Deletion of Nhlh2 Results in a Defective Torpor Response and Reduced Beta Adrenergic Receptor Expression in Adipose Tissue

Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2), display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO) mouse a useful model to study the role of central nervous system (CNS) control on peripheral tissue such as adipose tissue.Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes.We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for beta3-adrenergic receptors (beta3-AR), beta2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice.These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals

NPY and MC4R Signaling Regulate Thyroid Hormone Levels during Fasting through Both Central and Peripheral Pathways

SummaryFasting-induced suppression of the hypothalamic-pituitary-thyroid (HPT) axis is an adaptive response to decrease energy expenditure during food deprivation. Previous studies demonstrate that leptin communicates nutritional status to the HPT axis through thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus. Leptin targets TRH neurons either directly or indirectly via the arcuate nucleus through pro-opiomelanocortin (POMC) and agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons. To evaluate the role of these pathways in vivo, we developed double knockout mice that lack both the melanocortin 4 receptor (MC4R) and NPY. We show that NPY is required for fasting-induced suppression of Trh expression in the PVN. However, both MC4R and NPY are required for activation of hepatic pathways that metabolize T4 during the fasting response. Thus, these signaling pathways play a key role in the communication of fasting signals to reduce thyroid hormone levels both centrally and through a peripheral hepatic circuit

Arcuate AgRP, but not POMC neurons, modulate paraventricular CRF synthesis and release in response to fasting

Background: The activation of the hypothalamic–pituitary–adrenal (HPA) axis is essential for metabolic adaptation in response to fasting. However, the neurocircuitry connecting changes in the peripheral energy stores to the activity of hypothalamic paraventricular corticotrophin-releasing factor (CRFPVN) neurons, the master controller of the HPA axis activity, is not completely understood. Our main goal was to determine if hypothalamic arcuate nucleus (ARC) POMC and AgRP neurons can communicate fasting-induced changes in peripheral energy stores, associated to a fall in plasma leptin levels, to CRFPVN neurons to modulate the HPA axis activity in mice. Results: We observed increased plasma corticosterone levels associate with increased CRFPVN mRNA expression and increased CRFPVN neuronal activity in 36 h fasted mice. These responses were associated with a fall in plasma leptin levels and changes in the mRNA expression of Agrp and Pomc in the ARC. Fasting-induced decrease in plasma leptin partially modulated these responses through a change in the activity of ARC neurons. The chemogenetic activation of POMCARC by DREADDs did not affect fasting-induced activation of the HPA axis. DREADDs inhibition of AgRPARC neurons reduced the content of CRFPVN and increased its accumulation in the median eminence but had no effect on corticosterone secretion induced by fasting. Conclusion: Our data indicate that AgRPARC neurons are part of the neurocircuitry involved in the coupling of PVNCRF activity to changes in peripheral energy stores induced by prolonged fasting.Fil: Alves Fernandes, Alan Carlos. University Of Ribeirao Preto; BrasilFil: Pereira de Oliveira, Franciane. Universidade Federal de Sao Paulo.; BrasilFil: Fernandez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: da Guia Vieira, Luane. University Of Ribeirao Preto; BrasilFil: Gugelmin Rosa, Cristiane. University Of Ribeirao Preto; BrasilFil: do Nascimento, Taís. University Of Ribeirao Preto; BrasilFil: de Castro França, Suzelei. University Of Ribeirao Preto; BrasilFil: Donato Jr, Jose. Universidade de Sao Paulo. Departamento de Fisiología; BrasilFil: Vella, Kristen R.. Weill Cornell Medical College; Estados UnidosFil: Antunes Rodrigues, Jose. University Of Ribeirao Preto; BrasilFil: Mecawi , André. Universidade Federal de Sao Paulo.; BrasilFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Leico Kagohara Elias, Lucila. University Of Ribeirao Preto; BrasilFil: Rorato, Rodrigo. Universidade Federal de Sao Paulo.; Brasi

Modulation of Nhlh2 expression by energy availability leads to downstream effects on body weight regulation

Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 (N2KO) display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 co-localizes with the hypothalamic thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus (PVN) and proopiomelanocortin (POMC) neurons in the arcuate nucleus. N2KO mice become obese due to reduced physical activity in the absence of hyperphagia making them a unique mouse model for the study weight gain, obesity and energy expenditure. Signals that regulate Nhlh2 and the effects of Nhlh2 on peripheral tissues remain largely unknown. The research presented here utilized numerous techniques to investigate the effects of changes in energy availability on Nhlh2 expression. We show that Nhlh2 expression decreases significantly with food deprivation and cold exposure. Nhlh2 expression is stimulated with food return or leptin injection following food deprivation or return to room temperature following cold exposure. These data suggest that Nhlh2 gene expression responds positively to increased energy availability and negatively to reduced energy availability. These findings combined with the phenotype of N2KO mice led us to propose that Nhlh2 integrates energy availability inputs in various hypothalamic nuclei to drive expression of genes required for body weight maintenance. Investigation into peripheral tissues in N2KO mice revealed that responses of genes in the hypothalamus-pituitary-thyroid axis, muscle, and brown and white adipose tissue to changes in energy availability require Nhlh2 expression. The responses of serum total T4 levels and UCP1 mRNA and UCP3 mRNA to energy availability signals are altered in N2KO mice. In addition, N2KO mice maintain body temperature with cold exposure but are unable to maintain body weight. In summary, my work provides new insight into the role of Nhlh2 in coordinating energy availability signals to downstream genes required for body weight maintenance and thermoregulation

Expression profile in BAT and WAT.

<p>Relative quantitative expression levels for b-adrenergic receptors (β1AR, β2AR and β3AR) and uncoupling proteins (UCP1 and UCP2) in RNA isolated from BAT (<b>A.</b>) and WAT (<b>B.</b>). The data is reported as the mean expression level relative to WT expression ± SEM. (*p≤0.05, **p≤0.01).</p

Histology and immunohistochemistry of Brown and White Adipose Tissues from N2KO and WT mice.

<p>H&E staining of Brown Adipose Tissue (<b>A</b>) or White Adipose Tissue (<b>C</b>) from WT, (<b>B</b>) and (<b>D</b>) from N2KO. Scale bars for whole and inset pictures are given. Immunohistochemistry of WAT using F4/80, a macrophage specific marker staining of WT (<b>E</b>) and N2KO (<b>F</b>).</p

Expression profile for pro-inflammatory genes from WT and N2KO mice.

<p><b>A</b>. Relative quantitative expression in RNA from WAT isolated from ad lib fed WT and N2KO for interleukin-6 (IL-6), Cluster of Differentiation 68 (CD68) and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). The data are reported as mean expression level relative to WT expression ± SEM. (*p≤0.05). <b>B</b>. Serum IL-6 levels were measured from ad lib fed WT and N2KO mice. The data is reported as the serum level (pg/ml) ± SEM (*p≤0.05). <b>C.</b> Western analysis of WAT for F4/80 expression in protein extracted from ad lib fed mice. Equal total protein amounts were added for N = 3 WT mice, and N = 4 N2KO mice.</p

Primer sequences used for quantitative real-time RT-PCR assays.

<p>All sequences are to the mouse genes, and the forward and reverse primers are indicated.</p

WAT metabolic gene expression profile in WT and N2KO mice.

<p>Relative quantitative expression levels of carnitine palmitoyltransferase-1α (CPT-1α), adiponectin, hormone sensitive lipase (HSL), and peroxisome proliferator-activated receptor alpha (PPARα) and delta (PPARδ) in WAT from ad lib fed WT and N2KO. All samples were normalized to β-actin expression. The data is reported as the mean expression level relative to WT expression ± SEM. (*p≤0.05, **p≤0.01).</p