Mitochondrial uncoupling proteins and obesity: Molecular and ... - Nature

International Journal of Obesity (1999) 23, Suppl 6, S33±S37 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo

Mitochondrial uncoupling proteins and obesity: Molecular and genetic aspects of UCP1 LP Kozak1* and RA Koza1 1

Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808, USA

Genetic variation in brwon fat speci®c mitochondrial uncoupling protein-1 (UCP1) expression and brown adipocyte morphology, have provided models to test the hypothesis that nonshivering thermogenesis is associated with the regulation of body weight. Genetic manipulation using transgenic animals and gene targeting, has resulted in mice with an over-expression of UCP1. These variant animals consistently show that over-expression of UCP1 reduced adiposity. On the other hand, less agreement is found in models that reduce nonshivering thermogenesis. Inactivation of the UCP1 gene, by gene targeting, does not increase adiposity when compared to control animals; however, a mouse expressing the UCP1-DTA transgene (UCPI-diphtheria toxin A chain), in which there is a modest reduction in the number of brown adipocytes, becomes obese. Other phenotypes of this mouse, the hyperphagia, extreme resistance to leptin administration, retinopathy and high residual content of brown adipocytes, suggest that the effects of the transgene may be more extensive than simply a 60% reduction in the number of brown adipocytes. Ectopic expression of UCP1-DTA in the brain could explain the phenotype of this mouse in a manner more consistent with the results of other models with altered UCP1 and brown adipocyte expression. Keywords: transgene; UCP-DTA; aP2-UCP1; UCP1-KO; PGC1; brown adipose tissue

Introduction Experimental manipulations of brown adipose tissue (BAT) with genetic and pharmacological techniques in several animal models, have provided strong evidence that an increase in nonshivering thermogenesis is an effective mechanism for the regulation of body weight. Reductions in the capacity for thermogenesis, generally lead to increased adiposity, while an increased thermogenic capacity reduces adiposity. Although the experimental evidence from these studies strongly support a role for the brown fat speci®c mitochondrial uncoupling protein (UCP1) as the principle thermogenic mechanism causing a change in adiposity, it is possible that some of the effects are due to other thermogenic mechanisms. Of particular interest is the need to evaluate the role the other members in the UCP family of genes the thermogenic processes involved in the regulation of body weight.1 ± 5 For example, mice in which the Ucp1 gene has been inactivated by gene targeting, are sensitive to cold, but they are not more obese than the control mice that have a fully functioning Ucp1 gene.6 Since the UCP1de®cient mice have elevated levels of UCP2 mRNA,

it has been suggested that UCP2 compensates for the loss of UCP1 in maintaining a normal body weight.6 However, if UCP2 is able to compensate for UCP1 in regulating body weight, then why does it not compensate for Ucp1 de®ciency in protecting mice from acute exposure to cold? Our ability to detect these relationships is dependent on several parameters, including genetic background, gender and age. Of particular importance is the genetic background of the animal, since this can in¯uence the development of obesity by variant genes that are independent of nonshivering thermogenesis. The interaction of other variant genes can affect thermogenesis and have a large impact on the response of the animal to beta adrenergic agonists. It is important to emphasize that for a genetically de®ned model of obesity like dietinduced obesity of the A=J and C57BL=6J inbred strains,7 the susceptibility to obesity is almost certainly the consequence of the interactions of several sub-phenotypes and each of these sub-phenotypes will be determined by several genes. Given our focused agenda, we will discuss only the genetic variation associated with UCP1.

Knockout and transgenic models

*Correspondence: Dr Leslie Kozak, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808, USA. E-mail: [email protected]

We would like to review the genetic evidence that elevated UCP1 concentrations increase energy expenditure, with a resulting reduction in adiposity and, conversely, that reduced UCP1 leads to increased adiposity. The ®rst genetic study to address this

Molecular and genetic aspects of UCP1 LP Kozak and RA Koza

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issue, sought to reduce non-shivering thermogenesis by speci®cally expressing the cytotoxic diphtheria toxin in the brown adipocyte, by creating a transgenic mouse in which the diphtheria toxin gene was driven by the UCP1 promoter.8 It was hypothesized that the ablation of brown fat would reduce energy expenditure and cause obesity. As predicted, the Ucp1-DTA mice became obese, however, several aspects of the overall phenotype of Ucp1-DTA and the results of subsequent genetic models have made the original, simple interpretation untenable. Unexpectedly the Ucp1-DTA mice are hyperphagic. Accordingly, it is not possible to conclude that the obesity is caused by reduced brown fat-derived thermogenesis. Experiments by Melnyk et al 9 showing that Ucp1-DTA mice raised at thermoneutrality were neither more hyperphagic nor more obese than the normal controls, strongly suggests that the obesity is derived from the hyperphagia. Despite the fact that Ucp1-DTA mice have only a 50% reduction in brown adipocytes, it is becoming embedded in the literature that the hyperphagia of Ucp1-DTA mice is caused by a de®ciency of a satiety factor that is normally secreted by brown adipocytes.10 More recently, the Ucp1-DTA mouse has been found to be leptin resistant and this phenotype is also being ascribed to brown fat ablation.11 If expression of Ucp1-DTA is speci®c for brown fat, than these phenotypes of Ucp1-DTA mice must be accounted for by a 50 ± 60% reduction in the amount of brown fat. Two more recent genetic studies of mice indicate that reductions in brown fat non-shivering thermogenesis cause neither hyperphagia nor leptin resistance in mice. First, in contrast to the Ucp1-DTA mice, UCP1 KO mice on a hybrid C57BL=6J X 129=SvJ genetic background, have no increase in either adiposity or hyperphagia when compared to the normal littermate.6 The UCP1 KO mouse has no detectable immunoreactive UCP1, consumes less oxygen when stimulated with a b3-adrenergic agonist and is much more sensitive to the cold than the Ucp1-DTA mouse, indicating that the capacity for thermogenesis in the Ucp1-KO mouse in much less than the Ucp1-DTA mouse. One could speculate that the phenotype of a mouse with an inactivated Ucp1 gene will be different to a mouse in which the mass of brown fat has been reduced, although it seems most improbable that inactivation of Ucp1 would not affect other phenotypes associated with energy balance. Brown fat could have functions that can only be served by the presence of the brown adipocyte, for example the secretion of a satiety factor. A second transgenic model addresses this speculation of satiety factors and brown fat ablation. The aP2-Ucp1 transgenic mouse has ectopic expression of UCP1 in white fat and constitutive expression in brown fat.12 The increase in UCP1 concentrations in the hemizygous mouse compared to the nontransgenic controls is only about 15 ± 20% overall; however, the effects on adiposity are quite dramatic. This mouse is able to resist obesity caused

by either the Avy gene or a high fat diet. The antiobesity phenotype of aP2-Ucp1 mice is remarkably similar to that of mice homozygous for the targeted mutation of the RIIb regulatory subunit of protein kinase A, that selectively up-regulates UCP1 in brown fat.13 However, if the aP2-Ucp1 mouse is homozygous, it maintains its resistance to obesity and in addition becomes sensitive to the cold in a manner indistinguishable from the UCP1 KO mouse.14 This new phenotype is caused by the almost complete absence of brown adipose tissue (BAT). Importantly, there is neither obesity nor hyperphagia in this animal, strongly indicating that the ablation of brown fat is not associated with hyperphagia caused by the absence of a brown fat satiety factor. At this time it is dif®cult to know what causes the phenotype of the Ucp1-DTA mouse. The hyperphagia, the leptin resistance and the retinopathy, point to a central nervous system (CNS) defect that could have been caused by an insertional mutation in a gene of the CNS or expression of DTA in the brain. The Ucp1-DTA mouse shows features of LepR mutant homozygotes, with exception of the resistance to cold in the Ucp1-DTA mice.

Over-expression vs underexpression Reductions in adiposity as a consequence of increased concentrations of UCP1, are observed in both aP2Ucp1 transgenic and PKA-RIIb knockout mice. In the absence of an obesity stress caused by a high fat diet or a mutant obesity gene, the aP2-Ucp1 transgene does not cause a loss of body weight in lean mice. On a C57BL=6J background, there is no difference in body weight between young adult transgenic and nontransgenic mice when they are fed a standard lab chow containing 6% fat. On a low fat chow diet, normal C57BL=6J mice show a signi®cant age-related increase in adiposity when they are more than one year of age, but the aP2-Ucp1 transgene prevents this age-related increase the adiposity. The mechanism by which the body resists excessive loss of fat by the genetic manipulation of the genes for energy metabolism, is not understood. It is not simply an artifact of the over-expression of UCP1, since similar effects have previously been seen with a transgenic mouse that over-expressed the cytoplasmic glycerol 3-phosphate dehydrogenase gene, an enzyme with a role in carbohydrate and lipid metabolism.15 Given the ability of elevated concentrations of UCP1 to prevent obesity, we expected that the targeted inactivation of Ucp1 would cause the development of obesity, particularly in mice fed a high fat diet. No increase in adiposity has been detected in UCP1 targeted mice compared to normal controls, fed either a standard low fat laboratory chow or a high fat diet (58 kcal% as fat). The genetic background of


these mice (C57BL=6J129=SvJ hybrids) renders males susceptible to dietary obesity, while the females are relatively resistant to the obesity. However, neither the male or female targeted mice are any more obese than the corresponding controls.

The genetics of the induction of brown adipocytes in white adipose tissue (WAT) Our laboratory has recently initiated a genetic analysis to determine the mechanism by which an increase in the numbers of brown adipocytes occurs in the traditional white fat tissues following stimulation of the animal with cold or b-adrenergic agonists. This phenomenon has been known for a long time; Young et al16 ®rst showed that an increase in cells with the morphology of brown adipocytes and immunoreactive uncoupling protein, occurred in the parametrial fat pad of mice exposed to the cold. Subsequently, similar observations have been reported in other species following adrenergic stimulation by cold exposure or adrenergic17 ± 20 and, recently, human adipocytes treated with a b3-adrenergic agonist in culture were found to express elevated concentrations of UCP1 mRNA.21 Many studies have established that with increasing age, a requirement for non-shivering thermogenesis to protect the animal from the cold is reduced.22 Concomitantly, the maintenance of a brown adipocyte faction is diminished and the white adipocyte becomes the predominant adipocyte. The cellular mechanisms determining this transformation are still uncertain. On the one hand, the brown adipocytes could die and white adipocytes emerge by de novo cell proliferation. On the other hand, without adrenergic stimulation to the brown adipocyte, the Ucp1 gene becomes inactive, mitochondrial structures are lost and lipid utilization is minimized. Under these circumstances, the cell resembles a white adipocyte, even though it has been programmed genetically to be a brown adipocyte. The emergence of brown adipocytes in traditional white fat depots, is a reversal of the above process and may be explained simply as the action of the adrenergic signalling to the white adipocytes. The fact that not all white adipocytes are converted to brown adipocytes and that the tissue has a distinctly mosaic pattern, suggests that a conversion of a subpopulation of white adipocytes to brown adipocytes occurs.18 Additional comments on this issue will be discussed below. Variation in the induction of brown adipocyte expression in inbred stains of mice has made it possible to pursue a genetic analysis of the phenotype with a view to identifying genes that control the increased expression of brown adipocytes in white fat tissues.23 Adrenergic stimulation of C56BL=6J and

A=J mice by placing them in the cold (4 ) or administering a b3-adrenergic agonist, resulted in the differential induction of UCP1 mRNA in traditional white fat depots. Since the retroperitoneal fat showed the largest difference between strains and had the highest level of induction of UCP1 mRNA, we selected this regional fat as the primary focus of efforts to identify genes critical for the induction of brown adipocytes in white fat depots. The initial genetic step was based upon the analysis of the AXB recombinant inbred (RI) strains that were derived from A=J and C57BL=6J. This analysis shows that UCP1 mRNA concentrations in the retroperitoneal fat of RI lines, following seven days at 4 C, vary enormously > 130-fold, which is even greater than the differences between the parental B6 and A=J lines. If differences in UCP1 induction were controlled by one gene, then the RI lines would have separated into two groups, those with UCP1 mRNA concentrations like one parent and those with concentrations like the other parent. Since some strains had intermediate concentrations, (for example, AXB5), and other strains had concentrations that were greater than A=J (for example, AXB8), the data indicate that two or more genes control the differences in induced UCP1 mRNA. To determine whether the concentration of UCP1 mRNA is a necessary condition for the induction of brown adipocytes in white fat depots, RI mice that varied in their capacity to induce UCP1 mRNA, were kept in the cold for 17 d and the amount of brown adipocyte expression evaluated. The latter was assessed by image analysis of histological sections stained with an antibody speci®c for UCP1. AXB8 mice which had the highest level of UCP1 mRNA, also had the highest amount of brown fat, whereas AXB1 and AXB10 had low concentrations of both UCP1 mRNA and low amounts of brown fat. However, there were deviations from this strict relationship; AXB15 had levels of UCP1 mRNA in the retroperitoneal fat that were as high as in AXB8, but the amount of brown fat was much less. One interpretation of this result is that the accumulation of UCP1 mRNA is necessary, but not suf®cient, for the formation of brown adipocytes. AXB15 mice may have variant alleles at genes that do not provide conditions necessary for the facile development of the brown adipocyte in the retroperitoneal fat pad. Factors that are required for the biogenesis of mitochondria, if limited in concentration, could result in reduced maturation of a cell into a brown adipocyte. The availability of a series of strains in mice that vary in their capacity to induce brown fat, could provide additional insights into the role of brown fat in the regulation of body weight. First, do differences in this phenotype in¯uence the susceptibility to obesity in the absence of sympathetic stimulation and second, will a higher capacity to induce brown fat sensitize an animal to the weight-reducing effects of a b3-adrenergic agonist? C57BL=6J and A=J parental strains and six RI strains that varied in their capacity

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to induce brown adipocytes, were fed a high=fat high sucrose diet for 18 weeks to induce an obese condition. Some strains became obese and others did not; however, the susceptibility to develop obesity was independent of the inherited capacity of a strain to induce brown fat when stimulated by adrenergic signalling. The results suggest that a high fat diet, in the absence of an additional source of adrenergic signalling, is not capable of stimulating brown adipocyte formation in the retroperitoneal fat depot. To test the second question, eight strains of mice that varied in their obese condition, were treated with C1 316,243, a b3-selective adrenergic agonist, and with continued feeding of the high fat=high sucrose diet. After 24 d of treatment, the effects of the adrenergic treatment on adiposity and UCP1 brown fat induction were assessed. The results show a strong and highly signi®cant correlation between the induction of brown fat in the traditional white fat deposits and reduced adiposity. It should be emphasized that the induction of the brown adipocyte in white fat is almost certainly not the only mechanism operating in these inbred strains to affect the development of obesity and the response of the mice to b3-adrenergic stimulation. For example, Surwit and colleagues have presented evidence that in response to a high fat diet, C57BL=6J and A=J vary in the development of adiposity,7 in UCP2 mRNA concentrations in white fat and in UCP1 mRNA concentrations in brown fat.24 These studies underscore the fact that while the obesity phenotype is extremely complex, mouse strains like A=J and C57BL=6J are providing extremely useful models by identi®cation of sub-phenotypes, each of which are controlled by different gene combinations. The subphenotypes can interact in RI strains to provide new types of phenotypes. Cloning of these genes will provide powerful reagents for the understanding of human obesity.

The molecular biological approach Studies to determine the structural-functional basis for regulation of the UCP1 gene have utilized both transgenic mice and transient expression analysis of enhancer=promoter-reporter DNA constructs in cell cultures. The goal of this research has been to identify mechanisms that can explain the basis of the speci®c expression of the UCP1 gene in brown adipocytes and the mechanism for elevated expression in response to the demands of the organism for increases in thermogenesis. While the major mechanism for induction is probably the sympathetic nervous system acting through beta adrenergic receptors, there are certainly other signalling molecules that act in concert with adrenergic signalling to regulate transcription, translation and processing of the protein in the inner membrane.

The Ucp1 gene in mice encompasses 8 kb of sequence encoding six exons and 16 kb of 50 ¯anking sequence as de®ned by the presence of a 50 DNAse hypersensitive site at that distal location.25 However, regulatory motifs associated with brown fat speci®c expression are localized to a 200 bp region lying 2.6 kb upstream from the start of transcription. The importance of this small region for brown fat speci®c expression, has been established by the ability of this region to direct expression to the brown fat in transgenic mice.26 Within the 200 bps is a half-site cAMP responsive element (CRE2) ¯anked by an essential ETS domain and a DR1 motif.27,28 This intriguing motif has been the focus of some extraordinary research by the Graves and Spiegelman labs showing that a DR1 element (also referred to as URE) is the binding site of a PPARg2-RXR heterodimer.28 This heterodimer interacts with a nuclear coactivator, PGC1, to determine brown fat speci®c expression of UCP1.29 The effectiveness of PGC-1 in directing the brown fat speci®c expression, was illustrated by experiments showing that transfection of PGC-1 into 3T3 F422A preadipocytes, enables these cells to dramatically induce expression of UCP1 and other genes associated with mitochondrial biogenesis. With these experiments, major strides have been made in determining the mechanisms determining the brown fat differentiation program.

Prospects of UCP1-linked brown fat thermogenesis as an antiobesity target Pharmacological experiments in which adrenergic agonists has been administered to obese rodents and dogs, have shown that these agents reduce both adiposity and increase brown fat in the recipients. In addition, the genetic manipulations, described above, indicate that enhanced UCP1 expression can reduce adiposity in obese mice. An encouraging aspect of these studies is that mice with increased UCP1 expression do not signi®cantly delete adipose tissue to the point where the animal fails to thrive; however, a caveat to this conclusion is that the animals with potentially harmful levels of expression of a transgene may have been selected against during early development. Given the striking effects of over-expression to reduce adiposity, it needs to be explained why the Ucp1 KO mice do not exhibit enhanced adiposity compared to normal litter-mate controls. We are evaluating whether the absence of a strong effect of UCP1-de®ciency on the development of adiposity is due to the fact that the background genotype of the mice being tested already makes them very susceptible to diet-induced obesity. In other words, the ability of a high fat diet to induce obesity in the absence of over-feeding has already reached a


maximum. UCP1tm1 congenic lines resistant to dietinduced obesity are being developed to test this idea.

Conclusion The major barrier to the use of UCP1-linked brown fat thermogenesis for the problem of obesity, is the perception that insuf®cient brown fat resides in the human to make this an effective mechanism for the dissipation of fat. If the genetic variability for the induction of brown adipocytes in traditional white fat tissue of the mouse is also a property of the human genome, then a subpopulation of obese humans may also respond to beta adrenergic agonists. Furthermore, new targets for increasing brown adipocytes in an individual could emerge from recent progress in determining the transcriptional mechanisms controlling the differentiation of the brown adipocyte. References

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