OBJECTIVEPrevious animal studies suggest a functional relationship between metabolism, type 2 diabetes, and the amplitude of daily rhythms in white adipose tissue (WAT). However, data interpretation is confounded by differences in genetic background and diet or limited sampling points. We have taken the novel approach of analyzing serial human WAT biopsies across a 24-h cycle in controlled laboratory conditions.RESEARCH DESIGN AND METHODSLean (n = 8), overweight/obese (n = 11), or overweight/obese type 2 diabetic (n = 8) volunteers followed a strict sleep–wake and dietary regimen for 1 week prior to the laboratory study. They were then maintained in controlled light–dark conditions in a semirecumbent posture and fed hourly during wake periods. Subcutaneous WAT biopsies were collected every 6 h over 24 h, and gene expression was measured by quantitative PCR.RESULTSLean individuals exhibited significant (P < 0.05) temporal changes of core clock (PER1, PER2, PER3, CRY2, BMAL1, and DBP) and metabolic (REVERBα, RIP140, and PGC1α) genes. The BMAL1 rhythm was in approximate antiphase with the other clock genes. It is noteworthy that there was no significant effect (P > 0.05) of increased body weight or type 2 diabetes on rhythmic gene expression.CONCLUSIONSThe robust nature of these rhythms and their relative phasing indicate that WAT now can be considered as a peripheral tissue suitable for the study of in vivo human rhythms. Comparison of data between subject groups clearly indicates that obesity and type 2 diabetes are not related to the amplitude of rhythmic WAT gene expression in humans maintained under controlled conditions.

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.

Adipose tissue is central to metabolic homeostasis, signalling in part through the secretion of molecules termed adipokines. Circadian rhythms play an important role in adipose physiology, with plasma adipokine concentration and ~20% of the murine adipose transcriptome undergoing24 h variation. However, due to the heterogeneity of adipose tissue and rhythmical input from both neuronal and humoral signals, the cellular basis of adipose rhythms is unclear. We tested the hypothesis that adipocyte cells contain a circadian clock that drives rhythmic mRNA expression and adipokine secretion. From the murine preadipocyte 3T3-L1 cell line, we generated populations of both pre-adipocytes and differentiated adipocytes. Cells were then treated with a 2 h serum pulse and sampled every 4 h over a 48 h period. Expression of clock gene, 'metabolic' gene (PPARα, PPARγ, SREBP1) and adipokine mRNA was analyzed by quantitative real-time PCR, and secretion of the adipokines leptin and adiponectin was measured in culture medium from differentiated adipocytes. In pre-adipocytes, we observed robust rhythms of clock genes Per2, Rev-erbα, and Dbp, but not of Per1, Cry1, Bmal1, or any of the 'metabolic' genes. Adipocytes produced similar temporal profiles of mRNA expression, albeit with a markedly reduced amplitude of Per2 and Dbp rhythms. Despite no circadian rhythm of adipokine mRNA expression, leptin accumulation in the culture medium suggested circadian control of leptin secretion from adipocytes. Adiponectin secretion showed temporal variation, but without any apparent circadian rhythmicity. Our data, therefore, suggest that an endogenous adipocyte clock controls the rhythmic expression of only a subset of genes that are reported to exhibit 24 h rhythmicity in murine adipose tissue. Moreover, secretion of leptin may also be regulated by the adipocyte clock.

Circadian clocks time the daily occurrence of multiple aspects of behaviour and physiology. Through studies of chronic misalignment between our internal clocks and the environment (e.g. during shift work), it has long been postulated that disruption of circadian rhythms is detrimental to human health. Recent advances in understanding of the cellular and molecular basis of mammalian circadian timing mechanisms have identified many key genes involved in circadian rhythm generation and demonstrated the presence of clocks throughout the body. Furthermore, clear links between sleep, circadian rhythms and metabolic function have been revealed and much current research is studying these links in more detail. Here, we review the current evidence linking circadian rhythms, clock genes and adipose biology. We also highlight gaps in our understanding and finally suggest avenues for future research.

Adipose tissue has a major influence on insulin sensitivity. Stimulation of free fatty acid receptor 2 (FFAR2) has been proposed to influence adipocyte differentiation. We hypothesised that exposing preadipocytes to short chain fatty acids would induce earlier expression of nuclear receptors that co-ordinate adipogenesis, triglyceride accumulation and leptin secretion. 3T3-L1 preadipocytes were differentiated in the presence of 1 μM acetate, 0.1–10 μM propionate or vehicle control. In experiment 1, expression of Ffar2 and nuclear receptor mRNA was measured by quantitative PCR over 48 h following onset of differentiation. In experiment 2, extracellular leptin concentration and intracellular triglyceride content were measured at days 0, 2, 4, 6, 8 and 10 following the onset of differentiation. Control cells exhibited similar temporal dynamics of gene expression, triglyceride accumulation and leptin secretion as reported previously. We were unable to detect expression of Ffar3 mRNA at any stage of differentiation. Consistent with a lack of Ffar2 expression in the first 24 h of differentiation, acetate and propionate had no significant effect on nuclear receptor expression. Furthermore, acetate or propionate treatment did not alter leptin concentration or triglyceride content. In conclusion, we observed no significant effect of propionate or acetate on adipogenesis in 3T3-L1 cells using validated quantitative techniques.