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This manuscript adds to the literature of endocrine regulation of adaptive thermogenesis and energy balance. It specifically describes the role of pituitary adenylate cyclase-activating polypeptide on the regulation of brown adipose tissue via the sympathetic nervous system with a focus on compensatory mechanisms of thermogenesis. We highlight sex-specific differences in energy metabolism.

New findings What is the central question of this study? Can chronic treatment of pituitary adenylate cyclase-activating polypeptide (PACAP) deficient mice with the melanocortin agonist melanotan II during cold acclimation rescue the impaired thermogenic capacity previously observed in PACAP deficient mice? What is the main finding and its importance? Using a genetic model of PACAP deficiency, this study provides evidence that PACAP acts upstream of the melanocortin system in regulating sympathetic nerve activity to brown adipose tissue in mice. Abstract Impaired adipose tissue function in obesity, including reduced thermogenic potential, has detrimental consequences for metabolic health. Hormonal regulation of adaptive thermogenesis is being explored as a potential therapeutic target for human obesity. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide expressed in nuclei of the hypothalamus known to regulate energy expenditure, and functional studies reveal a role for PACAP in the central regulation of thermogenesis, although mechanisms are not well understood. We hypothesized that PACAP acts upstream of the melanocortin system to regulate sympathetic nerve activity to stimulate thermogenesis. To assess this, female PACAP-/- and PACAP+/+ mice were given daily peripheral injections of a melanocortin receptor agonist, melanotan II (MTII), for 3 weeks during cold acclimation, and the effect of MTII on thermogenic capacity and adipose tissue remodelling was examined by physiological and histological analyses. MTII partially rescued the impaired thermogenic capacity in PACAP-/- mice as compared to PACAP+/+ mice as determined by measuring noradrenaline-induced metabolic rate. In addition, MTII treatment during cold acclimation corrected the previously identified deficit in lipid utilization in response to adrenergic stimulation in PACAP-/- null mice, suggesting impaired lipid mobilization may contribute to the impaired thermogenic capacity of PACAP-/- mice. Results presented here provide physiological evidence to suggest that PACAP acts upstream of melanocortin receptors to facilitate sympathetically induced mechanisms of adaptive thermogenesis in response to cold acclimation.

Pituitary Adenylate Cyclase Activating Polypetptide (PACAP) is a peptide hormone known to regulate energy homeostasis1. Mice lacking PACAP are cold sensitive and have impaired adrenergic-induced thermogenesis2-4. Interestingly, Pacap null mice can survive cold housing if acclimated slowly, similar to what was observed in UCP1 deficient mice4,5. We hypothesized that Pacap-/- mice employ alternate thermogenic pathways to compensate for impaired adaptive thermogenesis and assessed shivering thermogenesis and UCP1-dependent and UCP1-independent adaptive thermogenesis in male and female Pacap-/- and Pacap+/+ with cold acclimation (4°C). Assessment of oxidative fibres in skeletal muscles and behavioural observations did not show evidence of prolonged shivering in male or female Pacap-/- mice during cold acclimation compared to Pacap+/+ mice. We did however observe morphological and molecular differences in adipose tissues of Pacap-/- mice compared to Pacap+/+ mice that were distinct in males and females. Cold-acclimated, female Pacap-/- mice had decreased induction of UCP1 protein in intrascapular brown fat (iBAT), yet had a significantly higher beiging and UCP1 immunoreactivity (ir) in gonadal white fat (gWAT) compared to female Pacap+/+ mice. Furthermore, beiging was observed in inguinal white fat (ingWAT) and gWAT of female Pacap-/- mice housed at thermoneutrality (30°C), a finding not observed in Pacap+/+ control mice. Unlike female mice, we did not observe impaired UCP1 induction in iBAT of male Pacap-/- mice compared to Pacap+/+ mice, and this was associated with negligible UCP1-ir in male gWAT similar to wildtype controls. Despite previous work that has established impaired adaptive thermogenesis in Pacap-/- mice4, we show here that UCP1 protein can be induced in adipose tissues of Pacap-/- mice during cold acclimation, although to a lesser degree or in a different pattern compared to Pacap+/+ control mice. Taken together, this work suggests that while PACAP is clearly involved in regulating thermoregulation, it is not required for cold-induced UCP1 expression. In addition, this work highlights sexual dimorphism in adipose tissue remodeling and induction of thermogenesis with cold acclimation. References: (1) Rudecki AP, et al. Trends Endocrinol Metab. 2016;27(9), 620–632. (2) Gray SL, et al. J Mol Endocrinol. 2001;15(10), 1739–1747. (3) Gray SL, et al. J Endocrinol. 2002;143(10), 3946–3954. (4) Diané A, et al. J Endocrinol. 2014;222, 327–339. (5) Golozoubova V, et al. FASEB J. 2001;15, 2048–2050.

Obesity arises from disrupted energy balance and is caused by chronically higher energy intake compared to expenditure via basal metabolic rate, exercise, and thermogenesis. The brown adipose tissue (BAT), the primary thermogenic organ, has received considerable attention as a potential therapeutic target due to its ability to burn lipids in the production of heat. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been identified as a key regulator of the physiological stress response both centrally and peripherally. While PACAP has been shown to increase thermogenesis by acting at the hypothalamus to increase sympathetic output to BAT, a peripheral role for PACAP-activated thermogenesis has not been studied. We identified PACAP receptor (PAC1, VPAC1/2) expression for the first time in murine BAT and confirmed their expression in white adipose tissues. PAC1 receptor expression was significantly altered in all three adipose tissues studied in response to 3.5-week cold acclimation, with expression patterns differing by depot type. In primary cell culture, VPAC1 was increased in differentiated compared to non-differentiated brown adipocytes, and the same trend was observed for the PACAP-specific receptor PAC1 in gonadal white fat primary cultures. The primary PAC1R mRNA splice variant in interscapular BAT was determined as isoform 2 by RNA-Seq. These results show that PACAP receptors are present in adipose tissues and may have important functional roles in adipocyte differentiation, lipid metabolism, or adipose sensitization to sympathetic signaling in response to thermogenic stimuli.

The sympathetic branch of the autonomic nervous system (ANS) regulates the body’s response to psychogenic and systemic stress. In response to stress, endocrine pathways are activated to rebalance physiological changes and restore homeostasis. Gene expression analysis through quantitative real-time PCR (qPCR) is a key method to assess differential gene expression in tissue and cell samples, with high sensitivity and specificity. To assess differential gene expression between samples, qPCR data must be normalized to two or more reference genes to account for technical variation in nucleic acid loading between samples, ensuring data represents target gene expression due to experimental treatments (1). Reference genes must be selected and tested for each experimental paradigm, showing stability of expression between samples and treatments. Reference genes are usually constitutively expressed genes required for cellular maintenance, such as well known “housekeeping” genes or genes encoding ribosomal RNAs. Ganglia of the sympathetic nervous system (SNS) are small in size, and thus total yields of RNA for qPCR experiments are limited. Our group has established a protocol for the isolation of adrenal glands, superior cervical, celiac, and stellate ganglia using a dissecting scope and anatomical landmarks in mice. Here we present optimization data assessing five commonly used reference genes for normalization of qPCR data in three ganglia of the SNS (superior cervical, stellate, and celiac) and adrenal glands. We have evaluated their suitability as reference genes in these tissues after acclimation to cold (4°C) stress compared to the same tissues from animals housed at thermoneutrality (30°C). Through qPCR reaction optimization and subsequent calculations of geNorm stability-measure M, we present optimized primer sequences and reaction conditions, and provide recommendations for combinations of three or more reference genes to use for normalization in gene expression analysis experiments in adrenal gland, superior cervical, stellate or celiac ganglia from male and female mice exposed to thermoneutrality or cold. This research will be valuable to groups interested in SNS physiology and facilitate a streamlined approach to gene expression analysis given the low RNA availability from these minute tissue samples, saving valuable sample, costs and time in carrying out qPCR experiments. Reference: (1) Bustin et al., Clin Chem. 2009 Jan 27;55(4):611-622.