Global loss of Bmal1 expression alters adipose tissue hormones, gene expression and glucose metabolism

Extent: 11p.The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.David John Kennaway, Tamara Jayne Varcoe, Athena Voultsios, Michael James Bode

The role of serotonin-2C receptors in the rat circadian system.

The suprachiasmatic nucleus receives dense serotonergic projections from the raphe nuclei and this input has been implicated in the modulation of circadian rhythms. This input appears to have many functions including the transmission of non-photic information during the day and the modulation of photic information at night. However, it has emerged that this input may also be involved in the transmission of light information with activation of 5-HT2C receptors at night having a photo-mimetic effect. The studies described in this thesis aim to clarify the role of 5-HT2C receptors in the control of circadian rhythms in the rat model and compare their actions to light. The acute effects of 5-HT2C receptor agonist administration on clock gene expression were investigated in the rat SCN. Systemic administration of the 5-HT2A/2C agonist DOI to rats during early night induced c-fos, Per1 and Per2 expression in a manner similar to light. This response was time of day dependent with maximal induction occurring in the early night, and no response during the day. The role of 5-HT2C receptors in this response was confirmed with the use of the selective 5-HT2C receptor agonist RO-60 0175. The effect of 5-HT2C receptor activation on the phase of expression of various circadian rhythms including temperature, melatonin and clock gene expression in the SCN and periphery was examined. Both DOI administration and light exposure at night phase delayed rhythms of melatonin and temperature. Similarly, the selective 5-HT2C receptor agonist RO-60 0175 phase delayed rhythms of 6-sulphatoxymelatonin, a response which was antagonised by the 5-HT2C receptor antagonist SB-242084. The expression of functional and clock genes within the pineal was also phase delayed following both light and 5-HT2C receptor agonist administration. However, the phase of expression of clock genes within the SCN or liver did not shift in response to either a single nocturnal light pulse or agonist administration. To investigate the site of action of 5-HT2C receptor agonists, rat SCN explants were maintained in culture allowing exposure of agonists to denervated tissue. The acute effect of DOI administration at various circadian times on c-fos and Per1 expression was assessed. 5-HT2C receptor activation significantly increased Per1 expression when administered during early subjective night, but had no effect during either subjective day or late subjective night, similar to that observed in vivo. Finally, the suitability of immortalised rat SCN cells for investigation of the intracellular actions of 5-HT2C receptors in the circadian system was assessed. Using RT-PCR the expression of various serotonin receptors in the SCN2.2 cell line was compared with that observed in punches of adult rat SCN. The mRNA for 5-HT1B and 5-HT2A receptor was expressed in both the SCN2.2 cell line and the adult rat SCN. However, 5-HT2C receptor mRNA along with 5-HT3 receptor, 5-HT5A receptor and 5-HT7 receptor mRNA was expressed in the adult rat SCN tissue but not the SCN2.2 cells. These significant differences in serotonin receptor expression limit the usefulness of this cell line for further investigation. Together these experiments further implicate 5-HT2C receptors in the control of circadian rhythms. The role of these receptors appears limited to early night, with activation showing photo-mimetic responses. Furthermore, the location of action appears to be post-synaptic within the SCN, altering the core clock genes, which in turn phase delay various circadian rhythms.Thesis(Ph.D.)-- School of Paediatrics and Reproductive Health, 200

Plasma metabolites, insulin, and adipokines in 8 week old wild-type and <i>Bmal1</i> null mice.

<p>Plasma glucose (a), free fatty acids (b), insulin (c), adiponectin (d) and leptin (e) levels. Data are the mean ± s.e.m. for <i>n = </i>4 mice of each genotype at each time point, wild-type mice (open circles) and <i>Bmal1</i> null mice (closed circles). The accompanying histograms represent the estimated marginal means ± s.e.m. of the individual gene expression as calculated from the ANOVA. The shaded areas represent the period of darkness. The symbol * indicates that there was a significant difference (<i>P</i><0.05) between the genotypes.</p

Primers used.

<p>Primers used.</p

The relative gene expression across 24 h of clock and liver enzyme genes in the liver of male wild-type and <i>Bmal1</i> null mice fed a normal rodent diet.

<p>(a) <i>Bmal1</i>, (b) <i>Per2</i>, (c) <i>Pfkfb3</i>, (d) <i>Pck</i>, (e) <i>Fbp1</i>, (f) <i>G6pc,</i> (g) <i>Gck</i> and (h) <i>Adipor2.</i> The data are the relative expression for each gene compared to <i>Actin</i> mRNA (mean ± s.e.m., n = 4 for each genotype), wild-type mice (open circles) and <i>Bmal1</i> null mice (closed circles). The highest expression of each gene for wild-type mice was set at one. The apparent absence of an SEM bar indicates that it is obscured by the symbol. The shaded areas represent the period of darkness.</p

The blood glucose response to the intra peritoneal administration of pyruvate (2 mg/g) to male and female wild-type and <i>Bmal1</i> null mice fed a normal rodent diet.

<p>The mean ± s.e.m. plasma glucose levels are shown for male (a) and female (c) wild-type (open circles) and <i>Bmal1</i> null (closed circles) mice. The mean area under the curve (± SEM) of the plasma glucose profiles up to 120 min post injection (b, d).</p