Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions.

Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.

Butyrate reduces appetite and activates brown adipose tissue via the gut-brain neural circuit.

OBJECTIVE: Butyrate exerts metabolic benefits in mice and humans, the underlying mechanisms being still unclear. We aimed to investigate the effect of butyrate on appetite and energy expenditure, and to what extent these two components contribute to the beneficial metabolic effects of butyrate. DESIGN: Acute effects of butyrate on appetite and its method of action were investigated in mice following an intragastric gavage or intravenous injection of butyrate. To study the contribution of satiety to the metabolic benefits of butyrate, mice were fed a high-fat diet with butyrate, and an additional pair-fed group was included. Mechanistic involvement of the gut-brain neural circuit was investigated in vagotomised mice. RESULTS: Acute oral, but not intravenous, butyrate administration decreased food intake, suppressed the activity of orexigenic neurons that express neuropeptide Y in the hypothalamus, and decreased neuronal activity within the nucleus tractus solitarius and dorsal vagal complex in the brainstem. Chronic butyrate supplementation prevented diet-induced obesity, hyperinsulinaemia, hypertriglyceridaemia and hepatic steatosis, largely attributed to a reduction in food intake. Butyrate also modestly promoted fat oxidation and activated brown adipose tissue (BAT), evident from increased utilisation of plasma triglyceride-derived fatty acids. This effect was not due to the reduced food intake, but explained by an increased sympathetic outflow to BAT. Subdiaphragmatic vagotomy abolished the effects of butyrate on food intake as well as the stimulation of metabolic activity in BAT. CONCLUSION: Butyrate acts on the gut-brain neural circuit to improve energy metabolism via reducing energy intake and enhancing fat oxidation by activating BAT.

A physiological glucocorticoid rhythm is an important regulator of brown adipose tissue function.

OBJECTIVE: Brown adipose tissue (BAT) displays a strong circadian rhythm in metabolic activity, but it is unclear how this rhythm is regulated. As circulating levels of corticosterone coincide with the rhythm of triglyceride-derived fatty acid (FA) uptake by BAT, we investigated whether corticosterone regulates BAT circadian rhythm. METHODS: Corticosterone levels were flattened by implanting mice with subcutaneous corticosterone-releasing pellets, resulting in constant circulating corticosterone levels. RESULTS: Flattened corticosterone rhythm caused a complete loss of circadian rhythm in triglyceride-derived fatty acid uptake by BAT. This effect was independent of glucocorticoid receptor expression in (brown) adipocytes and was not caused by deregulation of clock gene expression or overexposure to glucocorticoids, but rather seemed mediated by reduced sympathetic innervation of BAT. In a mouse model of hyperlipidemia and metabolic syndrome, long-term experimental flattening of corticosterone - and thus rhythm in BAT function - resulted in adiposity. CONCLUSIONS: This study highlights that a physiological rhythm in glucocorticoids is an important regulator of BAT function and essential for the maintenance of metabolic health.

Chronic infusion of taurolithocholate into the brain increases fat oxidation in mice.

Bile acids can function in the postprandial state as circulating signaling molecules in the regulation of glucose and lipid metabolism via the transmembrane receptor TGR5 and nuclear receptor FXR. Both receptors are present in the central nervous system, but their function in the brain is unclear. Therefore, we investigated the effects of intracerebroventricular (i.c.v.) administration of taurolithocholate (tLCA), a strong TGR5 agonist, and GW4064, a synthetic FXR agonist, on energy metabolism. We determined the effects of chronic i.c.v. infusion of tLCA, GW4064, or vehicle on energy expenditure, body weight and composition as well as tissue specific fatty acid uptake in mice equipped with osmotic minipumps. We found that i.c.v. administration of tLCA (final concentration in cerebrospinal fluid: 1 µM) increased fat oxidation (tLCA group: 0.083 ±â€…0.006 vs control group: 0.036 ±â€…0.023 kcal/h, F = 5.46, P = 0.04) and decreased fat mass (after 9 days of tLCA infusion: 1.35 ±â€…0.13 vs controls: 1.96 ±â€…0.23 g, P = 0.03). These changes were associated with enhanced uptake of triglyceride-derived fatty acids by brown adipose tissue and with browning of subcutaneous white adipose tissue. I.c.v. administration of GW4064 (final concentration in cerebrospinal fluid: 10 µM) did not affect energy metabolism, body composition nor bile acid levels, negating a role of FXR in the central nervous system in metabolic control. In conclusion, bile acids such as tLCA may exert metabolic effects on fat metabolism via the brain.

Selective Glucocorticoid Receptor Antagonist CORT125281 Activates Brown Adipose Tissue and Alters Lipid Distribution in Male Mice.

Glucocorticoids influence a wide range of metabolic processes in the human body, and excessive glucocorticoid exposure is known to contribute to the development of metabolic disease. We evaluated the utility of the novel glucocorticoid receptor (GR) antagonist CORT125281 for its potential to overcome adiposity, glucose intolerance, and dyslipidemia and compared this head-to-head with the classic GR antagonist RU486 (mifepristone). We show that, although RU486 displays cross-reactivity to the progesterone and androgen receptor, CORT125281 selectively inhibits GR transcriptional activity. In a mouse model for diet-induced obesity, rhythmicity of circulating corticosterone levels was disturbed. CORT125281 restored this disturbed rhythmicity, in contrast to RU486, which further inhibited endogenous corticosterone levels and suppressed adrenal weight. Both CORT125281 and RU486 reduced body weight gain and fat mass. In addition, CORT125281, but not RU486, lowered plasma levels of triglycerides, cholesterol, and free fatty acids and strongly stimulated triglyceride-derived fatty acid uptake by brown adipose tissue depots. In combination with reduced lipid content in brown adipocytes, this indicates that CORT125281 enhances metabolic activity of brown adipose tissue depots. CORT125281 was also found to increase liver lipid accumulation. Taken together, CORT125281 displayed a wide range of beneficial metabolic activities that are in part distinct from RU486, but clinical utility may be limited due to liver lipid accumulation. This warrants further evaluation of GR antagonists or selective modulators that are not accompanied by liver lipid accumulation while preserving their beneficial metabolic activities.

Selective Glucocorticoid Receptor Modulation Prevents and Reverses Nonalcoholic Fatty Liver Disease in Male Mice.

Medication for nonalcoholic fatty liver disease (NAFLD) is an unmet need. Glucocorticoid (GC) stress hormones drive fat metabolism in the liver, but both full blockade and full stimulation of GC signaling aggravate NAFLD pathology. We investigated the efficacy of selective glucocorticoid receptor (GR) modulator CORT118335, which recapitulates only a subset of GC actions, in reducing liver lipid accumulation in mice. Male C57BL/6J mice received a low-fat diet or high-fat diet mixed with vehicle or CORT118335. Livers were analyzed histologically and for genome-wide mRNA expression. Functionally, hepatic long-chain fatty acid (LCFA) composition was determined by gas chromatography. We determined very-low-density lipoprotein (VLDL) production by treatment with a lipoprotein lipase inhibitor after which blood was collected to isolate radiolabeled VLDL particles and apoB proteins. CORT118335 strongly prevented and reversed hepatic lipid accumulation. Liver transcriptome analysis showed increased expression of GR target genes involved in VLDL production. Accordingly, CORT118335 led to increased lipidation of VLDL particles, mimicking physiological GC action. Independent pathway analysis revealed that CORT118335 lacked induction of GC-responsive genes involved in cholesterol synthesis and LCFA uptake, which was indeed reflected in unaltered hepatic LCFA uptake in vivo. Our data thus reveal that the robust hepatic lipid-lowering effect of CORT118335 is due to a unique combination of GR-dependent stimulation of lipid (VLDL) efflux from the liver, with a lack of stimulation of GR-dependent hepatic fatty acid uptake. Our findings firmly demonstrate the potential use of CORT118335 in the treatment of NAFLD and underscore the potential of selective GR modulation in metabolic disease.

The effect of obesogenic diets on brain Neuropeptide Y.

Obesity is a major health problem characterized by accumulated fat mass. The availability of an energy-dense, highly palatable diet plays an important role in obesity development. Neuropeptide Y (NPY), an orexigenic peptide, is affected by dietary composition and NPY can affect dietary preference. The hypothalamic NPY system is well characterized and has been studied in several models of obesity. However, findings from models of diet-induced obesity are not straightforward. In addition, NPY plays a role in (food-)motivated behaviors and interacts with the mesolimbic dopamine system, both of which are altered in obesity. We here review the effect of obesogenic diets on NPY levels in the hypothalamus and reward-related regions.

Identification of a selective glucocorticoid receptor modulator that prevents both diet-induced obesity and inflammation.

BACKGROUND AND PURPOSE: High-fat diet consumption results in obesity and chronic low-grade inflammation in adipose tissue. Whereas glucocorticoid receptor (GR) antagonism reduces diet-induced obesity, GR agonism reduces inflammation, the combination of which would be desired in a strategy to combat the metabolic syndrome. The purpose of this study was to assess the beneficial effects of the selective GR modulator C108297 on both diet-induced weight gain and inflammation in mice and to elucidate underlying mechanisms. EXPERIMENTAL APPROACH: Ten-week-old C57Bl/6 J mice were fed a high-fat diet for 4 weeks while being treated with the selective GR modulator C108297, a full GR antagonist (RU486/mifepristone) or vehicle. KEY RESULTS: C108297 and, to a lesser extent, mifepristone reduced body weight gain and fat mass. C108297 decreased food and fructose intake and increased lipolysis in white adipose tissue (WAT) and free fatty acid levels in plasma, resulting in decreased fat cell size and increased fatty acid oxidation. Furthermore, C108297 reduced macrophage infiltration and pro-inflammatory cytokine expression in WAT, as well as in vitro LPS-stimulated TNF-α secretion in macrophage RAW 264.7 cells. However, mifepristone also increased energy expenditure, as measured by fully automatic metabolic cages, and enhanced expression of thermogenic markers in energy-combusting brown adipose tissue (BAT) but did not affect inflammation. CONCLUSIONS AND IMPLICATIONS: C108297 attenuates obesity by reducing caloric intake and increasing lipolysis and fat oxidation, and in addition attenuates inflammation. These data suggest that selective GR modulation may be a viable strategy for the reduction of diet-induced obesity and inflammation.

Neuronal Control of Brown Fat Activity.

Brown adipose tissue (BAT) activation reduces body fat and metabolic disorders by the enhanced combustion of lipids and glucose into heat. The thermogenic activity of brown adipocytes is primarily driven by the sympathetic nervous system (SNS) and controlled by the brain. In this review, we present recent advances in understanding how cues, such as temperature, light, and proteins, modulate the activity of brown fat by acting on the various hypothalamic nuclei. Given that activated BAT has a high capacity to take up and burn fatty acids (FAs) and glucose, pharmacological stimulation of brown fat in humans by either targeting the hypothalamus or mimicking outflow of the sympathetic nervous system might help improve glucose metabolism and insulin sensitivity, and also lower body fat.

Inhibitory Effect of the Melanocortin Receptor Agonist Melanotan-II (MTII) on Feeding Depends on Dietary Fat Content and not Obesity in Rats on Free-Choice Diets.

INTRODUCTION: Conflicting data exist on sensitivity changes of the melanocortin system during diet-induced obesity. We hypothesized that melanocortin sensitivity depends on diet composition, in particular on the fat content rather than the level of obesity. The aim of this study was to determine the influence of diet composition on feeding responses to a melanocortin receptor agonist, using free-choice diets that differ in food components. METHODS: Male Wistar rats were subjected to a chow (CHOW) diet or a free-choice (fc) diet of either chow, saturated fat and liquid sugar (fcHFHS), chow and saturated fat (fcHF), or chow and liquid sugar (fcHS) for 4 weeks. Melanocortin sensitivity was tested by measuring food intake following administration of the melanocortin 3/4 receptor agonist melanotan II (MTII) or vehicle in the lateral ventricle. In a separate experiment, proopiomelanocortin (POMC) and agouti-related protein (AgRP) mRNA levels were determined in the arcuate nucleus with in situ hybridization in rats subjected to the free-choice diets for 4 weeks. RESULTS: Rats on the fcHFHS diet for 4 weeks show increased caloric intake and body weight gain compared to rats on the CHOW, fcHS and fcHF diet. Caloric intake and body weight gain was comparable between rats on the fcHF, fcHS, and CHOW diet. After 4 weeks diet, POMC and AgRP mRNA levels were not different between diet groups. MTII inhibited caloric intake to a larger extent in rats on the fcHF diet compared to rats on the CHOW, fcHFHS or fcHS diet. Moreover, the fat component was the most inhibited by MTII, and the sugar component the least. CONCLUSION: Rats on the fcHF diet show stronger food intake inhibition to the melanocortin receptor agonist MTII than rats on the CHOW, fcHS, and fcHFHS diet, which is independent of caloric intake and body weight gain. Our data point toward an important role for diet composition, particularly the dietary fat content, and not obesity in the sensitivity of the melanocortin system.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

Neuropeptide Y activity in the nucleus accumbens modulates feeding behavior and neuronal activity.

BACKGROUND: Neuropeptide Y (NPY) is a hypothalamic neuropeptide that plays a prominent role in feeding and energy homeostasis. Expression of the NPY Y1 receptor (Y1R) is highly concentrated in the nucleus accumbens (Acb), a region important in the regulation of palatable feeding. In this study, we performed a number of experiments to investigate the actions of NPY in the Acb. METHODS: First, we determined caloric intake and food choice after bilateral administration of NPY in the Acb in rats on a free-choice diet of saturated fat, 30% sucrose solution, and standard chow and whether this was mediated by the Y1R. Second, we measured the effect of intra-Acb NPY on neuronal activity using in vivo electrophysiology. Third, we examined co-localization of Y1R with enkephalin and dynorphin neurons and the effect of NPY on preproenkephalin messenger RNA levels in the striatum using fluorescent and radioactive in situ hybridization. Finally, using retrograde tracing, we examined whether NPY neurons in the arcuate nucleus projected to the Acb. RESULTS: In rats on the free-choice, high-fat, high-sugar diet, intra-Acb NPY increased intake of fat, but not sugar or chow, and this was mediated by the Y1R. Intra-Acb NPY reduced neuronal firing, as well as preproenkephalin messenger RNA expression in the striatum. Moreover, Acb enkephalin neurons expressed Y1R and arcuate nucleus NPY neurons projected to the Acb. CONCLUSIONS: NPY reduces neuronal firing in the Acb resulting in increased palatable food intake. Together, our neuroanatomical, pharmacologic, and neuronal activity data support a role and mechanism for intra-Acb NPY-induced fat intake.

Differential modulation of arcuate nucleus and mesolimbic gene expression levels by central leptin in rats on short-term high-fat high-sugar diet.

OBJECTIVE: Leptin resistance is a common hallmark of obesity. Rats on a free-choice high-fat high-sugar (fcHFHS) diet are resistant to peripherally administered leptin. The aim of this study was to investigate feeding responses to central leptin as well as the associated changes in mRNA levels in hypothalamic and mesolimbic brain areas. DESIGN AND METHODS: Rats on a CHOW or fcHFHS diet for 8 days received leptin or vehicle intracerebro(lateral)ventricularly (ICV) and food intake was measured 5 h and 24 h later. Four days later, rats were sacrificed after ICV leptin or vehicle and mRNA levels were quantified for hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) and for preproenkephalin (ppENK) in nucleus accumbens and tyrosine hydroxylase (TH) in ventral tegmental area (VTA). RESULTS: ICV leptin decreased caloric intake both in CHOW and fcHFHS rats. In fcHFHS, leptin preferentially decreased chow and fat intake. Leptin increased POMC and decreased NPY mRNA in CHOW, but not in fcHFHS rats. In CHOW rats, leptin had no effect on ppENK mRNA and decreased TH mRNA. In fcHFHS, leptin decreased ppENK mRNA and increased TH mRNA. CONCLUSION: Despite peripheral and arcuate leptin resistance, central leptin suppresses feeding in fcHFHS rats. As the VTA and nucleus accumbens are still responsive to leptin, these brain areas may therefore, at least partly, account for the leptin-induced feeding suppression in rats on a fcHFHS diet.

AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.

An overview on how components of the melanocortin system respond to different high energy diets.

High energy diets are used to model the obesity epidemic. Moreover, from a variety of genetic studies, it has become clear that the melanocortin system plays an important role in the regulation of energy metabolism. Since most dietary interventions are not standardized, fat/sugar-induced effects on the melanocortin system vary distinctly among different studies. How components of the melanocortin system are affected by high energy diets remains unclear. Therefore, in this review, we first present an overview of the effects of high energy diets on different elements of the melanocortin system in both mice and rats. The effects of a high energy diet are most consistent for agouti related protein levels which were either not affected or decreased after consumption of a high energy diet, whereas for proopiomelanocortin and the melanocortin receptor expression (and binding) it was difficult to define an overall response to a high energy diet. Because of the complexity of the melanocortin system, it is important to measure more than one element of the system. Only a few studies measured both melanocortin peptide and receptor expression and show that a high fat diet consumed for a longer period of time starting at an early age increases melanocortin signaling, whereas in adulthood a very high fat diet decreases melanocortin signaling. Finally, we review our own data on diet-induced changes in peptide expression and melanocortin binding and show that short term exposure to a free-choice high-fat high-sugar diet also decreases melanocortin signaling which supports hyperphagia observed in this model.