Body weight homeostat that regulates fat mass independently of leptin in rats and mice.

Subjects spending much time sitting have increased risk of obesity but the mechanism for the antiobesity effect of standing is unknown. We hypothesized that there is a homeostatic regulation of body weight. We demonstrate that increased loading of rodents, achieved using capsules with different weights implanted in the abdomen or s.c. on the back, reversibly decreases the biological body weight via reduced food intake. Importantly, loading relieves diet-induced obesity and improves glucose tolerance. The identified homeostat for body weight regulates body fat mass independently of fat-derived leptin, revealing two independent negative feedback systems for fat mass regulation. It is known that osteocytes can sense changes in bone strain. In this study, the body weight-reducing effect of increased loading was lost in mice depleted of osteocytes. We propose that increased body weight activates a sensor dependent on osteocytes of the weight-bearing bones. This induces an afferent signal, which reduces body weight. These findings demonstrate a leptin-independent body weight homeostat ("gravitostat") that regulates fat mass.

Antioxidant treatment induces reductive stress associated with mitochondrial dysfunction in adipocytes.

ß-Adrenergic stimulation of adipose tissue increases mitochondrial density and activity (browning) that are associated with improved whole-body metabolism. Whereas chronically elevated levels of reactive oxygen species (ROS) in adipose tissue contribute to insulin resistance, transient ROS elevation stimulates physiological processes such as adipogenesis. Here, using a combination of biochemical and cell and molecular biology-based approaches, we studied whether ROS or antioxidant treatment affects ß3-adrenergic receptor (ß3-AR) stimulation-induced adipose tissue browning. We found that ß3-AR stimulation increases ROS levels in cultured adipocytes, but, unexpectedly, pretreatment with different antioxidants (N-acetylcysteine, vitamin E, or GSH ethyl ester) did not prevent this ROS increase. Using fluorescent probes, we discovered that the antioxidant treatments instead enhanced ß3-AR stimulation-induced mitochondrial ROS production. This pro-oxidant effect of antioxidants was, even in the absence of ß3-AR stimulation, associated with decreased oxygen consumption and increased lactate production in adipocytes. We observed similar antioxidant effects in WT mice: N-acetylcysteine blunted ß3-AR stimulation-induced browning of white adipose tissue and reduced mitochondrial activity in brown adipose tissue even in the absence of ß3-AR stimulation. Furthermore, N-acetylcysteine increased the levels of peroxiredoxin 3 and superoxide dismutase 2 in adipose tissue, indicating increased mitochondrial oxidative stress. We interpret this negative impact of antioxidants on oxygen consumption in vitro and adipose tissue browning in vivo as essential adaptations that prevent a further increase in mitochondrial ROS production. In summary, these results suggest that chronic antioxidant supplementation can produce a paradoxical increase in oxidative stress associated with mitochondrial dysfunction in adipocytes.

Glucagon-Like Peptide-1-, but not Growth and Differentiation Factor 15-, Receptor Activation Increases the Number of Interleukin-6-Expressing Cells in the External Lateral Parabrachial Nucleus.

Interleukin (IL)-6 in the hypothalamus and hindbrain is an important downstream mediator of suppression of body weight and food intake by glucagon-like peptide-1 (GLP-1) receptor stimulation. CNS GLP-1 is produced almost exclusively in prepro-glucagon neurons in the nucleus of the solitary tract. These neurons innervate energy balance-regulating areas, such as the external lateral parabrachial nucleus (PBNel); essential for induction of anorexia. Using a validated novel IL-6-reporter mouse strain, we investigated the interactions in PBNel between GLP-1, IL-6, and calcitonin gene-related peptide (CGRP, a well-known mediator of anorexia). We show that PBNel GLP-1R-containing cells highly (to about 80%) overlap with IL-6-containing cells on both protein and mRNA level. Intraperitoneal administration of a GLP-1 analogue exendin-4 to mice increased the proportion of IL-6-containing cells in PBNel 3-fold, while there was no effect in the rest of the lateral parabrachial nucleus. In contrast, injections of an anorexigenic peptide growth and differentiation factor 15 (GDF15) markedly increased the proportion of CGRP-containing cells, while IL-6-containing cells were not affected. In summary, GLP-1R are found on IL-6-producing cells in PBNel, and GLP-1R stimulation leads to an increase in the proportion of cells with IL-6-reporter fluorescence, supporting IL-6 mediation of GLP-1 effects on energy balance.

Preproglucagon neurons in the hindbrain have IL-6 receptor-α and show Ca2+ influx in response to IL-6.

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

Glucagon-like peptide 1 receptor induced suppression of food intake, and body weight is mediated by central IL-1 and IL-6.

Glucagon-like peptide 1 (GLP-1), produced in the intestine and the brain, can stimulate insulin secretion from the pancreas and alleviate type 2 diabetes. The cytokine interleukin-6 (IL-6) may enhance insulin secretion from ß-cells by stimulating peripheral GLP-1 production. GLP-1 and its analogs also reduce food intake and body weight, clinically beneficial actions that are likely exerted at the level of the CNS, but otherwise are poorly understood. The cytokines IL-6 and interleukin 1ß (IL-1ß) may exert an anti-obesity effect in the CNS during health. Here we found that central injection of a clinically used GLP-1 receptor agonist, exendin-4, potently increased the expression of IL-6 in the hypothalamus (11-fold) and the hindbrain (4-fold) and of IL-1ß in the hypothalamus, without changing the expression of other inflammation-associated genes. Furthermore, hypothalamic and hindbrain interleukin-associated intracellular signals [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and suppressor of cytokine signaling-1 (SOCS1)] were also elevated by exendin-4. Pharmacologic disruption of CNS IL-1 receptor or IL-6 biological activity attenuated anorexia and body weight loss induced by central exendin-4 administration in a rat. Simultaneous blockade of IL-1 and IL-6 activity led to a more potent attenuation of exendin-4 effects on food intake. Mice with global IL-1 receptor gene knockout or central IL-6 receptor knockdown showed attenuated decrease in food intake and body weight in response to peripheral exendin-4 treatment. GLP-1 receptor activation in the mouse neuronal Neuro2A cell line also resulted in increased IL-6 expression. These data outline a previously unidentified role of the central IL-1 and IL-6 in mediating the anorexic and body weight loss effects of GLP-1 receptor activation.

Dietary polyunsaturated fatty acids increase survival and decrease bacterial load during septic Staphylococcus aureus infection and improve neutrophil function in mice.

Severe infection, including sepsis, is an increasing clinical problem that causes prolonged morbidity and substantial mortality. At present, antibiotics are essentially the only pharmacological treatment for sepsis. The incidence of resistance to antibiotics is increasing; therefore, it is critical to find new therapies for sepsis. Staphylococcus aureus is a major cause of septic mortality. Neutrophils play an important role in the defense against bacterial infections. We have shown that a diet with high levels of dietary saturated fatty acids decreases survival in septic mice, but the mechanisms behind this remain elusive. The aim of the present study was to investigate how the differences in dietary fat composition affect survival and bacterial load after experimental septic infection and neutrophil function in uninfected mice. We found that, after S. aureus infection, mice fed a polyunsaturated high-fat diet (HFD-P) for 8 weeks had increased survival and decreased bacterial load during sepsis compared with mice fed a saturated high-fat diet (HFD-S), similar to mice fed a low-fat diet (LFD). Uninfected mice fed HFD-P had a higher frequency of neutrophils in bone marrow than mice fed HFD-S. In addition, mice fed HFD-P had a higher frequency of neutrophils recruited to the site of inflammation in response to peritoneal injection of thioglycolate than mice fed HFD-S. Differences between the proportion of dietary protein and carbohydrate did not affect septic survival at all. In conclusion, polyunsaturated dietary fat increased both survival and efficiency of bacterial clearance during septic S. aureus infection. Moreover, this diet increased the frequency and chemotaxis of neutrophils, key components of the immune response to S. aureus infections.

Improved Memory and Lower Stress Levels in Male Mice Co-Housed with Ovariectomized Female Mice.

Aggressiveness, expressed by fighting, is a frequent problem in group-housed laboratory male mice and results in increased stress, injury, and death. One way to prevent fighting is by pairing the male mice with ovariectomized female mice to provide a compatible companion. However, the effect of these housing conditions remains unclear. Therefore, we aimed to evaluate behavior and stress levels in two different housing conditions, pair-housed with an ovariectomized female and group-housed with other males. Behavioral tests were performed to assess stress and anxiety-like behavior. Moreover, the corticosterone levels in plasma were measured by ELISA. Based on home cage behavior assessment, pair-housed male mice showed no signs of fighting, not even after isolation and regrouping. Our results also showed that the pair-housed males had a better memory and demonstrated less anxiety-like behavior. Subsequently, the pair-housed male mice had a larger reduction in corticosterone levels compared to group-housed males. Overall, pair-housing reduced anxiety-like behavior and stress levels in male mice compared to standard group-housing.

Adiponectin stimulates Sca1+CD34--adipocyte precursor cells associated with hyperplastic expansion and beiging of brown and white adipose tissue.

BACKGROUND: The adipocyte hormone adiponectin improves insulin sensitivity and there is an inverse correlation between adiponectin levels and type-2 diabetes risk. Previous research shows that adiponectin remodels the adipose tissue into a more efficient metabolic sink. For instance, mice that overexpress adiponectin show increased capacity for hyperplastic adipose tissue expansion as evident from smaller and metabolically more active white adipocytes. In contrast, the brown adipose tissue (BAT) of these mice looks "whiter" possibly indicating reduced metabolic activity. Here, we aimed to further establish the effect of adiponectin on adipose tissue expansion and adipocyte mitochondrial function as well as to unravel mechanistic aspects in this area. METHODS: Brown and white adipose tissues from adiponectin overexpressing (APN tg) mice and littermate wildtype controls, housed at room and cold temperature, were studied by histological, gene/protein expression and flow cytometry analyses. Metabolic and mitochondrial functions were studied by radiotracers and Seahorse-based technology. In addition, mitochondrial function was assessed in cultured adiponectin deficient adipocytes from APN knockout and heterozygote mice. RESULTS: APN tg BAT displayed increased proliferation prenatally leading to enlarged BAT. Postnatally, APN tg BAT turned whiter than control BAT, confirming previous reports. Furthermore, elevated adiponectin augmented the sympathetic innervation/activation within adipose tissue. APN tg BAT displayed reduced metabolic activity and reduced mitochondrial oxygen consumption rate (OCR). In contrast, APN tg inguinal white adipose tissue (IWAT) displayed enhanced metabolic activity. These metabolic differences between genotypes were apparent also in cultured adipocytes differentiated from BAT and IWAT stroma vascular fraction, and the OCR was reduced in both brown and white APN heterozygote adipocytes. In both APN tg BAT and IWAT, the mesenchymal stem cell-related genes were upregulated along with an increased abundance of Lineage-Sca1+CD34- "beige-like" adipocyte precursor cells. In vitro, the adiponectin receptor agonist Adiporon increased the expression of the proliferation marker Pcna and decreased the expression of Cd34 in Sca1+ mesenchymal stem cells. CONCLUSIONS: We propose that the seemingly opposite effect of adiponectin on BAT and IWAT is mediated by a common mechanism; while reduced adiponectin levels are linked to lower adipocyte OCR, elevated adiponectin levels stimulate expansion of adipocyte precursor cells that produce adipocytes with intrinsically higher metabolic rate than classical white but lower metabolic rate than classical brown adipocytes. Moreover, adiponectin can modify the adipocytes' metabolic activity directly and by enhancing the sympathetic innervation within a fat depot.

Reduction of body weight by increased loading is associated with activation of norepinephrine neurones in the medial nucleus of the solitary tract.

We previously provided evidence supporting the existence of a novel leptin-independent body weight homeostat ("the gravitostat") that senses body weight and then initiates a homeostatic feed-back regulation of body weight. We, herein, hypothesize that this feed-back regulation involves a CNS mechanism. To identify populations of neurones of importance for the putative feed-back signal induced by increased loading, high-fat diet-fed rats or mice were implanted intraperitoneally or subcutaneously with capsules weighing ∼15% (Load) or ∼2.5% (Control) of body weight. At 3-5 days after implantation, neuronal activation was assessed in different parts of the brain/brainstem by immunohistochemical detection of FosB. Implantation of weighted capsules, both subcutaneous and intraperitoneal, induced FosB in specific neurones in the medial nucleus of the solitary tract (mNTS), known to integrate information about the metabolic status of the body. These neurones also expressed tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbH), a pattern typical of norepinephrine neurones. In functional studies, we specifically ablated norepinephrine neurones in mNTS, which attenuated the feed-back regulation of increased load on body weight and food intake. In conclusion, increased load appears to reduce body weight and food intake via activation of norepinephrine neurones in the mNTS.

The gravitostat protects diet-induced obese rats against fat accumulation and weight gain.

The gravitostat is a novel homeostatic body weight-regulating mechanism, mostly studied in mice, and recently confirmed in obese humans. In the present study, we explored the effect of weight loading on metabolic outcomes, meal patterns and parameters linked to energy expenditure in both obese and lean rats. Diet-induced obese (DIO) and lean rats were implanted with capsules weighing either 15% of biological body weight (load) or empty capsules (1.3% of body weight; controls). Loading protected against fat accumulation more markedly in the DIO group. In line with this, the obesity-related impairment in insulin sensitivity was notably ameliorated in DIO rats upon loading, as revealed by the reduction in serum insulin levels and homeostatic model assessment for insulin resistance index scores. Although 24-hour caloric intake was reduced in both groups, this effect was greater in loaded DIO rats than in loaded lean peers. During days 10-16, after recovery from surgery, loading: (i) decreased meal size in both groups (only during the light phase in DIO rats) but this was compensated in lean rats by an increase in meal frequency; (ii) reduced dark phase locomotor activity only in lean rats; and (iii) reduced mean caloric efficiency in DIO rats. Muscle weight was unaffected by loading in either group. Dietary-obese rats are therefore more responsive than lean rats to loading.

A Body Weight Sensor Regulates Prepubertal Growth via the Somatotropic Axis in Male Rats.

In healthy conditions, prepubertal growth follows an individual specific growth channel. Growth hormone (GH) is undoubtedly the major regulator of growth. However, the homeostatic regulation to maintain the individual specific growth channel during growth is unclear. We recently hypothesized a body weight sensing homeostatic regulation of body weight during adulthood, the gravitostat. We now investigated if sensing of body weight also contributes to the strict homeostatic regulation to maintain the individual specific growth channel during prepubertal growth. To evaluate the effect of increased artificial loading on prepubertal growth, we implanted heavy (20% of body weight) or light (2% of the body weight) capsules into the abdomen of 26-day-old male rats. The body growth, as determined by change in biological body weight and growth of the long bones and the axial skeleton, was reduced in rats bearing a heavy load compared with light load. Removal of the increased load resulted in a catch-up growth and a normalization of body weight. Loading decreased hypothalamic growth hormone releasing hormone mRNA, liver insulin-like growth factor (IGF)-1 mRNA, and serum IGF-1, suggesting that the reduced body growth was caused by a negative feedback regulation on the somatotropic axis and this notion was supported by the fact that increased loading did not reduce body growth in GH-treated rats. Based on these data, we propose the gravitostat hypothesis for the regulation of prepubertal growth. This states that there is a homeostatic regulation to maintain the individual specific growth channel via body weight sensing, regulating the somatotropic axis and explaining catch-up growth.

Testosterone reduces metabolic brown fat activity in male mice.

Brown adipose tissue (BAT) burns substantial amounts of mainly lipids to produce heat. Some studies indicate that BAT activity and core body temperature are lower in males than females. Here we investigated the role of testosterone and its receptor (the androgen receptor; AR) in metabolic BAT activity in male mice. Castration, which renders mice testosterone deficient, slightly promoted the expression of thermogenic markers in BAT, decreased BAT lipid content, and increased basal lipolysis in isolated brown adipocytes. Further, castration increased the core body temperature. Triglyceride-derived fatty acid uptake, a proxy for metabolic BAT activity in vivo, was strongly increased in BAT from castrated mice (4.5-fold increase vs sham-castrated mice) and testosterone replacement reversed the castration-induced increase in metabolic BAT activity. BAT-specific AR deficiency did not mimic the castration effects in vivo and AR agonist treatment did not diminish the activity of cultured brown adipocytes in vitro, suggesting that androgens do not modulate BAT activity via a direct, AR-mediated pathway. In conclusion, testosterone is a negative regulator of metabolic BAT activity in male mice. Our findings provide new insight into the metabolic actions of testosterone.

Metabolic profiles of fat and glucose differ by gender in healthy 8-year-olds.

OBJECTIVE: The aim was to investigate if metabolic markers were associated with anthropometry and weight increase in healthy 8-year-olds. METHODS: Ninety-seven healthy children, 66 of whom had been examined at the age of 4 years, were investigated. Dual energy X-ray absorptiometry was performed to determine fat (FM) and lean body mass (LBM). Plasma glucose and serum levels of insulin, cholesterol, triglycerides, adiponectin and leptin were analysed and HOMA-indices were calculated. RESULTS: Despite similar anthropometry, metabolic markers differed by gender. Sixteen % of the children were overweight or obese. Body mass index (BMI) was strongly correlated to FM. Anthropometric measures except LBM correlated to metabolic markers in the girls. Boys had higher concentrations of plasma glucose than girls. In overweight children, insulin was negatively associated with LBM. Leptin and the ratio between leptin and adiponectin, but not adiponectin, were significantly associated with HOMA-IR and body composition. CONCLUSION: The metabolic profile of plasma glucose, serum leptin, fasting insulin and related HOMA indices differed by gender, despite no difference in BMI or FM. LBM, but not FM correlated to the insulin concentration in the overweight children. Leptin was the best marker of overweight.

Increased weight loading reduces body weight and body fat in obese subjects - A proof of concept randomized clinical trial.

BACKGROUND: Recently we provided evidence for a leptin-independent homeostatic regulation, the gravitostat, of body weight in rodents. The aim of the present translational proof of concept study was to test the gravitostat hypothesis in humans. METHODS: We conducted a randomized controlled single center trial (ClinicalTrial.gov number, NCT03672903), to evaluate the efficacy of artificially increased weight loading on body weight in subjects with mild obesity (BMI 30-35 kg/m2). Subjects were either treated with a heavy (=high load; 11% of body weight) or light (=low load; 1% of body weight) weight vest for eight hours per day for three weeks. The primary outcome was change in body weight. Secondary outcomes included change in body fat mass and fat-free mass as measured using bioelectrical impedance analysis. FINDINGS: In total 72 participants underwent randomization and 69 (36 high load and 33 low load) completed the study for the primary outcome. High load treatment resulted in a more pronounced relative body weight loss compared to low load treatment (mean difference -1.37%, 95% confidence interval (CI), -1.96 to -0.79; p = 1.5 × 10-5). High load treatment reduced fat mass (-4.04%, 95% CI, -6,53 to -1.55; p = 1.9 × 10-3) but not fat free mass (0.43%, 95% CI, -1.47 to 2.34; p = 0.65) compared to low load treatment. INTERPRETATION: Increased weight loading reduces body weight and fat mass in obese subjects in a similar way as previously shown in obese rodents. These findings demonstrate that there is weight loading dependent homeostatic regulation of body weight, the gravitostat, also in humans. FUNDING: Funded by Jane and Dan Olsson (JADO) Foundation, the Torsten Söderberg Foundation, The Knut and Alice Wallenberg's Foundation and the Novo Nordisk Foundation.

BMP4 gene therapy enhances insulin sensitivity but not adipose tissue browning in obese mice.

OBJECTIVE: Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity. METHODS: Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17-18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10-12 weeks. Following termination, the mice underwent additional characterization in vitro. RESULTS: Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver. CONCLUSIONS: Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning.

Interactions Between the Gravitostat and the Fibroblast Growth Factor System for the Regulation of Body Weight.

Both fibroblast growth factors (FGFs), by binding to FGF receptors (FGFRs), and activation of the gravitostat, by artificial loading, decrease the body weight (BW). Previous studies demonstrate that both the FGF system and loading have the capacity to regulate BW independently of leptin. The aim of the current study was to determine the possible interactions between the effect of increased loading and the FGF system for the regulation of BW. We observed that the BW-reducing effect of increased loading was abolished in mice treated with a monoclonal antibody directed against FGFR1c, suggesting interactions between the two systems. As serum levels of endocrine FGF21 and hepatic FGF21 mRNA were increased in the loaded mice compared with the control mice, we first evaluated the loading response in FGF21 over expressing mice with constant high FGF21 levels. Leptin treatment, but not increased loading, decreased the BW in the FGF21-overexpressing mice, demonstrating that specifically the loading effect is attenuated in the presence of high activity in the FGF system. However, as FGF21 knockout mice displayed a normal loading response on BW, FGF21 is neither mediating nor essential for the loading response. In conclusion, the BW-reducing effect of increased loading but not of leptin treatment is blocked by high activity in the FGF system. We propose that both the gravitostat and the FGF system regulate BW independently of leptin and that pharmacologically enhanced activity in the FGF system reduces the sensitivity of the gravitostat.

Interleukin-6 in the central amygdala is bioactive and co-localised with glucagon-like peptide-1 receptor.

Neuronal circuits involving the central amygdala (CeA) are gaining prominence as important centres for regulation of metabolic functions. As a part of the subcortical food motivation circuitry, CeA is associated with food motivation and hunger. We have previously shown that interleukin (IL)-6 can act as a downstream mediator of the metabolic effects of glucagon-like peptide-1 (GLP-1) receptor (R) stimulation in the brain, although the sites of these effects are largely unknown. In the present study, we used the newly generated and validated RedIL6 reporter mouse strain to investigate the presence of IL-6 in the CeA, as well as possible interactions between IL-6 and GLP-1 in this nucleus. IL-6 was present in the CeA, mostly in cells in the medial and lateral parts of this structure, and a majority of IL-6-containing cells also co-expressed GLP-1R. Triple staining showed GLP-1 containing fibres co-staining with synaptophysin close to or overlapping with IL-6 containing cells. GLP-1R stimulation enhanced IL-6 mRNA levels. IL-6 receptor-alpha (IL-6Rα) was found to a large part in neuronal CeA cells. Using electrophysiology, we determined that cells with neuronal properties in the CeA could be rapidly stimulated by IL-6 administration in vitro. Moreover, microinjections of IL-6 into the CeA could slightly reduce food intake in vivo in overnight fasted rats. In conclusion, IL-6 containing cells in the CeA express GLP-1R, are close to GLP-1-containing synapses, and demonstrate increased IL-6 mRNA in response to GLP-1R agonist treatment. IL-6, in turn, exerts biological effects in the CeA, possibly via IL-6Rα present in this nucleus.

Interleukin-6 is important for regulation of core body temperature during long-term cold exposure in mice.

Interleukin-6 (IL6) is a cytokine important for inducing the fever response during infection and has been reported to uphold core body temperature during acute cold exposure. Recently it has also been indicated that IL6 in serum increases in cold-exposed mice. The aim of the present study was to investigate if IL6 is important for core body temperature regulation following a long-term cold exposure in mice. Experiments were performed with global IL6 deficient (-/-) mice, mice with conditional IL6 receptor α (IL6Rα) knockdown in the central nervous system (CNS; IL6RαNesCre) and appropriate wild-type (Wt) controls. All mice were placed in a cold environment (4°C) for 6 days. Core body temperature and oxygen consumption were measured by telemetry probes and indirect calorimetry at room temperature (20°C), and during the first and last day of cold exposure. Brain stem, hypothalamus and white and brown adipose tissues from the cold-exposed mice were subjected to gene expression analysis. After 6 days in 4°C, the IL6-/- mice exhibited significantly lower body temperature and oxygen consumption compared with Wt mice (P<0.05). The IL6RαNesCre mice also exhibited lower body temperature compared with WtNesCre controls during the last day of cold exposure (P<0.05). Furthermore, an increase in the mRNA level of brain-derived neurotrophic factor (Bdnf) was detected in the brain stem of both IL6-/- and IL6RαNesCre mice compared with the Wt groups (P<0.05). The finding that body temperature was decreased in IL6-/- and IL6RαNesCre mice indicates a decrease in thermogenesis in these animals. Bdnf has previously been indicated to increase body temperature and could in the present study be a mechanistic factor involved in counteracting the low body temperature in IL6-/- and IL6RαNesCre mice. These results suggest that IL6 is not only involved in body temperature regulation during infection, but also during long-term cold exposure, probably through mechanisms in the CNS.

The Gravitostat Regulates Fat Mass in Obese Male Mice While Leptin Regulates Fat Mass in Lean Male Mice.

Leptin has been the only known homeostatic regulator of fat mass, but we recently found evidence for a second one, named the gravitostat. In the current study, we compared the effects of leptin and increased loading (gravitostat stimulation) on fat mass in mice with different levels of body weight (lean, overweight, and obese). Leptin infusion suppressed body weight and fat mass in lean mice given normal chow but not in overweight or obese mice given a high-fat diet for 4 and 8 weeks, respectively. The maximum effect of leptin on body weight and fat mass was obtained already at <44 ng/mL of serum leptin. Increased loading using intraperitoneal capsules with different weights decreased body weight in overweight and obese mice. Although the implantation of an empty capsule reduced the body weight in lean mice, only a nonsignificant tendency of a specific effect of increased loading was observed in the lean mice. These findings demonstrate that the gravitostat regulates fat mass in obese mice, whereas leptin regulates fat mass only in lean mice with low endogenous serum leptin levels. We propose that activation of the gravitostat primarily protects against obesity, whereas low levels of leptin protect against undernutrition.

BMP4 Gene Therapy in Mature Mice Reduces BAT Activation but Protects from Obesity by Browning Subcutaneous Adipose Tissue.

We examined the effect of Bone Morphogenetic Protein 4 (BMP4) on energy expenditure in adult mature mice by targeting the liver with adeno-associated viral (AAV) BMP4 vectors to increase circulating levels. We verified the direct effect of BMP4 in inducing a brown oxidative phenotype in differentiating preadipocytes in vitro. AAV-BMP4-treated mice display marked browning of subcutaneous adipocytes, with increased mitochondria and Uncoupling Protein 1 (UCP1). These mice are protected from obesity on a high-fat diet and have increased whole-body energy expenditure, improved insulin sensitivity, reduced liver fat, and reduced adipose tissue inflammation. On a control diet, they show unchanged body weight but improved insulin sensitivity. In contrast, AAV-BMP4-treated mice showed beiging of BAT with reduced UCP1, increased lipids, and reduced hormone-sensitive lipase (HSL). Thus, BMP4 exerts different effects on WAT and BAT, but the overall effect is to enhance insulin sensitivity and whole-body energy expenditure by browning subcutaneous adipose tissue.