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Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs., (© 2018 British Society for Neuroendocrinology.)

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the α/β-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

Susceptibility to obesity changes during the course of life. We utilized the C57BL/6J (B6) and 129S mouse as a genetic model for variation in diet-induced obesity to define the adiposity phenotypes from birth to maturity at 8 weeks-of-age. From birth to 8 weeks-of-age, both male and female 129S mice had significantly higher fat mass and adiposity index than B6 mice, although they were not obese. After 8 weeks-of-age, B6 had greater adiposity/obesity than 129S mice in response to a high fat (HF). We sought to determine the mechanism activating the fat accumulation in B6 mice at 8-weeks-of-age. We used microarray analysis of gene expression during development of inguinal fat to show that molecular networks of lipogenesis were maximally expressed at 8 weeks-of-age. In addition, the DNA methylation analysis of the Sfrp5 promoter and binding of acetylated histones to Sfrp5 and Acly promoter regions showed that major differences in the expression of genes of lipogenesis and chromatin structure occur during development. Differences in lipogenesis networks could account for the strain-dependent differences in adiposity up to 8 weeks-of-age; however, changes in the expression of genes in these networks were not associated with the susceptibility to DIO in B6 male mice beyond 8 weeks-of-age., (© 2017 Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Evidence from animal studies continues to document the effectiveness of brown fat based thermogenesis in stimulating energy expenditure to reduce obesity. Evidence shows that the number of brown adipocytes in white fat is determined by developmental mechanisms, not the environment. The large variability in the capacity for brown fat thermogenesis comes from genetic variability in developmental mechanisms extent in the animal. This genetic variability ultimately drives the capacity for induction of the brown adipocyte phenotype in response to environmental signals in adult animals. We highlight recent studies that suggest a role for gut microbiota in the regulation of brown fat thermogenesis that is based, in part, upon the observation that bile acids can effectively induce thermogenesis by interscapular brown fat at thermoneutrality., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: Mouse fetuses up to 16 day of embryonic development and nude (Foxn1- deficient) mice are examples of animals that undergo regenerative (scar-free) skin healing. The expression of transcription factor Foxn1 in the epidermis of mouse fetuses begins at embryonic day 16.5 which coincides with the transition point from scar-free to scar-forming skin wound healing. In the present study, we tested the hypothesis that Foxn1 expression in the skin is an essential condition to establish the adult skin phenotype and that Foxn1 inactivity in nude mice keeps skin in the immature stage resembling the phenomena of neoteny., Results: Uninjured skin of adult C57BL/6J (B6) mice, mouse fetuses at days 14 (E14) and 18 (E18) of embryonic development and B6.Cg-Foxn1 nu (nude) mice were characterized for their gene expression profiles by RNA sequencing that was validated through qRT-PCR, Western Blot and immunohistochemistry. Differentially regulated genes indicated that nude mice were more similar to E14 (model of regenerative healing) and B6 were more similar to E18 (model of reparative healing). The up-regulated genes in nude and E14 mice were associated with tissue remodeling, cytoskeletal rearrangement, wound healing and immune response, whereas the down-regulated genes were associated with differentiation. E14 and nude mice exhibit prominent up-regulation of keratin (Krt23, -73, -82, -16, -17), involucrin (Ivl) and filaggrin (Flg2) genes. The transcription factors associated with the Hox genes known to specify cell fate during embryonic development and promote embryonic stem cells differentiation were down-regulated in both nude and E14. Among the genes enriched in the nude skin but not shared with E14 fetuses were members of the Wnt and matrix metalloproteinases (Mmps) families whereas Bmp and Notch related genes were down-regulated., Conclusions: In summary, Foxn1 appears to be a pivotal control element of the developmental program and skin maturation. Nude mice may be considered as a model of neoteny among mammals. The resemblance of gene expression profiles in the skin of both nude and E14 mice are direct or indirect consequences of the Foxn1 deficiency. Foxn1 appears to regulate the balance between cell proliferation and differentiation and its inactivity creates a pro-regenerative environment.

Maintenance of body temperature in cold-exposed animals requires induction of thermogenesis and management of fuel. Here, we demonstrated that reducing ambient temperature attenuated diet-induced obesity (DIO), which was associated with increased iBAT thermogenesis and a plasma bile acid profile similar to that of germ-free mice. We observed a marked shift in the microbiome composition at the phylum and family levels within 1 day of acute cold exposure and after 4 weeks at 12°C. Gut microbiota was characterized by increased levels of Adlercreutzia, Mogibacteriaceae, Ruminococcaceae, and Desulfovibrio and reduced levels of Bacilli, Erysipelotrichaceae, and the genus rc4-4. These genera have been associated with leanness and obesity, respectively. Germ-free mice fed a high-fat diet at room temperature gained less adiposity and improved glucose tolerance when transplanted with caecal microbiota of mice housed at 12°C compared to mice transplanted with microbiota from 29°C. Thus, a microbiota-liver-BAT axis may mediate protection against obesity at reduced temperature., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Reproductive success is dependent on development of hypothalamic circuits involving many hormonal systems working in concert to regulate gonadal function and sexual behavior. The timing of pubertal initiation and progression in mammals is likely influenced by the nutritional and metabolic state, leading us to the hypothesis that transient malnutrition experienced at critical times during development may perturb pubertal progression through successive generations. To test this hypothesis we have utilized a mouse model of undernutrition during suckling by exposing lactating mothers to undernutrition., Results: Using a combination of transcriptomic and biological approaches, we demonstrate that molecular programming of hypothalamus may perturb gender specific phenotypes across generations that are dependent on the nutritional environment of the lactation period. Lactation undernutrition in first (F1) generation offspring affected body composition, reproductive performance parameters and influenced the expression of genes responsible for hypothalamic neural circuits controlling reproductive function of both sexes. Strikingly, F2 offspring showed phenotypes similar to F1 progeny; however, they were sex and parental nutritional history specific. Here, we showed that deregulated expression of genes involved in kisspeptin signaling within the hypothalamus is strongly associated with a delay in the attainment of puberty in F1 and F2 male and female offspring., Conclusion: The early developmental plasticity of hypothalamus when challenged with undernutrition during postnatal development not only leads to altered expression of genes controlling hypothalamic neural circuits, altered body composition, delayed puberty and disturbed reproductive performance in F1 progeny, but also affects F2 offspring, depending on parental malnutrition history and in sexually dimorphic manner.

Obesity depends on a close interplay between genetic and environmental factors. However, it is unknown how these factors interact to cause changes in the obese condition during the progression of obesity from the neonatal to the aged individual. We have utilized Mest and Sfrp5 genes, two genes highly correlated with adipose tissue expansion in diet-induced obesity, to characterize the obese condition during development of 2 genetic models of obesity. A model for the early onset of obesity was presented by leptin-deficient mice (ob/ob), whereas late onset of obesity was induced with high-fat diet (HFD) consumption in C57BL/6J mice with inherent risk of obesity (DIO). We correlated obese and diabetic phenotypes with Mest and Sfrp5 gene expression profiles in subcutaneous fat during pre-weaning, pre-adulthood and adulthood. A rapid development of obesity began in ob/ob mice immediately after weaning at 21 days of age, whereas the obesity of DIO mice was not evident until after 2 months of age. Even after 5 months of HFD treatment, the adiposity index of DIO mice was lower than in ob/ob mice at 2 months of age. In both obesity models, the expression of Mest and Sfrp5 genes increased in parallel with fat mass expansion; however, gene expression proceeded to decrease when the adiposity reached a plateau. The reduction in the expression of genes of caveolae structure and glucose metabolism were also suppressed in the aging adipose tissue. The analysis of fat mass and adipocyte size suggests that reduction in Mest and Sfrp5 is more sensitive to the age of the fat than its morphology. The balance of factors controlling fat deposition can be evaluated in part by the differential expression profiles of Mest and Sfrp5 genes with functions linked to fat deposition as long as there is an active accumulation of fat mass., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

It has been proposed that diet-induced obesity at thermoneutrality (TN; 29°C) is reduced by a UCP1-dependent thermogenesis; however, it has not been shown how UCP1-dependent thermogenesis can be activated in the absence of sympathetic activity. A recent study provides such a mechanism by showing that dietary bile acids (BAs) suppress obesity in mice fed a high-fat diet (HFD) by a mechanism dependent on type 2 deiodinase (DIO2); however, neither a role for UCP1 nor the influence of sympathetic activity was properly assessed. To test whether the effects of BAs on adiposity are independent of Ucp1 and cold-activated thermogenesis, obesity phenotypes were determined in C57BL6/J.(+)/(+) (WT) and C57BL6/J.Ucp1.(-)/(-) mice (Ucp1-KO) housed at TN and fed a HFD with or without 0.5% (wt/wt) cholic acid (CA) for 9 wk. CA in a HFD reduced adiposity and hepatic lipogenesis and improved glucose tolerance in WT but not in Ucp1-KO mice and was accompanied by increases in food intake and energy expenditure (EE). In iBAT, CA increased Ucp1 mRNA and protein levels 1.5- and twofold, respectively, and increased DIO2 and TGR5 protein levels in WT mice. Despite enhanced Dio2 expression in Ucp1-KO and Ucp1-KO-CA treated mice, this did not enhance the ability of BAs to reduce obesity. By comparing the effects of BAs on WT and Ucp1-KO mice at TN, our study showed that BAs suppress diet-induced obesity by increasing EE through a mechanism dependent on Ucp1 expression, which is likely independent of adrenergic signaling., (Copyright © 2016 the American Physiological Society.)

Obesity has become a major public health problem. Given the current increase in life expectancy, the prevalence of obesity also raises steadily among older age groups. The increase in life expectancy is often accompanied with additional years of susceptibility to chronic ill health associated with obesity in the elderly. Both obesity and ageing are conditions leading to serious health problems and increased risk for disease and death. Ageing is associated with an increase in abdominal obesity, a major contributor to insulin resistance and the metabolic syndrome. Obesity in the elderly is thus a serious concern and comprehension of the key mechanisms of ageing and age-related diseases has become a necessary matter. Here, we aimed to identify similarities underlying mechanisms related to both obesity and ageing. We bring together evidence that age-related changes in body fat distribution and metabolism might be key factors of a vicious cycle that can accelerate the ageing process and onset of age-related diseases.

Objective: The current review summarizes recent advances in the origin of brown adipocytes in rodents and humans., Methods: This review describes recent insights into induction of the brown adipocyte phenotype (BAP) in white fat (WAT) revealed by murine studies during the early postnatal period and reversible temperature transitions. The origin of adipocytes and identity of progenitors as indicated by lineage tracing experiments are reviewed., Results: We describe a genetic model for brown adipocyte development that involves the appearance of brown adipocytes in WAT at 21 days of age and a mechanism of post-weaning involution relevant for acquisition of the BAP in fully functional WAT in mice. Under normal physiological conditions, the BAP is dormant with the potential to be stimulated by changes in the external environment. Current evidence for the acquisition of brown adipocytes by interconversion of mature adipocytes versus de novo recruitment of progenitors suggests that mechanisms for acquisition of the BAP in WAT in mice are depot-specific and controlled by allelic variation., Conclusions: Although the BAP is highly variable among mice, there is no information on genetic variability in the expression of brown adipocytes in humans. Thus, deeper understanding of genetic mechanisms underlying development of functional brown adipocytes is crucial., (© 2016 The Authors. Obesity published by Wiley Periodicals, Inc. on behalf of The Obesity Society (TOS).)

The brown adipocyte phenotype (BAP) in white adipose tissue (WAT) is transiently induced in adult mammals in response to reduced ambient temperature. Since it is unknown whether a cold challenge can permanently induce brown adipocytes (BAs), we reared C57BL/6J (B6) and AxB8/PgJ (AxB8) mice at 17 or 29°C from birth to weaning, to assess the BAP in young and adult mice. Energy balance measurements showed that 17°C reduced fat mass in the preweaning mice by increasing energy expenditure and suppressed diet-induced obesity in adults. Microarray analysis of global gene expression of inguinal fat (ING) from 10-day-old (D) mice indicates that expression at 17°C vs. 29°C was not different. Between 10 and 21 days of age, the BAP was induced coincident with morphologic remodeling of ING and marked changes in expression of neural development genes (e.g., Akap 12 and Ngfr). Analyses of Ucp1 mRNA and protein showed that 17°C transiently increased the BAP in ING from 21D mice; however, BAs were unexpectedly present in mice reared at 29°C. The involution of the BAP in WAT occurred after weaning in mice reared at 23°C. Therefore, the capacity to stimulate thermogenically competent BAs in WAT is set by a temperature-independent, genetically controlled program between birth and weaning., (© The Author(s).)

The mechanism by which mice, exposed to the cold, mobilize endogenous or exogenous fuel sources for heat production is unknown. To address this issue we carried out experiments using 3 models of obesity in mice: C57BL/6J+/+ (wild-type B6) mice with variable susceptibility to obesity in response to being fed a high-fat diet (HFD), B6. Ucp1-/- mice with variable diet-induced obesity (DIO) and a deficiency in brown fat thermogenesis and B6. Lep-/- with defects in thermogenesis, fat mobilization and hyperphagia. Mice were exposed to the cold and monitored for changes in food intake and body composition to determine their energy balance phenotype. Upon cold exposure wild-type B6 and Ucp1-/- mice with diet-induced obesity burned endogenous fat in direct proportion to their fat reserves and changes in food intake were inversely related to fat mass, whereas leptin-deficient and lean wild-type B6 mice fed a chow diet depended on increased food intake to fuel thermogenesis. Analysis of gene expression in the hypothalamus to uncover a central regulatory mechanism revealed suppression of the Npvf gene in a manner that depends on the reduced ambient temperature and degree of exposure to the cold, but not on adiposity, leptin levels, food intake or functional brown fat.

The importance of brown adipose tissue as a site of nonshivering thermogenesis has been well documented. Emerging studies suggest that skeletal muscle is also an important site of thermogenesis especially when brown adipose tissue function is lacking. We recently showed that sarcolipin (SLN), an uncoupler of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) pump, could contribute to heat production in skeletal muscle. In this study, we sought to understand how loss of UCP1 or SLN is compensated during cold exposure and whether they are both necessary for thermogenesis. Toward this goal, we generated a UCP1;SLN double knock-out (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Results from this study show that there is up-regulation of SLN expression in UCP1-KO mice, and loss of SLN is compensated by increased expression of UCP1 and browning of white adipose tissue. We found that the DKO mice were viable when reared at thermoneutrality. When challenged to acute cold, the DKO were extremely cold-sensitive and became hypothermic. Paradoxically, the DKO mice were able to survive gradual cold challenge, but these mice lost significant weight and depleted their fat stores, despite having higher caloric intake. These studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and that loss of both systems compromises survival of mice under cold stress., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To determine the differences between brown adipocytes from interscapular brown tissue (iBAT) and those induced in white adipose tissue (WAT) with respect to their thermogenic capacity, we examined two essential characteristics: the dynamics of mitochondrial turnover during reversible transitions from 29 °C to 4 °C and the quantitative relationship between UCP1 and selected subunits of mitochondrial respiratory complex in the fully recruited state. To follow the kinetics of induction and involution of mitochondria, we determined the expression pattern of UCP1 and other mitochondrial proteins as well as analyzed mtDNA content after cold stimulation and reacclimation to thermoneutrality. We showed that UCP1 turnover is very different in iBAT and inguinal WAT (ingWAT); the former showed minimal changes in protein content, whereas the latter showed major changes. Similarly, in iBAT both mtDNA content and the expression of mitochondrial proteins were stable and expressed at similar levels during reversible transitions from 29 °C to 4 °C, whereas ingWAT revealed dynamic changes. Further analysis showed that in iBAT, the expression patterns for UCP1 and other mitochondrial proteins resembled each other, whereas in ingWAT, UCP1 varied ∼100-fold during the transition from cold to warmth, and no other mitochondrial proteins matched UCP1. In turn, quantitative analysis of thermogenic capacity determined by estimating the proportion of UCP1 to respiratory complex components showed no significant differences between brown and brite adipocytes, suggesting similar thermogenic potentiality. Our results indicate that dynamics of brown adipocytes turnover during reversible transition from warm to cold may determine the thermogenic capacity of an individual in a changing temperature environment., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A cohort of genes was selected to characterize the adipogenic phenotype in primary cell cultures from three tissue sources. We compared the quantitative expression of biomarkers in culture relative to their expression in vivo because the mere presence or absence of expression is minimally informative. Although all biomarkers analyzed have biochemical functions in adipocytes, the expression of some of the biomarkers varied enormously in culture relative to their expression in the adult fat tissues in vivo, i.e. inguinal fat for white adipocytes and brite cells, interscapular brown adipose tissue for brown adipocytes, and ear mesenchymal stem cells for white adipocytes from adult mice. We propose that the pattern of expression in vitro does not reflect gene expression in the adult mouse; rather it is predominantly the expression pattern of adipose tissue of the developing mouse between birth and weaning. The variation in gene expression among fat depots in both human and rodent has been an extensively studied phenomenon, and as recently reviewed, it is related to subphenotypes associated with immune function, the inflammatory response, fat depot blood flow, and insulin sensitivity. We suggest that adipose tissue biology in the period from birth to weaning is not just a staging platform for the emergence of adult white fat but that it has properties to serve the unique needs of energy metabolism in the newborn. A case in point is the differentiation of brite cells that occurs during this period followed by their involution immediately following weaning., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The recent characterization of brown fat in humans has generated much excitement on the possibility that increased energy expenditure by heat production by this tissue will be able to reduce obesity. This expectation has largely been stimulated by studies with mice that show strong associations between increased brown fat activity and reductions in obesity and insulin resistance. Research in the mouse has been largely based upon the induction or suppression of brown fat and mitochondrial uncoupling protein by genetic methods. The review of this research literature underscores the idea that reductions in obesity in mice are secondary to the primary role of brown adipose tissue in the regulation of body temperature. Given that the variation in brown fat in humans, as detected by PET imaging, is highly associated with administration of adrenergic agonists and reductions in ambient temperature, the effects on obesity in humans may also be secondary to the regulation of body temperature. Induction of thermogenesis by reduced ambient temperature now becomes like muscle and physical activity, another natural method of increased energy expenditure to combat obesity. Furthermore, there is no evidence to indicate that heat production by adrenergic stimulation via cold exposure or drug treatment or the enriched physical environment is restricted to the thermogenic activity of the brown adipocyte. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A population of brown adipocytes emerges in white fat depots at weaning. The function of these adipocytes is not known, but at this late stage of development it is unlikely they are essential for body temperature regulation experienced during the cold stress at birth. A dietary protocol of under-nutrition during the perinatal period, causing hypoinsulinemia, hypoleptinemia and hypoglycemia, led to an 85% reduction in expression of brown fat biomarkers and genes encoding the components of the respiratory complex, the TCA cycle and fatty acid oxidation. Suppression of wBAT in 21-day-old mice showed no significant effect on diet-induced obesity or cold tolerance in adult mice. Analysis of gene expression indicated that capacity to induce the brown fat phenotype was normal. This suggests that the brown adipocytes in white fat of 21-day-old mice are highly plastic and able to recover from severe malnutrition or that a new population of brown adipocytes is induced de novo in adult mice.

Interscapular brown adipose tissue (iBAT) is formed during fetal development and stable for the life span of the mouse. In addition, brown adipocytes also appear in white fat depots (wBAT) between 10 and 21 days of age in mice maintained at a room temperature of 23 °C. However, this expression is transient. By 60 days of age the brown adipocytes have disappeared, but they can re-emerge if the adult mouse is exposed to the cold (5 °C) or treated with β3-adrenergic agonists. Since the number of brown adipocytes that can be induced in white fat influences the capacity of the mouse to resist the obese state, we determined the effects of the nutritional conditions on post-natal development (birth to 21 days) of wBAT and its long-term effects on diet-induced obesity (DIO). Under-nutrition caused essentially complete suppression of wBAT in inguinal fat at 21 days of age, as indicated by expression of Ucp1 and genes of mitochondrial structure and function based upon microarray and qRT-PCR analysis, whereas over-nutrition had no discernible effects on wBAT induction. Surprisingly, the suppression of wBAT at 21 days of age did not affect DIO in adult mice maintained at 23 °C, nor did it affect the reduction in obesity or cold tolerance when DIO mice were exposed to the cold at 5 °C for one week. Gene expression analysis indicated that mice raised under conditions that suppressed wBAT at 21 days of age were able to normally induce wBAT as adults. Therefore, neither severe hypoleptinemia nor hypoinsulinemia during suckling permanently impaired brown adipogenesis in white fat. In addition, energy balance studies of DIO mice exposed to cold indicates that mice with reduced adipose stores preferentially increased food intake, whereas those with larger adipose tissue depots preferred to utilize energy from their adipose stores.

Evidence that adult humans have functional brown adipose tissue has stirred interest in the possibility that the impressive effectiveness of induction of brown adipocytes to reduce obesity in mice may be translated to the human condition. A major focus recently on the identification of signaling and transcription factor that stimulate the induction of brown adipocytes has come from transgenic and gene KO models. However, these models have created a very complex picture of the regulatory mechanisms for brown fat induction. In this review insights into the critical regulatory pathways involved in brown adipocyte induction in the retroperitoneal fat depot of mice are described from quantitative trait locus (QTL) analysis of allelic variability determining Ucp1 levels and brown adipocyte induction in A/J vs. B6 mice. The key observation is that recombinant genotypes, found in recombinant inbred stains and backcross and intercross progeny, show transgressive variation for Ucp1 mRNA levels. These genetic crosses also show that the levels of Ucp1 mRNA are determined by interactions that control the levels of PPARα, PGC-1α, and type 2 deiodinase (DIO2) and that each factor is controlled by a subset of QTLs that also control Ucp1 expression. These results indicate that induction of Ucp1 in the retroperitoneal fat depot involves synergy between signaling and transcription factors that vary depending upon the environmental conditions. Inherent in this model is the idea that there is a high level of redundancy that can involve any factor with the potential to influence expression of the core factors, PPARα, PGC-1a, and DIO2.

An ATP-Mg(2+/)P(i) inner mitochondrial membrane solute transporter (SLC25A25), which is induced during adaptation to cold stress in the skeletal muscle of mice with defective UCP1/brown adipose tissue thermogenesis, has been evaluated for its role in metabolic efficiency. SLC25A25 is thought to control ATP homeostasis by functioning as a Ca(2+)-regulated shuttle of ATP-Mg(2+) and P(i) across the inner mitochondrial membrane. Mice with an inactivated Slc25a25 gene have reduced metabolic efficiency as evidenced by enhanced resistance to diet-induced obesity and impaired exercise performance on a treadmill. Mouse embryo fibroblasts from Slc25a25(-/-) mice have reduced Ca(2+) flux across the endoplasmic reticulum, basal mitochondrial respiration, and ATP content. Although Slc25a25(-/-) mice are metabolically inefficient, the source of the inefficiency is not from a primary function in thermogenesis, because Slc25a25(-/-) mice maintain body temperature upon acute exposure to the cold (4 °C). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to muscle function as evidenced from the physical endurance test of mutant mice on a treadmill. Consequently, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity. However, in the absence of UCP1-based thermogenesis, induction of Slc25a25 in mice with an intact gene may contribute to an alternative thermogenic pathway for the maintenance of body temperature during cold stress.

Physiological, pharmacological and genetic studies in dogs, mice and rats have established that the uncoupling protein-1 (UCP1)-based brown adipose tissue system has an important role in the regulation of body temperature. Although it may be possible to create laboratory conditions in which mice with inactivated Ucp1 can survive in a modestly cooled environment, data overwhelmingly support the conclusion that the UCP1/BAT system has evolved to maintain body temperature at 37 °C. The corollary to this conclusion is that any influence UCP1/BAT might have on body weight regulation is a secondary function. The idea that BAT prevents obesity by burning off excess energy to maintain energy balance seems incompatible with evolutionary biology. Premodern humans spent an enormous amount of energy either running to catch their meal or avoiding becoming a meal themselves; consequently, there was no obesity. Nevertheless, although secondary to body temperature regulation, UCP1/BAT is extraordinarily effective at reducing adiposity and insulin resistance in mice and rats. Variation among mice in susceptibility to diet-induced obesity is correlated with the induction of brown adipocytes in traditional white fat depots (wBAT). Both genetic and cell biology-based experimentation have shown that the cellular origins of wBAT are different from those of interscapular-like brown adipocytes (iBAT). Do they have different functions? We have analyzed the effects of the early nutritional environment on the induction of brown adipocytes in inguinal fat to test the hypothesis that wBAT is primarily involved in body weight regulation. Although undernutrition during lactation severely suppresses wBAT at 21 days of age, undernourished mice fed a normal chow diet ad libitum at weaning recovered their normal wBAT and iBAT systems as young adults. The function of wBAT does not seem to be uniquely devoted to body weight regulation.

While the phenomenon linking the early nutritional environment to disease susceptibility exists in many mammalian species, the underlying mechanisms are unknown. We hypothesized that nutritional programming is a variable quantitative state of gene expression, fixed by the state of energy balance in the neonate, that waxes and wanes in the adult animal in response to changes in energy balance. We tested this hypothesis with an experiment, based upon global gene expression, to identify networks of genes in which expression patterns in inguinal fat of mice have been altered by the nutritional environment during early post-natal development. The effects of over- and under-nutrition on adiposity and gene expression phenotypes were assessed at 5, 10, 21 days of age and in adult C57Bl/6J mice fed chow followed by high fat diet for 8 weeks. Under-nutrition severely suppressed plasma insulin and leptin during lactation and diet-induced obesity in adult mice, whereas over-nourished mice were phenotypically indistinguishable from those on a control diet. Food intake was not affected by under- or over-nutrition. Microarray gene expression data revealed a major class of genes encoding proteins of the caveolae and cytoskeleton, including Cav1, Cav2, Ptrf (Cavin1), Ldlr, Vldlr and Mest, that were highly associated with adipose tissue expansion in 10 day-old mice during the dynamic phase of inguinal fat development and in adult animals exposed to an obesogenic environment. In conclusion gene expression profiles, fat mass and adipocyte size in 10 day old mice predicted similar phenotypes in adult mice with variable diet-induced obesity. These results are supported by phenotypes of KO mice and suggest that when an animal enters a state of positive energy balance adipose tissue expansion is initiated by coordinate changes in mRNA levels for proteins required for modulating the structure of the caveolae to maximize the capacity of the adipocyte for lipid storage.

The notion that brown adipose tissue (BAT) in mice or humans maintains energy balance by burning off excess calories seems incompatible with evolutionary biology. Studies in obese rats and mice lacking UCP1 indicate that diet-induced thermogenesis by BAT is unlikely., (2010 Elsevier Inc. All rights reserved.)

Brown adipose tissue is highly differentiated and has evolved as a mechanism for heat production based upon uncoupling of mitochondrial oxidative phosphorylation. Additionally, large amounts of lipid can be stored in the cells to provide fuel necessary for heat production upon adrenergic stimulation from the central nervous system, and a highly developed vascular system evolved to rapidly deliver heat to vital organs. For unknown reasons, the development of brown adipocytes has two independent pathways: one originates from muscle progenitor cells in the fetus and leads to a fully functional cell at birth (interscapular-type brown fat), while the other transiently emerges in traditional white fat depots at weaning, regresses, and then can be induced in adult mice upon adrenergic stimulation. No genetic variants have been found for interscapular fat, but naturally occurring alleles at eight genetic loci in mice lead to over 100-fold variation for brown adipocytes in white fat upon adrenergic stimulation. The ability to activate this potential for energy expenditure is of great interest in obesity research., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mesoderm specific transcript (Mest), an imprinted gene associated with fat mass expansion under conditions of positive energy balance, shows highly variable expression (approximately 80-fold) in white adipose tissue (WAT) of C57BL/6J (B6) mice fed an obesogenic diet. Since B6 mice are essentially genetically invariant and Mest is known to be regulated by CpG methylation within its immediate proximal promoter, the large variability in its expression in adipose tissue has the hallmarks of being controlled via an epigenetic mechanism. In this study, bisulfite sequencing and allelic discrimination analyses were performed to determine whether variations in CpG methylation within the Mest promoter were associated with its expression. Results showed no relationship between CpG methylation in the Mest promoter and high versus low expression in either WAT or isolated adipocytes; and, experiments using a single nucleotide polymorphism in the Mest promoter region between B6 and Castaneus mice showed the expected pattern for an imprinted gene with all maternal alleles being methylated. These data suggest that mechanisms independent of the CpG methylation status of the Mest promoter must underlie the control of its expression during adipose tissue expansion.

In the 1970s and 1980s, it was observed that rodents could offset excess calories ingested when they were fed a human-like 'cafeteria diet'. Although it was erroneously concluded that this so-called diet-induced thermogenesis was because of brown adipose tissue (BAT), it led to efforts to test whether variations in brown fat in humans may explain the susceptibility to obesity. However, from evidence on the inability of ephedrine or beta-3 adrenergic agonists to induce BAT thermogenesis, it was concluded that the thermogenic role of BAT was unimportant in adult humans largely because humans had low numbers of brown adipocytes. Solid evidence on the actual numbers of brown adipocytes in humans was not available. We are now re-evaluating the role of BAT for the treatment of obesity given the following recent observations (i) studies in nuclear medicine by using PET/CT scanning reveal the presence of BAT in adult humans; and (ii) recent data suggest that a new transcription factor called PDRM16 may control the induction of BAT. These recent discoveries should revamp our effort to target the molecular development of brown adipogenesis in the treatment of obesity.

A 50-fold variation in mRNA and protein levels of the mesoderm-specific transcript gene (Mest) in white fat of C57BL/6J (B6) mice fed an obesogenic diet is positively correlated with expansion of fat mass. MEST protein was detected only in adipocytes, in which its induction occurred with both unsaturated and saturated dietary fat. To test the hypothesis that MEST modulates fat mass expansion, its expression was compared to that of stearoyl CoA desaturase (Scd1) in B6 mice exposed to diets and environmental temperatures that generated conditions separating the effects of food intake and adiposity. Under a range of conditions, Mest expression was always associated with variations in adiposity, whereas Scd1 expression was associated with the amount of saturated fat in the diet. Mest mRNA was expressed at its highest levels during early postnatal growth at the onset of the most rapid phase of fat mass expansion. MEST is localized to the endoplasmic reticulum/Golgi apparatus where its putative enzymatic properties as a lipase or acyltransferase, predicted from sequence homology with members of the alpha/beta fold hydrolase superfamily, can enable it to function in lipid accumulation under conditions of positive energy balance. Variations in adiposity and Mest expression in genetically identical mice also provides a model of epigenetic regulation.

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 degrees C, provided prior adaptation occurred at 20 degrees C. Obesity resistant Gdm(-/-).Ucp1(-/-) mice maintained at 28 degrees C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.

Cold exposure induces brown adipocytes in retroperitoneal fat (RP) of adult A/J mice but not in C57BL/6J (B6) mice. In contrast, induction of the mitochondrial uncoupling protein 1 gene (Ucp1) in interscapular brown adipose tissue (iBAT) shows no strain dependence. We now show that unlike iBAT, in which Ucp1 was expressed in the fetus and continued throughout life, in RP, Ucp1 was transiently expressed between 10 and 30 days of age and then disappeared. Similar to the lack of genetic variation in the expression of Ucp1 in iBAT during cold induction of adult mice, no genetic variation in Ucp1 expression in iBAT was detected during development. In contrast, UCP1-positive multilocular adipocytes, together with corresponding increases in Ucp1 expression, appeared in RP at 10 days of age in A/J and B6 mice, but with much higher expression in A/J mice. At 20 days of age, brown adipocytes represent the major adipocyte present in RP of A/J mice. The disappearance of brown adipocytes by 30 days of age suggested that tissue remodeling occurred in RP. Genetic variability in Ucp1 expression could not be explained by variation in the expression of selective transcription factors and signaling molecules of adipogenesis. In summary, the existence of genetic variability between A/J and B6 mice during the development of brown adipocyte expression in RP, but not in iBAT, suggests that developmental mechanisms for the brown adipocyte differentiation program are different in these adipose tissues.

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.

High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity., Competing Interests: Competing interests. Dr. Jihad S. Skaf is a paid employee of Applied Biosystems Inc.; his job title is Technical Applications Specialist.

We investigated the role of leptin in regulating energy metabolism through induction of uncoupling protein (UCP)-1-based brown fat thermogenesis by comparing phenotypes of energy balance in ob/ob and double-mutant ob/ob.Ucp1(-/-) mice. Measurements of adiposity and lean body mass (nuclear magnetic resonance), energy expenditure (indirect calorimetry), body weight, food intake, and core body temperature were determined in the two mutant stocks of 3-month-old mice maintained at an initial ambient temperature of 28 C for 21 d and then at 21 C for 16 d, and finally with leptin administration for 8 d at 21 C. No phenotypic differences between ob/ob and ob/ob.Ucp1(-/-) mice were detected, suggesting that UCP1-based thermogenesis is not essential for the regulation of adiposity in ob/ob mice at temperatures between 21 and 28 C. Although both Ucp1(-/-) and ob/ob mice can survive in extreme cold at 4 C, provided they are adapted to the cold by gradually lowering ambient temperature, ob/ob.Ucp1(-/-) mice could not adapt and survive at temperatures lower than 12 C unless they were administered leptin. As the ambient temperature was reduced from 20 to 16 C, ob/ob.Ucp1(-/-) mice treated with leptin have elevated levels of circulating T(3) that correlate with elevated sarcoendoplasmic reticulum Ca(2+) ATPase 2a mRNA levels in gastrocnemius muscle. Furthermore, ob/ob.Ucp1(-/-) mice, treated with T(3), were able to maintain body temperature and stimulate sarcoendoplasmic reticulum Ca(2+) ATPase 2a expression when the ambient temperature was gradually reduced to 4 C. Thus, in the absence of UCP1, leptin-induced thermogenesis protects body temperature in part through its action on the thyroid hormone axis.

Ultrastructural and metabolic changes were studied during chronic fluorine intoxication and low doses of radiation (total dose is 1 Gr) in liver cells and tissues in rats fed with amaranth oil and treated with intermittent hypoxic training (IHT). The obtained data detected the ordered and compact position of mitochondria, peroxisomes, lipoprotein droplets with light electronic density, glycogen granules and also agranular endoplasmatic retuculum channels, which may be linked with reorganization of metabolic pathway of energy supply from fat acids via gluconeogenesis. Simultaneously the decrease of TBA-reactive substances accumulation with the considerable increase in activity of the antioxidant enzymes (catalase, glutathione peroxidase) and index of general antioxidant activity have been established. Therefore, the combined effect of IHT and amaranth oil on adequate occurence of free radical reactions provides the effective adaptation of organism to fluorine intoxication and ionizing radiation via the restoration of homeostasis on metabolic and ultrastructural levels. The obtained results allow to recommend IHT and amaranth oil for complex correction of changes, induced by fluorine intoxication and ionizing radiation.

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

Transcription factors that regulate gene expression during adipogenesis also control the expression of genes of thermogenesis in brown adipose tissue, in particular, the mitochondrial uncoupling protein gene (Ucp1). There is evidence that a plasticity exists among adipocytes in which activation of the Ucp1 gene together with mitochondrial biogenesis can increase the brown adipocyte character of white fat. To understand this process, we have characterized the changes in transcription that occur in interscapular brown adipocytes during development. We have found dramatic reductions in both DNA-binding activity to probes and immunoreactive protein for peroxisome proliferator-activated receptor, retinoid X receptor, CCAAT/enhancer binding protein, and cAMP-response element-binding protein regulatory motifs in nuclear extracts when mice reach adulthood. Exposure of adult mice to the cold, which reactivates Ucp1 expression, leads to a re-accumulation of factors in the nucleus. We propose that transcription factors are sequestered in the cytoplasm as mice age under conditions of reduced thermogenesis. Changes in isoform sub-types for peroxisome proliferator-activated receptor-gamma and cAMP-response element-binding proteins indicate an additional level of control on gene expression during thermogenesis. The increased movement of the RIIbeta protein kinase A regulatory subunit into the nucleus with age suggests a mechanism for regulating the phosphorylation of transcription factors in the nucleus in response to the thermogenic requirements of the animal. Nuclear factor-kappaB has been used as a model to demonstrate that the nuclear localization of transcription factors in brown fat are reduced during post-natal development. Furthermore, it was found by immunofluorescence that adrenergic stimulation of primary adipocyte cultures causes an increase of both the protein kinase A catalytic alpha-subunit and nuclear factor-kappaB into the nucleus.

New Zealand Obese (NZO) male mice develop a polygenic juvenile-onset obesity and maturity-onset hyperinsulinemia and hyperglycemia (diabesity). Here we report on metabolic and molecular changes associated with the antidiabesity action of CL316,243 (CL), a beta(3)-adrenergic receptor agonist. Dietary CL treatment initiated at weaning reduced the peripubertal rise in body weight and adiposity while promoting growth without suppressing hyperphagia. The changes in adiposity, in turn, suppressed development of hyperinsulinemia, hyperleptinemia, hyperlipidemia, and hyperglycemia. These CL-induced alterations were reflected by decreased adipose tissue mass, increased expression of transcripts for uncoupling protein-1 (UCP-1), peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferater-activated receptor coactivator-1 (PGC-1), and robust development of brown adipocyte function in white fat. Increased drug-mediated energy dissipation elicited a 1.5 degrees C increase in whole body temperature under conditions of increased food intake but with no change in physical activity. Indirect calorimetry of mice treated with CL showed both increased energy expenditure and a restoration of a prominent diurnal pattern in the respiratory exchange ratio suggesting improved nutrient sensing. Our data suggest that CL promotes increased energy dissipation in white and brown fat depots by augmenting thermogenesis and by metabolic re-partitioning of energy in a diabesity-protective fashion. This is the first report demonstrating the effects of dietary beta(3)-agonist in preventing the onset of diabesity in a polygenic rodent model of type 2 diabetes.

Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane. Whereas UCP1 is uniquely present in brown adipose tissue where it uncouples respiration from ATP synthesis and activates respiration and heat production, UCP2 is present in numerous tissues, and its exact function remains to be clarified. Two sets of data provided the rationale for this study: (i) the intriguing report that UCP1 is present in uterus of mice (Nibbelink, M., Moulin, K., Arnaud, E., Duval, C., Penicaud, L., and Casteilla, L. (2001) J. Biol. Chem. 276, 47291-47295); and (ii) an observation that Ucp2(-/-) female mice (homozygous matings) have smaller litters compared with Ucp2(+/+) animals (S. Rousset and A.-M. Cassard-Doulcier, unpublished observations). These data prompted us to examine the expression of UCP1 and UCP2 in the reproductive tract of female mice. Using wild type, Ucp1(-/-) mice, and Ucp2(-/-) mice, we were unable to detect UCP1 in uterus of mice with appropriate antibodies, and we conclude that the signal assigned to UCP1 by others was neither UCP1 nor UCP2. Using a polyclonal antibody against UCP2 and tissues from Ucp2(-/-) mice as controls, UCP2 was detected in ovary, oviduct, and uterus. Expression of Ucp2 mRNA was also observed in ovary and uterus using in situ hybridization analysis. Bone marrow transplantation experiments revealed that the UCP2 signal of the ovary was restricted to ovarian cells. UCP2 level in ovary decreased during follicular growth and increased during the pre-ovulatory period, during which aspects of an inflammatory process are known to exist. Because UCP2 down-regulates reactive oxygen species, a role in the regulation of inflammatory events linked to the preparation of ovulation is suggested.

C57BL/6J (B6) and AKR/J (AKR) inbred strains of mice develop a comparable degree of obesity when fed a high-fat diet. However, although obese B6 mice are more glucose intolerant, obese AKR mice are more insulin resistant. To understand the basis for these strain differences, we characterized features of adiposity and glucose homeostasis in mice fed a high-fat diet for 8 weeks. The results indicated that despite hyperglycemia and impaired glucose tolerance, B6 mice have lower plasma insulin and are more insulin sensitive than AKR mice. Compared with adipose tissue of AKR mice, adipose tissue of B6 mice contained about threefold higher levels of total membrane-bound GLUT4 protein, whereas in skeletal muscle the levels were similar. Uptake of 2-[(14)C]deoxyglucose in vivo was reduced by a high-fat diet in adipose tissue, but not in skeletal muscle. Surprisingly, no significant differences in uptake occurred between the strains, despite the differences in GLUT4; however, glucose flux was calculated to be slightly higher in B6 mice. Higher expression of PEPCK in the liver of B6 mice, under both standard-diet and high-fat-diet conditions, suggests a plausible mechanism for elevated glycemia in these mice. In conclusion, phenotypic variation in insulin resistance and glucose production in the B6 and AKR strains could provide a genetic system for the identification of genes controlling glucose homeostasis.

To identify novel regulatory factors controlling induction of the brown adipocyte-specific mitochondrial uncoupling protein (Ucp1) mRNA in the retroperitoneal white fat depot, we previously mapped quantitative trait loci (QTLs) that control this trait to chromosomes 2, 3, 8, and 19. Since the peroxisome proliferator activator receptor-gamma coactivator-1alpha (PGC-1alpha) regulates Ucp1 and other genes of energy metabolism, we have evaluated whether the QTLs controlling Ucp1 mRNA levels also modulate Pgc-1alpha mRNA levels by analysis of backcross progeny from the A/J and C57BL/6J strains of mice. The results indicate that a locus on chromosome 3 orchestrates expression of Pgc-1alpha and Ucp1 in retroperitoneal fat of mice fed a low-fat diet; however, the effect of this locus on Pgc-1alpha is lost, and a significant correlation between Ucp1 and Pgc-1alpha is severely reduced in mice fed a high-fat diet. An additional QTL located on chromosome 5 has also been identified for the selective regulation of Ucp1 mRNA levels. Similar to the effects of a high-fat diet on the chromosome 3 QTL, linkage of the chromosome 5 QTL is also lost in mice on a high-fat diet. Thus dietary fat has a profound influence on PGC-1alpha-regulated pathways controlling energy metabolism in white fat. The allelic variation observed in the regulation of Ucp1 and Pgc-1alpha expression in brown adipocytes of white fat but not interscapular brown fat suggests that fundamentally different regulatory mechanisms exist to control the thermogenic capacities of these tissues.

The availability of mice lacking the mitochondrial uncoupling protein UCP1, has provided an opportunity to analyze the relationship between the capacity for energy expenditure and the development of obesity in response to a high-fat, high-sucrose diet. Congenic UCP1-deficient mice on a C57BL/6J genetic background show a temperature-dependent resistance to diet-induced obesity when compared with wild-type mice. This resistance, which occurs at 20 degrees C, is quickly reversed when the ambient temperature is increased to 27 degrees C. At 20 degrees C, total oxygen consumption and physical activity of mutant and wild-type mice are indistinguishable; however, body temperature is higher in UCP1-deficient mice by 0.1-0.3 degrees C, and respiratory quotient is slightly reduced. A reduced respiratory quotient, together with elevated beta-hydroxybutyrate and reduced plasma fatty acid levels, suggests that the mutants oxidize a greater proportion of fat than wild-type mice, and that this possibly accounts for the resistance to diet-induced obesity. Although shivering is one alternative mechanism of thermogenesis that is probably used in UCP1-deficient mice, whether there are others remains to be determined. Nevertheless, our study underscores the paradox that elimination of the major thermogenic mechanism in the animal reduces rather than increases metabolic efficiency. We propose that in the absence of nonshivering thermogenesis, alternative, calorically more costly pathways of metabolism must be used to maintain body temperature.