Craig H. Warden

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma.

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

Identification of an obesity quantitative trait locus on mouse chromosome 2 and evidence of linkage to body fat and insulin on the human homologous region 20q.

Chromosomal synteny between the mouse model and humans was used to map a gene for the complex trait of obesity. Analysis of NZB/BINJ x SM/J intercross mice located a quantitative trait locus (QTL) for obesity on distal mouse chromosome 2, in a region syntenic with a large region of human chromosome 20, showing linkage to percent body fat (likelihood of the odds [LOD] score 3.6) and fat mass (LOD score 4.3). The QTL was confirmed in a congenic mouse strain. To test whether the QTL contributes to human obesity, we studied linkage between markers located within a 52-cM region extending from 20p12 to 20q13.3 and measures of obesity in 650 French Canadian subjects from 152 pedigrees participating in the Quebec Family Study. Sib-pair analysis based on a maximum of 258 sib pairs revealed suggestive linkages between the percentage of body fat (P < 0.004), body mass index (P < 0.008), and fasting insulin (P < 0.0005) and a locus extending approximately from ADA (the adenosine deaminase gene) to MC3R (the melanocortin 3 receptor gene). These data provide evidence that a locus on human chromosome 20q contributes to body fat and insulin in a human population, and demonstrate the utility of using interspecies syntenic relationships to find relevant disease loci in humans.

PEDIATRIC OBESITY: An Overview of Etiology and Treatment

Pediatric obesity is a chronic and growing problem for which new ideas about the biologic basis of obesity offer hope for effective solutions. Prevalence of pediatric and adult obesity is increasing despite a bewildering array of treatment programs and severe psychosocial and economic costs. The definition of obesity as an increase in fat mass, not just an increase in body weight, has profound influence on the understanding and treatment of obesity. In principle, body weight is determined by a balance between energy expenditure and energy intake, but this observation does not by itself explain obesity. There is surprisingly little evidence that the obese overeat and only some evidence that the obese are more sedentary. Understanding of the biologic basis of obesity has grown rapidly in the last few years, especially with the identification of a novel endocrine pathway involving the adipose tissue secreted hormone leptin and the leptin receptor that is expressed in the hypothalamus. Plasma leptin levels are strongly correlated with body fat mass and are regulated by feeding and fasting, insulin, glucocorticoids, and other factors, consistent with the hypothesis that leptin is involved in body weight regulation and may even be a satiety factor (Fig. 2, Table 1). Leptin injections have been shown to reduce body weight of primates, although human clinical trials will not be reported until summer 1997. So many peptides influencing feeding have been described that one or more may have therapeutic potential (Fig. 2, Table 1). Although the complexity of pathways regulating body weight homeostasis slowed the pace of understanding underlying mechanisms, these complexities now offer many possibilities for novel therapeutic interventions (Fig. 2). Obesity is a major risk factor for insulin resistance and diabetes, hypertension, cancer, gallbladder disease, and atherosclerosis. In particular, adults who were obese as children have increased mortality independent of adult weight. Thus, prevention programs for children and adolescents will have long-term benefits. Treatment programs focus on modification of energy intake and expenditure through decreased calorie intake and exercise programs. Behavior-modification programs have been developed to increase effectiveness of these intake and exercise programs. These programs can produce short-term weight loss. Long-term losses are more modest but achieved more successfully in children than in adults. Several drug therapies for obesity treatment recently have been approved for adults that produce sustained 5% to 10% weight losses but experience with their use in children is limited. Identification of the biochemical pathways causing obesity by genetic approaches could provide the theoretic foundation for novel, safe, and effective obesity treatments. The cloning of leptin in 1994 has already led to testing the efficacy of leptin in clinical trials that are now underway. Although novel treatments of obesity are being developed as a result of the new biology of obesity, prevention of obesity remains an important goal.

The case for strategic international alliances to harness nutritional genomics for public and personal health

Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countries.

Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

Brain mitochondrial uncoupling protein 2 (UCP2): a protective stress signal in neuronal injury

Mitochondrial uncoupling proteins (UCPs) can dissociate oxidative phosphorylation from respiration, and they appear to be critical for energy balance. One of these proteins, UCP2, is also expressed in neurons of subcortical brain regions of healthy subjects. Here, we report on the protective role of UCP2 in brain injury by revealing its early induction after lesions and its inverse relationship with activation of an apoptotic signal, caspase 3, in wild-type and UCP2 overexpressing transgenic mice.

Coincidence of genetic loci for plasma cholesterol levels and obesity in a multifactorial mouse model.

We have examined backcross progeny derived from a cross of Mus spretus with C57BL/6J, that range from 1 to 50% carcass lipid (n = 215), and from 22 to 130 mg/dl plasma total cholesterol (n = 238). Statistical analysis revealed that distal mouse chromosome 7 exhibits significant linkage both to plasma total cholesterol (likelihood of the odds [LOD] 5.8) and to carcass lipid (LOD 3.8). A locus on chromosome 6 also shows significant linkage to plasma total cholesterol (LOD 5.6), but no linkage to carcass lipid. Neither chromosomal region contains any previously mapped genes likely to influence lipoprotein metabolism, indicating that novel genetic factors contributing to plasma lipoprotein levels have been identified.

Comparisons of Diets Used in Animal Models of High-Fat Feeding

Animal models are invaluable resources for biomedical research, including research on the effects of diet on metabolism and disease. Usually, great care is taken to ensure comparable genetic backgrounds and environmental conditions when performing studies using animal models, since this minimizes introduction of variability that can confound detection of treatment-related phenotypic differences. However, many papers using animal models draw conclusions about dietary effects from comparisons of natural-ingredient chow with defined diets, despite marked differences in micro- and macronutrient content. When comparing the effects of a chow diet with a defined high-fat diet, the effects of the dietary fat will be confounded with the effects of other components in the diets. This issue is highlighted by a limited literature survey that was conducted to identify common problems in the use and reporting of rodent diets.All original research papers identified by the keywords “mouse high fat” published in 2007 in five high-impact journals were included in this evaluation. Of the 35 papers examined, only 14% (5 papers) compared diets using identical nutrients differing only in relative amounts of fat and carbohydrate (Figure 1Figure 1). Specific details regarding the dietary comparisons made were often lacking, and frequently conclusions were drawn from comparisons of defined high-fat diets to chow.Figure 1Diet Comparisons in Recent Research PapersPie chart showing the percentage of 35 original research papers evaluated that used appropriate diet comparisons (14%), that compared chow and defined high-fat diets (43%), and that presented insufficient information to evaluate diet comparisons (34%). In the “chow and defined” category (9%), both diet types were used but no direct comparison was made between them. The journals examined were Cell Metabolism (7 papers), Diabetes (the first 11 of 36 papers), The Journal of Clinical Investigation (12 papers), Nature (2 papers), and Nature Medicine (3 papers).View Large Image | View Hi-Res Image | Download PowerPoint SlideRegular chow is composed of agricultural byproducts such as ground wheat, corn, or oats, alfalfa and soybean meal, a protein source such as fish, and vegetable oil and is supplemented with minerals and vitamins. Thus, chow is a high-fiber diet containing complex carbohydrates, with fats from a variety of vegetable sources. Chow is inexpensive to manufacture and is palatable to rodents. In contrast, defined high-fat diets consist of amino acid-supplemented casein, corn starch, maltodextrose or sucrose, and soybean oil or lard, also supplemented with minerals and vitamins. Fiber is often provided by cellulose. Chow and defined diets may exert significant separate and independent unintended effects on the measured phenotypes in any research protocol.Two important differences between chow and defined diets are the phytoestrogen content from soy, which is high but variable in chow diets but absent from defined diets (reviewed in Thigpen et al., 2004xThigpen, J.E., Setchell, K.D., Saunders, H.E., Haseman, J.K., Grant, M.G., and Forsythe, D.B. ILAR J. 2004; 45: 401–416Crossref | PubMedSee all ReferencesThigpen et al., 2004), and from sucrose, which is used as a carbohydrate source in defined diets but is absent from chow. Dietary phytoestrogens influence food and water intake, anxiety-related behaviors, locomotor activity, learning and memory, fat deposition, blood insulin, leptin and thyroid levels, and lipogenesis and lipolysis in isolated rat adipocytes (Torre-Villalvazo et al., 2008xTorre-Villalvazo, I., Tovar, A.R., Ramos-Barragan, V.E., Cerbon-Cervantes, M.A., and Torres, N. J. Nutr. 2008; 138: 462–468PubMedSee all References, Lephart et al., 2004axLephart, E.D., Porter, J.P., Lund, T.D., Bu, L., Setchell, K.D., Ramoz, G., and Crowley, W.R. Nutr. Metab. (Lond). 2004; 1: 16Crossref | PubMed | Scopus (34)See all References; reviewed in Lephart et al., 2004bxLephart, E.D., Setchell, K.D., Handa, R.J., and Lund, T.D. ILAR J. 2004; 45: 443–454Crossref | PubMedSee all ReferencesLephart et al., 2004b). Sucrose is 50% fructose, and there is abundant evidence that fructose can exacerbate weight gain and contribute to insulin resistance and dyslipidemia (reviewed in Stanhope and Havel, 2008xStanhope, K.L. and Havel, P.J. Curr. Opin. Lipidol. 2008; 19: 16–24Crossref | PubMed | Scopus (121)See all ReferencesStanhope and Havel, 2008). Other effects that differ between chow and defined diets and may be related to either phytoestrogen or fructose content include bone-related changes, plasma estradiol, urinary calcium and corticosterone (Tou et al., 2005xTou, J.C., Arnaud, S.B., Grindeland, R., and Wade, C. Exp. Biol. Med. (Maywood). 2005; 230: 31–39PubMedSee all ReferencesTou et al., 2005), and solution taste preferences (Tordoff et al., 2002xTordoff, M.G., Pilchak, D.M., Williams, J.A., McDaniel, A.H., and Bachmanov, A.A. J. Nutr. 2002; 132: 2288–2297PubMedSee all ReferencesTordoff et al., 2002).When comparing the effects of chow with a defined high-fat diet, the effects of the dietary fat will be confounded with the effects of other components that differ between the diets. Just as it is essential that mouse strains be specified, constituents of experimental diets must be specified (Thigpen et al., 2004xThigpen, J.E., Setchell, K.D., Saunders, H.E., Haseman, J.K., Grant, M.G., and Forsythe, D.B. ILAR J. 2004; 45: 401–416Crossref | PubMedSee all References, Tordoff et al., 1999xTordoff, M.G., Bachmanov, A.A., Friedman, M.I., and Beauchamp, G.K. Science. 1999; 285: 2069PubMedSee all References, Everitt and Foster, 2004xEveritt, J.I. and Foster, P.M. ILAR J. 2004; 45: 417–424Crossref | PubMedSee all References). Specifics to be considered are the objectives of the study and whether the endpoints are affected by several diet components, the maintenance diet, or the palatability of the diet (Thigpen et al., 2004xThigpen, J.E., Setchell, K.D., Saunders, H.E., Haseman, J.K., Grant, M.G., and Forsythe, D.B. ILAR J. 2004; 45: 401–416Crossref | PubMedSee all ReferencesThigpen et al., 2004). Although this correspondence focuses on high-fat diet comparisons, these issues are applicable to any specific nutrient comparisons.

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci.

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice.DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates.RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice.Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks.CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.