Exercise is necessary to maintain body weight, but most individuals do not get enough of it. This fundamental precept is illustrated by N2KO mice created by the PI in 1997, that contain a targeted deletion of the Nhlh2 transcription factor (Good et al, 1997).  These animals develop an adult onset overweight phenotype, characterized by increased body weight and body fat after 7 (females) to 12 (males) weeks of age. Weight gain is not due to increased food intake but due, rather, to failure to partake in high levels of physical activity. Thus, failure to exercise leads to adult-onset obesity in N2KO mice (Coyle et al., 2002). This problem extends directly to humans, as being overweight is a worldwide problem that affects nearly 60% of the U.S. adult population, due in part to sedentary lifestyles.

Body weight control is ultimately determined by the orchestration of gene regulation through activation of signal transduction pathways. Targets of these signal transduction pathways are transcription factors and the genes they regulate. Although many hypothalamic ?obesity genes? have been identified, relatively few laboratories are studying the molecular mechanisms underlying transcriptional and post-transcriptional regulation of these genes in response to changes in energy balance. N2KO mice are perfectly suited for this type of research because they contain a targeted deletion of Nhlh2, which impacts energy balance through regulation of exercise (Coyle et al., 2002). Two target genes of Nhlh2 are the post-translational neuropeptide processing enzymes, PC1 and PC2 (Jing et al., 2004). Reduced expression of PC1 and PC2 in N2KO mice leads to reduced synthesis of fully processed pro-opiomelanocortin (POMC) and thyrotropin-releasing hormone (TRH), and ultimately to adult-onset obesity. N2KO mice were the first animals developed in which targeted deletion of a neuronal transcription factor resulted in obesity, and they still remain one of the few models for studying transcriptional regulation of genes using an obese knockout mouse model. Thus, studies using this model and involving the Nhlh2 transcription factor bring fundamental insights to the transcriptional mechanisms controlling obesity and overweight phenotypes.

In a long-term study of the phenotypic changes in N2KO mice, our laboratory showed normal food intake in pre-obese N2KO mice. Slight hyperphagia was evident only when the animals became overweight and was proportional to body weight (Coyle et al., 2002). Body temperature was normal in these animals. Spontaneous voluntary exercise was decreased by more than 50% in both male and female N2KO mice given access to computerized running wheels, with no difference in the circadian pattern of running and no motor deficiencies in standard rotarod apparatus tests, indicating that lack of physical exercise was not due to motor or balance problems (Coyle et al., 2002). Reduced spontaneous activity in these animals preceded increased body weight by more than 7 weeks in male N2KO mice. When animals were tested for activity levels beginning at 5 weeks of age, and continuing through 10 weeks of age, N2KO mice were less active at all pre-obese ages compared to same-aged WT mice (Coyle et al., 2002). In studies of home cage ambulatory activity, N2KO mice show a 2-fold reduction in basal activity compared to WT mice. Thus, reduced physical activity in N2KO mice is at least one of the main causes of the adult-onset overweight phenotype in this animal model.

These results suggest that some of the genes that are direct targets of Nhlh2 must be involved in the regulation of exercise and activity. There is evidence that melanocortins, such as αMSH, or downstream effectors of the melanocortin receptors are involved in physical activity behavior, and we have shown that N2KO mice have a 40% reduction in αMSH production (Jing et al., 2004). Mice with a targeted deletion of the melanocortin-4-receptor (MC4RKO), the receptor for αMSH, show adult-onset obesity characterized by lack of physical activity and reduced oxygen consumption, but without lowered body temperature or increased food intake relative to lean mass. This phenotype is very similar to N2KO mice. In humans, functional polymorphisms in MC4R is one of the most common causes of genetic obesity, and some polymorphisms have been shown to affect exercise level in humans18. Recently two unrelated human families with obesity were shown to carry a polymorphism in the promoter of MC4R within an E-box motif that binds human NHLH2 (Valli-Jaakola et al., 2006). These data link expression of MC4R with requirement for Nhlh2 transcription factor in the control of body weight.

Transcription factors are grouped into different families by the motifs they use to regulate gene expression. The Nhlh2 transcription factor belongs to a family of bHLH factors that bind to DNA through their basic domain at an E-box motif (CANNTG) and interact with other transcription factors through their HLH domain. Nhlh2 shares almost 90% homology with another bHLH family member, Nhlh1, which can interact with the E-box motif CAGCTG. Nhlh1 and Nhlh2 share 100% identity in their bHLH domains (Lipkowitz et al., 1992). While both Nhlh1 and Nhlh2 are expressed in overlapping areas of the developing nervous system, adult expression of both genes becomes regionalized. Nhlh2 is expressed in the hypothalamus?particularly the arcuate (ARC), paraventricular (PVN), ventromedial (VMH) dorsalmedial (DMH), and lateral hypothalamus (LH) (Jing et al., 2004; Vella et al., in press; Figure 2) while Nhlh1 is expressed in the hippocampus and cerebellum with low hypothalamic expression (Cogliati et al., 2002). Many of the neuropeptides and receptors involved in body weight regulation are expressed in the same regions as Nhlh2, but not Nhlh1. Nhlh1 knockout mice are not obese (Cogliati et al., 2002).

There are at least eight transcription factors, including Nhlh2, whose activity or expression levels are altered by changes in energy availability (Burnside and Good, 2005). We have evidence that Nhlh2 is one of these energy availability-regulated transcription factors. The proximal promoter region of Nhlh2 contains five putative STAT3 binding motifs (Good, 2000), two NFκB sites and one AP-1 site. Leptin injection and food ingestion by mice result in increased levels of hypothalamic Nhlh2 expression (Vella et al., in press).

Using electrophoretic mobility shift assays (EMSA), we can detect only weak interaction of STAT3 with a putative STAT3 binding motif on the Nhlh2 promoter (Vella et al., in press). Thus, we are considering other putative sites on the Nhlh2 promoter and other possible factors activated by leptin.

Prohormone Convertase 1 (PC1) is a member of a large family of subtilisin-like calcium-dependent serine proteinases. In the CNS, PC1 is expressed in neuropeptide-rich tissues including the hypothalamus, thalamus, hippocampus, and cerebral cortex of the brain. Within the hypothalamus, PC1 expression is co-localized with POMC neurons in the ARC and TRH neurons in the PVN. We have found that levels of PC1 are reduced by up to 50% in ad lib fed N2KO mice (Jing et al., 2004) and that leptin stimulation of PC1 expression is reduced in N2KO mice (Fox et al., unpublished). Using published sequences of mouse and human PC1 promoters, we have found that their proximal promoters contain multiple E-box and STAT binding motifs (Fox et al., in press). Two of the STAT and E-box motifs on both the human and mouse PC1 promoters practically overlap each other. Is this a coincidence or does it imply that there may be coordinate regulation of PC1 by Nhlh2 and STAT3? We are using molecular, proteomic and in vivo approaches to ask if Nhlh2 and STAT3 coordinately regulate PC1 expression in response to leptin.

Nhlh2 is a member of the basic helix-loop-helix transcription factor family. Characterization of the Nhlh2 knockout mouse led us to hypothesize that this transcription factor controls the expression of genes necessary for body weight control. We have published results identifying two possible direct targets of Nhlh2, prohormone convertase I and II (PC1 and PC2) (Jing et al., 2004). Recent work from our laboratory using microarray technology on WT and N2KO animals treated with ad lib feeding, food deprivation, and food deprivation followed by leptin stimulation has identified over 300 gene targets that are significantly different between N2KO and WT in at least two of the conditions. The results, shown in the form of a pie chart (Figure 3), display the types of genes that are potentially regulated by Nhlh2 in the hypothalamus (Fox et al., submitted). These data gives us even more potential targets to examine in the future.

Fox, D.L., Fox, D.L.G., Jensen R.V., and Good, D.J. (2007) “Microarray Technology Uncovers Biological Pathways Involved in the Development of Obesity in Nhlh2 Knockout Mice” in Genes, Genomes and Genomics, Vol 2, Thangadurai D, Tang W., and Pullaiah T., eds Regency Publications, New Delhi, invited chapter, submission date January 15, 2007.

Good, D.J. (2007) ”Mouse Models of Obesity and Overweight”, in “Sourcebook of Models for Biomedical Research”, invited chapter, submission date September 15, 2006

Fox, D. L., Vella, K.R., and Good, D.J. (2007) “Energy Balance Pathways Converging in the Nhlh2 Transcription Factor”, in Frontiers in Bioscience, Peptides Controlling Energy Balance, invited chapter, under review

Coyle, C.A., Strand, S. C., and Good, D. J. (2007) Reduced physical activity contributes to obesity in Tub Mutant Mice, Obes. Res., in preparation.

Fox, D., Vella, K. R., Strand, S. C., and Good, D.J. (2007) Non-canonical of Nhlh2 Knockout Mice to Food Deprivation, Obes. Res., in revision

Vella, K.A., Burnside, A.S. Brennan, K.A. and Good, D.J.  (2007)  Nhlh2 is a target of energy balance signals, J. Neuroendocrinology, in review

Kevrekidis, P. G., Whitaker, N., and Good, D.J. (2006) A minimal model for tumor angiogenesis, Physical Review Letters, 73(6 Pt 1):061926. PubMed

Brennan, K.M., Vella, K.R. and Good, D.J. (2006) Genetic analysis of NHLH2 and its putative role in bovine body weight control, Animal Genetics, 37, Suppl 1:24-7. Pubmed

A.S., and Good, D.J. (2006) Genetic Diversity of Genes Involved in Body Weight Regulation in Genes, Genomes and Genomics, Vol 1, Thangadurai D, Tang W., and Pullaiah T., eds Regency Publications, New Delhi, pp74-97. ISBN 81-89233-38-6 Publisher’s Link

Kevrekidis, P. G., Whitaker, N., and Good, D.J. (2005) Towards a reduced model for angiogenesis: A Hybrid Approach, Mathematics and Computer Modeling, 41: 987-996.

Good, D.J. (2005) Using Obese Mouse Models in Research:  Special considerations for IACUC members, animal care technicians and researchers. Lab Animal 34: 30-37. PubMed

Good, D.J.  (2004) The use of Flash animations within a WebCT environment: Enhancing comprehension of experimental procedures in a biotechnology laboratory. International Journal of Instructional Media, 31: 355-370. Amazon.com link

Burnside, A.S. and Good, D.J. (2004) Mind Over Matter:  Transcriptional regulation of body weight by Hypothalamic Neurons.  In Recent Research Developments in Molecular and Cellular Biology, Vol 5 S. Pandali, ed., Research Signpost Publishers, pp 23-39. ISBN 81-7736-213-5 Publisher’s Link

Johnson, S.A., Miele, M., Marin-Bivens, C., Coyle, C.A., Fissore, R., and Good, D.J.,  (2004) The Nhlh2 transcription factor is required for female sexual behavior and reproductive longevity, Hormones and Behavior, 46(4):420-7   PubMed

Jing E., Nillni, E.A., Sanchez, V.C., Stuart, R., and Good, D.J., (2004) Reduced levels of hypothalamic prohormone convertase I mRNA in obese the Nhlh2 transcription factor knockout mouse. Endocrinology, 145: 1503-1513  PubMed

Jing, E., and Good, D.J.,  (2003) “Decreased Expression of Protein Convertase I and II mRNA in Obese Nhlh2 Knockout Mice”, The Melanocortine System, New York Academy of Sciences. (published abstract)

Coyle, C.A., Jing, E., Hosmer, T., Powers, J.B., Wade, G., and Good, D.J. (2002)  Reduced voluntary activity precedes adult-onset obesity in Nhlh2 knockout mice, Physiol. Behavior, 77: 387-402. PubMed

Cogliati, T.*, Good, D.J.*, Haigney, M., Delgado-Romero, P., Eckhaus, M.A., Koch, W.J., and Kirsch, I.R., (2002) Predisposition to arrhythmia and autonomic dysfunction in Nhlh1-deficient mice.  Mol. Cell. Biol, 22: 4977-4983  PubMed  Note: T. Colgiati and D.J. Good contributed equally to this paper

Good, D.J., Jing, E., Coyle, C.A., Powers, B. and Wade, G. (2000) Mechanism of obesity in the Nhlh2 transcription factor knockout mouse. Obesity Res. 8: Suppl. 1, 15S. (published abstract)

Good, D.J.(2000) How tight are your genes?  Transcriptional and posttranscriptional regulation of the leptin receptor, POMC and NPY genes. Hormones and Behavior, 37:  284-298. PubMed

Izraeli, S., Lowe, L.A., Bertness, V. L., Good, D.J., Dorward, D.W., Kirsch, I.R., and Kuehn, M.R., (1999)  The SIL gene is required for mouse embryonic axial development and left-right specification, Nature, 399: 691-694 PubMed

Good, D.J. (1998) Obesity…is it all in your head?  Accents 2:1-2. (invited review)

Good, D.J., Porter, F.D., Mahon, K.A., Parlow, A., Westphal, H., and Kirsch, I.R., (1997) Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene.  Nature Genetics 15:  397-401. PubMed

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Izraeli S, Lowe L, Bertness V, Good DJ, Kuehn MR, Kirsch IR. (1997) The immediate early gene SIL is required for embryonic development and determination of left/right axis.; Dev. Biol. (1997) 186:B93 (published abstract)