PET/MRI-evaluated brown adipose tissue activity may be related to dietary MUFA and omega-6 fatty acids intake

An investigation of new ways to activate brown adipose tissue (BAT) is highly valuable, as it is a possible tool for obesity prevention and treatment. The aim of our study was to evaluate the relationships between dietary intake and BAT activity. The study group comprised 28 healthy non-smoking males aged 21–42 years. All volunteers underwent a physical examination and 75-g OGTT and completed 3-day food intake diaries to evaluate macronutrients and fatty acid intake. Body composition measurements were assessed using DXA scanning. An FDG-18 PET/MR was performed to visualize BAT activity. Brown adipose tissue was detected in 18 subjects (67% normal-weight individuals and 33% overweight/obese). The presence of BAT corresponded with a lower visceral adipose tissue (VAT) content (p = 0.04, after adjustment for age, daily kcal intake, and DXA Lean mass). We noted significantly lower omega-6 fatty acids (p = 0.03) and MUFA (p = 0.02) intake in subjects with detected BAT activity after adjustment for age, daily average kcal intake, and DXA Lean mass, whereas omega-3 fatty acids intake was comparable between the two groups. BAT presence was positively associated with the concentration of serum IL-6 (p = 0.01) during cold exposure. Our results show that BAT activity may be related to daily omega-6 fatty acids intake.


Results
In the studied group, brown adipose tissue was detected in 18 volunteers (BAT-positive) ( Table 1), with a mean age of 24 years and a mean BMI of 25 kg/m 2 . A total of 67% of BAT-positive subjects had normal BMI, whereas 33% were overweight or obese. In 10 volunteers who underwent a 2 h cold exposure test, we did not observe the activity of brown adipose tissue in PET/MR images. These subjects were included in the BAT negative group (BAT-negative) (Figs. 1, 2).
In the BAT-positive group, the mean volume of brown adipose tissue was estimated as 26 355 ± 43 202 mm 3 and the mean activity of BAT was 19.1 ± 4.3 µmol × (100 g −1 ) × min −1 . We noticed a lower mass and volume of visceral adipose tissue (p = 0.02, after adjustment for age, daily kcal intake, and DXA lean mass) in the BATpositive group, as well as a lower percentage of VAT content (p = 0.007, after adjustment for age, daily average kcal intake, and DXA lean mass). No significant association was found linking BAT volume and 18F-FDG uptake with fat-free mass (FFM) (p = 0.57) and lean mass (lean mass) (p = 0.54). We did not observe any differences between studied groups in FFM, lean mass, fat mass, and OGTT results (data not shown).
We also observed that omega-6 fatty acids intake and MUFA intake was significantly lower in BAT-positive subjects (p = 0.03 and p = 0.02, respectively, Table 1) (Figs. 3,4), compared to BAT-negative, after adjustment for age, daily average kcal intake, and DXA Lean Mass, whereas omega-3 fatty acids intake did not differ significantly between the studied groups. We did not discover any differences between groups BAT-positive and BAT-negative in the total kcal energy intake and consumption of carbohydrates, protein, and fat (data not presented). Also, BAT-positive group was characterized by a higher concentration of IL-6 during 2 h cold exposure (p = 0.01 after adjustment for age, daily average kcal intake, and DXA Lean mass) (Fig. 5).
The BAT volume was positively associated with omega-3 fatty acids intake (Est. 0.00001, R 2 = 0.69, p = 0.00007) and long-chain PUFA intake (Est. 0.0001, R 2 = 0.38, p = 0.0005). It was negatively associated with an omega-6/ omega-3 fatty acids ratio (Est. − 0.00002, R 2 = 0.53, p = 0.001, after adjustment for age, daily kcal intake, and DXA Lean mass). We did not notice any differences in the AUC of REE measurements during cold exposure tests, except for the slight tendency (p = 0.07) of a higher Δ of REE between 0 and 120 min of the cold exposure test in BAT-positive subjects.

Discussion
In our study, we evaluated the relationship between dietary intake and the activity of brown adipose tissue in healthy males aged . Subjects in which the presence of BAT was found presented a lower daily consumption of MUFA and PUFA omega-6 fatty acids. Moreover, the BAT volume was related to higher omega-3 fatty acids intake, long-chain PUFA, and a lower ratio of omega-6/omega-3 fatty acids. A lower percentage of visceral www.nature.com/scientificreports/ adipose tissue was observed in subjects with detectable brown adipose tissue, along with a higher serum IL-6 concentration during the cold exposure test. This finding is consistent with previously published studies 36 . However, Monfort-Pires et al. 21 reported that MUFA-rich olive intake induces the activation of BAT, which is in contrary to our observation; nevertheless, it has been noted only in lean subjects.  www.nature.com/scientificreports/ Our analysis of dietary omega-3 and omega-6 fatty acids intake produced valuable insight as well. Notably, omega-3 and omega-6 fatty acids belong to polyunsaturated fatty acids. The proportion between omega-3 and omega-6 fatty acids is crucial for health, and the proper ratio should be 1-4:1 of omega-6 to omega-3 fatty acids 37 . In all diets, especially Western diets, omega-6 fatty acids cover the majority of PUFAs food supply. Moreover, dietary changes over recent decades have resulted in a significant increase in the intake of omega-6 fatty acids, thus altering the omega-6 to omega-3 fatty acids ratio to around ~ 15:1 38 . An imbalance in the omega-6/omega-3 fatty acids ratio may enhance an immune response. Omega-3 fatty acids are considered to be an anti-inflammatory agents, while omega-6 fatty acids have an opposite function. Derivatives from both polyunsaturated fatty acids-eicosapentaenoic acid (EPA), which belongs to omega-3 fatty acids, and arachidonic acid (ARA), which belongs to omega-6 fatty acids-compete for the same enzymes in the prostaglandin biosynthesis 39 .  The long-chain omega-3 fatty acids depressed the production of proinflammatory prostaglandin 40 , while the omega-6 fatty acids are known for their proinflammatory properties. ARA can be metabolized to prostaglandins (A2, E2, I2, and thromboxane A2) by cyclooxygenases-2 (COX-2), while leukotrienes (B4, C4, and E4) are biosynthesized from ARA by lipoxygenases (5-LOX) 41 . The influence of omega-3 and omega-6 fatty acids on the immune system has been widely surveyed 42 . PUFA show also a significant impact on the regulation of adipocyte differentiation and their function. The beneficial role of the omega-6 and omega-3 fatty acids and long chain PUFA dietary intake on adipose tissue development and function has been already shown 43 Diet enriched in particular in the omega-3 PUFA, may decrease adipose tissue content, however, the physiological and cellular effects of PUFA may depend on many factors, and it has been noted that omega-6 PUFAs may exert either an anti-or a proadipogenic effects 44 . Recently, studies on animal models reported that high-fat diets rich in PUFA affect the expression of uncoupling protein 1 mRNA in brown adipose tissue. The increase was more significant with the supplementation of omega-3 PUFA than with omega-6 PUFA 45 . In line with previous results, outcomes from the intervention study with supplementation with omega-3 long-chain PUFA showed that omega-3 fatty acids enhanced thermogenesis via the activation of brown adipose tissue (BAT) 46,47 . A recently published paper on metabolite profiling by liquid chromatography-mass spectrometry (LC-MS) in humans with detectable BAT showed a unique systemic PUFA and oxylipin profile with increased levels of anti-inflammatory omega-3 fatty acids 48 . The above-listed papers supported the relationship between PUFAs and brown adipose tissue. We did not notice any differences in omega-3 fatty acids intake between subjects with and without identified BAT activity, but the linear regression models showed that the BAT volume was positively associated with omega-3 fatty acids intake. Moreover, as mentioned above, the proper ratio between omega-6 and omega-3 fatty acids may also play a crucial role in metabolism 49 . A lower dietary omega-6/omega-3 fatty acids was noted to improve the thermogenic response of BAT and WAT under β3-adrenergic stimulation 50 . Based on our results, we can hypothesize that a lower amount of omega-6 fatty acids in a person's diet may have a beneficial effect on brown adipose tissue activity, possibly due to the enzyme competition between omega-3 and omega-6 fatty acids, which need further investigation. Dietary omega-6 fatty acids intake could be one of the potential mechanisms underlying the activity of brown adipose tissue. www.nature.com/scientificreports/ PUFA and their metabolites may have an impact not only on the BAT activity but on the conversion of white into brite adipocytes as well 51 . It was noted that diets rich in ARA favor WAT formation by preventing the "browning" process 52 . However, recently published results suggest no effect of dietary fish oil supplementation on the recruitment of brown and brite adipocytes in mice or humans under thermoneutral conditions 53 . The thermoneutral conditions could be an explanation of noted conflicting results, since different circulating PUFA and oxylipins (being the lipid mediators produced from PUFA) profiles in BAT-positive and BAT-negative subjects were noted 54 and cold exposure significantly increased plasma lipid composition only in BAT-positive individuals, strongly supporting the relationship between BAT and PUFA. The presence of BAT was also characterized by increased concentrations of omega-3 fatty acids and their precursor molecules 54 .
We did not notice any differences between the studied groups in regard to total energy and macronutrient intake. Our results are in line with Sanchez-Delgato et al. 's study in which associations between BAT volume or 18F-FDG uptake and energy intake, assessed via either the ad libitum meal or the habitual dietary intake, were not observed 55 .
In our study, we also observed that subjects with detectable brown adipose tissue are characterized by lower visceral adipose tissue and lower BMI. The fact that individuals with identified BAT activity were significantly younger could also affect this observation. Nevertheless, our results are in line with Matsushita's study, which similarly reported that subjects with BAT are younger and have less abdominal fat 56 . Several studies observed a relationship between body composition, adiposity-related parameters, such as BMI, central body fat distribution, and BAT, thus indicating a reduced amount of brown adipose tissue in obese subjects [57][58][59] . Obesity is characterized by a chronic low-grade inflammatory state in adipose tissue maintained by the secretion of a wide range of inflammatory proteins. Systemic inflammation, especially TNF alfa, suppresses the thermogenic activity of brown fat's capacity to reduce energy expenditure 60 . Moreover, data suggested its contribution to the whitening of BAT that occurs after the prolonged consumption of high-fat foods 61 . In the 52-week-old insulin receptor knockout mice, a significant decrease of BAT mass was observed with a significant increase of visceral WAT mass compared to 33-week-old mice 62 . www.nature.com/scientificreports/ We observed a slight tendency of higher Δ of the resting energy expenditure (REE) between baseline and 120 min of the cold exposure in BAT-positive subjects, but we did not observe any differences between BATpositive and BAT-negative individuals in the REE during the cold exposure test. These results correspond with the outcomes of Orava et al. 's research 59 .
In our study, we noticed an increase of serum IL-6 during 2 h of cold exposure, which is in line with the results of other authors 63 . It may seem confusing, as IL-6 is known as a proinflammatory cytokine, while its effect and associations with BAT need more investigations since it is suggested that IL-6 may increase its activity 64 . The lack of IL-6 expression impaired the beneficial effects of BAT transplantation on metabolic health through the interaction with FGF21 65 . Additionally, IL-6 is indispensable for the induction of WAT browning in response to a cold environment 66 . Moreover, another anti-inflammatory role of interleukin-6 has also been shown. It is released by skeletal muscle in response to exercise and promotes insulin sensitivity 67 .
To the best of our knowledge, our study is one of the first to assess the relationship between daily nutrient intake assessed by the 3-day food diary and the activity of brown adipose tissue. The relationship between diet-induced thermogenesis has been evaluated in the interventional studies [68][69][70] . It is worth highlighting the significant outcomes from a systematic review (PROSPERO) and meta analyzes which showed no differences in standardized uptake value of BAT following a single meal or after 6 weeks of l-Arginine supplementation. Resting energy expenditure, however, was increased following a single meal and after supplementation of capsinoid and catechin when compared to a control condition 16 . The topic is still relevant and needs to be further investigated. The results from our study indicate an association between BAT volume/activity and omega-3 and omega-6 polyunsaturated fatty acids. Moreover, the results suggest that further attention should be directed toward the right balance between omega-6 and omega-3 fatty acids in brown adipose tissue activity. Researchers should evaluate whether polyunsaturated fatty acids directly influence the activation of BAT or if they indirectly do so through the beneficial effect of omega-3 fatty acids on body fat, weight loss, or the reduction of an inflammatory state [71][72][73] . Maintaining an adequate proportion of body fat with a normal body index may promote the activation of brown adipose tissue. Future studies should investigate how do omega-3 and omega-6 fatty acids activate BAT, if directly or through particular mechanisms.
It is worth to notice also, that previous research on BAT primarily used PET/CT as a tool for imaging human brown adipose tissue 74 . PET/MR is the preferred imaging source because of the lack of ionizing radiation, feasibility, and higher spatial resolution. PET/MR imaging has previously been used to detect the presence of BAT in adults as well as in children 75,76 .
Our study has some limitations. The major limitation is a relatively small sample size, but the number of subjects enrolled in the study is comparable to the previous survey 77 . The main reason of limitation for conducting a large-scale trial is the high costs associated with PET/MR scanning and a tracer purchase. Therefore, if possible, our findings should be further tested in a larger population and different ethnic groups. The other important fact is that in our study, only the BAT glucose uptake was measured, and it is important to note that the main source of energy for BAT are fatty acids 78,79 . Therefore, we overlook the possibility that some of the BAT-negative subjects, defined by the glucose rate, might have a significant fatty acid uptake by the BAT tissue. Moreover, FDG allow to analyze and to localize BAT but it could be less informative for BAT activity. Indeed, BAT thermogenic activity, as mentioned above, is mainly due to fatty acid oxidation and uptake 77 . Because of the difficulties associated with obtaining a tracer to investigate fatty acids metabolism in humans, we used 18F-FDG in our study. Moreover, brown adipocytes are interspersed within white adipose tissue. Therefore, through PET detection, BAT regions could contain both, BAT and some white adipocytes 57 . It is also possible that the cooling was not optimal for some of the subjects, especially for those who were obese, thus resulting in falsenegative results related to BAT activity. In our study, the water-perfused blankets were used, and the many cold exposures to large skin areas, such as via water-perfused suits or vests, seem to demonstrate minor variation in BAT activation 80,81 , what also should be considered.

Conclusions
In conclusion, we noted lower visceral fat accumulation in subjects with identified BAT, which confirms the protective role of brown adipose tissue and indicates that BAT shows strong potential as a means to combat obesity and its metabolic consequences. Moreover, our results suggest that both, dietary MUFA, as well as omega-3 and omega-6 fatty acods intake, may be associated with the volume and activity of BAT in healthy males aged 21-42, which deserve further investigations.

Materials and methods
Study participants. The study group comprised of 28 healthy, non-smoking Caucasian males aged 21-42 years (mean age 26.75 ± 5.11 years old). Sixteen participants had normal body weight (BMI < 25 kg/m 2 ) and 12 were obese/overweight (BMI > 25 kg/m 2 ). Volunteers to this study were recruited from the other cohort study group, described in detail previously 82,83 . The participants in this study were without any comorbidities (e.g., hypo-or hyperthyroidism, asthma, cardiovascular disease, renal or liver failure, and any acute or chronic diseases) and were not taking any medications (e.g., beta-blockers) or dietary supplements that could have had an impact on the results. Outside and shift workers were excluded from the study as well. Subjects were enrolled in the study, and all study procedures were performed during the October-April periods of 2016-2018.

Screening of subjects.
During the screening visit, the medical history and metabolic status of all volunteers were reviewed. They underwent a physical examination, routine laboratory tests (hematology, TSH, creatinine, liver enzymes, Na, K, CRP), blood pressure measurement, an electrocardiogram (ECG), and an oral Dietary assessments. All subjects completed a 3-day food diary. Subjects were asked to compare their portion sizes with each portion size's color photographs from "Album of Photographs of Food Products and Dishes" developed by the National Food and Nutrition Institute 84 and weigh food, if possible. Subjects were asked to record the amount and the type of fats and oils used for cooking as well. Daily total energy, macronutrients, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and omega-3 and omega-6 fatty acids intake were analyzed using Dieta 6 software (National Food and Nutrition Institute, Warsaw, Poland), which was developed and which is continuously updated by the National Food and Nutrition Institute (Warsaw, Poland). This software is used to calculate the nutritional value of food and diets based on tables of the nutritional value of local food products and dishes and is commonly used to evaluate the fatty acid dietary intake 85 .
Anthropometric measurements. The body height and weight of participants were measured using a standardized method. Bodyweight was measured in a standard way (InBody 220, Biospace, Korea). Body composition and body fat distribution measurements were assessed using DXA scanning (enCORE™, iDXA Lunar GE Healthcare). In further analyses, the following parameters were evaluated: visceral adipose tissue mass (VAT mass), visceral adipose tissue volume (VAT volume), the visceral adipose tissue percentage of body weight (VAT BW %), the visceral adipose tissue percentage of adipose tissue (VAT AT%), the android fat to gynoid fat ratio (DXA A/G ratio), free fat mass (FFM), and lean mass (Lean mass).
Cold exposure and PET/MR scanning. During the second visit, in the fasting state, all volunteers underwent 2 h of cold exposure. Water perfused blankets were used as part of the applied protocol for cooling. Blood samples were also taken in the 60th and 120th min of cooling. After this procedure, a fluorodeoxyglucose F 18 injection (18F-FDG) (4 MBq/kg of body mass) was given, and a PET/MRI scan (Biograph mMR 3 T, Siemens Healthcare, Erlangen, Germany) of the whole body was performed during the autumn and winter periods.
Regions of interest (ROIs) were manually outlined in fusion images composed of a summed dynamic 18F-FDG PET image and magnetic resonance (MR). The software Carimas, developed at the Turku PET Centre in Finland, was applied for the image analyses. ROIs were drawn in image planes with a defined structure of brown adipose tissue and in the aortic arch in the time frame with the highest first-pass concentration of the tracer. Regional time-activity curves (TACs) were generated, and glucose uptake rate data for the regions were assessed. The influx rate constant (Ki) of FDG-F18 for BAT was determined using the Gjedde-Patlak model. A lumped constant (LC) value of 1.14 86 was used for all adipose tissues. The glucose uptake rate was calculated as follows: plasma glucose concentration × Ki × LC −1 . The activation of BAT was defined as a glucose uptake rate higher than 2.0 µmol × (100 g −1 ) × min −1 , which was chosen after a visual interpretation of PET images and the determination of the BAT glucose uptake rate at warm conditions, where it was always lower than 1.7 µmol × (100 g −1 ) × min −187 . Individuals in which BAT was detected were matched to the BAT positive group (BAT-positive), while subjects without detectable BAT in PET/MR images were classified as BAT negative (BAT-negative).
Resting metabolic rate measurements. During the cold exposure, whole-body resting energy expenditure (REE) was assessed using a computed open-circuit indirect calorimetry method based on the consumption of O 2 and the production of CO 2 . The 30-min long measurements of resting oxygen uptake and resting carbon dioxide production were performed using a ventilated canopy Vmax Encore 29n system (Viasys HealthCare, Yorba Linda, CA, USA) at the baseline (− 30 to 0 min) and every 30 min until 120 min of cold exposure.
Blood collection and biochemical measurements. During the cold exposure, blood samples were collected and stored at − 60 °C for further analyses. The serum IL-6 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) (ELISA Kit for Human Interleukin 6 (Human IL-6); R&D Systems, Inc., Minneapolis, MN 55413, Canada, D6050) according to the manufacturer's protocol and based on observing the principles of internal laboratory control for the performed determinations. The serum glucose level was measured using the colorimetric methods of the Cobas c111 analyzer (Roche Diagnostics, Basel, Switzerland). Samples and controls were measured in the same run using the blind analysis method.
Statistical analyses. Numerical data were summarized with a number of observations (N), arithmetic means, and standard deviations (SD). For categorical data, the number of observations and frequencies were presented. Study participants were divided into two groups based on the presence of brown adipose tissue: BATpositive and BAT-negative. Continuous parameters were examined for normality with the Shapiro-Wilk test and thorough visual inspection. The homogeneity of variance across groups was studied using the Levene test. Nonparametric tests were used for response variables that failed these two tests. The differences between the selected responses and BAT groups were then compared using an analysis of variance (ANOVA) or the Kruskal-Wallis test for numerical variables, with, respectively, a Tukey or Dunn post hoc test with a Holm p-value adjustment (in case multiple pairwise tests were performed or when there were multiple grouping variables). In order to study the hypothesis that there is a significant association between the presence of brown adipose tissue and body composition, as well as the hypothesis that the average daily consumption of omega-3 and omega-6 fatty acids can significantly alter brown adipose tissue activation, we studied its association using multivariate linear regression models. In all two-group comparisons and regression models, an adjustment for age, daily average energy intake (kcal/day), and DXA lean mass) was made to eliminate the potential effect of the covariates. The