Immunometabolic Signatures of Circulating Monocytes in Humans With Obesity and Insulin Resistance.

Obesity is associated with chronic inflammation and metabolic complications, including insulin resistance (IR). Immune cells drive inflammation through the rewiring of intracellular metabolism. However, the impact of obesity-related IR on the metabolism and functionality of circulating immune cells, like monocytes, remains poorly understood. To increase insight into the interindividual variation of immunometabolic signatures among individuals and their role in the development of IR, we assessed systemic and tissue-specific IR and circulating immune markers, and we characterized metabolic signatures and cytokine secretion of circulating monocytes from 194 individuals with a BMI ≥25 kg/m2. Monocyte metabolic signatures were defined using extracellular acidification rates (ECARs) to estimate glycolysis and oxygen consumption rates (OCRs) for oxidative metabolism. Although monocyte metabolic signatures and function based on cytokine secretion varied greatly among study participants, they were strongly associated with each other. The ECAR-to-OCR ratio, representing the balance between glycolysis and oxidative metabolism, was negatively associated with fasting insulin levels, systemic IR, and liver-specific IR. These results indicate that monocytes from individuals with IR were relatively more dependent on oxidative metabolism, whereas monocytes from more insulin-sensitive individuals were more dependent on glycolysis. Additionally, circulating CXCL11 was negatively associated with the degree of systemic IR and positively with the ECAR-to-OCR ratio in monocytes, suggesting that individuals with high IR and a monocyte metabolic dependence on oxidative metabolism also have lower levels of circulating CXCL11. Our findings suggest that monocyte metabolism is related to obesity-associated IR progression and deepen insights into the interplay between innate immune cell metabolism and IR development in humans.

Physical activity and sedentary behavior show distinct associations with tissue-specific insulin sensitivity in adults with overweight.

AIM: The aim of this study is to investigate associations between the physical activity (PA) spectrum (sedentary behavior to exercise) and tissue-specific insulin resistance (IR). METHODS: We included 219 participants for analysis (median [IQR]: 61 [55; 67] years, BMI 29.6 [26.9; 32.0] kg/m2 ; 60% female) with predominant muscle or liver IR, as determined using a 7-point oral glucose tolerance test (OGTT). PA and sedentary behavior were measured objectively (ActivPAL) across 7 days. Context-specific PA was assessed with the Baecke questionnaire. Multiple linear regression models (adjustments include age, sex, BMI, site, season, retirement, and dietary intake) were used to determine associations between the PA spectrum and hepatic insulin resistance index (HIRI), muscle insulin sensitivity index (MISI) and whole-body IR (HOMA-IR, Matsuda index). RESULTS: In fully adjusted models, objectively measured total PA (standardized regression coefficient ß = 0.17, p = 0.020), light-intensity PA (ß = 0.15, p = 0.045) and moderate-to-vigorous intensity PA (ß = 0.13, p = 0.048) were independently associated with Matsuda index, but not HOMA-IR (p > 0.05). A higher questionnaire-derived sport index and leisure index were associated with significantly lower whole-body IR (Matsuda, HOMA-IR) in men but not in women. Results varied across tissues: more time spent sedentary (ß = -0.24, p = 0.045) and a higher leisure index (ß = 0.14, p = 0.034) were respectively negatively and positively associated with MISI, but not HIRI. A higher sport index was associated with lower HIRI (ß = -0.30, p = 0.007, in men only). CONCLUSION: While we confirm a beneficial association between PA and whole-body IR, our findings indicate that associations between the PA spectrum and IR seem distinct depending on the primary site of insulin resistance (muscle or liver).

Cardiometabolic health improvements upon dietary intervention are driven by tissue-specific insulin resistance phenotype: A precision nutrition trial.

Precision nutrition based on metabolic phenotype may increase the effectiveness of interventions. In this proof-of-concept study, we investigated the effect of modulating dietary macronutrient composition according to muscle insulin-resistant (MIR) or liver insulin-resistant (LIR) phenotypes on cardiometabolic health. Women and men with MIR or LIR (n = 242, body mass index [BMI] 25-40 kg/m2, 40-75 years) were randomized to phenotype diet (PhenoDiet) group A or B and followed a 12-week high-monounsaturated fatty acid (HMUFA) diet or low-fat, high-protein, and high-fiber diet (LFHP) (PhenoDiet group A, MIR/HMUFA and LIR/LFHP; PhenoDiet group B, MIR/LFHP and LIR/HMUFA). PhenoDiet group B showed no significant improvements in the primary outcome disposition index, but greater improvements in insulin sensitivity, glucose homeostasis, serum triacylglycerol, and C-reactive protein compared with PhenoDiet group A were observed. We demonstrate that modulating macronutrient composition within the dietary guidelines based on tissue-specific insulin resistance (IR) phenotype enhances cardiometabolic health improvements. Clinicaltrials.gov registration: NCT03708419, CCMO registration NL63768.068.17.

Corrigendum: The PERSonalized Glucose Optimization Through Nutritional Intervention (PERSON) Study: Rationale, Design and Preliminary Screening Results.

[This corrects the article DOI: 10.3389/fnut.2021.694568.].

The PERSonalized Glucose Optimization Through Nutritional Intervention (PERSON) Study: Rationale, Design and Preliminary Screening Results.

Background: It is well-established that the etiology of type 2 diabetes differs between individuals. Insulin resistance (IR) may develop in different tissues, but the severity of IR may differ in key metabolic organs such as the liver and skeletal muscle. Recent evidence suggests that these distinct tissue-specific IR phenotypes may also respond differentially to dietary macronutrient composition with respect to improvements in glucose metabolism. Objective: The main objective of the PERSON study is to investigate the effects of an optimal vs. suboptimal dietary macronutrient intervention according to tissue-specific IR phenotype on glucose metabolism and other health outcomes. Methods: In total, 240 overweight/obese (BMI 25 - 40 kg/m2) men and women (age 40 - 75 years) with either skeletal muscle insulin resistance (MIR) or liver insulin resistance (LIR) will participate in a two-center, randomized, double-blind, parallel, 12-week dietary intervention study. At screening, participants undergo a 7-point oral glucose tolerance test (OGTT) to determine the hepatic insulin resistance index (HIRI) and muscle insulin sensitivity index (MISI), classifying each participant as either "No MIR/LIR," "MIR," "LIR," or "combined MIR/LIR." Individuals with MIR or LIR are randomized to follow one of two isocaloric diets varying in macronutrient content and quality, that is hypothesized to be either an optimal or suboptimal diet, depending on their tissue-specific IR phenotype (MIR/LIR). Extensive measurements in a controlled laboratory setting as well as phenotyping in daily life are performed before and after the intervention. The primary study outcome is the difference in change in disposition index, which is the product of insulin sensitivity and first-phase insulin secretion, between participants who received their hypothesized optimal or suboptimal diet. Discussion: The PERSON study is one of the first randomized clinical trials in the field of precision nutrition to test effects of a more personalized dietary intervention based on IR phenotype. The results of the PERSON study will contribute knowledge on the effectiveness of targeted nutritional strategies to the emerging field of precision nutrition, and improve our understanding of the complex pathophysiology of whole body and tissue-specific IR. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03708419, clinicaltrials.gov as NCT03708419.

Fasting and postprandial remnant-like particle cholesterol concentrations in obese participants are associated with plasma triglycerides, insulin resistance, and body fat distribution.

Elevated plasma concentrations of remnant-like particle cholesterol (RLP-C) are atherogenic. However, factors that determine RLP-C are not fully understood. This study evaluates which factors affect RLP-C in the fasting and postprandial state, using multiple regression analyses in a large cohort of lean and obese participants. All participants (n = 740) underwent a test meal challenge containing 95 energy % (en%) fat (energy content 50% of predicted daily resting metabolic rate). Fasting and postprandial concentrations of circulating metabolites were measured over a 3-h period. Obese participants (n = 613) also participated in a 10-wk weight loss program (-2510 kJ/d), being randomized to either a low-fat or a high-fat diet (20-25 vs. 40-45en% fat). Postprandial RLP-C was associated with fasting RLP-C, waist:hip ratio (WHR), HOMA(IR) (homeostasis model assessment index for insulin resistance) (P < 0.001), and age, independently of BMI and gender [adjusted R(2) (adj. R(2)) = 0.70). These factors were also related to fasting RLP-C (P < 0.010), along with gender and physical activity (adj. R(2) = 0.23). The dietary intervention resulted in significantly lower fasting RLP-C concentrations, independently mediated by weight loss, improvements in HOMA(IR), and the fat content of the prescribed diet. However, after inclusion of plasma triglyceride (TG), HDL-cholesterol, and FFA concentrations in the models, HOMA(IR) and WHR no longer significantly predicted fasting RLP-C, although WHR remained a predictor of postprandial RLP-C (P = 0.002). Plasma TG was strongly associated with both fasting and postprandial RLP-C (P < 0.001). In conclusion, plasma RLP-C concentrations are mainly associated with plasma TG concentrations. Interestingly, the high-fat diet was more effective at decreasing fasting RLP-C concentrations in obese participants than the low-fat diet.

Adipose triglyceride lipase and hormone-sensitive lipase protein expression is decreased in the obese insulin-resistant state.

AIM/HYPOTHESIS: Obesity is associated with increased triacylglycerol (TAG) storage in adipose tissue and insulin resistance. The mobilization of stored TAG is mediated by hormone-sensitive lipase (HSL) and the recently discovered adipose triglyceride lipase (ATGL). The aim of the present study was to examine whether ATGL and HSL mRNA and protein expression are altered in insulin-resistant conditions. In addition, we investigated whether a possible impaired expression could be reversed by a period of weight reduction. METHODS: Adipose tissue biopsies were taken from obese subjects (n = 44) with a wide range of insulin resistance, before and just after a 10-wk hypocaloric diet. ATGL and HSL protein and mRNA expression was determined by Western blot and quantitative RT-PCR, respectively. RESULTS: Fasting insulin levels and the degree of insulin resistance (using the homeostasis model assessment index for insulin resistance) were negatively correlated with ATGL and HSL protein expression, independent of age, gender, fat cell size, and body composition. Both mRNA and protein levels of ATGL and HSL were reduced in insulin-resistant compared with insulin-sensitive subjects (P < 0.05). Weight reduction significantly decreased ATGL and HSL mRNA and protein expression. A positive correlation between the decrease in leptin and the decrease in ATGL protein level after weight reduction was observed. Finally, ATGL and HSL mRNA and protein levels seem to be highly correlated, indicating a tight coregulation and transcriptional control. CONCLUSIONS: In obese subjects, insulin resistance and hyperinsulinemia are strongly associated with ATGL and HSL mRNA and protein expression, independent of fat mass. Data on weight reduction indicated that also other factors (e.g. leptin) relate to ATGL and HSL protein expression.

Effects of 3 diets with various calcium contents on 24-h energy expenditure, fat oxidation, and adipose tissue message RNA expression of lipid metabolism-related proteins.

BACKGROUND: Evidence from molecular and animal research and epidemiologic investigations indicates that calcium intake may be inversely related to body weight, possibly through alterations in 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] metabolism. OBJECTIVE: We tested whether energy and substrate metabolism and adipose tissue enzyme messenger RNA (mRNA) expression can be altered by dietary calcium intake in healthy, nonobese, human volunteers consuming an isocaloric diet. DESIGN: Twelve healthy men [age: 28 +/- 2 y; body mass index (BMI; in kg/m(2)): 25.2 +/- 06] received 3 isocaloric diets [high calcium (1259 +/- 9 mg/d), high dairy (high/high); high calcium (1259 +/- 9 mg/d), low dairy (high/low); and low calcium (349 +/- 8 mg/d), low dairy (low/low)] in a randomized crossover design. At the end of the 7-d dietary periods, 24-h energy expenditure and substrate metabolism were measured, and fat biopsy specimens were obtained to determine mRNA expression in genes involved in the lipolytic and lipogenic pathways. RESULTS: The 24-h energy expenditure was 11.8 +/- 0.3, 11.6 +/- 0.3, and 11.7 +/- 0.3 MJ/24 h in the high/high, high/low, and low/low conditions, respectively. Fat oxidation in these conditions was 108 +/- 7, 105 +/- 9, and 100 +/- 6 g/24 h. These differences were not statistically significant. mRNA concentrations of UCP2, FAS, GPDH2, HSL, and PPARG did not differ significantly. Serum 1,25(OH)(2)D(3) concentrations changed from 175 +/- 16 to 138 +/- 15, 181 +/- 23 to 159 +/- 19, and 164 +/- 13 to 198 +/- 19 pmol/L in the high/high, high/low, and low/low conditions, respectively, and was significantly different between the high/high and low/low conditions (P < 0.05). CONCLUSION: Altering the dietary calcium content for 7 d does not influence substrate metabolism, energy metabolism, or gene expression in proteins related to fat metabolism, despite significant changes in 1,25(OH)(2)D(3) concentrations.

Acute effect of L-796568, a novel beta 3-adrenergic receptor agonist, on energy expenditure in obese men.

OBJECTIVE: Our objective was to investigate the thermogenic efficacy of single oral doses of the novel beta(3)-adrenergic receptor agonist L-796568 [(R )-N -[4-[2-[[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]-phenyl]-4-[4-[4-(trifluoromethyl)phenyl]thiazol-2-yl]-benzenesulfonamide, dihydrochloride] in humans. METHODS: Twelve healthy overweight to obese men participated in this 2-center, 3-period, randomized, placebo-controlled, crossover trial. In each period subjects received 250 mg L-796568, 1000 mg L-796568, or placebo. Energy expenditure and respiratory quotient were determined by indirect calorimetry; blood samples were taken; and ear temperature, heart rate, and blood pressure were measured at baseline and during the 4-hour period after administration. RESULTS: Energy expenditure increased significantly after the 1000-mg dose (about 8%) and this was accompanied by an increase in plasma glycerol and free fatty acid concentrations. Systolic blood pressure also increased significantly. No changes in heart rate, diastolic blood pressure, ear temperature, plasma catecholamine, potassium, or leptin were found. CONCLUSIONS: Single-dose administration of 1000 mg of the novel beta(3)-adrenergic receptor agonist L-796568 increased lipolysis and energy expenditure in overweight men. This is the first study to show such an effect of beta(3)-adrenergic receptor agonists in humans without significant evidence for beta(2)-adrenergic receptor involvement.

Long-term effects of low-intensity exercise training on fat metabolism in weight-reduced obese men.

The aim of the present study was to investigate the effect of long-term continuation of low-intensity exercise training on weight maintenance, substrate metabolism, and beta-adrenergic-mediated fat oxidation in weight-reduced obese men. Preceding this part of the study, subjects lost 15 +/- 6 kg of body weight by energy restriction with or without low-intensity exercise training. Twenty-nine subjects (diet group, n = 15; diet + exercise group, n = 14) participated in the follow-up study of 40 weeks in which the former diet + exercise group continued their exercise training program. Pre- and postfollow-up, measurements of body weight, body composition, maximal aerobic capacity and substrate oxidation during rest, exercise, and recovery with or without infusion of the beta-adrenergic antagonist, propranolol (PRP), were performed. Over the follow-up period, body weight, fat mass, and fat free mass increased in both groups (P <.0001) without differences between groups. Attendance at exercise training sessions was negatively correlated with regain of body weight (r = -.6, P <.05). Relative fat oxidation, energy expenditure, and beta-adrenergic-mediated fat oxidation during rest, exercise, and recovery were maintained over the follow-up period in both groups. Continuation of low-intensity exercise training after weight reduction did not limit regain of body weight, unless exercise training was frequently performed. Relative (beta-adrenergic-mediated) fat oxidation and energy expenditure were maintained at postdiet level whether or not low-intensity exercise training was performed during follow-up.

Skeletal muscle fatty acid handling in insulin resistant men.

Disturbances in skeletal muscle lipid metabolism may precede or contribute to the development of whole body insulin resistance. In this study, we examined fasting and postprandial skeletal muscle fatty acid (FA) handling in insulin resistant (IR) men. Thirty men with the metabolic syndrome (MetS) (National Cholesterol Education Program-Adult Treatment Panel III) were included in this sub-study to the LIPGENE study, and divided in two groups (IR and control) based on the median of insulin sensitivity (S(I) = 2.06 (mU/l(-1))·min(-1)·10(-4)). Fasting and postprandial skeletal muscle FA handling were examined by combining the forearm balance technique with stable isotopes of palmitate. [(2)H(2)]-palmitate was infused intravenously to label endogenous triacylglycerol (TAG) and free FAs (FFAs) in the circulation and [U-(13)C]-palmitate was incorporated in a high-fat mixed meal (2.6 MJ, 61 E% fat) to label chylomicron-TAG. Muscle biopsies were taken to determine muscle TAG, diacylglycerol (DAG), FFA, and phospholipid (PL) content, their fractional synthetic rates (FSRs) and degree of saturation, as well as messenger RNA (mRNA) expression of genes involved in lipid metabolism. In the first 2 h after meal consumption, forearm muscle [(2)H(2)]-labeled TAG extraction was higher in IR vs. control (P = 0.05). Fasting percentage saturation of muscle DAG was higher in IR vs. control (P = 0.016). No differences were observed for intramuscular TAG, DAG, FFA, and PL content, FSR, and muscle mRNA expression. In conclusion, increased muscle (hepatically derived) TAG extraction during postprandial conditions and increased saturation of intramuscular DAG are associated with insulin resistance, suggesting that disturbances in skeletal muscle FA handling could play a role in the development of whole body insulin resistance and type 2 diabetes.

Differential valine metabolism in adipose tissue of low and high fat-oxidizing obese subjects.

Differences in fat metabolism are of importance in relation to energy balance. Low fat-oxidizers (LFO) are thought to be more prone for developing obesity. We studied whether LFO have different fasting adipose tissue (AT) protein profiles than high fat-oxidizers (HFO). Six LFO and six HFO subjects were selected from an obese group (n = 99, body mass index>30 kg/m(2) ) taking part in a multi-center study (Nutrient-Gene interaction in human obesity) based on the postprandial fat oxidation capacity after a high fat load. AT protein profiles were studied by 2-DE. Differential proteins were clustered with MAPPfinder according to their function. Protein profiles of purified blood cells and adipocytes served to confine the comparison to adipocyte-specific proteins in AT profiles of LFO and HFO subjects. LFO had increased mitochondrial ROS scavengers possibly related to long-chain unsaturated fatty acid-induced increases in mitochondrial ROS-production. Carbohydrate oxidation seemed to be reduced since expression of several proteins from the glycolysis pathway was lower in LFO. Up-regulation of the valine catabolism at the level of methylmalonate-semialdehyde dehydrogenase appeared to be (part of) the compensatory mechanism. In conclusion, the fasting AT protein profile of LFO and HFO differ at the level of ROS scavenging, the glycolysis pathway and valine metabolism.

Intramyocellular lipid and glycogen content are reduced following resistance exercise in untrained healthy males.

Resistance exercise has recently been shown to improve whole-body insulin sensitivity in healthy males. Whether this is accompanied by an exercise-induced decline in skeletal muscle glycogen and/or lipid content remains to be established. In the present study, we determined fibre-type-specific changes in skeletal muscle substrate content following a single resistance exercise session. After an overnight fast, eight untrained healthy lean males participated in a approximately 45 min resistance exercise session. Muscle biopsies were collected before, following cessation of exercise, and after 30 and 120 min of post-exercise recovery. Subjects remained fasted throughout the test. Conventional light and (immuno)fluorescence microscopy were applied to assess fibre-type-specific changes in intramyocellular triacylglycerol (IMTG) and glycogen content. A significant 27+/-7% net decline in IMTG content was observed in the type I muscle fibres (P<0.05), with no net changes in the type IIa and IIx fibres. Muscle glycogen content decreased with 23+/-6, 40+/-7 and 44+/-7% in the type I, IIa and IIx muscle fibres, respectively (P<0.05). Fibre-type-specific changes in intramyocellular lipid and/or glycogen content correlated well with muscle fibre-type oxidative capacity. During post-exercise recovery, type I muscle fibre lipid content returned to pre-exercise levels within 120 min. No changes in muscle glycogen content were observed during recovery. We conclude that intramyocellular lipid and glycogen stores are readily used during resistance exercise and this is likely associated with the reported increase in whole-body insulin sensitivity following resistance exercise.

The effects of increasing serum calcitriol on energy and fat metabolism and gene expression.

OBJECTIVE: Evidence from a number of investigations indicates that calcium intake could be inversely related to body weight through alterations in the 1,25-OH(2)-D(3) metabolism. The objective of this study was to test whether energy and substrate metabolism and adipose tissue enzyme mRNA expression can be altered by changes in serum 1,25-OH(2)-D(3) through oral cholecalciferol supplementation in non-obese human subjects. RESEARCH METHODS AND PROCEDURES: An intervention study was used with a treatment period of 7 days. During this intervention, energy expenditure (EE) and substrate metabolism were measured using indirect calorimetry at t = 0, 1, 3, and 7 days, and blood samples were obtained at t = -1, 0, 1, 2, 3, 5 and 7 days. Fat biopsies were obtained at t = 0 and 7 days for determination of expression of genes involved in lipolytic and lipogenic pathways. Subjects from the general community were studied in an ambulatory setting at a university hospital. Ten healthy young men (age, 28 +/- 3 years; BMI, 25.5 +/- 0.5 kg/m(2)) were recruited by local announcement, and all completed the study. All subjects received 2000 IU cholecalciferol/d for 7 days, and they were instructed to consume a low-cholecalciferol, low-calcium diet. EE, fat oxidation, and adipose tissue enzyme mRNA were the main outcome measures. RESULTS: Despite a significant increase in serum 1,25-OH(2)-D(3) concentration at t = 5 and 7 days, no significant differences in substrate and energy metabolism nor mRNA concentrations of different lipid metabolism-related proteins were observed. DISCUSSION: Seven-day supplementation with 2000 IU cholecalciferol/d together with a decrease in dietary calcium intake does not affect EE or substrate metabolism nor gene expression of proteins related to fat metabolism, despite a significant increase in serum 1,25-OH(2)-D(3) concentration.

A single session of resistance exercise enhances insulin sensitivity for at least 24 h in healthy men.

The aim of the present study was to determine whether a single session of resistance exercise improves whole-body insulin sensitivity in healthy men for up to 24 h. Twelve male subjects (23 +/- 1 years) were studied over a period of 4 days during which they consumed a standardized diet, providing 0.16 +/- 0.01 MJ.kg(-1).day(-1) containing 15 +/- 0.1 energy% (En%) protein, 29 +/ -0.1 En% fat and 55 +/- 0.3 En% carbohydrate. Insulin sensitivity was determined 24 h before and 24 h after a single resistance exercise session (8 sets of 10 repetitions at 75% of 1 repetition maximum for two leg exercise tasks) using an intravenous insulin tolerance test. Insulin sensitivity index was calculated by the decline in arterial blood glucose concentration following intravenous administration of a single bolus of human insulin (0.075 IU.kg(-1) fat free mass). Basal glucose and insulin concentrations were not changed up to 24 h after the resistance exercise. However, a substantial 13+/-5% improvement in whole-body insulin sensitivity was observed, 24 h after the resistance exercise (P < 0.05). This study shows that even a single session of resistance exercise improves whole-body insulin sensitivity for up to 24 h in healthy men, which is consistent with earlier observations following endurance exercise tasks.

Effect of beta1- and beta2-adrenergic stimulation on energy expenditure, substrate oxidation, and UCP3 expression in humans.

In humans, beta-adrenergic stimulation increases energy and fat metabolism. In the case of beta1-adrenergic stimulation, it is fueled by an increased lipolysis. We examined the effect of beta2-adrenergic stimulation, with and without a blocker of lipolysis, on thermogenesis and substrate oxidation. Furthermore, the effect of beta1-and beta2-adrenergic stimulation on uncoupling protein 3 (UCP3) mRNA expression was studied. Nine lean males received a 3-h infusion of dobutamine (DOB, beta1) or salbutamol (SAL, beta2). Also, we combined SAL with acipimox to block lipolysis (SAL+ACI). Energy and substrate metabolism were measured continuously, blood was sampled every 30 min, and muscle biopsies were taken before and after infusion. Energy expenditure significantly increased approximately 13% in all conditions. Fat oxidation increased 47 +/- 7% in the DOB group and 19 +/- 7% in the SAL group but remained unchanged in the SAL+ACI condition. Glucose oxidation decreased 40 +/- 9% upon DOB, remained unchanged during SAL, and increased 27 +/- 11% upon SAL+ACI. Plasma free fatty acid (FFA) levels were increased by SAL (57 +/- 11%) and DOB (47 +/- 16%), whereas SAL+ACI caused about fourfold lower FFA levels compared with basal levels. No change in UCP3 was found after DOB or SAL, whereas SAL+ACI downregulated skeletal muscle UCP3 mRNA levels 38 +/- 13%. In conclusion, beta2-adrenergic stimulation directly increased energy expenditure independently of plasma FFA levels. Furthermore, this is the first study to demonstrate a downregulation of skeletal muscle UCP3 mRNA expression after the lowering of plasma FFA concentrations in humans, despite an increase in energy expenditure upon beta2-adrenergic stimulation.