Dexamethasone Inhibits White Adipose Tissue Browning.

White adipose tissue (WAT) regulates energy balance through energy storage, adipokines secretion and the thermogenesis process. Beige adipocytes are responsible for WAT thermogenesis. They are generated by adipogenesis or transdifferentiation during cold or ß3-adrenergic agonist stimulus through a process called browning. Browning has gained significant interest for to its preventive effect on obesity. Glucocorticoids (GCs) have several functions in WAT biology; however, their role in beige adipocyte generation and WAT browning is not fully understood. The aim of our study was to determine the effect of dexamethasone (DXM) on WAT thermogenesis. For this purpose, rats were treated with DXM at room temperature (RT) or cold conditions to determine different thermogenic markers. Furthermore, the effects of DXM on the adipogenic potential of beige precursors and on mature beige adipocytes were evaluated in vitro. Our results showed that DXM decreased UCP-1 mRNA and protein levels, mainly after cold exposure. In vitro studies showed that DXM decreased the expression of a beige precursor marker (Ebf2), affecting their ability to differentiate into beige adipocytes, and inhibited the thermogenic response of mature beige adipocytes (Ucp-1, Dio2 and Pgc1α gene expressions and mitochondrial respiration). Overall, our data strongly suggest that DXM can inhibit the thermogenic program of both retroperitoneal and inguinal WAT depots, an effect that could be exerted, at least partially, by inhibiting de novo cell generation and the thermogenic response in beige adipocytes.

Role of glucocorticoid receptor (GR) in white adipose tissue beiging.

AIM: Glucocorticoids (GCs) play a crucial role in energy homeostasis including white adipose tissue function; however, chronic GC excess is detrimental to mammals' health. White hypertrophic adiposity is a main factor for neuroendocrine-metabolic dysfunctions in monosodium L-glutamate (MSG)-damaged hypercorticosteronemic rat. Nevertheless, little is known about the receptor path in endogenous GC impact on white adipose tissue-resident precursor cells to bring them into beige lineage. Thus, our aim was to explore whether transient/chronic endogenous hypercorticosteronemia affects browning capacity in white adipose tissue pads from MSG rats during development. MAIN METHODS: Control and MSG male rats aged 30 and 90 days were 7-day exposed to cold conditions in order to stimulate wet white epidydimal adipose tissue (wEAT) beiging capacity. This procedure was also replicated in adrenalectomized rats. KEY FINDINGS: Data indicated that whereas epidydimal white adipose tissue pads from prepubertal hypercorticosteronemic rats retained full expression of GR/MR genes resulting in a drastic reduction in wEAT beiging capacity, conversely, chronic hypercorticosteronemic adult MSG rats developed down-regulation of corticoid genes (and reduced GR cytosolic mediators) in wEAT pads and consequently partially restored local beiging capacity. Finally, wEAT pads from adrenalectomized rats revealed up-regulation of GR gene accompanied by full local beiging capacity. SIGNIFICANCE: This study strongly supports a GR-dependent inhibitory effect of GC excess on white adipose tissue browning, an issue strongly supporting a key role of GR in the non-shivering thermogenic process. As a consequence, normalizing the GC milieu could be a relevant factor to handle dysmetabolism in white hyperadipose phenotypes.

Inhibitory effect of androgens on white adipose tissue thermogenic capacity.

White adipose tissue (WAT) browning has gained interest due to its impact in obesity. Here, we evaluated the effect of androgens on the Ucp1-dependent thermogenic process from inguinal (IAT) and retroperitoneal (RPAT) WAT. Surgically androgens depleted rats (ODX) showed basal thermogenic activation (room temperature) in both WAT depots, which expressed higher levels of Ucp1, Prdm16 and Pgc1a. WAT pads from ODX cold-exposed rats (ODX-C) expressed increased levels of Ucp1 and Pgc1a and showed high UCP1 protein content. In primary beige adipocyte cultures, testosterone decreased the mitochondrial marker Cox8b and mitochondrial content. Finally, testosterone and dihydrotestosterone (DHT) decreased the expression of Ucp1, Pcg1a and Prdm16 in forskolin-stimulated beige adipocytes, an effect that was prevented by the antiandrogen flutamide. In conclusion, androgen deficient rats developed WAT depots with enhanced basal and cold-stimulated thermogenic activity. Additionally, in vitro androgen treatments inhibited the thermogenic program, effect which was mediated by the androgen receptor pathway.

Dexamethasone primes adipocyte precursor cells for differentiation by enhancing adipogenic competency.

AIM: Dexamethasone (DXM) is a synthetic glucocorticoid whose effects in early and terminal adipogenesis have been addressed. In this study, we evaluated if DXM affects adipocyte precursor cells (APCs), priming them for further adipogenic differentiation. For this purpose, we analyzed APCs number and competency after DXM treatment. MATERIALS AND METHODS: Adult male rats were injected for 2 or 7 days with either DXM (30 µg/kg of weight, sc.) or vehicle. Stromal vascular fraction (SVF) cells from retroperitoneal adipose tissue (RPAT) were isolated to quantify APCs by flow cytometry (CD34+/CD45-/CD31-). Also, expression of competency markers (PPARγ2 and Zfp423) was assessed. Additionally, SVF cells from control rats were incubated with DXM (0.25 µM) alone or combined with a mineralocorticoid receptor (MR) antagonist (Spironolactone 10 µM) and/or a glucocorticoid receptor (GR) antagonist (RU486 1 µM) to assess APCs competency and adipocyte differentiation. KEY FINDINGS: APCs from 2 days DXM-treated rats showed increased expression of PPARγ2 and Zfp423 (competency markers), but did not affect APCs percentage by FACS analysis (CD34+/CD45-/CD31-). Additionally, we found that DXM treatment in SVF also increased APCs competency in vitro, predisposing APCs to further adipocyte differentiation. These effects on APCs were abrogated only when both, MR and GR, were blocked. SIGNIFICANCE: Overall, our results suggest that DXM primes APCs for differentiation mainly by enhancing Zfp423 and PPARγ2 expressions. Also, we showed that the inhibition of MR and GR was necessary for the complete abolishment of DXM effects.

"Spexin improves adipose tissue inflammation and macrophage recruitment in obese mice".

Spexin (SPX) is a novel adipokine related to many metabolic effects, such as gastrointestinal movements, insulin and glucose homeostasis, lipid metabolism and energy balance. This study evaluates the role of SPX in the improvement of the metabolic and inflammatory profile in fructose-rich-diet obese mice. Adult Swiss mice were supplemented or not with fructose (20% in tap water, FRD and CTR, respectively) for 10 weeks. The last ten days, mice were treated or not with SPX (ip. 29 µg/Kg/day, FRD-SPX and CTR-SPX, respectively). A positive correlation was observed between body weight prior to treatment and weight loss after SPX challenge. Moreover, plasma and liver triglycerides and adipose tissue (AT) features (mass, adipocyte hypertrophy, mRNA of leptin) were improved. SPX also induced a reduction in epididymal AT (EAT) expression of TNFα, IL1ß and IL6 and an improvement in IL10 and CD206. M1 macrophages in EAT, principally the Ly6C- populations (M1a and M1b), were decreased. Adipocytes from FRD-SPX mice induced less macrophage activation (IL6, mRNA and secretion) than FRD after overnight co-culture with the monocyte cell line (RAW264.7) in stimulated conditions (M1 activation, LPS 100 ng/mL). Finally, in vitro, monocytes pre-incubated with SPX and stimulated with LPS showed decreased inflammatory mRNA markers compared to monocytes with LPS alone. In conclusion, SPX decreased body weight and improved the metabolic profile and adipocyte hypertrophy. Inflammatory Ly6C- macrophages decreased, together with inflammatory marker expression. In vitro studies demonstrate that SPX induced a decrease in M1 macrophage polarization directly or through mature adipocytes.

M1 macrophage subtypes activation and adipocyte dysfunction worsen during prolonged consumption of a fructose-rich diet.

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1ß, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C+/-CD11c+CD206- and M1b: Ly6C+/-CD11c+CD206+) and M2 subtypes (Ly6C+/-CD11c-CD206+). M1a phenotype increased from 6 weeks onward, while Ly6C+/- M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro.

Deleterious Metabolic Effects of High Fructose Intake: The Preventive Effect of Lactobacillus kefiri Administration.

Modern lifestyle and diets have been associated with metabolic disorders and an imbalance in the normal gut microbiota. Probiotics are widely known for their health beneficial properties targeting the gut microbial ecosystem. The aim of our study was to evaluate the preventive effect of Lactobacillus kefiri (L. kefiri) administration in a fructose-rich diet (FRD) mice model. Mice were provided with tap water or fructose-added (20% w/v) drinking water supplemented or not with L. kefiri. Results showed that probiotic administration prevented weight gain and epidydimal adipose tissue (EAT) expansion, with partial reversion of the adipocyte hypertrophy developed by FRD. Moreover, the probiotic prevented the increase of plasma triglycerides and leptin, together with the liver triglyceride content. Leptin adipocyte secretion was also improved by L. kefiri, being able to respond to an insulin stimulus. Glucose intolerance was partially prevented by L. kefiri treatment (GTT) and local inflammation (TNFα; IL1ß; IL6 and INFγ) was completely inhibited in EAT. L. kefiri supplementation generated an impact on gut microbiota composition, changing Bacteroidetes and Firmicutes profiles. Overall, our results indicate that the administration of probiotics prevents the deleterious effects of FRD intake and should therefore be promoted to improve metabolic disorders.

Relationship between the Balance of Hypertrophic/Hyperplastic Adipose Tissue Expansion and the Metabolic Profile in a High Glucocorticoids Model.

Adipose tissue (AT) expansion is the result of two processes: hyperplasia and hypertrophy; and both, directly or indirectly, depend on the adipogenic potential of adipocyte precursor cells (APCs). Glucocorticoids (GCs) have a potent stimulatory effect on terminal adipogenesis; while their effects on early stages of adipogenesis are largely unknown. In the present work, we study, in a model of high GC levels, the adipogenic potential of APCs from retroperitoneal AT (RPAT) and its relationship with RPAT mass expansion. We employed a model of hyper-adiposity (30- and 60-day-old rats) due to high endogenous GC levels induced by neonatal treatment with l-monosodium glutamate (MSG). We found that the RPAT APCs from 30-day-old MSG rats showed an increased adipogenic capacity, depending on the APCs' competency, but not in their number. Analyses of RPAT adipocyte diameter revealed an increase in cell size, regardless of the rat age, indicating the prevalence of a hypertrophic process. Moreover, functional RPAT alterations worsened in 60-day-old rats, suggesting that the hyperplastic AT expansion found in 30-day-old animals might have a protective role. We conclude that GCs chronic excess affects APCs' adipogenic capacity, modifying their competency. This change would modulate the hyperplastic/hypertrophic balance determining healthy or unhealthy RPAT expansion and, therefore, its functionality.

Long-Term Fructose Intake Increases Adipogenic Potential: Evidence of Direct Effects of Fructose on Adipocyte Precursor Cells.

We have previously addressed that fructose rich diet (FRD) intake for three weeks increases the adipogenic potential of stromal vascular fraction cells from the retroperitoneal adipose tissue (RPAT). We have now evaluated the effect of prolonged FRD intake (eight weeks) on metabolic parameters, number of adipocyte precursor cells (APCs) and in vitro adipogenic potential from control (CTR) and FRD adult male rats. Additionally, we have examined the direct fructose effects on the adipogenic capacity of normal APCs. FRD fed rats had increased plasma levels of insulin, triglyceride and leptin, and RPAT mass and adipocyte size. FACS studies showed higher APCs number and adipogenic potential in FRD RPAT pads; data is supported by high mRNA levels of competency markers: PPARγ2 and Zfp423. Complementary in vitro experiments indicate that fructose-exposed normal APCs displayed an overall increased adipogenic capacity. We conclude that the RPAT mass expansion observed in eight week-FRD fed rats depends on combined accelerated adipogenesis and adipocyte hypertrophy, partially due to a direct effect of fructose on APCs.

Apocynin administration prevents the changes induced by a fructose-rich diet on rat liver metabolism and the antioxidant system.

In the present study, we investigated the role of NADPH oxidase in F (fructose)-rich-diet-induced hepatic OS (oxidative stress) and metabolic changes, and their prevention by apocynin co-administration. Wistar rats were fed for 21 days on (i) a control diet, (ii) a control diet plus 10% F in the drinking water, (iii) a control diet with apocynin in the drinking water (CA) and (iv) F plus apocynin in the drinking water (FA). Glycaemia, triglyceridaemia, NEFAs (non-esterified fatty acids) and insulinaemia were determined. In the liver, we measured (i) NADPH oxidase activity, and gene and protein expression; (ii) protein carbonyl groups, GSH and TBARSs (thiobarbituric acid-reactive substances); (iii) catalase, CuZn-SOD (superoxide dismutase) and Mn-SOD expression; (iv) liver glycogen and lipid content; (v) GK (glucokinase), G6Pase (glucose-6-phosphatase) and G6PDH (glucose-6-phosphate dehydrogenase) activities; (vi) FAS (fatty acid synthase), GPAT (glycerol-3-phosphate acyltransferase), G6Pase and G6PDH, IL-1ß (interleukin-1ß), PAI-1 (plasminogen-activator inhibitor-1) and TNFα (tumour necrosis factor α) gene expression; and (vii) IκBα (inhibitor of nuclear factor κB α) protein expression. F-fed animals had high serum TAG (triacylglycerol), NEFA and insulin levels, high liver NADPH oxidase activity/expression, increased OS markers, reduced antioxidant enzyme expression, and increased glycogen, TAG storage and GK, G6Pase and G6PDH activities. They also had high G6Pase, G6PDH, FAS, GPAT, TNFα and IL-1ß gene expression and decreased IκBα expression. Co-administration of apocynin to F-fed rats prevented the development of most of these abnormalities. In conclusion, NADPH oxidase plays a key role in F-induced hepatic OS production and probably also in the mechanism of liver steatosis, suggesting its potential usefulness for the prevention/treatment of T2DM (Type 2 diabetes mellitus).