Mardi S. Byerly

Myonectin (CTRP15), a novel myokine that links skeletal muscle to systemic lipid homeostasis

Background: Skeletal muscle-derived myokines modulate metabolic, inflammatory, and other processes. Results: Myonectin, a novel myokine whose expression and circulating levels are regulated by diet, metabolic state, and exercise, functions to control lipid metabolism. Conclusion: Myonectin is a potential nutrient-responsive metabolic regulator secreted by muscle. Significance: Myonectin links muscle to systemic lipid metabolism via its action on adipocytes and hepatocytes, providing insight into complex tissue cross-talk. Skeletal muscle plays important roles in whole-body glucose and fatty acid metabolism. However, muscle also secretes cytokines and growth factors (collectively termed myokines) that can potentially act in an autocrine, a paracrine, and/or an endocrine manner to modulate metabolic, inflammatory, and other processes. Here, we report the identification and characterization of myonectin, a novel myokine belonging to the C1q/TNF-related protein (CTRP) family. Myonectin transcript was highly induced in differentiated myotubes and predominantly expressed by skeletal muscle. Circulating levels of myonectin were tightly regulated by the metabolic state; fasting suppressed, but refeeding dramatically increased, its mRNA and serum levels. Although mRNA and circulating levels of myonectin were reduced in a diet-induced obese state, voluntary exercise increased its expression and circulating levels. Accordingly, myonectin transcript was up-regulated by compounds (forskolin, epinephrine, ionomycin) that raise cellular cAMP or calcium levels. In vitro, secreted myonectin forms disulfide-linked oligomers, and when co-expressed, forms heteromeric complexes with other members of the C1q/TNF-related protein family. In mice, recombinant myonectin administration reduced circulating levels of free fatty acids without altering adipose tissue lipolysis. Consistent with this, myonectin promoted fatty acid uptake in cultured adipocytes and hepatocytes, in part by up-regulating the expression of genes (CD36, FATP1, Fabp1, and Fabp4) that promote lipid uptake. Collectively, these results suggest that myonectin links skeletal muscle to lipid homeostasis in liver and adipose tissue in response to alterations in energy state, revealing a novel myonectin-mediated metabolic circuit.

Lhx1 Controls Terminal Differentiation and Circadian Function of the Suprachiasmatic Nucleus

SUMMARY Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillators role in physiology and cognition.

Vertebrate retina and hypothalamus development

The vertebrate retina and hypothalamus, which emerge from adjacent regions of the ventral diencephalon, provide accessible experimental systems for analysis of the molecular mechanisms by which neuronal subtype diversity is specified, and how this neuronal subtype diversity regulates perception and behavior. Although the retina emerges as a lateral extension of the hypothalamus prior to the onset of neurogenesis, the retina and hypothalamus go on to eventually be comprised of almost entirely different cell types, and differ extensively in the spatial organization, function, and connectivity of these cells. Despite these differences in cell composition, there are a number of mechanistic and molecular similarities in the process of cell fate specification in both organs, including a stereotyped temporal sequence in which major cell types are generated. Although a handful of genes have been identified in both systems that direct cell fate specification, many more remain to be characterized, and large numbers of candidate genes have been identified in recent high‐throughput screens, particularly in retina. Experimental challenges for the near future include functional analysis of the genes identified so far, and the use of the molecular pathways gained from analysis of the development of specific neuronal lineages to study the contribution of these cells to perception and behavior. Copyright

Rax regulates hypothalamic tanycyte differentiation and barrier function in mice.

The wall of the ventral third ventricle is composed of two distinct cell populations: tanycytes and ependymal cells. Tanycytes regulate many aspects of hypothalamic physiology, but little is known about the transcriptional network that regulates their development and function. We observed that the retina and anterior neural fold homeobox transcription factor (Rax) is selectively expressed in hypothalamic tanycytes, and showed a complementary pattern of expression to markers of hypothalamic ependymal cells, such as Rarres2 (retinoic acid receptor responder [tazarotene induced] 2). To determine whether Rax controls tanycyte differentiation and function, we generated Rax haploinsufficient mice and examined their cellular and molecular phenotype in adulthood. These mice appeared grossly normal, but careful examination revealed a thinning of the third ventricular wall and reduction of both tanycyte and ependymal markers. These experiments show that Rax is required for hypothalamic tanycyte and ependymal cell differentiation. Rax haploinsufficiency also resulted in the ectopic presence of ependymal cells in the α2 tanycytic zone, where few ependymal cells are normally found, suggesting that Rax is selectively required for α2 tanycyte differentiation. These changes in the ventricular wall were associated with reduced diffusion of Evans Blue tracer from the ventricle to the hypothalamic parenchyma, with no apparent repercussion on the gross anatomical or behavioral phenotype of these mice. In conclusion, we have provided evidence that Rax is required for the normal differentiation and patterning of hypothalamic tanycytes and ependymal cells, as well as for maintenance of the cerebrospinal fluid–hypothalamus barrier. J. Comp. Neurol. 522:876–899, 2014.

A central role for C1q/TNF-related protein 13 (CTRP13) in modulating food intake and body weight.

C1q/TNF-related protein 13 (CTRP13), a hormone secreted by adipose tissue (adipokines), helps regulate glucose metabolism in peripheral tissues. We previously reported that CTRP13 expression is increased in obese and hyperphagic leptin-deficient mice, suggesting that it may modulate food intake and body weight. CTRP13 is also expressed in the brain, although its role in modulating whole-body energy balance remains unknown. Here, we show that CTRP13 is a novel anorexigenic factor in the mouse brain. Quantitative PCR demonstrated that food restriction downregulates Ctrp13 expression in mouse hypothalamus, while high-fat feeding upregulates expression. Central administration of recombinant CTRP13 suppressed food intake and reduced body weight in mice. Further, CTRP13 and the orexigenic neuropeptide agouti-related protein (AgRP) reciprocally regulate each other’s expression in the hypothalamus: central delivery of CTRP13 suppressed Agrp expression, while delivery of AgRP increased Ctrp13 expression. Food restriction alone reduced Ctrp13 and increased orexigenic neuropeptide gene (Npy and Agrp) expression in the hypothalamus; in contrast, when food restriction was coupled to enhanced physical activity in an activity-based anorexia (ABA) mouse model, hypothalamic expression of both Ctrp13 and Agrp were upregulated. Taken together, these results suggest that CTRP13 and AgRP form a hypothalamic feedback loop to modulate food intake and that this neural circuit may be disrupted in an anorexic-like condition.

Identification of hypothalamic neuron-derived neurotrophic factor as a novel factor modulating appetite

Disruption of finely coordinated neuropeptide signals in the hypothalamus can result in altered food intake and body weight. We identified neuron-derived neurotrophic factor (NENF) as a novel secreted protein through a large-scale screen aimed at identifying novel secreted hypothalamic proteins that regulate food intake. We observed robust Nenf expression in hypothalamic nuclei known to regulate food intake, and its expression was altered under the diet-induced obese (DIO) condition relative to the fed state. Hypothalamic Nenf mRNA was regulated by brain-derived neurotrophic factor (BDNF) signaling, itself an important regulator of appetite. Delivery of purified recombinant BDNF into the lateral cerebral ventricle decreased hypothalamic Nenf expression, while pharmacological inhibition of trkB signaling increased Nenf mRNA expression. Furthermore, recombinant NENF administered via an intracerebroventricular cannula decreased food intake and body weight and increased hypothalamic Pomc and Mc4r mRNA expression. Importantly, the appetite-suppressing effect of NENF was abrogated in obese mice fed a high-fat diet, demonstrating a diet-dependent modulation of NENF function. We propose the existence of a regulatory circuit involving BDNF, NENF, and melanocortin signaling. Our study validates the power of using an integrated experimental and bioinformatic approach to identify novel CNS-derived proteins with appetite-modulating function and reveals NENF as an important central modulator of food intake.

C1q/TNF-related protein 4 (CTRP4) is a unique secreted protein with two tandem C1q domains that functions in the hypothalamus to modulate food intake and body weight.

Background: CTRP4 is a conserved member of the C1q family of secreted proteins with poorly defined function. Results: CTRP4 acts in the hypothalamus to modulate food intake and body weight by regulating the expression of orexigenic neuropeptide expression. Conclusion: CTRP4 plays a role in food intake regulation. Significance: This study establishes the first known in vivo function of CTRP4. CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.

Estrogen-related receptor β deletion modulates whole-body energy balance via estrogen-related receptor γ and attenuates neuropeptide Y gene expression

Estrogen‐related receptors (ERRs) α, β and γ are orphan nuclear hormone receptors with no known ligands. Little is known concerning the role of ERRβ in energy homeostasis, as complete ERRβ‐null mice die mid‐gestation. We generated two viable conditional ERRβ‐null mouse models to address its metabolic function. Whole‐body deletion of ERRβ in Sox2‐Cre:ERRβlox/lox mice resulted in major alterations in body composition, metabolic rate, meal patterns and voluntary physical activity levels. Nestin‐Cre:ERRβlox/lox mice exhibited decreased expression of ERRβ in hindbrain neurons, the predominant site of expression, decreased neuropeptide Y (NPY) gene expression in the hindbrain, increased lean body mass, insulin sensitivity, increased energy expenditure, decreased satiety and decreased time between meals. In the absence of ERRβ, increased ERRγ signaling decreased satiety and the duration of time between meals, similar to meal patterns observed for both the Sox2‐Cre:ERRβlox/lox and Nestin‐Cre:ERRβlox/lox strains of mice. Central and/or peripheral ERRγ signaling may modulate these phenotypes by decreasing NPY gene expression. Overall, the relative expression ratio between ERRβ and ERRγ may be important in modulating ingestive behavior, specifically satiety, gene expression, as well as whole‐body energy balance.

Estrogen-related receptor β deficiency alters body composition and response to restraint stress

BackgroundEstrogen-related receptors (ERRs) are orphan nuclear hormone receptors expressed in metabolically active tissues and modulate numerous homeostatic processes. ERRs do not bind the ligand estrogen, but they are able to bind the estrogen response element (ERE) embedded within the ERR response elements (ERREs) to regulate transcription of genes. Previous work has demonstrated that adult mice lacking Errβ have altered metabolism and meal patterns. To further understand the biological role of Errβ, we characterized the stress response of mice deficient for one or both alleles of Errβ.ResultsSox2-Cre:Errβ mice lack Errβ expression in all tissues of the developing embryo. Sox2-Cre:Errβ+/lox heterozygotes were obese, had increased Npy and Agrp gene expression in the arcuate nucleus of the hypothalamus, and secreted more corticosterone in response to stress. In contrast, Sox2-Cre:Errβlox/lox homozygotes were lean and, despite increased Npy and Agrp gene expression, did not secrete more corticosterone in response to stress. Sox2-Cre:Errβ+/lox and Sox2-Cre:Errβlox/lox mice treated with the Errβ and Errγ agonist DY131 demonstrated increased corticotropin-releasing hormone (Crh) expression in the paraventricular nucleus of the hypothalamus, although corticosterone levels were not affected. Nes-Cre:Errβlox/lox mice, which selectively lack Errβ expression in the nervous system, also demonstrated elevated stress response during an acoustic startle response test and decreased expression of both Crh and corticotropin-releasing hormone receptor 2 (Crhr2).ConclusionsLoss of Errβ affects body composition, neuropeptide levels, stress hormones, and centrally-modulated startle responses of mice. These results indicate that Errβ alters the function of the hypothalamic-pituitary-adrenocortical axis and indicates a role for Errβ in regulating stress response.

Seasonal oscillation of liver-derived hibernation protein complex in the central nervous system of non-hibernating mammals.

Mammalian hibernation elicits profound changes in whole-body physiology. The liver-derived hibernation protein (HP) complex, consisting of HP-20, HP-25 and HP-27, was shown to oscillate circannually, and this oscillation in the central nervous system (CNS) was suggested to play a role in hibernation. The HP complex has been found in hibernating chipmunks but not in related non-hibernating tree squirrels, leading to the suggestion that hibernation-specific genes may underlie the origin of hibernation. Here, we show that non-hibernating mammals express and regulate the conserved homologous HP complex in a seasonal manner, independent of hibernation. Comparative analyses of cow and chipmunk HPs revealed extensive biochemical and structural conservations. These include liver-specific expression, assembly of distinct heteromeric complexes that circulate in the blood and cerebrospinal fluid, and the striking seasonal oscillation of the HP levels in the blood and CNS. Central administration of recombinant HPs affected food intake in mice, without altering body temperature, physical activity levels or energy expenditure. Our results demonstrate that HP complex is not unique to the hibernators and suggest that the HP-regulated liver–brain circuit may couple seasonal changes in the environment to alterations in physiology.