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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 31, 2005 as doi:10.1096/fj.04-3216fje. |
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,2,
* Sub-Department of Animal Behaviour, University of Cambridge, Madingley, UK;
Department of Physiology, University of Cambridge, Cambridge, UK;
Department of Physiology, Gothenburg, Sweden; and
Wellcome Trust/Cancer Research UK, University of Cambridge, Cambridge, UK
1Correspondence: Sub-Department of Animal Behavior, University of Cambridge, Madingley, Cambridge CB3 8AA, UK. E-mail: jpc38{at}cam.ac.uk
SPECIFIC AIMS
The paternally expressed gene Peg3 encodes a C2H2 type zinc finger protein that is implicated in regulating various apoptotic pathways. Previous studies demonstrated that mice with a targeted mutation of the Peg3 gene were growth retarded both in utero and postnatally. Recently, Peg3 has been identified from a whole genome scan as a candidate gene for the regulation of fat content and has been shown to have altered expression in a high-fat diet-induced mouse model of obesity. In the course of our studies of Peg3+/– mutants, we observed they were hypophagic but had elevated fat levels, a finding that merited further metabolic studies.
PRINCIPAL FINDINGS
1. Adult Peg3+/– mutants have elevated abdominal, subcutaneous, and intrascapular fat despite being growth retarded and hypophagic
Both male and female Peg3+/– mice weigh significantly less at birth than wild-type mice (1.20±0.02 g cf 1.40±0.03 g). As adults, despite having a smaller mean body weight (28.3±0.3 g cf 32.7±0.4 g), mutant males had significantly more white fat (1.78±0.11 g cf 0.97±0.06 g) than wild-type males (Fig. 1
a). The relationship between body weight and total fat (abdominal+subcutaneous+intrascapular) in wild-type and mutant animals is cumulative with the slope showing greatest divergence from controls at higher body weights. The inverse situation was observed when examining gastrocnemius muscle in adult Peg3+/– and wild-types (Fig. 1b
), as mutants had significantly smaller muscle weight than wild-types (0.11±0.00 g cf 0.15±0.01 g). Despite the higher levels of body fat, mutant Peg3+/– animals were hypophagic compared with controls. Over 4 months, adult mutant males did not consume as much food as wild-type males (Fig. 1c
, mean weekly intake: 29.8±0.8 g cf 36.8±0.6 g) and had a significantly slower increase in body weight (Fig. 1d
). This increased body fat phenotype appears to emerge postpubertally, as prepubertal (day35pn) Peg3+/– females have less body fat than wild-type females (0.08±0.01 g cf 0.22±0.01 g). Hence, there appears to be a postpubertal switch from a low- to high-fat phenotype (Fig. 1e, f
).
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2. Peg3+/– mutants have a reduced metabolic rate and body temperature and cannot respond to a cold challenge
Mutants had a significantly lower oxygen consumption than wild-type males (0.97±0.05 mL/kg/minVO2 cf 1.41±0.06 mL/kg/minVO2) even when body weight is accounted for (42.4±2.7 mL/kg/minVO2/weight cf 52.2±1.2 mL/kg/minVO2/weight), although no difference between wild-type and Peg3+/– males was recorded in wheel running activity (revolutions/24 h). Peg3+/– males and females had a significantly lower resting body temperature than wild-types and were unable to increase their body temperature when confronted with a cold challenge, unlike wild-types. However, peripheral noradrenaline challenge produced similar increases in body temperature in wild-type and mutant males.
3. Peg3+/– mutants have elevated leptin levels and appear to be leptin resistant
The composition of adult wild-type and Peg3+/– male serum was analyzed to determine whether the body fat phenotype was related to any gross differences in hormones or metabolites. No significant differences in levels of insulin, glucose, corticosterone, free fatty acids, IGF-I, T3, or T4 were observed between adult genotypes. However, consistent with having higher body fat, the Peg3+/– mutants had significantly elevated levels of leptin hormone than wild-type males (2124±382pg/mL cf 285±101pg/mL). Consistent with these elevated leptin levels, Peg3+/– males were less responsive to exogenous leptin than controls. Both Peg3+/– and wild-type males significantly decreased their food intake levels during leptin administration, but wild-type males did so by significantly more than mutants. After the termination of leptin treatment, wild-type males increased their food intake by significantly more than did Peg3+/– males. Significant differences in body weight changes were also observed, with wild-type males losing more weight when treated with leptin and gaining more weight post-treatment.
4. Prepubertal mutant Peg3+/– hypothalamic neurons express higher levels of NPY, MCH, and orexin mRNA and lower levels of POMC mRNA than wild-types
Expression of the orexigenic neuropeptides NPY, MCH, and orexin and the anorexigenic neuropeptide POMC was measured in hypothalamic neurons of prepubertal and adult males. In the arcuate nucleus of the hypothalamus, prepubertal mutant Peg3+/– neurons expressed significantly less POMC mRNA and significantly more NPY mRNA than wild-type neurons. In the lateral hypothalamus, prepubertal mutant Peg3+/– neurons expressed significantly more MCH mRNA and orexin mRNA than wild-type neurons.
In adult animals, there was no significant difference between mutant and wild-type males in the levels of expression of POMC mRNA in the arcuate nucleus. However, adult mutants did express significantly more POMC mRNA than prepubertal mutants, while there was no difference between adult and prepubertal expression of POMC mRNA in wild-types. In contrast to the findings in prepubertal animals, adult mutant Peg3+/– arcuate neurons actually expressed significantly less NPY mRNA than those of adult wild-type males. Congruent with this change, adult Peg3+/– arcuate neurons expressed significantly less NPY than prepubertal mutant arcuate neurons. No difference was found between adult and prepubertal wild-type males in the expression of NPY mRNA.
In the lateral hypothalamus, adult Peg3+/– neurons expressed significantly more MCH than wild-type adults but significantly less MCH than neurons in prepubertal mutant mice. There was no difference in the expression of MCH between prepubertal and adult wild-type mice. In addition, adult Peg3+/– males expressed significantly less orexin mRNA in the lateral hypothalamus than prepubertal mutant males, but there was no change in the expression of orexin mRNA between prepubertal and adult wild-type males.
CONCLUSIONS AND SIGNIFICANCE
Previous work has shown that a lack of Peg3 expression in fetal and extraembryonic tissues leads to growth retardation during embryogenesis in mutant animals. Postnatally, mutants continue to be growth retarded and are impaired in suckling, thereby receiving fewer resources during the lactational period than wild-type animals. They are also slower than controls to make the transition from milk to solid food and later to enter puberty and thermoregulate. Here we show that postpubertally, mutant Peg3+/– males and females injest fewer calories, eating 80% of the food consumed by wild-type animals (Fig. 1c
). Despite experiencing significantly less energy intake via the placenta during embryogenesis and by ingestion throughout lactation, postpubertal mutants have elevated levels of abdominal, subcutaneous, and intrascapular body fat.
Given that Peg3+/– mice are hypophagic, the increased body fat is likely to be a consequence of reduced energy expenditure. Although Peg3+/– animals do not show any significant difference in their levels of running activity, they do have metabolic dysfunctions. They have a lower core body temperature than wild-type animals and a lower metabolic rate as shown by reduced oxygen consumption. In addition, the smaller size of gastrocnemius skeletal muscle in mutants compared with controls (Fig. 1b
) suggests that Peg3+/– animals are less able to increase their body temperature through shivering thermogenesis. The deficits in temperature regulation in mutants appear to be related to hypothalamic dysfunction rather than deficits in adaptive thermogenesis in brown fat. Body temperature is regulated by the medial preoptic area (MPOA) of the hypothalamus, which promotes heat gain and inhibits heat loss through activation of brown adipose tissue thermogenesis. Noradrenaline release from the sympathetic nervous system operates via ß-adrenergic receptors and cyclic AMP to initiate adaptive thermogenesis and an increase in metabolic rate and oxygen consumption. Mutant Peg3+/– males are unable to increase their body temperature when presented with a 30 min cold challenge, but are able to increase their body temperature when administered noradrenaline. Peg3+/– animals are therefore able to respond to sympathetic noradrenaline challenge, albeit at a lower level, but there exists a deficit in activating this system via the hypothalamus. The apparent leptin resistance of adult mutants, and the inability of prepubertal mutants to respond appropriately by increasing food intake to neuropeptide signals are evidence of other hypothalamic dysfunctions.
The Peg3 gene appears to be regulating developmental processes that predispose the brain to a lower set-point in energy balance. Understanding how such a mechanism coordinates many components of energy homeostasis makes this mutant unique for the study of increased adiposity and the changing set-point with respect to fat deposition on aging. The likeliest explanation is that the mutation in the Peg3 gene disrupts normal hypothalamic development leading to the cascade of energy balance dysfunctions. The hypothalamus has long been known to be critical for energy homeostasis through its actions on food intake, body temperature, metabolic rate, and physical activity. Peg3 is expressed at high levels throughout the embryonic, prepubescent, and adult brain in several of these brain areas. The expression is particularly high in the arcuate, ventromedial, dorsomedial, paraventricular (PVN), supraoptic (SON), and suprachiasmatic nuclei of the hypothalamus, MPOA, BNST, and amygdala. Through its role in various apoptotic pathways the Peg3 gene has the capacity to regulate multiple events that relate to energy homeostasis. This has already been demonstrated in Peg3+/– mutant females in a previous study where a deficit in maternal behavior is associated with a decrease in neurons expressing the neuropeptide oxytocin in the MPOA, SON, and PVN of the hypothalamus postpartum.
A further consideration is the effects on energy balance regulation that may indirectly result from the action of the Peg3 gene in the placenta. There is increasing evidence that events before birth can have a strong causal effect upon metabolic phenotypes, and numerous studies demonstrate that intrauterine growth retardation is a high-risk factor for developing obesity later in life. The so-called "thrifty phenotype" or "fetal origins" hypothesis argues that adaptations are made by a developing organism in response to a poor in utero environment, leading to an increased risk of adiposity and other diseases in adulthood. A variation of this hypothesis is that it is not low birth weight per se that leads to the increased risk of adult obesity, but rather that a common factor influences intrauterine growth and establishes the set-point of energy-regulating homeostasis. Indeed, there is good evidence that individual genes (e.g., the glucose sensor enzyme glucokinase) can regulate both prenatal growth and insulin homeostasis. Four imprinted genes, the paternally expressed Peg3, Pref1/Dlk1, Gnasxl and the maternally expressed Grb10, also have significant roles in embryonic development and postnatal energy regulation.
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FOOTNOTES
2 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3216fje;
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