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Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Conjugated linoleic acid (CLA) has been reported to prevent body weight gain and fat accumulation in part by improving physical activity in mice. However, the effects of postweaning administration of CLA on the development of obesity later in life have not yet been demonstrated. The current study investigated the role of postweaning CLA treatment on skeletal muscle energy metabolism in genetically induced inactive adult-onset obese model, nescient basic helix-loop-helix 2 knockout (N2KO) mice. Four-week-old male N2KO and wild type mice were fed either control or a CLA-containing diet (0.5%) for 4 weeks, and then CLA was withdrawn and control diet provided to all mice for the following 8 weeks. Postweaning CLA supplementation in wild type animals, but not N2KO mice, may activate AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-δ (PPARδ) as well as promote desensitization of phosphatase and tensin homologue (PTEN) and sensitization of protein kinase B (AKT) at threonine 308 in gastrocnemius skeletal muscle, improving voluntary activity and glucose homeostasis. We suggest that postweaning administration of CLA may in part stimulate the underlying molecular targets involved in muscle energy metabolism to reduce weight gain in normal animals, but not in the genetically induced inactive adult-onset animal model.

Nescient helix-loop-helix 2 (NHLH2/NSCL2) is a neuronal transcription factor originally thought to be involved in neuronal development and childhood neuroblastomas. Accumulating evidence has since identified roles for NHLH2 in adult phenotypes of obesity and fertility. We summarize these findings here and attempt to link genotype with phenotype in mouse models and humans. In particular, NHLH2 (Nhlh2 in mice) is one of only two genes that are genetically linked to physical activity levels. Nhlh2 also controls obesity and fertility, with strong sexual dimorphism for both phenotypes in Nhlh2 mutant animals. We propose that Nhlh2 might function as a molecular sensor in different adult hypothalamic neurons to regulate energy balance, leading to normal body weight and reproduction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Conjugated linoleic acid (CLA) has been extensively studied during the last two decades with regard to its effects on controlling body composition. As a cognate to CLA, conjugated nonadecadienoic acid (CNA) has been previously reported to reduce body fat more effectively than CLA. However, it is not known whether CNA supplementation can influence adult-onset obesity. Thus, the purpose of this study was to evaluate the effects of dietary CNA on the prevention of adult-onset inactivity-induced obesity using nescient basic helix-loop-helix 2 knockout (N2KO) mice. CNA supplementation at 0.1 w/w% level starting in the preobese state significantly prevented the reduction of voluntary movement and the increase in weight gain in N2KO mice during the experimental period compared to wild-type animals. In both wild-type and N2KO mice, respiratory exchange ratio was significantly reduced by CNA treatment during light and dark cycles, and dietary CNA significantly increased energy expenditure in N2KO mice. Selected gene expression profiles in white adipose tissue, muscle or liver showed a beneficial action of CNA on lipid metabolism and energy expenditure. These findings suggest that CNA could prevent adult-onset obesity by enhancing voluntary activity and energy expenditure in N2KO mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The purpose of this study was to evaluate whether conjugated linoleic acid (CLA) exposure during the developmental period increases voluntary activity, which would influence obesity outcome later in life. The effects of dietary supplementation of 0.5% CLA in a high fat diet were evaluated in nescient basic helix-loop-helix 2 (Nhlh2) knock-out (N2KO) mice, which is a unique animal model representing inactivity-induced obesity in a pre-obese condition. Male wild type and N2KO mice were fed either control or CLA (0.5%) diet for 8 weeks. As expected, control diet fed N2KO animals showed greater body weight with decreased physical activity in the late stage of the experimental period compared with wild type control. Dietary CLA significantly decreased body weight and adipose depots in both wild type and N2KO mice, and the body weights of both genotypes fed CLA were similar during the experimental period. CLA exposure during the developmental period significantly improved the impairment of physical activity in N2KO mice, but the wild type did not show any effect of CLA. In both genotypes, CLA significantly reduced serum triglycerides levels and down-regulated the mRNA expressions of CCAAT/enhancer binding protein α (C/EBPα) and leptin in white adipose tissue. These findings suggest that early CLA exposure could prevent obesity with improved voluntary physical activity in N2KO mice.

Conjugated linoleic acid (CLA) has shown a number of biologically beneficial effects, including prevention of obesity. The purpose of this study was to test effects of dietary supplementation of 0.5% trans-10,cis-12 CLA in a high fat diet in neuronal basic helix-loop-helix 2 knock-out animals (N2KO), which is a unique animal model representing adult-onset inactivity-related obesity. Eight wild-type (WT) and eight N2KO female mice were fed either 0.5% trans-10,cis-12 CLA-containing diet or control diet (with 20% soybean oil diet) for 12 weeks. Body weights, food intake, adipose tissue weights, body compositions, and blood parameters were analyzed. Overall, N2KO animals had greater body weights, food intake, adipose tissue weights, and body fat compared to WT animals. CLA supplementation decreased overall body weights and total fat, and the effect of dietary CLA on adipose tissue reduction was greater in N2KO than in WT mice. Serum leptin and triglyceride levels were reduced by CLA in both N2KO and WT animals compared to control animals, while there was no effect by CLA on serum cholesterol. The effect of CLA to lower fat mass, increase lean body mass, and lower serum leptin and triglycerides in sedentary mice supports the possibility of using CLA to prevent or alleviate ailments associated with obesity.

The Tub gene was originally identified as a spontaneous mutation in C57Bl/6J mice, and associated with adult-onset obesity (Tub MUT mice). Although the original Tub MUT mouse was identified over 15 years ago, there have been few reports on the animal's food intake, body fat percentage or energy expenditure. In this study, we report food intake, body weight from 5-20 weeks, body fat, body temperature and three different measures of physical activity behavior. Tub MUT mice display reduced food intake, uncharacteristic of many obese mouse models, and reduced voluntary wheel running with normal home cage ambulatory behavior. We conclude that motivation for food and exercise is an underlying defect in TUB MUT mice.

The mouse is the animal most commonly used to study the underlying causes of and treatments for obesity. The author reviews many of the issues that should be considered by all involved in research with mice expressing this phenotype, and describes some procedures exclusive to obesity research.

Body weight is controlled by the activation of signal transduction pathways in both the brain and peripheral tissues. Interestingly, although many hypothalamic neuropeptides and receptors have been implicated in the regulation of body weight, the transcriptional and posttranscriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Our laboratory studies a mouse in which targeted deletion of the neuronal basic helix-loop-helix (bHLH) transcription factor, nescient helix-loop-helix 2 protein (Nhlh2), results in adult-onset obesity. The aim of this work was to use the phenotype of the Nhlh2 knockout mouse and the expression pattern of Nhlh2 to identify genes that are regulated by this transcription factor. In this article, we show that Nhlh2 is expressed throughout the adult hypothalamus. Using dual-label in situ hybridization, we demonstrate that, in the arcuate nucleus of the adult hypothalamus (ARC), Nhlh2 expression can be found in rostral proopiomelanocortin (POMC) neurons, whereas in the paraventricular nucleus (PVN), Nhlh2 is expressed in TRH neurons. In addition, we find that hypothalamic POMC-derived alphaMSH in the ARC and TRH in the PVN are regulated posttranscriptionally via Nhlh2-mediated control of prohormone convertase I and II mRNA levels. This is the first report in which regulation of body weight is linked to the action of a neuronal bHLH transcription factor on prohormone convertase mRNA levels. Furthermore, this work supports a direct role for transcriptional control of neuropeptide processing enzymes in the etiology of adult-onset obesity.

Targeted deletion of the neuronal basic helix-loop-helix transcription factor Nhlh2 results in adult-onset obesity in mice. Measurement of body weight and body composition in animals aged 3-25 weeks indicates that while male and female Nhlh2 knockout (N2KO) animals both show adult-onset obesity, the time frame for development of obesity is different, with females becoming obese by 7 weeks of age and males becoming obese by 10 weeks of age. Heterozygous (HET) animals also become obese but with a slower onset, indicating a dosage effect for the activity of the Nhlh2 transcription factor. Food intake, body temperature, and voluntary activity were measured in both preobese and obese N2KO, HET, and wild-type (WT) animals to determine which factors contributed to weight gain. While increased food intake and decreased body temperature were found in older obese N2KO animals, only reduced physical activity preceded the onset of obesity in N2KO mice. N2KO animals had no deficit in either circadian rhythm or balance and motor control, indicating that reduced voluntary activity is the result of a behavioral change. These data demonstrate a role for the Nhlh2 transcription factor in controlling genes important to energy expenditure, and more specifically voluntary physical activity of animals.

Objective: To assess the role of body mass index (BMI) and exercise levels in self-perception of critical thinking skills. Participants: Three hundred forty-seven students from an upper-division nutrition class over two consecutive years. Methods: A pre/post survey with a 15-week intervention assessed perceived critical thinking skills in a blended classroom. Results: Students gained in perceived critical thinking skills in six areas over the semester. A higher BMI was associated with decreased perception of one's ability to think logically, along with increased perception that memorization was the key to success. Those that exercised reported that they had strong critical thinking skills compared to those that exercised less frequently. Conclusions: A blended classroom approach was effective in increasing multiple areas of perceptions of critical thinking. However, some perceptions of critical thinking are viewed differently for those of different BMIs and exercise frequency. Consequently, designing interventions specifically targeting those with higher BMIs, could work to erase these inequities.

Recently, conjugated linoleic acid (CLA) has been reported to prevent body weight gain and fat accumulation in part by improving physical activity in mice. The current study was conducted to determine the role of CLA on skeletal muscle metabolism in nescient basic helix-loop-helix 2 knock-out (N2KO) mice, an adult-onset inactivity induced obese model. Five-week-old female N2KO and wild type mice were fed either control or CLA containing diet (0.5%) for 10 wk. Voluntary activity was determined biweekly and markers for muscle metabolisms were determined from the gastrocnemius muscle. CLA fed N2KO animals showed significant increased voluntary movement and gastrocnemius muscle mass compared to control group, whereas in wild type animals, no differences were observed. CLA treatment up-regulated AMP-activated protein kinase (AMPK), mitochondria biogenic markers, peroxisome proliferator-activated receptor-δ (PPARδ), and mitochondrial transcription factor A (Tfam) compared to control animals. These observations indicate that CLA supplementation activates AMPKα-PPARδ and/or -Tfam signaling cascade for stimulating mitochondria biogenesis. Taken together, these results suggest that CLA may in part activate the underlying biomarkers involved in muscle metabolism via stimulation of mitochondrial biogenesis, resulting in increased voluntary activity and muscle mass, potentially contributing to regulating weight gain. Practical applications: Lack of physical activity is a global public health problem, which induces obesity and its associated pathologies. Approximately, 3.2 million deaths per year are attributable to lack of physical activity. CLA has previously been reported to increase voluntary and endurance activities in mice. However, the exact mode of action is not completely understood. Thus, the purpose of the study was to shed light on positive effects of CLA on physical activity through modulation of molecular targets in skeletal muscle of Nhlh2 knockout mice, which are an adult-onset inactivity induced obesity model. The current results suggest that CLA acts as a potential exercise-mimetic, resulting in increased voluntary activity and muscle mass, potentially contributing to regulating weight gain. Conjugated linoleic acid partially improves muscle metabolism via stimulation of mitochondrial biogenesis, resulting in increased voluntary activity. [ABSTRACT FROM AUTHOR]

Prior research has demonstrated a genetic basis for motivated exercise, with evidence of a role for nescient helix-loop-helix-2 (NHLH2/Nhlh2). Nhlh2 transcriptionally regulates the monoamine oxidase A (MAO-A) gene. This article examines the evidence for the hypothesis that polymorphisms in NHLH2 or MAO-A contribute to differences in the human motivation for exercise and physical activity. The genetic pathways that link exercise and motivation are discussed. [ABSTRACT FROM AUTHOR]

Prader–Willi Syndrome (PWS) is a human genetic condition that affects up to 1 in 10,000 live births. Affected infants present with hypotonia and developmental delay. Hyperphagia and increasing body weight follow unless drastic calorie restriction is initiated. Recently, our laboratory showed that one of the genes in the deleted locus causative for PWS, Snord116, maintains increased expression of hypothalamic Nhlh2, a basic helix–loop–helix transcription factor. We have previously also shown that obese mice with a deletion of Nhlh2 respond to a conjugated linoleic acid (CLA) diet with weight and fat loss. In this study, we investigated whether mice with a paternal deletion of Snord116 (Snord116m+/p−) would respond similarly. We found that while Snord116m+/p− mice and mice with a deletion of both Snord116 alleles were not significantly obese on a high-fat diet, they did lose body weight and fat on a high-fat/CLA diet, suggesting that the genotype did not interfere with CLA actions. There were no changes in food intake or metabolic rate, and only moderate differences in exercise performance. RNA-seq and microbiome analyses identified hypothalamic mRNAs, and differentially populated gut bacteria, that support future mechanistic analyses. CLA may be useful as a food additive to reduce obesity in humans with PWS. [ABSTRACT FROM AUTHOR]

Abstract: Melanocortin 4 receptor (Mc4r/MC4R) is a G-Protein coupled receptor that is expressed in the hypothalamus and implicated in body weight control. Mutations in MC4R are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation underlying the melanocortin pathway are far from being fully understood. The transcription factor nescient helix-loop-helix 2 (Nhlh2) influences the melanocortin pathway through transcriptional regulation of prohormone convertase I, which influences the production of melanocortin peptides. In the present study, Nhlh2’s role as a transcriptional regulator of Mc4r has been demonstrated. Nhlh2 knockout mice have reduced hypothalamic expression of Mc4r mRNA, suggesting that it could be a direct or indirect transcriptional regulator of the Mc4r promoter. To demonstrate direct transcriptional regulation, chromatin immunoprecipitation and electrophoretic gel shift assays show that Nhlh2 binds to the E-Boxes located at −551, −366 and +54 on the Mc4r promoter. Leptin-induced transactivation of the Mc4r promoter is significantly higher in the presence of exogenously added Nhlh2. siRNA knockdown of Nhlh2 leads to significantly reduced endogenous Mc4r mRNA expression levels in N29/2 cell line. Transactivation using promoters with mutations in each of the E-Boxes results in significantly reduced transactivation efficiency compared to the WT Mc4r promoter, suggesting that Nhlh2 regulates Mc4r transcription through these sites. Findings from these studies, combined with previous work implicating Nhlh2 as a transcriptional regulator of both the Mc4r gene and the melanocortin pathway, suggest that Nhlh2’s transcriptional activity directly influences the human and rodent body weight control pathways. [Copyright &y& Elsevier]

Abstract: Nescient helix-loop-helix-2 (NHLH2) is a basic helix-loop-helix transcription factor, which has been implicated, using mouse knockouts, in adult body weight regulation and fertility. A scan of the known single nucleotide polymorphisms (SNPs) in the NHLH2 gene revealed one in the 3′ untranslated region (3′UTR), which lies within an AUUUA RNA stability motif. A second SNP is nonsynonymous within the coding region of NHLH2, and was found in a genome-wide association study for obesity. Both of these SNPs were examined for their effect on NLHL2 by creating mouse mimics and examining mRNA stability, and protein function in mouse hypothalamic cell lines. The 3′UTR SNP causes increased instability and, when the SNP-containing Nhlh2 3′UTR is attached to luciferase mRNA, reduced protein levels in cells. The nonsynonymous SNP at position 83 in the protein changes an alanine residue, conserved in NHLH2 orthologs through the Drosophila sp. to a proline residue. This change affects migration of the protein on an SDS-PAGE gel, and appears to alter secondary structure of the protein, as predicted using in silico methods. These results provide functional information on two rare human SNPs in the NHLH2 gene. One of these has been linked to human obese phenotypes, while the other is present in a relatively high proportion of individuals. Given their effects on NHLH2 protein levels, both SNPs deserve further analysis in whether they are causative and/or additive for human body weight and fertility phenotypes. [Copyright &y& Elsevier]