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Summary: Inguinal white adipose tissue (iWAT) is essential for the beneficial effects of exercise training on metabolic health. The underlying mechanisms for these effects are not fully understood, and here, we test the hypothesis that exercise training results in a more favorable iWAT structural phenotype. Using biochemical, imaging, and multi-omics analyses, we find that 11 days of wheel running in male mice causes profound iWAT remodeling including decreased extracellular matrix (ECM) deposition and increased vascularization and innervation. We identify adipose stem cells as one of the main contributors to training-induced ECM remodeling, show that the PRDM16 transcriptional complex is necessary for iWAT remodeling and beiging, and discover neuronal growth regulator 1 (NEGR1) as a link between PRDM16 and neuritogenesis. Moreover, we find that training causes a shift from hypertrophic to insulin-sensitive adipocyte subpopulations. Exercise training leads to remarkable adaptations to iWAT structure and cell-type composition that can confer beneficial changes in tissue metabolism.

Summary: The Scandinavian winter-swimming culture combines brief dips in cold water with hot sauna sessions, with conceivable effects on body temperature. We study thermogenic brown adipose tissue (BAT) in experienced winter-swimming men performing this activity 2–3 times per week. Our data suggest a lower thermal comfort state in the winter swimmers compared with controls, with a lower core temperature and absence of BAT activity. In response to cold, we observe greater increases in cold-induced thermogenesis and supraclavicular skin temperature in the winter swimmers, whereas BAT glucose uptake and muscle activity increase similarly to those of the controls. All subjects demonstrate nocturnal reduction in supraclavicular skin temperature, whereas a distinct peak occurs at 4:30–5:30 a.m. in the winter swimmers. Our data leverage understanding of BAT in adult human thermoregulation, suggest both heat and cold acclimation in winter swimmers, and propose winter swimming as a potential strategy for increasing energy expenditure.

Objective: Increasing the amounts of functionally competent brown adipose tissue (BAT) in adult humans has the potential to restore dysfunctional metabolism and counteract obesity. In this study, we aimed to characterize the human perirenal fat depot, and we hypothesized that there would be regional, within-depot differences in the adipose signature depending on local sympathetic activity. Methods: We characterized fat specimens from four different perirenal regions of adult kidney donors, through a combination of qPCR mapping, immunohistochemical staining, RNA-sequencing, and pre-adipocyte isolation. Candidate gene signatures, separated by adipocyte morphology, were recapitulated in a murine model of unilocular brown fat induced by thermoneutrality and high fat diet. Results: We identified widespread amounts of dormant brown adipose tissue throughout the perirenal depot, which was contrasted by multilocular BAT, primarily found near the adrenal gland. Dormant BAT was characterized by a unilocular morphology and a distinct gene expression profile, which partly overlapped with that of subcutaneous white adipose tissue (WAT). Brown fat precursor cells, which differentiated into functional brown adipocytes were present in the entire perirenal fat depot, regardless of state. We identified SPARC as a candidate adipokine contributing to a dormant BAT state, and CLSTN3 as a novel marker for multilocular BAT. Conclusions: We propose that perirenal adipose tissue in adult humans consists mainly of dormant BAT and provide a data set for future research on factors which can reactivate dormant BAT into active BAT, a potential strategy for combatting obesity and metabolic disease. Keywords: Perirenal adipose tissue, Brown fat precursor cells, Human brown fat, Sympathetic activation, Dormant brown fat

Summary: Glutamine is a central nutrient for many cancers, contributing to the generation of building blocks and energy-promoting signaling necessary for neoplastic proliferation. In this study, we hypothesized that lowering systemic glutamine levels by exercise may starve tumors, thereby contributing to the inhibitory effect of exercise on tumor growth. We demonstrate that limiting glutamine availability, either pharmacologically or physiologically by voluntary wheel running, significantly attenuated the growth of two syngeneic murine tumor models of breast cancer and lung cancer, respectively, and decreased markers of atrophic signaling in muscles from tumor-bearing mice. In continuation, wheel running completely abolished tumor-induced loss of weight and lean body mass, independently of the effect of wheel running on tumor growth. Moreover, wheel running abolished tumor-induced upregulation of muscular glutamine transporters and myostatin signaling. In conclusion, our data suggest that voluntary wheel running preserves muscle mass by counteracting muscular glutamine release and tumor-induced atrophic signaling. : Cancer; Physiology; Specialized Functions of Cells Subject Areas: Cancer, Physiology, Specialized Functions of Cells

Objective: Interleukin (IL)-18 plays a crucial role in maintaining metabolic homeostasis and levels of this cytokine are influenced by gender, age, and sex hormones. The role of gender on IL-18 signaling, however, is unclear. We hypothesized that the presence of female sex hormone could preserve the metabolic phenotype of the IL-18R−/− animals. Methods: We studied female mice with a global deletion of the α isoform of the IL-18 receptor (IL-18R−/−) and littermates control. Three studies were done: 1) animals fed a high fat diet (HFD) for 16 weeks; 2) animals fed chow diet for 72 weeks and 3) animals (3 weeks-old) randomized to either bilateral ovariectomy (OVX) or control surgery (SHAM) and followed for 16 weeks. Results: Female IL-18R−/− mice gained less weight and maintained glucose homeostasis on a chow diet compared with HFD, but no differences between genotypes were observed. The maintenance of body weight and glucose homeostasis in IL-18R−/− mice was lost with aging. By 72 weeks of age, IL-18R−/− mice became heavier compared with WT mice due to an increase in both visceral and subcutaneous adiposity and displayed glucose intolerance. OVX did not affect body weight in IL-18R−/− mice but exacerbated glucose intolerance and impaired liver insulin signaling when compared with SHAM mice. Conclusions: Female mice harboring a global deletion of the IL-18R, only present the same phenotype as reported in male IL-18R−/− mice if they are aged or have undergone OVX, in which circulating estrogen is likely to be blunted. The role of estrogen signaling in the protection against altered metabolic homeostasis in IL-18R−/− mice appears to be mediated by liver insulin signaling. We therefore suggest that the metabolic effects mediated by loss of IL-18 signaling are only present in a female sex hormone free environment. Keywords: IL-18, Obesity, Insulin resistance, Gender

Background: Low physical activity level is associated with poor prognosis in patients with colorectal cancer (CRC). To increase physical activity, technology-based platforms are emerging and provide intriguing opportunities to prescribe and monitor active lifestyle interventions. The “Interval Walking in Colorectal Cancer“(I-WALK-CRC) study explores the feasibility and efficacy a home-based interval-walking intervention delivered by a smart-phone application in order to improve cardio-metabolic health profile among CRC survivors. The aim of the present report is to describe the design, methods and recruitment results of the I-WALK-CRC study. Methods/Results: The I-WALK-CRC study is a randomized controlled trial designed to evaluate the feasibility and efficacy of a home-based interval walking intervention compared to a waiting-list control group for physiological and patient-reported outcomes. Patients who had completed surgery for local stage disease and patients who had completed surgery and any adjuvant chemotherapy for locally advanced stage disease were eligible for inclusion. Between October 1st, 2015, and February 1st, 2017, 136 inquiries were recorded; 83 patients were eligible for enrollment, and 42 patients accepted participation. Age and employment status were associated with participation, as participants were significantly younger (60.5 vs 70.8 years, P

Objective: Angiopoietin-like protein-4 (ANGPTL4) is a circulating protein that is highly expressed in liver and implicated in regulation of plasma triglyceride levels. Systemic ANGPTL4 increases during prolonged fasting and is suggested to be secreted from skeletal muscle following exercise. Methods: We investigated the origin of exercise-induced ANGPTL4 in humans by measuring the arterial-to-venous difference over the leg and the hepato-splanchnic bed during an acute bout of exercise. Furthermore, the impact of the glucagon-to-insulin ratio on plasma ANGPTL4 was studied in healthy individuals. The regulation of ANGPTL4 was investigated in both hepatic and muscle cells. Results: The hepato-splanchnic bed, but not the leg, contributed to exercise-induced plasma ANGPTL4. Further studies using hormone infusions revealed that the glucagon-to-insulin ratio is an important regulator of plasma ANGPTL4 as elevated glucagon in the absence of elevated insulin increased plasma ANGPTL4 in resting subjects, whereas infusion of somatostatin during exercise blunted the increase of both glucagon and ANGPTL4. Moreover, activation of the cAMP/PKA signaling cascade let to an increase in ANGPTL4 mRNA levels in hepatic cells, which was prevented by inhibition of PKA. In humans, muscle ANGPTL4 mRNA increased during fasting, with only a marginal further induction by exercise. In human muscle cells, no inhibitory effect of AMPK activation could be demonstrated on ANGPTL4 expression. Conclusions: The data suggest that exercise-induced ANGPTL4 is secreted from the liver and driven by a glucagon-cAMP-PKA pathway in humans. These findings link the liver, insulin/glucagon, and lipid metabolism together, which could implicate a role of ANGPTL4 in metabolic diseases.

Objective: MicroRNAs (miRNAs) are increasingly recognized as fine-tuning regulators of metabolism, and are dysregulated in several disease conditions. With their capacity to rapidly change gene expression, miRNAs are also important regulators of development and cell differentiation. In the current study, we describe an impaired myogenic capacity of muscle stem cells isolated from humans with type 2 diabetes (T2DM) and assess whether this phenotype is regulated by miRNAs. Methods: We measured global miRNA expression during in vitro differentiation of muscle stem cells derived from T2DM patients and healthy controls. Results: The mir-23b/27b cluster was downregulated in the cells of the patients, and a pro-myogenic effect of these miRNAs was mediated through the p53 pathway, which was concordantly dysregulated in the muscle cells derived from humans with T2DM. Conclusions: Our results indicate that we have identified a novel pathway for coordination of myogenesis, the miR-23b/27b-p53 axis that, when dysregulated, potentially contributes to a sustained muscular dysfunction in T2DM.

Objective: Dietary medium-chain fatty acids (MCFAs), characterized by chain lengths of 8–12 carbon atoms, have been proposed to have beneficial effects on glucose and lipid metabolism, yet the underlying mechanisms remain elusive. We hypothesized that MCFA intake benefits metabolic health by inducing the release of hormone-like factors. Methods: The effects of chow diet, high-fat diet rich in long-chain fatty acids (LCFA HFD) fed ad libitum or pair-fed to a high-fat diet rich in MCFA (MCFA HFD) on glycemia, hepatic gene expression, circulating fibroblast growth factor 21 (FGF21), and liver fat content in both wildtype and Fgf21 knockout mice were investigated. The impact of a single oral dose of an MCFA-rich oil on circulating FGF21 and hepatic Fgf21 mRNA expression was assessed. In flag-tagged Crebh knockin mice and liver-specific Crebh knockout mice, fed LCFA HFD or MCFA HFD, active hepatic CREBH and hepatic Fgf21 mRNA abundance were determined, respectively. Results: MCFA HFD improves glucose tolerance, enhances glucose clearance into brown adipose tissue, and prevents high-fat diet-induced hepatic steatosis in wildtype mice. These benefits are associated with increased liver expression of CREBH target genes (Apoa4 and Apoc2), including Fgf21. Both acute and chronic intake of dietary MCFAs elevate circulating FGF21. Augmented hepatic Fgf21 mRNA following MCFA HFD intake is accompanied by higher levels of active hepatic CREBH; and MCFA-induced hepatic Fgf21 expression is blocked in mice lacking Crebh. Notably, while feeding male and female Fgf21 wildtype mice MCFA HFD results in reduced liver triacylglycerol (TG) levels, this liver TG-lowering effect is blunted in Fgf21 knockout mice fed MCFA HFD. The reduction in liver TG levels observed with MCFA HFD was independent of weight loss. Conclusions: Dietary MCFAs reduce liver fat accumulation via activation of a CREBH-FGF21 signaling axis.

Objective: Medium chain fatty acids (MCFAs), which are fatty acids with chain lengths of 8–12 carbon atoms, have been shown to reduce food intake in rodents and humans, but the underlying mechanisms are unknown. Unlike most other fatty acids, MCFAs are absorbed from the intestine into the portal vein and enter first the liver. We thus hypothesized that MCFAs trigger the release of hepatic factors that reduce appetite. Methods: The liver transcriptome in mice that were orally administered MCFAs as C8:0 triacylglycerol (TG) was analyzed. Circulating growth/differentiation factor 15 (GDF15), tissue Gdf15 mRNA and food intake were investigated after acute oral gavage of MCFAs as C8:0 or C10:0 TG in mice. Effects of acute and subchronic administration of MCFAs as C8:0 TG on food intake and body weight were determined in mice lacking either the receptor for GDF15, GDNF Family Receptor Alpha Like (GFRAL), or GDF15. Results: Hepatic and small intestinal expression of Gdf15 and circulating GDF15 increased after ingestion of MCFAs, while intake of typical dietary long-chain fatty acids (LCFAs) had no effect. Plasma GDF15 levels also increased in the portal vein with MCFA intake, indicating that in addition to the liver, the small intestine contributes to the rise in circulating GDF15. Acute oral provision of MCFAs decreased food intake over 24 h compared with a LCFA-containing bolus, and this anorectic effect required the GDF15 receptor, GFRAL. Moreover, subchronic oral administration of MCFAs reduced body weight over 7 days, an effect that was blunted in mice lacking either GDF15 or GFRAL. Conclusions: We have identified ingestion of MCFAs as a novel nutritional approach that increases circulating GDF15 in mice and have revealed that the GDF15-GFRAL axis is required for the full anorectic effect of MCFAs.

This study investigated the aminoacidemia after ingestion of a poultry protein hydrolysate (PPH) and whey protein in healthy young and old participants. Protein-drinks were also digested using the INFOGEST static in vitro digestion model to simulate gastrointestinal changes in young and old adults. In fasted state, 10 young (20-40y) and 10 old (70-80y) ingested PPH or whey as a 20 g protein-drink and blood samples were collected. Plasma leucine concentration increased more when ingesting whey than PPH (young 62 ± 27 vs. 48 ± 27%, old 94 ± 57 vs. 66 ± 26%) but the peak concentration was reached faster after drinking PPH (p

Summary: Lack of interleukin-6 (IL-6) leads to expansion of adipose tissue mass in rodents and humans. The exact underlying mechanisms have not been identified. In this placebo-controlled, non-randomized, participant-blinded crossover study, we use the IL-6 receptor antibody tocilizumab to investigate the role of endogenous IL-6 in regulating systemic energy metabolism at rest and during exercise and recovery in lean and obese men using tracer dilution methodology. Tocilizumab reduces fatty acid appearance in the circulation under all conditions in lean and obese individuals, whereas lipolysis (the rate of glycerol appearance into the circulation) is mostly unaffected. The fact that fatty acid oxidation is unaffected by IL-6 receptor blockade suggests increased re-esterification of fatty acids. Glucose kinetics are unaffected. We find that blocking endogenous IL-6 signaling with tocilizumab impairs fat mobilization, which may contribute to expansion of adipose tissue mass and, thus, affect the health of individuals undergoing anti-IL-6 therapy (Clinicaltrials.gov: NCT03967691).

Excessive circulating FAs have been proposed to promote insulin resistance (IR) of glucose metabolism by increasing the oxidation of FAs over glucose. Therefore, inhibition of FA oxidation (FAOX) has been suggested to ameliorate IR. However, prolonged inhibition of FAOX would presumably cause lipid accumulation and thereby promote lipotoxicity. To understand the glycemic consequences of acute and prolonged FAOX inhibition, we treated mice with the carnitine palmitoyltransferase 1 (CPT-1) inhibitor, etomoxir (eto), in combination with short-term 45% high fat diet feeding to increase FA availability. Eto acutely increased glucose oxidation and peripheral glucose disposal, and lowered circulating glucose, but this was associated with increased circulating FAs and triacylglycerol accumulation in the liver and heart within hours. Several days of FAOX inhibition by daily eto administration induced hepatic steatosis and glucose intolerance, specific to CPT-1 inhibition by eto. Lower whole-body insulin sensitivity was accompanied by reduction in brown adipose tissue (BAT) uncoupling protein 1 (UCP1) protein content, diminished BAT glucose clearance, and increased hepatic glucose production. Collectively, these data suggest that pharmacological inhibition of FAOX is not a viable strategy to treat IR, and that sufficient rates of FAOX are required for maintaining liver and BAT metabolic function.

Objective: The goal of this study was to investigate the importance of central hormone-sensitive lipase (HSL) expression in the regulation of food intake and body weight in mice to clarify whether intracellular lipolysis in the mammalian hypothalamus plays a role in regulating appetite. Methods: Using pharmacological and genetic approaches, we investigated the role of HSL in the rodent brain in the regulation of feeding and energy homeostasis under basal conditions during acute stress and high-fat diet feeding. Results: We found that HSL, a key enzyme in the catabolism of cellular lipid stores, is expressed in the appetite-regulating centers in the hypothalamus and is activated by acute stress through a mechanism similar to that observed in adipose tissue and skeletal muscle. Inhibition of HSL in rodent models by a synthetic ligand, global knockout, or brain-specific deletion of HSL prevents a decrease in food intake normally seen in response to acute stress and is associated with the increased expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Increased food intake can be reversed by adeno-associated virus-mediated reintroduction of HSL in neurons of the mediobasal hypothalamus. Importantly, metabolic stress induced by a high-fat diet also enhances the hyperphagic phenotype of HSL-deficient mice. Specific deletion of HSL in the ventromedial hypothalamic nucleus (VMH) or AgRP neurons reveals that HSL in the VMH plays a role in both acute stress-induced food intake and high-fat diet-induced obesity. Conclusions: Our results indicate that HSL activity in the mediobasal hypothalamus is involved in the acute reduction in food intake during the acute stress response and sensing of a high-fat diet.

Objective: Exercise is a cornerstone in the management of skeletal muscle insulin-resistance. A well-established benefit of a single bout of exercise is increased insulin sensitivity for hours post-exercise in the previously exercised musculature. Although rodent studies suggest that the insulin-sensitization phenomenon involves enhanced insulin-stimulated GLUT4 cell surface translocation and might involve intramuscular redistribution of GLUT4, the conservation to humans is unknown. Methods: Healthy young males underwent an insulin-sensitizing one-legged kicking exercise bout for 1 h followed by fatigue bouts to exhaustion. Muscle biopsies were obtained 4 h post-exercise before and after a 2-hour hyperinsulinemic-euglycemic clamp. Results: A detailed microscopy-based analysis of GLUT4 distribution within seven different myocellular compartments revealed that prior exercise increased GLUT4 localization in insulin-responsive storage vesicles and T-tubuli. Furthermore, insulin-stimulated GLUT4 localization was augmented at the sarcolemma and in the endosomal compartments. Conclusions: An intracellular redistribution of GLUT4 post-exercise is proposed as a molecular mechanism contributing to the insulin-sensitizing effect of prior exercise in human skeletal muscle.

Objective: Acute administration of the main protein component of high-density lipoprotein, apolipoprotein A-I (ApoA-1), improves glucose uptake in skeletal muscle. The molecular mechanisms mediating this are not known, but in muscle cell cultures, ApoA-1 failed to increase glucose uptake when infected with a dominant-negative AMP-activated protein kinase (AMPK) virus. We therefore investigated whether AMPK is necessary for ApoA-1-stimulated glucose uptake in intact heart and skeletal muscle in vivo. Methods: The effect of injection with recombinant human ApoA-1 (rApoA-1) on glucose tolerance, glucose-stimulated insulin secretion, and glucose uptake into skeletal and heart muscle with and without block of insulin secretion by injection of epinephrine (0.1 mg/kg) and propranolol (5 mg/kg), were investigated in 8 weeks high-fat diet-fed (60E%) wild-type and AMPKα2 kinase-dead mice in the overnight-fasted state. In addition, the effect of rApoA-1 on glucose uptake in isolated skeletal muscle ex vivo was studied. Results: rApoA-1 lowered plasma glucose concentration by 1.7 mmol/l within 3 h (6.1 vs 4.4 mmol/l; p

Objective: A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in prior exercised muscle. Our objective was to illuminate molecular mechanisms underlying this phenomenon in skeletal muscle of man. Methods: We studied the temporal regulation of glycogen supercompensation in human skeletal muscle during a 5 day recovery period following a single bout of exercise. Nine healthy men depleted (day 1), normalized (day 2) and supercompensated (day 5) muscle glycogen in one leg while the contralateral leg served as a resting control. Euglycemic hyperinsulinemic clamps in combination with leg balance technique allowed for investigating insulin-stimulated leg glucose uptake under these 3 experimental conditions. Cellular signaling in muscle biopsies was investigated by global proteomic analyses and immunoblotting. We strengthened the validity of proposed molecular effectors by follow-up studies in muscle of transgenic mice. Results: Sustained activation of glycogen synthase (GS) and AMPK in combination with elevated expression of proteins determining glucose uptake capacity were evident in the prior exercised muscle. We hypothesize that these alterations offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen. In line with key roles of AMPK and GS seen in the human experiments we observed abrogated ability for glycogen supercompensation in muscle with inducible AMPK deletion and in muscle carrying a G6P-insensitive form of GS in muscle. Conclusion: Our study demonstrates that both AMPK and GS are key regulators of glycogen supercompensation following a single bout of glycogen-depleting exercise in skeletal muscle of both man and mouse. Keywords: AMP-activated protein kinase (AMPK), TBC1 domain family member 4 (TBC1D4), Glycogen synthase (GS), Glucose uptake, Exercise, Insulin action

Objective: The growth differentiation factor 15 (GDF15) is a stress-sensitive circulating factor that regulates systemic energy balance. Since exercise is a transient physiological stress that has pleiotropic effects on whole-body energy metabolism, we herein explored the effect of exercise on a) circulating GDF15 levels and b) GDF15 release from skeletal muscle in humans. Methods: Seven healthy males either rested or exercised at 67% of their VO2max for 1 h and blood was sampled from the femoral artery and femoral vein before, during, and after exercise. Plasma GDF15 concentrations were determined in these samples. Results: Plasma GDF15 levels increased 34% with exercise (p

Objective: Dietary protein dilution (PD) has been associated with metabolic advantages such as improved glucose homeostasis and increased energy expenditure. This phenotype involves liver-induced release of FGF21 in response to amino acid insufficiency; however, it has remained unclear whether dietary dilution of specific amino acids (AAs) is also required. Circulating branched chain amino acids (BCAAs) are sensitive to protein intake, elevated in the serum of obese humans and mice and thought to promote insulin resistance. We tested whether replenishment of dietary BCAAs to an AA-diluted (AAD) diet is sufficient to reverse the glucoregulatory benefits of dietary PD. Methods: We conducted AA profiling of serum from healthy humans and lean and high fat-fed or New Zealand obese (NZO) mice following dietary PD. We fed wildtype and NZO mice one of three amino acid defined diets: control, total AAD, or the same diet with complete levels of BCAAs (AAD + BCAA). We quantified serum AAs and characterized mice in terms of metabolic efficiency, body composition, glucose homeostasis, serum FGF21, and tissue markers of the integrated stress response (ISR) and mTORC1 signaling. Results: Serum BCAAs, while elevated in serum from hyperphagic NZO, were consistently reduced by dietary PD in humans and murine models. Repletion of dietary BCAAs modestly attenuated insulin sensitivity and metabolic efficiency in wildtype mice but did not restore hyperglycemia in NZO mice. While hepatic markers of the ISR such as P-eIF2α and FGF21 were unabated by dietary BCAA repletion, hepatic and peripheral mTORC1 signaling were fully or partially restored, independent of changes in circulating glucose or insulin. Conclusions: Repletion of BCAAs in dietary PD is sufficient to oppose changes in somatic mTORC1 signaling but does not reverse the hepatic ISR nor induce insulin resistance in type 2 diabetes during dietary PD.

Objective: Fibroblast growth factor 21 (FGF21) shows great potential for the treatment of obesity and type 2 diabetes, as its long-acting analogue reduces body weight and improves lipid profiles of participants in clinical studies; however, the intracellular mechanisms mediating these effects are poorly understood. AMP-activated protein kinase (AMPK) is an important energy sensor of the cell and a molecular target for anti-diabetic medications. This work examined the role of AMPK in mediating the glucose and lipid-lowering effects of FGF21. Methods: Inducible adipocyte AMPK β1β2 knockout mice (iβ1β2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received long-acting FGF21 for two weeks. Results: Consistent with previous studies, FGF21 treatment significantly reduced body weight, adiposity, and liver lipids in HFD fed mice. To add, FGF21 improved circulating lipids, glycemic control, and insulin sensitivity. These effects were independent of adipocyte AMPK and were not associated with changes in browning of white (WAT) and brown adipose tissue (BAT). Lastly, we assessed whether FGF21 exerted its effects through the AMPK/ACC axis, which is critical in the therapeutic benefits of the anti-diabetic medication metformin. ACC DKI mice had improved glucose and insulin tolerance and a reduction in body weight, body fat and hepatic steatosis similar to WT mice in response to FGF21 administration. Conclusions: These data illustrate that the metabolic improvements upon FGF21 administration are independent of adipocyte AMPK, and do not require the inhibitory action of AMPK on ACC. This is in contrast to the anti-diabetic medication metformin and suggests that the treatment of obesity and diabetes with the combination of FGF21 and AMPK activators merits consideration. Keywords: FGF21, AMPK, ACC, Adipocyte, Brown fat, Obesity, Diabetes

Objective: Fibroblast-growth factor 21 (FGF21) is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF21 levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must be increased to meet eucaloric balance. This raises the possibility that intake of a diet rich in carbohydrates may induce an increase in plasma FGF21 levels per se. Here we studied the role of dietary carbohydrates on the levels of circulating FGF21 and concomitant physiologic effects by feeding healthy men a carbohydrate rich diet without reducing protein intake. Methods: A diet enriched in carbohydrates (80 E% carbohydrate; CHO) and a eucaloric control diet (CON) were provided to nine healthy men for three days. The energy intake during the CHO diet was increased (+75% energy) to ensure similar dietary protein intake in CHO and CON. To control for the effect of caloric surplus, we similarly overfed (+75% energy) the same subjects for three days with a fat-rich diet (78 E% fat; FAT), consisting of primarily unsaturated fatty acids. The three diets were provided in random order. Results: After CHO, plasma FGF21 concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml−1, p

Objective: We have recently shown that acute inhibition of both mTOR complexes (mTORC1 and mTORC2) increases whole-body lipid utilization, while mTORC1 inhibition had no effect. Therefore, we tested the hypothesis that mTORC2 regulates lipid metabolism in skeletal muscle. Methods: Body composition, substrate utilization and muscle lipid storage were measured in mice lacking mTORC2 activity in skeletal muscle (specific knockout of RICTOR (Ric mKO)). We further examined the RICTOR/mTORC2-controlled muscle metabolome and proteome; and performed follow-up studies in other genetic mouse models and in cell culture. Results: Ric mKO mice exhibited a greater reliance on fat as an energy substrate, a re-partitioning of lean to fat mass and an increase in intramyocellular triglyceride (IMTG) content, along with increases in several lipid metabolites in muscle. Unbiased proteomics revealed an increase in the expression of the lipid droplet binding protein Perilipin 3 (PLIN3) in muscle from Ric mKO mice. This was associated with increased AMPK activity in Ric mKO muscle. Reducing AMPK kinase activity decreased muscle PLIN3 expression and IMTG content. AMPK agonism, in turn, increased PLIN3 expression in a FoxO1 dependent manner. PLIN3 overexpression was sufficient to increase triglyceride content in muscle cells. Conclusions: We identified a novel link between mTORC2 and PLIN3, which regulates lipid storage in muscle. While mTORC2 is a negative regulator, we further identified AMPK as a positive regulator of PLIN3, which impacts whole-body substrate utilization and nutrient partitioning. Keywords: PLIN3, RICTOR, mTOR, Metabolism, Akt

Background & aims: Fibroblast growth factor 21 (FGF-21) is a liver-derived metabolic regulator induced by energy deprivation. However, its regulation in humans is incompletely understood. We addressed the origin and regulation of FGF-21 secretion in humans. Methods: By determination of arterial-to-venous differences over the liver and the leg during exercise, we evaluated the organ-specific secretion of FGF-21 in humans. By four different infusion models manipulating circulating glucagon and insulin, we addressed the interaction of these hormones on FGF-21 secretion in humans. Results: We demonstrate that the splanchnic circulation secretes FGF-21 at rest and that it is rapidly enhanced during exercise. In contrast, the leg does not contribute to the systemic levels of FGF-21. To unravel the mechanisms underlying the regulation of exercise-induced hepatic release of FGF-21, we manipulated circulating glucagon and insulin. These studies demonstrated that in humans glucagon stimulates splanchnic FGF-21 secretion whereas insulin has an inhibitory effect. Conclusions: Collectively, our data reveal that 1) in humans, the splanchnic bed contributes to the systemic FGF-21 levels during rest and exercise; 2) under normo-physiological conditions FGF-21 is not released from the leg; 3) a dynamic interaction of glucagon-to-insulin ratio regulates FGF-21 secretion in humans.

Skeletal myocytes are metabolically active and susceptible to insulin resistance and are thus implicated in type 2 diabetes (T2D). This complex disease involves systemic metabolic changes, and their elucidation at the systems level requires genome-wide data and biological networks. Genome-scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism, connected through the downregulated dihydrolipoamide dehydrogenase. Strikingly, the gene signature underlying this metabolic regulation successfully classifies the disease state of individual samples, suggesting that regulation of these pathways is a ubiquitous feature of myocytes in response to T2D.

Summary: The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-κB; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LTβR-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans. Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive. : Proteolytic precursor processing is a hallmark of the NF-κB system. Yilmaz et al. show that in lymphotoxin-stimulated cells p100 acts upstream of p105, resulting in concurrent production of p52 and p50. Both precursors form complexes and bind to segregase (p97/VCP), which promotes proteasomal processing. The findings are supported by mass spectrometry and incorporated in quantitative mathematical models.

The primary aim of the present study was to investigate in which cellular compartments fatty acid trans­locase CD36 (FAT/CD36) is localized. Intact and fully functional skeletal muscle mitochondria were isolated from lean and obese female Zucker rats and from 10 healthy male individuals. FAT/CD36 could not be detected in the isolated mitochondria, whereas the mitochondrial marker F1ATPase-β was clearly detected using immunoblotting. Lack of markers for other membrane structures indicated that the mitochondria were not contaminated with membranes known to contain FAT/CD36. In addition, fluorescence immunocytochemistry was performed on single muscle fibers dissected from soleus muscle of lean and obese Zucker rats and from the vastus lateralis muscle from humans. Costaining against FAT/CD36 and MitoNEET clearly show that FAT/CD36 is highly present in sarcolemma and it also associates with some vesicle-like intracellular compartments. However, FAT/CD36 protein was not detected in mitochondrial membranes, supporting the biochemical findings. Based on the presented data, FAT/CD36 seems to be abundantly expressed in sarcolemma and in vesicle-like structures throughout the muscle cell. However, FAT/CD36 is not present in mitochondria in rat or human skeletal muscle. Thus, the functional role of FAT/CD36 in lipid transport seems primarily to be allocated to the plasma membrane in skeletal muscle.

FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.

FAT/CD36 is a transmembrane protein that is thought to facilitate cellular long-chain fatty acid uptake. However, surprisingly little is known about the localization of FAT/CD36 in human skeletal muscle. By confocal immunofluorescence microscopy, we demonstrate high FAT/CD36 expression in endothelial cells and weaker but significant FAT/CD36 expression in sarcolemma in human skeletal muscle. No apparent intracellular staining was observed in the muscle cells. There are indications in the literature that caveolae may be involved in the uptake of fatty acids, possibly as regulators of FAT/CD36 or other fatty acid transporters.We show that in sarcolemma, FAT/CD36 colocalizes with the muscle-specific caveolae marker protein caveolin-3, suggesting that caveolae may regulate cellular fatty acid uptake by FAT/CD36. Furthermore, we provide evidence that FAT/CD36 expression is significantly higher in type 1 compared with type 2 fibers, whereas caveolin-3 expression is significantly higher in type 2 fibers than in type 1 fibers.

Vipera berus (V. berus) bites are associated with high morbidity, including kidney injury, in dogs. Although antivenom is often used and perceived effective to treat this type of snakebite, it is costly and associated with adverse events and specific diagnostics for this type of snakebite are lacking. We sought to improve diagnostics in V. berus envenomation by using currently available tools, including evaluating urinary albumin as a biomarker for snakebite-associated kidney injury. Additionally, we planned to adapt a method from human medicine for venom detection in clinical samples from bitten dogs and describe the composition of Norwegian V. berus venom. Serum biochemical analytes and urine albumin (ELISA) were measured in samples collected at 24 hours and two weeks after bite in 29 envenomated dogs. An adapted ELISA was applied to detect venom in urine and plasma collected from 25 cases between presentation and 24 hours after bite, using a commercial antivenom as the capture and detection antibody. Proteomic analysis of venom collected from 11 V. berus was performed using liquid chromatography-tandem mass spectrometry. Elevated serum C-reactive protein, creatine kinase, and aspartate aminotransferase were common for the case group. Although no case dogs showed acute kidney injury with azotemia and/or reduced urine output, elevated urinary albumin concentrations may indicate early or mild kidney injury in some case dogs. The venom ELISA detected positive signals in both plasma and urine for up to 24 hours after bite. However, with false positives detected in plasma, urine seemed to be the most appropriate body fluid for this assay. The venom proteome identified L-amino acid oxidases as the dominant component. In conclusion, serum biochemical and urinary albumin analyses are useful tools for evaluating canine V. berus envenomation. The venom ELISA is proposed as a promising tool for studies of V. berus envenomation and future diagnostic test development. Venom from the studied Norwegian V. berus was shown to differ considerably from previous reports from other countries, implying geographical variation in composition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Weight reduction may reduce the severity of psoriasis, but little is known about the long-term effects., Objective: We aimed to investigate long-term effects of weight reduction in psoriasis., Design: We previously conducted a randomized trial (n = 60) involving patients with psoriasis who were allocated to a control group or a low-energy diet (LED) group. Here we followed the participants for an additional 48-wk period. In total, 56 patients with psoriasis [mean ± SD body mass index (in kg/m(2)): 34.4 ± 5.3] underwent a 64-wk weight-loss program consisting of an initial 16-wk randomized phase with an LED for 8 wk and 8 wk of normal food intake combined with 2 LED products/d, followed by a 48-wk period of weight maintenance with the latter diet. After the randomization phase, the control group received the same 8 + 8-wk LED intervention, and all patients were then followed for 48 wk while on the weight-loss maintenance diet. The main outcome was the Psoriasis Area and Severity Index (PASI), and secondary outcome was the Dermatology Life Quality Index (DLQI)., Results: For the present study, 56 patients were eligible, 38 agreed to participate, and 32 completed. After the 16-wk LED-only period, the mean weight loss was -15.0 kg (95% CI: -16.6, -13.4 kg), and PASI and DLQI were reduced by -2.3 (95% CI: -3.1, -1.5) and -2.3 (95% CI: -3.2, -1.4), respectively. At week 64, the mean weight loss compared with baseline was -10.1 kg (95% CI: -12.0, -8.1 kg), and PASI and DLQI were maintained at -2.9 (95% CI: -3.9, -1.9) and -1.9 (95% CI: -3.0, -0.9), respectively., Conclusion: Long-term weight loss in patients with psoriasis has long-lasting positive effects on the severity of psoriasis. This trial was registered at clinicaltrials.gov as NCT01137188., (© 2016 American Society for Nutrition.)

Introduction: Biomechanical tests on bones are frequently accomplished in anatomically fixed tissues. The effects of ethanol or formaldehyde based fixation in bone material properties are subject to controversial discussions, regarding their appropriateness and usability to answer clinical questioning or biomechanical issues. We hypothesized that ethanol and formaldehyde irreversibly change bone material properties, and that this effect is mainly related to the bone's organic matrix., Material and Methods: Fixation related alterations in material properties were investigated in six fresh and two macerated human coxal bones by means of three-dimensional laser vibrometry based modal analysis. Ethanol or formaldehyde fixation were performed in one macerated and three unfixed specimens each. Changes in specimen weight and modal frequencies related to fixation, rinsing and drying were obtained. Modal assurance criterion (MAC) values were recorded to determine altered bone anisotropy., Results: Due to fixation, modal frequencies were irreversibly altered in unfixed specimens, indicating weight loss in ethanol and structural changes in formaldehyde fixed specimens. In the macerated and inorganic controls, fixation related weight and modal frequency changes were reversible by rinsing. In the unfixed specimens, bone anisotropy was irreversibly altered by both modes of fixation, whereas the fixation related changes in bony anisotropy were reversible in the macerated controls after rinsing., Discussion: Anatomical fixation that includes ethanol or formaldehyde irreversibly alters material properties of unfixed bones and impacts bone anisotropic properties, caused by changes in the organic matrix. In macerated bones that exclusively consisted of inorganic mineral salts, the observed effects on material properties and anisotropy were reversible. Conclusively, anatomical fixation on basis of ethanol or formaldehyde cannot be recommended, if material characteristics close to the vital state are of interest. Modal analysis is a potential method to gain insight into material properties, revealing the influence of the organic bone matrix on coxal bone elasticity., (Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.)

In a non-hypothesis driven metabolomics approach plasma samples collected at six different time points (before, during and after an exercise bout) were analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS). Since independent component analysis (ICA) does not need a priori information on the investigated process and moreover can separate statistically independent source signals with non-Gaussian distribution, we aimed to elucidate the analytical power of ICA for the metabolic pattern analysis and the identification of key metabolites in this exercise study. A novel approach based on descriptive statistics was established to optimize ICA model. In the GC-TOF MS data set the number of principal components after whitening and the number of independent components of ICA were optimized and systematically selected by descriptive statistics. The elucidated dominating independent components were involved in fuel metabolism, representing one of the most affected metabolic changes occurring in exercising humans. Conclusive time dependent physiological changes of the metabolic pattern under exercise conditions were detected. We conclude that after optimization ICA can successfully elucidate key metabolite pattern as well as characteristic metabolites in metabolic processes thereby simplifying the explanation of complex biological processes. Moreover, ICA is capable to study time series in complex experiments with multi-levels and multi-factors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Obese and lean humans treated with leptin have not experienced convincing weight-loss results compared with the dramatic weight losses observed in obese rodents., Objective: We sought to investigate the effect of acutely elevating leptin to concentrations observed in obese individuals on muscle and adipose tissue metabolism and muscle signaling in healthy lean males., Design: Healthy, lean, postabsorptive males were infused with either recombinant human leptin (rhleptin; n = 8) or saline (control; n = 8) for 4 h, which elicited leptin concentrations of ~ 20 and ~ 1 ng/mL, respectively. Systemic, skeletal muscle, and adipose tissue fat and glucose metabolism in vivo were assessed before, during, and 2 h after cessation of the infusion. Skeletal muscle biopsy specimens were obtained to quantify changes in signal transducers and activators of transcription-5'AMP-activated protein kinase (STAT-AMPK) signaling., Results: During the infusion of rhleptin, no differences in either systemic, skeletal muscle, or adipose tissue glucose or fat metabolism were observed. These observations were made despite increased activation of STAT (~ 17-fold) and AMPK (1.43-fold) after 1 h of rhleptin infusion. After the rhleptin infusion, an increase in systemic palmitate and fat oxidation was observed (P < 0.0003), which likely was caused by a concomitant increase in skeletal muscle palmitate oxidation (P < 0.02). This was observed despite lowered leptin concentrations and basal skeletal muscle STAT-AMPK signaling., Conclusions: Elevating circulating leptin concentrations to concentrations comparable with those of obese individuals increases human in vivo skeletal muscle signaling through the AMPK pathway and causes an increase in skeletal muscle fatty acid oxidation. Abdominal adipose tissue was unaffected by the acute physiologic increase in leptin concentrations.

Physical inactivity has recently been identified as a major and independent risk factor for the development of dementia and cognitive decline. In addition to the effect of exercise with regard to protection against neurodegenerative diseases, it is well-established that physical inactivity increases the risk of type 2 diabetes, cardiovascular diseases (CVD), colon cancer and postmenopausal breast cancer. These diseases constitute a network of related diseases, also called "the diseasome of physical inactivity". In this review, physical inactivity is given the central role as an independent and strong risk factor for accumulation of visceral fat and consequently the activation of a network of systemic inflammatory pathways, which promote development of neurodegeneration as well as insulin resistance, atherosclerosis, and tumour growth. The recent finding that muscles produce and release myokines provides a conceptual basis for understanding some of the molecular mechanisms underlying organ cross talk, including muscle-fat cross talk. Accumulating data suggest that contracting skeletal muscles release myokines, which may work in a hormone-like fashion, exerting specific endocrine effects on visceral fat or mediating direct anti-inflammatory effects. Other myokines work locally within the muscle via paracrine mechanisms, exerting their effects on signalling pathways involved in fat oxidation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To investigate the association of the levels of methylglyoxal-derived hydroimidazolone AGE modified proteins (MG-H1-AGE) with cardiovascular disease (CVD) mortality in an 18-year follow-up study in Finnish nondiabetic and diabetic subjects., Methods: The study design was a nested case-control study. Serum MG-H1-AGE levels in samples drawn at baseline were measured with a DELFIA type immunoassay in 220 diabetic subjects and 61 nondiabetic subjects who died from CVD during the follow-up, and age- and gender-matched 157 diabetic subjects and 159 nondiabetic subjects who did not die from CVD., Results: In type 2 diabetic subjects serum MG-H1-AGE levels were similar in subjects who died from CVD and in subjects who did not, 32.6 (24.6-42.1) (median (interquartile range)) vs. 31.3 (22.5-40.7)U/mL (p=0.281). In nondiabetic subjects serum MG-H1 levels were significantly higher in subjects who died from CVD than in subjects who were alive, 35.4 (28.1-44.7) vs. 31.3 (24.2-38.6)U/mL (p=0.025). Corresponding MG-H1 levels were 41.2 (35.6-58.7) vs. 31.1 (26.7-35.7)U/mL, p=0.003, in women, and 34.4 (26.3-41.2) vs. 32.0 (22.8-40.3)U/mL, p=0.270, in men. Multivariate logistic regression analysis showed a significant association of serum levels of MG-H1-AGE with CVD mortality in nondiabetic women (adjusted p=0.021), but not in nondiabetic men., Conclusions: Our 18-year follow-up study shows that high baseline serum levels of MG-H1 type of AGE modified proteins were associated with CVD mortality in nondiabetic women, but not in nondiabetic men or in diabetic subjects.

Background: The importance of the postprandial glycemic and insulinemic responses for appetite and energy intake (EI) is controversial., Objective: The aim of the study was to test the hypothesis that postprandial appetite sensations and subsequent EI are determined by postprandial glycemic and insulinemic responses after the intake of a range of breakfast meals., Design: The study was a randomized, crossover meal test including 28 healthy young men, each of whom tested 10 of 14 breakfast meals. Each meal contained 50 g carbohydrate with various glycemic index and energy and macronutrient contents. Blood samples were taken, and appetite sensations were measured 3 h after the meals. Subsequently, EI at lunch (EI(lunch)) was recorded., Results: The glycemic response was unrelated to appetite sensations, whereas the insulinemic response was positively associated with postprandial fullness (R2 = 0.33, P < 0.05). In contrast, the insulinemic response was unrelated to the subsequent EI(lunch), whereas the glycemic response was positively associated with EI(lunch) (R2 = 0.33, P < 0.05). Although no significant difference in EI(lunch) was observed between different breakfast conditions, a low breakfast EI was associated with a high EI(lunch) (R2 = 0.60, P < 0.001)., Conclusions: The current study does not support the contention that the postprandial glycemic response has an important effect on short-term appetite sensations, but a low-glycemic index meal may reduce subsequent EI. In contrast, postprandial insulin seems to affect short-term appetite sensations.

Heat shock protein (Hsp) 72 is a cytosolic stress protein that is highly inducible by several factors including exercise. Hsp60 is primarily mitochondrial in cellular location, plays a key role in the intracellular protein translocation and cytoprotection, is increased in skeletal muscle by exercise, and is found in the peripheral circulation of healthy humans. Glucose deprivation increases Hsp72 in cultured cells, whereas reduced glycogen availability elevates Hsp72 in contracting human skeletal muscle. To determine whether maintained blood glucose during exercise attenuates the exercise-induced increase in intramuscular and circulating Hsp72 and Hsp60, 6 males performed 120 minutes of semirecumbent cycling at approximately 65% maximal oxygen uptake on 2 occasions while ingesting either a 6.4% glucose (GLU) or sweet placebo (CON) beverage throughout exercise. Muscle biopsies, obtained before and immediately after exercise, were analyzed for Hsp72 and Hsp60 protein expression. Blood samples were simultaneously obtained from a brachial artery, a femoral vein, and the hepatic vein before and during exercise for the analysis of serum Hsp72 and Hsp60. Leg and hepatosplanchnic blood flow were measured to determine Hsp72-Hsp60 flux across these tissue beds. Neither exercise nor glucose ingestion affected the Hsp72 or Hsp60 protein expression in, or their release from, contracting skeletal muscle. Arterial serum Hsp72 increased (P < 0.05) throughout exercise in both trials but was attenuated (P < 0.05) in GLU. This may have been in part because of the increased (P < 0.05) hepatosplanchnic Hsp72 release in CON, being totally abolished (P < 0.05) in GLU. Serum Hsp60 increased (P < 0.05) after 60 minutes of exercise in CON before returning to resting levels at 120 minutes. In contrast, no exercise-induced increase in serum Hsp60 was observed in GLU. We detected neither hepatosplanchnic nor contracting limb Hsp60 release in either trial. In conclusion, maintaining glucose availability during exercise attenuates the circulating Hsp response in healthy humans.

Following exercise, AMP-activated protein kinase (AMPK) activity is increased several fold in rat liver and adipose tissue as well as muscle; however, the mechanism by which this occurs is not known. Interleukin-6 (IL-6) is released from muscle in large amounts during and after sustained physical activity resulting in up to 100-fold increases in its plasma concentration, from 1-2 ng/ml to 50-100 ng/ml. We report here that incubation with IL-6 (30-120 ng/ml) increases the phosphorylation of AMPK (an indicator of its activation) and that of its target molecule, acetyl CoA carboxylase (ACC), in both extensor digitorum longus muscle and cultured F422a adipocytes. To assess more directly whether IL-6 regulates AMPK in vivo during exercise, measurements were carried out in skeletal muscle, liver, and adipose tissue of 3-month-old IL-6 knockout (IL-6(-/-)) and C57 black control mice. In agreement with previous studies in the rat, in control mice P-AMPK and P-ACC abundance was increased by 30-150% in the three tissues in response to exercise with the greatest increases in skeletal muscle. In contrast, in IL-6(-/-) mice, we found that the abundance of both P-AMPK and P-ACC was lower (60-90%) in muscle and adipose tissue at rest. Also the absolute increases in P-AMPK caused by exercise were diminished compared to those in control mice, although percentage increases were similar. In liver, decreases in P-AMPK and P-ACC in the IL-6(-/-) mice were more modest and the increases in their abundance caused by exercise were indistinguishable from those of control mice. The results indicate that IL-6 can activate AMPK in muscle and adipose tissue, and that this contributes to, but does not fully account for, the increase in AMPK activity in these tissues in response to exercise. They also suggest that a genetic lack of IL-6 is associated with a decrease in AMPK activity.

Interleukin-6 (IL-6) is a cytokine with immuno-regulatory functions. However, contracting skeletal muscle expresses and subsequently releases IL-6 in high amounts, and recent evidence in IL-6 deficient mice suggests a role of IL-6 in metabolism. Since IL-6 mRNA levels also increase in abdominal adipose tissue in response to exercise, we wanted to examine the possible existence of a positive feedback mechanism between muscle and adipose tissue. We obtained biopsies from human skeletal muscle and abdominal subcutaneous adipose tissue in relation to either 3h of bicycle exercise or recombinant human IL-6 infusion (rhIL-6) or saline infusion. In muscle, IL-6 mRNA increased (p<0.05) immediately after exercise, peaking at this time-point, whereas IL-6 mRNA in adipose tissue increased 1.5-h post exercise (p<0.05) displaying a different kinetic of induction. During rhIL-6 infusion, IL-6 mRNA increased 120-fold in muscle (p<0.05). In conclusion, the present study demonstrates that muscle IL-6 is regulated by an autocrine mechanism at the transcriptional level.

Inflammatory and oxidative stresses play a pivotal role in atherogenesis. Vitamin E and vitamin C are the two most important dietary antioxidants; moreover, vitamin E has anti-inflammatory effects. Combined supplementations with vitamin E and vitamin C twice daily for 3 y reduced lipid peroxidation and retarded the progression of common carotid atherosclerosis in healthy men in the Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study. To further elucidate the underlying mechanisms that retarded the progression of atherosclerosis in the ASAP study, we investigated the effect of a combined intake of vitamin E and vitamin C on inflammatory markers in vivo. Circulating levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and C reactive protein (CRP) were measured in 45- to 69-y-old men from the ASAP study with cholesterol >5.0 mmol/L before and after treatment with either placebo (n = 52) or a combined supplementation with 91 mg (136 IU) alpha-tocopherol and 250 mg of slow-release vitamin C twice a day (n = 55) for 3 y. Antioxidant treatment for 36 mo had no effect on circulating levels of TNF-alpha, IL-6 or CRP. In conclusion, long-term combined supplementations with alpha-tocopherol and vitamin C in reasonable doses have no detectable systemic anti-inflammatory effects in a healthy population of men with slight hypercholesterolemia and no overt signs of inflammation.