high-throughput-danio-rerio-energy-expenditure-assay

High Throughput <em>Danio Rerio</em> Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

University of Arizona

The influence of dietary restriction on the negative feedback potency of 17-beta-estradiol (E2) was evaluated in both castrated male (wethers) and female sheep (OVX ewes) during the breeding season. In study 1, OVX ewes received maintenance or restricted dietary energy for 7 weeks or maintenance energy for 6 weeks prior to a 5 day fast (n=12ewes/feeding group). Estradiol (0.31microg E2/50kg/h) or vehicle (10% EtOH-saline) was continuously infused into half the animals in each dietary treatment for the final 54h of the study. The dynamic pattern of LH secretion was assessed during the final 6h of infusion. Estradiol inhibited luteinizing hormone (LH) pulse amplitude independent of nutrition (P=0.02); fasting increased mean LH, LH peak height, and LH nadir in the absence of E2 (P=0.004, P=0.02, and P=0.02, respectively); while E2 inhibited pulse frequency (P=0.02) and increased peak width (P=0.04) in restricted ewes. Interestingly, despite uniform E2 delivery, serum concentrations of E2 differed with feeding status. Therefore, 12 wethers were infused with 0.31microg E2/50kg/h (6 fed, 6 fasted) and six wethers received 0.19microg E2/50kg/h (fasted) to establish similar serum concentrations of E2 in fed (0.31microg/50kg/h) and fasted (0.19microg/50kg/h) wethers. When fed and fasted wethers had uniform serum concentrations of E2 LH pulse frequency was suppressed (P<0.05) in fasted relative to fed animals, supporting the postulate that energy restriction enhances the E2 negative feedback potency. Collectively, these studies demonstrate that nutrition affects E2 feedback potency and clearance.

Circulating estradiol suppresses luteinizing hormone pulse frequency during dietary restriction.

The melanocortin MC(3) receptor remains the most enigmatic of the melanocortin receptors with regard to its physiological functions. The receptor is expressed both in the CNS and in multiple tissues in the periphery. It appears to be an inhibitory autoreceptor on proopiomelanocortin neurons, yet global deletion of the receptor causes an obesity syndrome. Knockout of the receptor increases adipose mass without a readily measurable increase in food intake or decrease in energy expenditure. And finally, no melanocortin MC(3) receptor null humans have been identified and associations between variant alleles of the melanocortin MC(3) receptor and diseases remain controversial, so the physiological role of the receptor in humans remains to be determined.

Physiological roles of the melanocortin MC₃ receptor.

The melanocortin-3 receptor-deficient (MC3-R(-/-)) mouse exhibits mild obesity without hyperphagia or hypometabolism. MC3-R deletion is reported to increase adiposity, reduce lean mass and white adipose tissue inflammation, and increase sensitivity to salt-induced hypertension. We show here that the MC3-R(-/-) mouse exhibits defective fasting-induced white adipose tissue lipolysis, fasting-induced liver triglyceride accumulation, fasting-induced refeeding, and fasting-induced regulation of the adipostatic and hypothalamic-adrenal-pituitary axes. Close examination of the hypothalamic-pituitary-adrenal axis showed that MC3-R(-/-) mice exhibit elevated nadir corticosterone as well as a blunted fasting-induced activation of the axis. The previously described phenotypes of this animal and the reduced bone density reported here parallel those of Cushing syndrome. Thus, MC3-R is required for communicating nutritional status to both central and peripheral tissues involved in nutrient partitioning, and this defect explains much of the metabolic phenotype in the model.

Melanocortin-3 receptor regulates the normal fasting response.

Energy homeostasis is maintained by balancing energy intake and expenditure. Many signals regulating energy intake are conserved between the human and teleost. However, before this work, there was no sensitive high-throughput system to monitor energy expenditure in the teleost. We exploit the nonfluorescent and fluorescent properties of resazurin and its reduced form resorufin (alamarBlue(®)) to monitor energy expenditure responses to drug application and genetic manipulation. We show that leptin, insulin, and alpha-melanocyte-stimulating hormone (α-MSH) increase energy expenditure dose dependently in the larval zebrafish. As previously established in the mouse, etomoxir, a carnitine palmitoyl transferase I inhibitor, blocks leptin-induced energy expenditure in the zebrafish. Metformin, the most commonly prescribed insulin sensitizer, increases the insulin-induced metabolic rate. Using genetic knockdown, we observed that α-MSH treatment increases the metabolic rate, as does knockdown of the melanocortin antagonist, agouti-related protein. The agouti-related protein and multiple melanocortin receptors are shown to be involved in these effects. These studies confirm that aspects of hormonal regulation of energy expenditure are conserved in the teleost, and suggest that this assay may provide a unique tool to perform in vivo screens for drugs or genes that affect the metabolic rate, including insulin or leptin sensitizers.

Benjamin J. Renquist

School of Animal and Comparative Biomedical Sciences

University of Arizona

High Throughput Danio Rerio Energy Expenditure Assay

Benjamin J. Renquist

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University of Arizona

High Throughput Danio Rerio Energy Expenditure Assay

School of Animal and Comparative Biomedical Sciences