Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

Aims

to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression.

Subjects and Methods

VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m²) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.

Results

CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.

Conclusions

24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.     

High‐fat Diet Followed by Fasting Disrupts Circadian Expression of Adiponectin Signaling Pathway in Muscle and Adipose Tissue

The circadian clock controls energy homeostasis by regulating circadian expression of proteins involved in metabolism. Disruption of circadian rhythms leads to obesity and metabolic disorders. Little is known regarding the control of the biological clock over adiponectin signaling pathway in adipose tissue, the adiponectin producer, and muscle, an adiponectin target tissue under fasting, low‐fat (LF), or high‐fat (HF) diet. Mice were fed LF or HF diet for 7 weeks and fasted on the last day. The circadian mRNA expression of clock genes and components of adiponectin metabolic pathway (mAdipoR1, mAdipoR2, mPparα, mPparγ, mAmpk, and mAcc) in the muscle and adipose tissue were tested. Using average daily levels of multiple time points around the circadian cycle, we assessed mRNA levels of the different adiponectin signaling components. In addition, serum glucose, adiponectin, and insulin were measured. Under LF diet, adiponectin signaling pathway components exhibited circadian rhythmicity at the mRNA levels. Fasting and HF diet followed by fasting disrupted this circadian expression causing a phase advance or delay, respectively. Changes were also found in the expression levels of adiponectin receptor, mAmpk, mAcc, mPparα, and mPparγ reflecting a defect in adiponectin signaling. As both peroxisome proliferator‐activated receptor α (PPARα) and mAMPK are linked to the core clock mechanism, they could mediate the disruptions seen in clock gene expression under HF diet. In turn, the circadian clock affects the daily rhythm of these adiponectin signaling components.     

Adiponectin and leptin up-regulate extracellular matrix production by dermal fibroblasts

Adipocytes were recently shown to secrete adipocytokines, such as adiponectin and leptin, which may have an endocrine role. Subcutaneous adipose tissue lies just beneath the dermis, and dermal condition is correlated with body mass index (BMI). However, it is not clear whether adipocytokines released by adipocytes in subcutaneous adipose tissue influence the adjacent dermis. We found that human dermal fibroblasts express genes encoding receptors for adiponectin and leptin, and that those cytokines both significantly increase production of hyaluronic acid (HA), a major extracellular matrix component (ECM) of dermis, by dermal fibroblasts. This effect is accompanied with up-regulation of HA synthase 2 gene expression. Moreover, adiponectin significantly increases production of collagen, the most abundant component of ECM in dermis, by dermal fibroblasts. These results suggest that subcutaneous adipocytes influence dermal condition by up-regulating collagen and HA production by dermal fibroblasts via secretion of adiponectin and leptin.     

Differences in adiponectin protein expression: effect of fat depots and type 2 diabetic status.

Adiponectin is an adipocyte-derived hormone associated with insulin sensitivity and atherosclerotic risk. As central rather than gluteofemoral fat is known to increase the risk of type 2 diabetes and cardiovascular disease, we investigated the mRNA and protein expression of adiponectin in human adipose tissue depots. RNA was extracted from 46 human adipose tissue samples from non-diabetic subjects aged 44.33 +/- 12.4 with a BMI of 28.3 +/- 6.0 (mean +/- SD). The samples were as follows: 21 abdominal subcutaneous, 13 omentum, 6 thigh; samples were also taken from diabetic subjects aged 66.6 +/- 7.5 with BMI 28.9 +/- 3.17; samples were: 6 abdominal subcutaneous; 3 thigh. Quantitative PCR and Western analysis was used to determine adiponectin content. Protein content studies determined that when compared with non-diabetic abdominal subcutaneous adipose tissue (Abd Sc AT) (values expressed as percentage relative to Abd Sc AT -100 %). Adiponectin protein content was significantly lower in non-diabetic omental AT (25 +/- 1.6 %; p < 0.0001, n = 6) and in Abd Sc AT from diabetic subjects (36 +/- 1.5 %; p < 0.0001, n = 4). In contrast, gluteal fat maintained high adiponectin protein content from non-diabetic patients compared with diabetic patients. An increase in BMI was associated with lower adiponectin protein content in obese ND Abd Sc AT (25 +/- 0.4 %; p < 0.0001). These findings were in agreement with the mRNA expression data. In summary, this study indicates that adiponectin protein content in non-diabetic subjects remains high in abdominal subcutaneous fat, including gluteal fat, explaining the high serum adiponectin levels in these subjects. Omental fat, however, expresses little adiponectin. Furthermore, abdominal and gluteal subcutaneous fat appears to express significantly less adiponectin once diabetic status is reached. In conclusion, the adipose tissue depot-specific expression of adiponectin may influence the pattern of serum adiponectin concentrations and subsequent disease risk.     

Daily rhythms of plasma melatonin, but not plasma leptin or leptin mRNA, vary between lean, obese and type 2 diabetic men

Melatonin and leptin exhibit daily rhythms that may contribute towards changes in metabolic physiology. It remains unclear, however, whether this rhythmicity is altered in obesity or type 2 diabetes (T2DM). We tested the hypothesis that 24-hour profiles of melatonin, leptin and leptin mRNA are altered by metabolic status in laboratory conditions. Men between 45-65 years old were recruited into lean, obese-non-diabetic or obese-T2DM groups. Volunteers followed strict sleep-wake and dietary regimes for 1 week before the laboratory study. They were then maintained in controlled light-dark conditions, semi-recumbent posture and fed hourly iso-energetic drinks during wake periods. Hourly blood samples were collected for hormone analysis. Subcutaneous adipose biopsies were collected 6-hourly for gene expression analysis. Although there was no effect of subject group on the timing of dim light melatonin onset (DLMO), nocturnal plasma melatonin concentration was significantly higher in obese-non-diabetic subjects compared to weight-matched T2DM subjects (p<0.01) and lean controls (p<0.05). Two T2DM subjects failed to produce any detectable melatonin, although did exhibit plasma cortisol rhythms comparable to others in the group. Consistent with the literature, there was a significant (p<0.001) effect of subject group on absolute plasma leptin concentration and, when expressed relative to an individual's 24-hour mean, plasma leptin showed significant (p<0.001) diurnal variation. However, there was no difference in amplitude or timing of leptin rhythms between experimental groups. There was also no significant effect of time on leptin mRNA expression. Despite an overall effect (p<0.05) of experimental group, post-hoc analysis revealed no significant pair-wise effects of group on leptin mRNA expression. Altered plasma melatonin rhythms in weight-matched T2DM and non-diabetic individuals supports a possible role of melatonin in T2DM aetiology. However, neither obesity nor T2DM changed 24-hour rhythms of plasma leptin relative to cycle mean, or expression of subcutaneous adipose leptin gene expression, compared with lean subjects.     

Expression of Obesity Markers and Persistent Organic Pollutants Levels in Adipose Tissue of Obese Patients: Reinforcing the Obesogen Hypothesis?

Introduction

Persistent Organic Pollutants (POPs) accumulate in adipose tissue and some are described to possess endocrine disrupting capacities. Therefore, it is important to evaluate their effects on key endocrine pathways in adipose tissue (AT), to further evaluate their potential role in metabolic pathologies such as obesity.

Objectives

The aim is twofold: (i) evaluate gene expression levels of obesity marker genes, i.e. the adipokines leptin (LEP), adiponectin (ADIPOQ) and Tumor Necrosis Factor α (TNFα) and the nuclear receptor, Peroxisome Proliferator Activated Receptor γ (PPARγ) in paired subcutaneous (SAT) and visceral (VAT) AT of obese subjects (n = 50) and to relate these values to serum concentrations of LEP and ADIPOQ (ii) evaluate the association of expression levels of marker genes in AT and serum with POP concentrations in AT.

Results and Conclusions

Leptin and adiponectin levels in serum were positively correlated to respectively expression levels of leptin in SAT and adiponectin in VAT. Our study shows more significant correlations between gene expression of obesity marker genes and POP concentrations in VAT compared to SAT. Since VAT is more important than SAT in pathologies associated with obesity, this suggests that POPs are able to influence the association between obesity and the development of associated pathologies. Moreover, this finding reveals the importance of VAT when investigating the obesogen hypothesis. Concerning PPARγ expression in VAT, negative correlations with polychlorinated biphenyls (PCBs) concentrations were found in non T2D patients. LEP serum concentrations correlated with several PCBs in women whereas in men no correlations were found. This strengthens the potential importance of gender differences in obesity and within the obesogen hypothesis.     

Fetal life malnutrition was not reflected in the relative abundances of adiponectin and leptin mRNAs in adipose tissue in male mink kits at 9.5 weeks of age

Background

Malnutrition in fetal life and during suckling have in some animal studies resulted in adaptive changes related to the fat and glucose metabolism, which in the long term might predispose the offspring for metabolic disorders such as obesity later in life. The objective was to study the effect of fetal life malnutrition in male mink on the gene expression of leptin and adiponectin in different adipose tissue sites.

Results

Thirty-two male mink, strict carnivore species, exposed to low (FL) or adequate (FA) protein provision the last 16.3 ± 1.8 days of fetal life and randomly assigned to a low (LP) or adequate (AP) protein diet from 7 to 9.5 weeks of age were used. Adipose tissues (subcutaneous, perirenal and mesenteric) were analyzed using qPCR. Fetal life or post-weaning protein provision did not affect the relative abundances of leptin and adiponectin mRNAs in adipose tissue at 9.5 weeks of age. Relative abundances of leptin and adiponectin mRNAs were different between adipose tissue sites and were significantly higher in subcutaneous than in perirenal and mesenteric tissues.

Conclusion

Fetal life protein malnutrition in male mink, did not result in adaptive changes in the gene expression of leptin and adiponectin mRNAs in adipose tissue at 9.5 weeks of age as found in rodents. However, both leptin and adiponectin mRNAs were significantly differently expressed between tissue sites.

     

Circulating cortisol‐associated signature of glucocorticoid‐related gene expression in subcutaneous fat of obese subjects

Objective:

Serum cortisol concentrations fluctuate in a circadian fashion, and glucocorticoids exert strong effects on adipose tissue and induce obesity through the glucocorticoid receptor.

Design and Methods:

To examine the impact of physiologic levels of circulating cortisol on subcutaneous adipose tissue, 25 overweight and obese subjects were employed, and their serum levels of morning (AM) and evening (PM) cortisol, AM/PM cortisol ratios, and 24‐h urinary‐free cortisol (UFC) were compared with their clinical parameters, serum cytokine levels, and mRNA expression of 93 receptor action‐regulating and 93 glucocorticoid‐responsive genes in abdominal subcutaneous fat.

Results and Conclusions:

AM cortisol levels did not correlate with mRNA expression of the all genes examined, whereas PM cortisol levels, AM/PM cortisol ratios, and 24‐h UFC were associated with distinct sets of these genes. Body mass index did not significantly correlate with the four cortisol parameters employed. These results suggest that physiologic levels of AM serum cortisol do not solely represent biological effects of circulating cortisol on the expression of glucocorticoid‐related genes in subcutaneous adipose tissue, whereas PM levels, amplitude, and net amounts of the diurnally fluctuating serum cortisol have distinct effects. Through the genes identified in this study, glucocorticoids appear to influence intermediary metabolism, energy balance, inflammation, and local circadian rythmicity in subcutaneous fat. Our results may also explain in part the development of metabolic abnormality and obesity in subjects under stress or patients with melancholic/atypical depression who demonstrate elevated levels of PM serum cortisol.     

Chronopharmacological Study of an Antimicrobial Agent,Norfloxacin, in the Rat

Circadian rhythms in physiological processes may affect pharmacological actions of drugs. The purpose of this study was to determine whether pharmacokinetics or acute lethality (LD 50) of norfloxacin, exhibited circadian rhythmicity. Female Sprague- Dawley prepuberal rats (weight 115.8 ± 10.2 g) synchronized with a 12-h-light/ 12-h-dark cycle (lights on 7:00h) were used throughout the study. Norfloxacin pharmacokinetics after intraperitoneal administration at 4:00, 10:00, 16:00 and 22:00h was characterized. Intraperitoneal norfloxacin LD 50 was administered at 2:00, 6:00, 10:00, 14:00, 18:00 and 22:00 h. Pharmacokinetic parameters and lethality percentages were analyzed by the cosinor method for the presence of circadian rhythmicity. The results showed evidence of circadian rhythmicity for norfloxacin k abs, t ½abs, t max, MRT abs, Cl t /f and AUC. Absorption was higher when the drug was administered during the rest (16:00 h) period, meanwhile elimination was higher when administered during the activity (22:00 h) period. No rhythmicity was determined for norfloxacin lethality. It is concluded that, in this study, time of administration modifies the pharmacokinetics of norfloxacin.