Publisher Summary This chapter demonstrates that there are many expressed non-protein coding regi... more Publisher Summary This chapter demonstrates that there are many expressed non-protein coding regions in the mammalian genome. The core power of positional genetics approaches is that one can identify the causative factor whether it is an expressed gene, microRNA, antisense, or other unknown mechanisms. New phenotyping resources may also facilitate gene discovery programs. The Mouse Phenome Database includes extensive phenotyping information on dozens of strains. One possibility is that this phenotyping can be combined with high-density mouse haplotyping to find genes for many complex traits. Using genetic approaches leads to unpredictable results; only a small fraction may identify genes that can be targeted by weight loss drugs. Although each obesity gene may have its own interesting story, not all genes have human orthologs that cause obesity; and not all human obesity genes provide safe and effective drug targets. It is not always clear that weight gain or loss in mice can be predicted from measurements of food intake and energy expenditure, possibly because the assays used are not accurate enough to capture the small imbalances of intake and expenditure that cause fat accumulation or because the assays are used for cross-sectional studies at one time point, but the obesity-causing energy imbalance occurs at another time. Settling these questions will allow for a more precise understanding of the mechanisms by which alleles cause fat accumulation.
The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidy... more The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidylcho Iine biosynthesis by choline kinase have been investi gated in 3T3 fibroblasts. Treatment of quiescent 3T3 fibroblasts with serum was shown in previous work to increase phosphocholine pool size and phosphatidyl choline biosynthesis. We now report that treatment of 3T3 cells with serum increased intracellular choline kinase activity by 2-3-fold with a concomitant 2-3- fold decrease of intracellular free choline concentra tions. Initial rates of choline transport were the same in quiescent and serum-treated cells, whereas choline kinase activity was 2-3-fold higher in serum-treated cells. As a consequence, free choline concentrations were 2-3-fold lower in serum-stimulated cells than in control quiescent cells. Phosphocholine turnover rates were increased 2-fold by serum treatment both as a consequence of a serum-dependent increase of phos phocholine pools and as a result of a serum-dependent lowering of the phosphocholine half-life. Thus, the overall response of 3T3 cells to serum stimulation in cluded decreased choline pools and increased choline kinase activity, phosphocholine pool size, phosphocho line turnover, and phosphatidylcholine biosynthesis.
Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degree... more Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2-4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of 13H)choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of 13H)choline into acid-soluble material by up to 2-fold. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.
Automated phosphate analysis of acid-soluble pools of phosphate esters was employed to reveal pos... more Automated phosphate analysis of acid-soluble pools of phosphate esters was employed to reveal possible biochemical changes during the transition of Swiss 3T3 mouse fibroblasts from quiescence to active replication of DNA. After 1 2 hours of stimulation with 10% fetal bovine serum the most notable were 3-fold increases in pools of phospholipid precursors and derivatives. These included glycerophosphocholine, glycerophosphoethanolamine, phosphocholine and phosphoethanolamine. Concurrent but less dramatic increases in pools of ATP, CTP and fructose 1 ,6-diphosphate were also obtained.
The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is... more The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is poorly understood. Previous studies of naturally occurring variations of apoA-II in mice have, however, suggested that apoA-II expression influences HDL size and concentration. We now provide definitive evidence of this using transgenic mice overexpressing mouse apoA-II. Thus, as compared with nontransgenic littermates, transgenic mice possessing 4-fold elevations of apoA-II mRNA and 2-3-fold elevations in plasma apoA-II levels exhibited more than a 2-fold increase in HDL levels as well as about a 25% increase in average HDL diameter. Overexpression of mouse apoA-II also resulted in a substantial decrease in the levels of apolipoprotein E associated with HDL, suggesting that apoA-II can displace apoE from HDL. The apoA-II transgenic mice also exhibited a 2-4-fold increase in plasma very low density and low density lipoprotein-cholesterol levels, suggesting novel interactions among the classes of plasma lipoproteins.
The LLC/CKC mouse was developed from a hybrid stock derived by cross-breeding C57BL/6J, C57BR/CdJ... more The LLC/CKC mouse was developed from a hybrid stock derived by cross-breeding C57BL/6J, C57BR/CdJ, A/J, BALB/cJ, LLC/Ckc and SM/Ckc mice screened for low leukocyte counts (4-5000/mm3) over 22 generations. 100% develop amyloidosis at 10-18 months (spleen>adrenals>kidney>liver). The amyloid subunit protein (ALLC) has a MW of 8.7kD and is distinct from AA protein on 2D Gels and by Western Blot analysis. ALLC has a blocked amino- terminus; following deblocking, the N-terminal sequence of mouse ApoAII was obtained to position 17, with a substitution of Gln for Pro at position 5. The remainder of the sequence of ApoAII was obtained from ALLC following cyanogen bromide cleavage, and by isolation of tryptic peptides by HPLC. A monospecific rabbit antibody to ALLC recognized LLC amyloid in tissue section by immunohistology as well as amorphous forms near reticular cells by immuno EM. Anti-ALLC reacted with a serum protein with alpha globulin mobility that copurified with SAA by formic acid gel filtration. LLC ApoAII was fully sequenced at the DNA level following reverse transcription of poly A+ mRNA isolated from liver and amplification by the Polymerase Chain Reaction (PCR) to further confirm the Pro5→Gln substitution. Anchor PCR amplification and sequencing of ApoAII mRNA from 11 different inbred mouse strains revealed considerable polymorphism of this protein, with common representation of the position 5 Pro→Gln substitution. SJL, a strain reported to develop non-AA spontaneous amyloidosis (Lab Invest 35:47, 1976) was also found to have this polymorphism. It thus appears that identical variant ApoAII molecules are the major constituents of age-related amyloid developing in SAM/P, LLC/Ckc and possibly SJL mice. Further studies can now be targeted to the physiologic correlates of ApoAII polymorphism, as well as the elucidation of separate but interactive genetic determinants of amyloidogenesis.
The American Journal of Clinical Nutrition, Dec 1, 2006
An epidemic of overweight and obesity is occurring in both industrial and developing societies. A... more An epidemic of overweight and obesity is occurring in both industrial and developing societies. According to the National Health and Nutrition Examination Survey (NHANES), the prevalence of obesity doubled in the adult population, and the prevalence of overweight tripled in children and adolescents between 1982 and 2002 (Internet: www.cdc.gov/nchs/nhanes.htm). The most recent prevalence estimates from weight and height data collected by NHANES in 2003-2004 indicate that the prevalence of overweight in children and adolescents continues to increase and that 18.2% of boys and 16.0% of girls between the ages of 2 and 19 y are now overweight (1). Prevalence estimates differ by ethnicity, eg, 22.0% of Mexican American boys and 16.2% of Mexican American girls are overweight (1). These recent rapid increases in obesity are due to environmental changes-including readily available palatable and energydense foods and a sedentary lifestyle (2). Comorbidities associated with obesity include the metabolic syndrome (hypertension, hyperlipidemia, and insulin resistance), type 2 diabetes, certain cancers, orthopedic problems, and sleep apnea. Because childhood overweight tracks with obesity in adulthood (3), the dramatic increase in the frequency of overweight in children and adolescents bodes ill for the health status of our population in future years. Thus, there is considerable interest in understanding the genetic and environmental contributions to obesity to aid in the development of more effective interventions. As reviewed by Comuzzie and Allison (4), 40-70% of the within-population variation in obesity is due to genetic variation. The proportion of the total variance in a trait that is due to genetic factors is defined as the heritability coefficient (h 2). Population or family studies tend to have lower and twin studies to have higher heritability values for body mass index (BMI), a common surrogate measure for obesity. Data from adult nondiabetic Mexican American participants in the San Antonio Diabetes Study showed the additive genetic heritability coefficient for BMI in that population to be 0.65 (5). Although there are few estimates of the heritability of overweight or obesity in children and adolescents, genetic influences contributed 0.75-0.80 of the variation in percentage body fat in a pediatric twin study (6). New heritability estimates for childhood obesity-related phenotypes and risk factors for metabolic diseases are available in the Hispanic population from the Viva la Familia Study, as reported by Butte et al (7) in this issue of the Journal. In this study, the heritability coefficients (adjusted for sex, age, and environmental covariates) of anthropometric indexes range from 0.24 to 0.75, whereas the heritability coefficients for body composition are somewhat lower. For example, the h 2 for fat mass is 0.18.
American Journal of Physiology-endocrinology and Metabolism, Aug 1, 1999
and B. A. Horwitz. T 3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphor... more and B. A. Horwitz. T 3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E380-E389, 1999.-The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and-3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T 3) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and-3 mRNAs. T 3 stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and-3 expression. Mitochondrial respiration was not affected by T 3 and did not correlate with UCP-2 and-3 mRNAs. Gastrocnemius UCP-2 and-3 expression did correlate with RMR. We conclude 1) T 3 did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and-3 expression may partly explain variations in RMR. One possible explanation for these data is that T 3 stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and-3 is not retained in the mitochondrial fraction. proton leak; thyroid hormone; mitochondria; uncoupling protein
High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of ... more High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans.
Nutrition in the Prevention and Treatment of Disease, 2017
Most genetic human obesity is due to multiple genes interacting with each other and with environm... more Most genetic human obesity is due to multiple genes interacting with each other and with environment. Sequencing exomes, genome-wide associations with single nucleotide polymorphisms, sequencing methylations, and sequencing genomic DNA have revealed hundreds of obesity-causing polymorphisms that all together account for a small percent of total heritability. Pathway analyses reveal that most body mass index genes are expressed in the brain whereas most waist-to-hip ratio fat distribution genes are expressed in adipose tissue. Most mouse obesity genes are also human obesity genes and vice versa. Studies in mice demonstrate that quantitating fat mass, individual fat depots, responses of individual fat depots to dieting and exercise, and parental effects will reveal many new obesity genes. These topics have been explored superficially or not at all in studies of humans. Genetic and randomized controlled diet data are presently inadequate for development of personalized obesity therapy.
Macrophages possess a number of surface receptors that are capable of mediating the internalizati... more Macrophages possess a number of surface receptors that are capable of mediating the internalization of lipoproteins. The low-density lipoprotein (LDL) receptor of human monocyte macrophages recognizes apolipoprotein B-100 and apolipoprotein E and is rapidly regulated in response to changes in intracellular cholesterol levels. In contrast, in J774 macrophages LDL receptor regulation is defective and LDL can cause massive cholesterol accumulation. The ft migrating very low density lipoprotein (/3-VLDL) receptor is poorly regulated by cellular cholesterol concentrations, readily recognizes apolipoprotein E, poorly recognizes apolipoprotein B-100, and is immunologically related to the LDL receptor. The scavenger receptor (acetyl-LDL receptor) appears to have a molecular weight of 250 000 and is not regulated by cellular cholesterol levels. This receptor recognizes LDL that has been chemically or biologically altered. LDL complexes can also enter macrophages and cause cholesterol accumulation. Examples of such complexes are LDL-dextran sulphate complexes, LDL-proteoglycan aggregates, LDL-mast cell granule complexes, LDL-heparin-fibronectin-denatured collagen complexes, and LDL-antibody complexes. The entry of lipoprotein into macrophages by a pathway that is poorly regulated or is not regulated by cellular cholesterol concentrations appears to be a prerequisite for the formation of arterial foam cells.
The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes m... more The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala→Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3′-untranslated region of exon 8 of UCP2 and a C→T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 ± 7 years), unrelated, fullblooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium (P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.
which permits unrestricted use, distribution, and reproduction in any medium, provided the origin... more which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. There has been intense interest in defining the functions of UCP2 and UCP3 during the nine years since the cloning of these UCP1 homologues. Current data suggest that both UCP2 and UCP3 proteins share some features with UCP1, such as the ability to reduce mitochondrial membrane potential, but they also have distinctly different physiological roles. Human genetic studies consistently demonstrate the effect of UCP2 alleles on type-2 diabetes. Less clear is whether UCP2 alleles influence body weight or body mass index (BMI) with many studies showing a positive effect while others do not. There is strong evidence that both UCP2 and UCP3 protect against mitochondrial oxidative damage by reducing the production of reactive oxygen species. The evidence that UCP2 protein is a negative regulator of insulin secretion by pancreatic β-cells is also strong: increased UCP2 d...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
Oxidative damage of mitochondrial compartments contribute to a range of degenerative diseases. He... more Oxidative damage of mitochondrial compartments contribute to a range of degenerative diseases. Here protective effect of quinones and their derivatives on mitochondrial proteins and lipids under oxidative stress was analysed. The most effective quinone analogues appear to be conjugates of lipophilic cations ("Skulachev cations", by D. Green) with ubiquinone (Murphy et al., 2007) or plastoquinone (Skulachev et al., 2008, in press), so called "mitochondrially targeted antioxidants" (MTAO). MTAO effectively inhibit lipid peroxidation and protect membrane cardiolipin from oxidation. Using dihydroethidium and Amplex Red as a probes for ROS detection, we found that MTAO effectively suppress ROS production from different sites of the mitochondrial respiratory chain. In succinate-energised mitochondria there is a lag-period for the MTAO effect, but not in pyruvate/malateenergised mitochondria. When mitochondria isolated from the heart of a mouse strain mutated on mitochondrial DNA polymerase PolgA (Trifunovic et al., 2004) were used, we found that the mitochondria of PolgA mice did not produce more ROS than wild type, but the inhibitory effect of MTAO was stronger and more pronounced. Taken together, these data provided evidence that MTAO could be useful for treatment of genetic disorders, manifested degenerative diseases and aging complications.
The use of simple sequence repeat (SSR) loci lacking length polymorphisms among their alleles (de... more The use of simple sequence repeat (SSR) loci lacking length polymorphisms among their alleles (defined here as homozygotes) in systematic studies has thus far been largely ignored due to their apparent lack of genetic heterogeneity. However, in this paper we show that point mutations, insertions, and deletions within SSR loci of identical size can be highly informative for population genetic and evolutionary studies. Our preliminary study of the von Willebrand factor (vWF) locus shows that there is a significant level of divergence among the Bornean Orangutans which implies that present orangutan subpopulations in Borneo are genetically isolated from each other. However, our findings do not confirm that the Bornean and Sumatran Orangutans form two separate clades.
Pediatric obesity is a chronic and growing problem for which new ideas about the biologic basis o... more 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.
Publisher Summary This chapter demonstrates that there are many expressed non-protein coding regi... more Publisher Summary This chapter demonstrates that there are many expressed non-protein coding regions in the mammalian genome. The core power of positional genetics approaches is that one can identify the causative factor whether it is an expressed gene, microRNA, antisense, or other unknown mechanisms. New phenotyping resources may also facilitate gene discovery programs. The Mouse Phenome Database includes extensive phenotyping information on dozens of strains. One possibility is that this phenotyping can be combined with high-density mouse haplotyping to find genes for many complex traits. Using genetic approaches leads to unpredictable results; only a small fraction may identify genes that can be targeted by weight loss drugs. Although each obesity gene may have its own interesting story, not all genes have human orthologs that cause obesity; and not all human obesity genes provide safe and effective drug targets. It is not always clear that weight gain or loss in mice can be predicted from measurements of food intake and energy expenditure, possibly because the assays used are not accurate enough to capture the small imbalances of intake and expenditure that cause fat accumulation or because the assays are used for cross-sectional studies at one time point, but the obesity-causing energy imbalance occurs at another time. Settling these questions will allow for a more precise understanding of the mechanisms by which alleles cause fat accumulation.
The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidy... more The regulation of choline kinase activity by fetal bovine serum and the regulation of phosphatidylcho Iine biosynthesis by choline kinase have been investi gated in 3T3 fibroblasts. Treatment of quiescent 3T3 fibroblasts with serum was shown in previous work to increase phosphocholine pool size and phosphatidyl choline biosynthesis. We now report that treatment of 3T3 cells with serum increased intracellular choline kinase activity by 2-3-fold with a concomitant 2-3- fold decrease of intracellular free choline concentra tions. Initial rates of choline transport were the same in quiescent and serum-treated cells, whereas choline kinase activity was 2-3-fold higher in serum-treated cells. As a consequence, free choline concentrations were 2-3-fold lower in serum-stimulated cells than in control quiescent cells. Phosphocholine turnover rates were increased 2-fold by serum treatment both as a consequence of a serum-dependent increase of phos phocholine pools and as a result of a serum-dependent lowering of the phosphocholine half-life. Thus, the overall response of 3T3 cells to serum stimulation in cluded decreased choline pools and increased choline kinase activity, phosphocholine pool size, phosphocho line turnover, and phosphatidylcholine biosynthesis.
Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degree... more Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2-4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of 13H)choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of 13H)choline into acid-soluble material by up to 2-fold. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.
Automated phosphate analysis of acid-soluble pools of phosphate esters was employed to reveal pos... more Automated phosphate analysis of acid-soluble pools of phosphate esters was employed to reveal possible biochemical changes during the transition of Swiss 3T3 mouse fibroblasts from quiescence to active replication of DNA. After 1 2 hours of stimulation with 10% fetal bovine serum the most notable were 3-fold increases in pools of phospholipid precursors and derivatives. These included glycerophosphocholine, glycerophosphoethanolamine, phosphocholine and phosphoethanolamine. Concurrent but less dramatic increases in pools of ATP, CTP and fructose 1 ,6-diphosphate were also obtained.
The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is... more The role of apolipoprotein A-II (apoA-II) in the metabolism of high density lipoproteins (HDL) is poorly understood. Previous studies of naturally occurring variations of apoA-II in mice have, however, suggested that apoA-II expression influences HDL size and concentration. We now provide definitive evidence of this using transgenic mice overexpressing mouse apoA-II. Thus, as compared with nontransgenic littermates, transgenic mice possessing 4-fold elevations of apoA-II mRNA and 2-3-fold elevations in plasma apoA-II levels exhibited more than a 2-fold increase in HDL levels as well as about a 25% increase in average HDL diameter. Overexpression of mouse apoA-II also resulted in a substantial decrease in the levels of apolipoprotein E associated with HDL, suggesting that apoA-II can displace apoE from HDL. The apoA-II transgenic mice also exhibited a 2-4-fold increase in plasma very low density and low density lipoprotein-cholesterol levels, suggesting novel interactions among the classes of plasma lipoproteins.
The LLC/CKC mouse was developed from a hybrid stock derived by cross-breeding C57BL/6J, C57BR/CdJ... more The LLC/CKC mouse was developed from a hybrid stock derived by cross-breeding C57BL/6J, C57BR/CdJ, A/J, BALB/cJ, LLC/Ckc and SM/Ckc mice screened for low leukocyte counts (4-5000/mm3) over 22 generations. 100% develop amyloidosis at 10-18 months (spleen>adrenals>kidney>liver). The amyloid subunit protein (ALLC) has a MW of 8.7kD and is distinct from AA protein on 2D Gels and by Western Blot analysis. ALLC has a blocked amino- terminus; following deblocking, the N-terminal sequence of mouse ApoAII was obtained to position 17, with a substitution of Gln for Pro at position 5. The remainder of the sequence of ApoAII was obtained from ALLC following cyanogen bromide cleavage, and by isolation of tryptic peptides by HPLC. A monospecific rabbit antibody to ALLC recognized LLC amyloid in tissue section by immunohistology as well as amorphous forms near reticular cells by immuno EM. Anti-ALLC reacted with a serum protein with alpha globulin mobility that copurified with SAA by formic acid gel filtration. LLC ApoAII was fully sequenced at the DNA level following reverse transcription of poly A+ mRNA isolated from liver and amplification by the Polymerase Chain Reaction (PCR) to further confirm the Pro5→Gln substitution. Anchor PCR amplification and sequencing of ApoAII mRNA from 11 different inbred mouse strains revealed considerable polymorphism of this protein, with common representation of the position 5 Pro→Gln substitution. SJL, a strain reported to develop non-AA spontaneous amyloidosis (Lab Invest 35:47, 1976) was also found to have this polymorphism. It thus appears that identical variant ApoAII molecules are the major constituents of age-related amyloid developing in SAM/P, LLC/Ckc and possibly SJL mice. Further studies can now be targeted to the physiologic correlates of ApoAII polymorphism, as well as the elucidation of separate but interactive genetic determinants of amyloidogenesis.
The American Journal of Clinical Nutrition, Dec 1, 2006
An epidemic of overweight and obesity is occurring in both industrial and developing societies. A... more An epidemic of overweight and obesity is occurring in both industrial and developing societies. According to the National Health and Nutrition Examination Survey (NHANES), the prevalence of obesity doubled in the adult population, and the prevalence of overweight tripled in children and adolescents between 1982 and 2002 (Internet: www.cdc.gov/nchs/nhanes.htm). The most recent prevalence estimates from weight and height data collected by NHANES in 2003-2004 indicate that the prevalence of overweight in children and adolescents continues to increase and that 18.2% of boys and 16.0% of girls between the ages of 2 and 19 y are now overweight (1). Prevalence estimates differ by ethnicity, eg, 22.0% of Mexican American boys and 16.2% of Mexican American girls are overweight (1). These recent rapid increases in obesity are due to environmental changes-including readily available palatable and energydense foods and a sedentary lifestyle (2). Comorbidities associated with obesity include the metabolic syndrome (hypertension, hyperlipidemia, and insulin resistance), type 2 diabetes, certain cancers, orthopedic problems, and sleep apnea. Because childhood overweight tracks with obesity in adulthood (3), the dramatic increase in the frequency of overweight in children and adolescents bodes ill for the health status of our population in future years. Thus, there is considerable interest in understanding the genetic and environmental contributions to obesity to aid in the development of more effective interventions. As reviewed by Comuzzie and Allison (4), 40-70% of the within-population variation in obesity is due to genetic variation. The proportion of the total variance in a trait that is due to genetic factors is defined as the heritability coefficient (h 2). Population or family studies tend to have lower and twin studies to have higher heritability values for body mass index (BMI), a common surrogate measure for obesity. Data from adult nondiabetic Mexican American participants in the San Antonio Diabetes Study showed the additive genetic heritability coefficient for BMI in that population to be 0.65 (5). Although there are few estimates of the heritability of overweight or obesity in children and adolescents, genetic influences contributed 0.75-0.80 of the variation in percentage body fat in a pediatric twin study (6). New heritability estimates for childhood obesity-related phenotypes and risk factors for metabolic diseases are available in the Hispanic population from the Viva la Familia Study, as reported by Butte et al (7) in this issue of the Journal. In this study, the heritability coefficients (adjusted for sex, age, and environmental covariates) of anthropometric indexes range from 0.24 to 0.75, whereas the heritability coefficients for body composition are somewhat lower. For example, the h 2 for fat mass is 0.18.
American Journal of Physiology-endocrinology and Metabolism, Aug 1, 1999
and B. A. Horwitz. T 3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphor... more and B. A. Horwitz. T 3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E380-E389, 1999.-The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and-3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T 3) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and-3 mRNAs. T 3 stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and-3 expression. Mitochondrial respiration was not affected by T 3 and did not correlate with UCP-2 and-3 mRNAs. Gastrocnemius UCP-2 and-3 expression did correlate with RMR. We conclude 1) T 3 did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and-3 expression may partly explain variations in RMR. One possible explanation for these data is that T 3 stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and-3 is not retained in the mitochondrial fraction. proton leak; thyroid hormone; mitochondria; uncoupling protein
High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of ... more High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans.
Nutrition in the Prevention and Treatment of Disease, 2017
Most genetic human obesity is due to multiple genes interacting with each other and with environm... more Most genetic human obesity is due to multiple genes interacting with each other and with environment. Sequencing exomes, genome-wide associations with single nucleotide polymorphisms, sequencing methylations, and sequencing genomic DNA have revealed hundreds of obesity-causing polymorphisms that all together account for a small percent of total heritability. Pathway analyses reveal that most body mass index genes are expressed in the brain whereas most waist-to-hip ratio fat distribution genes are expressed in adipose tissue. Most mouse obesity genes are also human obesity genes and vice versa. Studies in mice demonstrate that quantitating fat mass, individual fat depots, responses of individual fat depots to dieting and exercise, and parental effects will reveal many new obesity genes. These topics have been explored superficially or not at all in studies of humans. Genetic and randomized controlled diet data are presently inadequate for development of personalized obesity therapy.
Macrophages possess a number of surface receptors that are capable of mediating the internalizati... more Macrophages possess a number of surface receptors that are capable of mediating the internalization of lipoproteins. The low-density lipoprotein (LDL) receptor of human monocyte macrophages recognizes apolipoprotein B-100 and apolipoprotein E and is rapidly regulated in response to changes in intracellular cholesterol levels. In contrast, in J774 macrophages LDL receptor regulation is defective and LDL can cause massive cholesterol accumulation. The ft migrating very low density lipoprotein (/3-VLDL) receptor is poorly regulated by cellular cholesterol concentrations, readily recognizes apolipoprotein E, poorly recognizes apolipoprotein B-100, and is immunologically related to the LDL receptor. The scavenger receptor (acetyl-LDL receptor) appears to have a molecular weight of 250 000 and is not regulated by cellular cholesterol levels. This receptor recognizes LDL that has been chemically or biologically altered. LDL complexes can also enter macrophages and cause cholesterol accumulation. Examples of such complexes are LDL-dextran sulphate complexes, LDL-proteoglycan aggregates, LDL-mast cell granule complexes, LDL-heparin-fibronectin-denatured collagen complexes, and LDL-antibody complexes. The entry of lipoprotein into macrophages by a pathway that is poorly regulated or is not regulated by cellular cholesterol concentrations appears to be a prerequisite for the formation of arterial foam cells.
The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes m... more The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala→Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3′-untranslated region of exon 8 of UCP2 and a C→T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 ± 7 years), unrelated, fullblooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium (P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.
which permits unrestricted use, distribution, and reproduction in any medium, provided the origin... more which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. There has been intense interest in defining the functions of UCP2 and UCP3 during the nine years since the cloning of these UCP1 homologues. Current data suggest that both UCP2 and UCP3 proteins share some features with UCP1, such as the ability to reduce mitochondrial membrane potential, but they also have distinctly different physiological roles. Human genetic studies consistently demonstrate the effect of UCP2 alleles on type-2 diabetes. Less clear is whether UCP2 alleles influence body weight or body mass index (BMI) with many studies showing a positive effect while others do not. There is strong evidence that both UCP2 and UCP3 protect against mitochondrial oxidative damage by reducing the production of reactive oxygen species. The evidence that UCP2 protein is a negative regulator of insulin secretion by pancreatic β-cells is also strong: increased UCP2 d...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
Oxidative damage of mitochondrial compartments contribute to a range of degenerative diseases. He... more Oxidative damage of mitochondrial compartments contribute to a range of degenerative diseases. Here protective effect of quinones and their derivatives on mitochondrial proteins and lipids under oxidative stress was analysed. The most effective quinone analogues appear to be conjugates of lipophilic cations ("Skulachev cations", by D. Green) with ubiquinone (Murphy et al., 2007) or plastoquinone (Skulachev et al., 2008, in press), so called "mitochondrially targeted antioxidants" (MTAO). MTAO effectively inhibit lipid peroxidation and protect membrane cardiolipin from oxidation. Using dihydroethidium and Amplex Red as a probes for ROS detection, we found that MTAO effectively suppress ROS production from different sites of the mitochondrial respiratory chain. In succinate-energised mitochondria there is a lag-period for the MTAO effect, but not in pyruvate/malateenergised mitochondria. When mitochondria isolated from the heart of a mouse strain mutated on mitochondrial DNA polymerase PolgA (Trifunovic et al., 2004) were used, we found that the mitochondria of PolgA mice did not produce more ROS than wild type, but the inhibitory effect of MTAO was stronger and more pronounced. Taken together, these data provided evidence that MTAO could be useful for treatment of genetic disorders, manifested degenerative diseases and aging complications.
The use of simple sequence repeat (SSR) loci lacking length polymorphisms among their alleles (de... more The use of simple sequence repeat (SSR) loci lacking length polymorphisms among their alleles (defined here as homozygotes) in systematic studies has thus far been largely ignored due to their apparent lack of genetic heterogeneity. However, in this paper we show that point mutations, insertions, and deletions within SSR loci of identical size can be highly informative for population genetic and evolutionary studies. Our preliminary study of the von Willebrand factor (vWF) locus shows that there is a significant level of divergence among the Bornean Orangutans which implies that present orangutan subpopulations in Borneo are genetically isolated from each other. However, our findings do not confirm that the Bornean and Sumatran Orangutans form two separate clades.
Pediatric obesity is a chronic and growing problem for which new ideas about the biologic basis o... more 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.
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