline. These subjects were included in all data analysis presented here, since their exclusion made no difference to the results.
Effects of the intervention
All anthropometric outcomes showed significant time*group effects (P<0.0001). There were no significant changes in body weight in the control group, while both WLPO and WLFO groups showed a significant reduction in body weight at 12 weeks and 24 weeks compared to baseline (P<0.0001, Table 2). There were no significant changes in body weight in the WLPO or WLFO groups during the weight maintenance phase of the study (between 12 and 24 weeks). There were no significant differences in body weight at any timepoint between the WLPO and WLFO groups. The results for waist circumference, fat mass and abdominal fat mirror those of body weight (Table 2, Figure 3). There were no significant changes in lean mass in any of the groups with the intervention.
Plasma and adipose tissue fatty acid composition
Both plasma and adipose tissue fatty acid profiles were measured (shown in online Supplementary Table). There were no significant changes in any plasma or adipose tissue fatty acids in the control group.
There were significant time*group effects for all plasma n-3 PUFA outcome variables (P<0.0001). Detailed exploration showed significant increases in plasma LC n-3 PUFA, EPA and DHA, at 12 weeks and 24 weeks vs baseline, in the WLFO group (P<0.0001, Figure 2). The proportion of DHA in plasma increased between 12 and 24 weeks, resulting in significant increases in measures of total n-3 PUFA, LC n-3 PUFA and n-6:n-3 PUFA (P<0.05). There were no significant time*group effects for total n-6 PUFA, saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), although the data suggests non-significant reciprocal decreases in the proportion of total n-6 PUFA and MUFA in the WLFO group. Between-group comparisons showed no significant differences in plasma fatty acid profiles between the groups at baseline, but at 12 and 24 weeks all n-3 PUFA outcome variables were significantly higher in the WLFO group compared to both the WLPO and control groups.
There were significant time*group effects for changes in n-3 PUFA in adipose tissue. The WLFO group showed a similar increase in adipose DHA status between baseline and both 12 and 24 weeks as that in plasma (P<0.0001, Figure 2). There was also a further increase in DHA status between 12 and 24 weeks of the intervention (P<0.0001). In the between-group comparison, the WLFO group showed significantly higher DHA status than both the WLPO and control groups at 12 and 24 weeks (P<0.05).
However, in contrast to the results of the plasma fatty acid analysis, both weight-loss groups (WLPO and WLFO) showed similar significant increases in adipose tissue EPA status (P<0.0001). The WLPO group also showed significant increases in adipose tissue DHA status compared to the control group, although these were significantly smaller than the increases in the WLFO group (P<0.01).
Clinical and biochemical data
Table 3 shows clinical and biochemical outcomes at baseline and 24 weeks, to compare the effects of both weight-loss and the LC n-3 PUFA intervention after a period of weight stability and the maximal attained incorporation of DHA into both plasma and adipose tissue.
There was a significant time*group effect for AUC insulin, an index of insulin sensitivity (P<0.0001) and AUC glucose (P<0.01). Detailed exploration showed significant improvements in AUC insulin at 24 weeks, for both weight-loss groups, within group (P<0.0001) and compared to the control group (P<0.01, Figure 3). There were significant within group differences in AUC glucose in the WLFO and WLPO group (P<0.0001), but no significant differences compared to the control group. There were no significant time*group effects for any of the fasting insulin sensitivity indices, including HOMA (Table 3).
Significant time*group effects were seen for triglycerides and HDL cholesterol (P<0.05), but not for LDL cholesterol, total cholesterol, systolic or diastolic blood pressure (Table 3). Triglycerides were significantly lower in the WLFO group at 24 weeks, relative to the control group (P<0.01, Figure 3). Importantly, while the WLPO group also showed a significant decrease in triglycerides, it was significantly smaller than the decrease in the WLFO group (P<0.01) and not significantly different from the control group. HDL cholesterol data showed a significant within group increase in the WLFO group (P<0.01), but no significant differences in any between group comparisons.
A significant time*group effect was seen for adiponectin (P<0.0001). Specifically, the WLFO, but not the WLPO group showed a significant increase in adiponectin (P<0.0001), and this increase was significantly different from the control group (P<0.05, Figure 3, Table 3). Leptin showed a significant time*group effect and was significantly lower in the WLPO and WLFO within group (P<0.0001, Table 3) and compared to the placebo group (P<0.05, Table 3). None of the inflammatory outcome variables (sialic acid, CRP, IL6, and TNFα) showed significant time*group effects in the comparison of all three intervention groups.
Low-fat/high-carbohydrate diets are widely promoted for weight management. However, reductions in total fat may include important reductions in essential fatty acids, and proportional increases in carbohydrate, which if dominated by simple sugars, have been imputed to attenuate improvements in health outcomes as a result of weight-loss.29, 30 This study confirms that weight-loss of 10% body weight, is associated with improvements in insulin sensitivity, risk factors for cardiovascular disease, which are maintained after a period of weight-loss maintenance. Furthermore, supplementation of a low-fat/high-carbohydrate diet with LC n-3 PUFA, during and after weight reduction, is related to additional benefits in lipid profile and the adipocyte-derived protein adiponectin.
There is evidence from studies in rodents of a relative protective effect of LC n-3 PUFA supplementation on weight gain in animals fed a high-fat diet, suggesting the potential for fish oil to promote greater weight-loss.31 Additionally, in human studies, LC n-3 PUFA protected against loss of fat-free mass with 10% weight-loss in obese hypertensive men.22 However, the present study does not support greater weight-loss or differential changes in body composition with LC n-3 PUFA supplementation.
The principal hypothesis of this study was that increases in LC n-3 PUFA intake would result in increase in these fatty acids at the tissue level, leading to improvement in insulin sensitivity and risk factors for cardiovascular disease, over and above weight-loss alone. High-dose LC n-3 PUFA supplements led to a significant increase in LC n-3 PUFA status in target tissues. It is important to note that DHA increased significantly from 12 to 24 weeks indicating a slow turnover of this compartment. The time course of change and maximal response needs further investigation. The predominant increase in DHA may reflect the greater DHA content of the capsules, or metabolic factors leading to preferential incorporation of DHA vs EPA.32 There were no significant changes in specific fatty acids in the WLPO group with the exception of adipose tissue EPA. This may be due to proportional reductions in tissue SFA with preservation of PUFA, which may account for the proportional increase in tissue EPA in this group. This provides reassurance that low-fat diets are not associated with significant reductions in essential fatty acids in the short term.
The clinical benefits of modest weight-loss on glucose metabolism were confirmed in this study. Glycated haemoglobin, glucose excursion during the OGTT and insulin sensitivity were improved with weight-loss and retained with maintenance of lower body weight.
Studies in animals clearly demonstrate improved whole body insulin sensitivity with LC n-3 PUFA.33 This is thought to be related to a combination of effects in the liver with reduced hepatic triglyceride synthesis and release, and also a general increase in fatty acid oxidation in liver and skeletal muscle34 and upregulation of uncoupling proteins. In humans, the situation is controversial and made more complex by differences in effects observed in different states of glucose tolerance. Some previous studies have demonstrated detrimental effects of fish oil on fasting glucose or insulin concentration in hyperlipidaemic men or subjects with diabetes.35, 36, 37, 38, 39, 40 This study, and others, has not shown detrimental effects, in healthy volunteers, hypertensive or dyslipidaemic subjects or those with diabetes.36, 41, 42 In obese hyperinsulinaemic women, we found there are no adverse effects of LC n-3 PUFA on glucose, HbA1c or insulin sensitivity either during weight-loss or in a stable weight-reduced state. Moreover, there was a significant reduction glucose excursion during the OGTT in both the WLFO and WLPO groups.
Other studies show mixed effects. Mori et al. indicated greater benefits of LC n-3 PUFA combined with weight-loss in a study incorporating a daily meal of oily fish into a weight-loss programme, compared with weight-loss without frequent fish consumption.43 There were non-significantly greater reductions in fasting and AUC insulin and glucose during an OGTT with a daily fish meal and mean weight-loss of −5.6 kg in hypertensive men and women aged 40–70 years. In contrast, the ‘KANWU study’, which compared the effects on insulin sensitivity on an isoenergetic diet rich in SFA vs MUFA, with additional randomisation to supplemental LC n-3 PUFA, did not show any effect of increases in LC n-3 PUFA in weight stable subjects.44 The absence of a LC n-3 PUFA effect may also relate to the dose of LC n-3 PUFA, and the subjects studied. There may also be interactions with inflammation and we have previously shown a beneficial effect of LC n-3 PUFA supplementation on insulin sensitivity in overweight women with increased background inflammatory status.45
Changes in lipid profile with weight-loss are complex and inconsistent.7 Triglycerides decreased significantly with weight-loss and this effect was significantly greater in the WLFO vs WLPO group, confirming existing evidence of triglyceride lowering effects of LC n-3 PUFA in a wide range of situations.20 The effects of fish oil on HDL cholesterol have been inconsistent, but in weight stable subjects appear to cause small increases in HDL, mostly the protective HDL2 subtype.35, 43 HDL cholesterol increased with weight-loss in WLFO group in this study.
Blood pressure was reduced with weight-loss and improvements maintained with subsequent weight maintenance. There was no significant additional benefit on blood pressure with LC n-3 PUFA supplements. In a group of hypertensive men undertaking a weight-loss programme, Bao et al.46 demonstrated that a daily meal of oily fish was related to additional reductions in blood pressure over and above weight-loss. Contrasting this with the present study suggests that the greater the underlying blood pressure, the more likely that LC n-3 PUFA will provide additional benefits or that there may be gender differences in the response.
This study also explored the hypothesis that improvements in insulin sensitivity and cardiovascular disease risk factors with weight-loss and LC n-3 PUFA may be mediated by changes in adipose tissue-derived factors. There was no significant change in adiponectin in the WLPO group, but a significant increase in the WLFO group, suggesting a beneficial effect of LC n-3 PUFA. Increases in adiponectin have previously been demonstrated with large weight-loss (20% body weight) induced by gastric bypass surgery,47 although the smaller reductions in weight in this study were not linked to increases in adiponectin in the absence of supplemental LC n-3 PUFA. Changes in adiponectin were also related to changes in EPA and DHA in the pooled data (data not shown), suggesting that LC n-3 PUFA could be involved in the regulation of adiponectin levels.
Weight-loss was associated with reductions in circulating inflammatory markers in both weight-loss and weight-maintenance phases, though these did not reach statistical significance, unlike previous reports.48, 49, 50, 51 There were no clear differences with LC n-3 PUFA supplementation in spite of the known anti-inflammatory effect of LC n-3 PUFA.52
There are some limitations to this study. The ability of the study to determine the relative contributions of weight-loss and of increased LC n-3 PUFA levels to changes in study end points would have been greater if an additional non-weight-loss group taking LC n-3 PUFA had been included. In the final analysis, statistical power was also reduced by the number of dropouts, particularly in the control group. The nature of the study design meant that despite careful recruitment strategies, many subjects dropped out after being randomised into the non-weight-loss group. The effect of reduced statistical power is seen where comparisons within or between groups are significant using t-tests, but not with repeated measures or ANOVA, both of which are adjusted for multiple comparisons. Finally, the fasting insulin concentration in some subjects was considerably lower at baseline than that taken at screening. This meant that some subjects were not as insulin resistant at baseline as intended in the study design, which is likely to have increased the variance. However, this study provides good evidence from which to plan a larger and more powerful study to assess the impact of LC n-3 PUFA on insulin sensitivity.
In conclusion, this study confirms the metabolic benefits of sustained modest weight-losses of 10% body weight in a population of obese hyperinsulinaemic women who are at increased risk of disease and identified additional benefits of LC n-3 PUFA supplementation. However, these effects were obtained with a dose of LC n-3 PUFA considerably higher than can realistically be achieved through dietary change. Further research is required to assess the dose–response relationship for each of the benefits observed and to assess whether similar effects can be achieved with a food-based programme.
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This study was funded by the Medical Research Council. None of the authors had any conflicts of interest. We thank Ms J Cooke, HNR volunteer suite staff and Dieticians at Addenbrooke's Hospital for the volunteer work, Mr N Matthews for the fatty acid analysis, Mr I Halsall for the insulin analysis, Mr C Charalambos and the Nutritional Biochemistry Laboratory for other biochemical analyses. We also thank Pronova (Norway) for supplying the capsules for the intervention and SMILES for funding the development of the dietary advice programme.
Author contributions: JD Krebs was responsible for all aspects of the study design, data collection, analysis and writing the manuscript. SA Jebb was involved in study design, data analysis and writing the manuscript. LM Browning was involved in data collection, data analysis and writing the manuscript. N McLean was involved in the developing the weight-loss programme and reviewing the manuscript. JL Rothwell, CS Moore and GD Mishra were involved in data collection, analysis and reviewing the manuscript.
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Krebs, J., Browning, L., McLean, N. et al. Additive benefits of long-chain n-3 polyunsaturated fatty acids and weight-loss in the management of cardiovascular disease risk in overweight hyperinsulinaemic women. Int J Obes 30, 1535–1544 (2006). https://doi.org/10.1038/sj.ijo.0803309
- insulin sensitivity
- fish oil
- n-3 PUFA
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