Biomedical subjects
Publications and source records attributed to C Bogardus.
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Selected candidate genes have been analyzed in the Pima Indians of Arizona based on evidence that insulin resistance and type 2 diabetes have significant genetic determinants. An amino acid substitution at codon 905 of the glycogen-targeting subunit of type 1 protein phosphatase that regulates skeletal muscle glycogenesis was recently reported to be associated with changes in insulin action in Danish subjects. In addition to the variant at 905, we report here a novel substitution at codon 883 and common variant of an "ATTTA" element in the 3'-untranslated region (UTR) of the corresponding gene (PPP1R3). The 3'-UTR variant resembled the mRNA-destabilizing AT(AU)-rich elements (AREs) and resulted in a 10-fold difference in reporter mRNA half-life, was correlated with PPP1R3 transcript and protein concentrations in vivo, and was associated with insulin resistance and type 2 diabetes in the Pimas. The variant is more common in Pimas (0.56) than in Caucasians (0.40). Because of its apparent effect on expression of PPP1R3, it may, in part, contribute to the higher prevalence of type 2 diabetes in this Native American population.
To investigate the possible role of impaired sympathetic nervous system and/or adrenal medullary function in the etiology of human obesity, we studied 64 Pima Indian men (28 +/- 6 years, 101 +/- 25 kg, 34 +/- 9% body fat, mean +/- SD) in whom sympathoadrenal function was estimated at baseline by measurements of 24-hour urinary norepinephrine (NE) and epinephrine (Epi) excretion rates under weight-maintenance conditions. Body weight, body composition (hydrodensitometry), and body fat distribution (waist-to-thigh circumference ratio, W/T) were measured at baseline and follow-up. Follow-up data were available on 44 subjects who gained on average 8.4 +/- 9.5 kg over 3.3 +/- 2.1 years. In these subjects, baseline NE excretion rate, adjusted for its determinants (i.e., fat free mass, fat mass, and W/T), correlated negatively with bodyweight gain (r = -0.38; p = 0.009). Baseline Epi excretion rate correlated negatively with changes in W/T (r = -0.44; p = 0.003). In conclusion, our data show for the first time that a flow sympathetic nervous system activity is associated with body weight gain in humans. Also, a low activity of the adrenal medulla is associated with the development of central adiposity.
Leptin, the product of the ob gene, is a hormone, produced by adipose cells, that inhibits food intake and increases energy expenditure in rodents. In humans, plasma leptin concentrations correlate closely with the size of the adipose tissue depot; however, there is considerable variation in plasma leptin concentrations at any given degree of fatness. To investigate whether individuals prone to weight gain are hypoleptinemic, we measured fasting plasma leptin concentrations in two groups of weight-matched nondiabetic Pima Indians followed for approximately 3 years, 19 of whom subsequently gained weight and 17 of whom maintained their weight. After we adjusted for initial percent body fat, mean plasma leptin concentration was lower in those who gained weight than in those whose weight was stable. These data indicate that relatively low plasma leptin concentrations may play a role in the development of obesity in Pima Indians, a population prone to obesity.
The cloning of human and mouse cDNAs from brain that encode high affinity leptin receptors was recently reported. We have physically localized the human leptin receptor gene (LEPR) to a region at 1p31, between the anonymous microsatellite markers D1S515 and D1S198. The genomic structure of the human leptin receptor gene, corresponding to the published human brain cDNA sequence, spans over 70 kb and includes 20 exons. Since the leptin receptor gene is a candidate gene for obesity, and because of its proximity to D1S198, a marker previously linked to insulin secretion, the LEPR gene was sequenced in 20 non-diabetic Pima Indians chosen for extremes in percent body fat and in their acute insulin response to intravenous glucose. Seven polymorphic sites were identified. Two of these polymorphisms, Lys109Arg and Gln223Arg, are amino acid substitutions in the extracellular domain of the leptin receptor, one polymorphism is a silent substitution, and four occur in non-coding regions of the leptin receptor. Four of these sites are in linkage disequilibrium with one another. Nucleotides at three noncoding polymorphic sites were found exclusively in obese Pima Indians. This demonstrates an association between variation at the leptin receptor gene and obesity in humans.
Leptin is believed to play a role in the regulation of energy balance, but little is known about factors influencing plasma leptin concentrations. To determine the effect of short-term changes in energy balance, we measured plasma leptin concentrations as well as plasma glucose, insulin, triglyceride, nonesterified fatty acid concentrations, and metabolic rate in response to a standard test meal followed by a 24-h fast in 21 healthy Pima Indians. Plasma leptin concentrations decreased by 8% (P < 0.05) 2-4 h after the test meal. They returned to baseline 6-12 h after the subjects ate, then subsequently decreased, and, by the end of the fast, were an average of 37% below baseline (P < 0.0001). Changes in plasma leptin concentrations did not correlate with changes in plasma glucose, insulin, triglyceride, or nonesterified fatty acid concentrations or with changes in metabolic rate. The results of this study indicate that plasma leptin concentrations decrease in response to short-term energy restriction. These changes were not due to changes in glucose, insulin, triglycerides, or nonesterified fatty acids, nor did they relate to changes in metabolic rate. The decrease in plasma leptin concentrations with fasting may be an important homeostatic response to an energy deficit, stimulating food intake and thus restoring energy balance.
In animal studies, increased amounts of triglyceride associated with skeletal muscle (mTG) correlate with reduced skeletal muscle and whole body insulin action. The aim of this study was to test this relationship in humans. Subjects were 38 nondiabetic male Pima Indians (mean age 28 +/- 1 years). Insulin sensitivity at physiological (M) and supraphysiological (MZ) insulin levels was assessed by the euglycemic clamp. Lipid and carbohydrate oxidation were determined by indirect calorimetry before and during insulin administration. mTG was determined in vastus lateralis muscles obtained by percutaneous biopsy. Percentage of body fat (mean 29 +/- 1%, range 14-44%) was measured by underwater weighing. In simple regressions, negative relationships were found between mTG (mean 5.4 +/- 0.3 micromol/g, range 1.3-1.9 micromol/g) and log10M (r = -0.53, P < or = 0.001), MZ (r = -0.44, P = 0.006), and nonoxidative glucose disposal (r = -0.48 and -0.47 at physiological and supraphysiological insulin levels, respectively, both P = 0.005) but not glucose or lipid oxidation. mTG was not related to any measure of adiposity. In multiple regressions, measures of insulin resistance (log10M, MZ, log10[fasting insulin]) were significantly related to mTG independent of all measures of obesity (percentage of body fat, BMI, waist-to-thigh ratio). In turn, all measures of obesity were related to the insulin resistance measures independent of mTG. The obesity measures and mTG accounted for similar proportions of the variance in insulin resistance in these relationships. The results suggest that in this human population, as in animal models, skeletal muscle insulin sensitivity is strongly influenced by local supplies of triglycerides, as well as by remote depots and circulating lipids. The mechanism(s) underlying the relationship between mTG and insulin action on skeletal muscle glycogen synthesis may be central to an understanding of insulin resistance.
On the basis of accumulating evidence that obesity has a substantial genetic component, a genomewide search for linkages of DNA markers to percent body fat is ongoing in Pima Indians, a population with a very high prevalence of obesity. An initial screen of the genome (>600 markers in 874 individuals) has been completed using highly polymorphic markers (mean heterozygosity = .67). Reported here are the sib-pair linkage results for percent body fat (277 siblings), the best available indicator of overall obesity. Single-marker linkages to percent body fat were evaluated by sib-pair analysis for quantitative traits. From these analyses, the best evidence of genes influencing body fat came from markers at chromosome 11q21-q22 and 3p24.2-p22 (P = .001; LOD = 2.0). Regions flanking these markers were further investigated by multipoint linkage. The evidence for linkage at 11q21-q22 increased to P = .0002 (LOD = 2.8), peaking between markers D11S2000 and D11S2366. Evidence for linkage at 3p24.2-p22 did not change. No association was detected for any marker in the region. Although several genes are known in the 11q21-q22 region, none have been implicated as candidate genes for obesity.
Susceptibility to non-insulin-dependent diabetes mellitus (NIDDM) is largely genetically determined. In Pima Indians, obesity, insulin resistance, and a low acute insulin response (AIR) to an intravenous glucose infusion are each predictors of the disease. To ascertain whether these phenotypes are genetically determined, we estimated their familiality in nondiabetic Pima Indians with a maximum-likelihood method. Percentage body fat (PFAT) was highly familial (h2 =.76), whereas waist/ thigh circumference ratio (W/T ratio) was not significantly familial after controlling for PFAT (h2 = .16). AIR was also highly familial (h2 = .80 at 10 min), even after controlling for PFAT and insulin action (h2 = .70). Insulin action at physiologic plasma insulin concentrations was familial (h2 = .61) but less so after controlling for PFAT and W/T ratio (h2 = .38). At maximally stimulating insulin concentrations, insulin action was familial (h2 = .45) and was less influenced by controlling for PFAT and W/T ratio (h2 = .49). We conclude that in Pima Indians (1) PFAT and AIR are highly familial traits, (2) central distribution of fat is not a familial trait when controlled for PFAT, (3) 38%-49% of the variance in insulin action, independent of the effect of obesity, is familial, and (4) PFAT, AIR, and insulin action are useful traits to study genetic susceptibility to NIDDM. Because genetic parameter estimates are applicable only to the populations from which they were estimated, it is important to determine whether these estimates of familialities in Pima Indians can be confirmed in other populations before the utility of these traits in searching for NIDDM susceptibility genes in those populations can be fully advocated.
Different isoenzymes of pyruvate dehydrogenase kinase (PDK) inhibit the mitochondrial pyruvate dehydrogenase complex by phosphorylation of the E1alpha subunit, thus contributing to the regulation of glucose metabolism. By positional cloning in the 7q21.3-q22.1 region linked with insulin resistance and non-insulin-dependent diabetes mellitus in the Pima Indians, we identified a gene encoding an additional human PDK isoform, as evidenced by its amino acid sequence identity (>65%) with other mammalian PDKs, and confirmed by biochemical analyses of the recombinant protein. We performed detailed comparative analyses of the gene, termed PDK4, in insulin-resistant and insulin-sensitive Pima Indians, and detected five DNA variants with comparable frequencies in both subject groups. Using quantitative reverse transcription polymerase chain reaction, we found that the variants identified in the promoter and 5'-untranslated region did not correlate with differences in mRNA level in skeletal muscle and adipose tissue. We conclude that alterations in PDK4 are unlikely to be the molecular basis underlying the observed linkage at 7q21.3-q22.1 in the Pima Indians. Information about the genomic organization and promoter sequences of PDK4 will be useful in studies of other members of this family of mitochondrial protein kinases that are important for the regulation of glucose metabolism.
The human intestinal fatty acid binding protein (IFABP) binds long-chain fatty acids in vitro, but its intracellular function has remained speculative. A polymorphism in the gene that encodes IFABP results in an alanine (Ala54) to threonine (Thr54) substitution at codon 54 that alters the in vitro binding affinity of the protein for long-chain fatty acids. To identify potential functional variability between Ala54 and Thr54 IFABP, we established permanently transfected Caco-2 cell lines that express either Ala54 or Thr54 IFABP. We found that Caco-2 cells expressing Thr54 IFABP transport long-chain fatty acids and secrete triglycerides to a greater degree than Caco-2 cells expressing Ala54 IFABP. These results provide the first demonstration that IFABP participates in the intracellular transport of long-chain fatty acids. In addition, the observed increase in transport of fatty acids across cells expressing Thr54 IFABP suggests a plausible physiologic mechanism for our prior observation that Pima Indians with a Thr54 IFABP genotype have increased post-absorptive lipid oxidation rates and are more insulin-resistant than Pimas with a Ala54 IFABP genotype.
Skeletal muscle glycogen synthase (encoded by GYS1 on chromosome 19q13.3) is the rate-limiting enzyme in insulin-mediated non-oxidative glucose disposal. Our previous studies have demonstrated an impairment of insulin-stimulated GYS1 activities in insulin-resistant Pima Indians, and associations of non-insulin-dependent diabetes mellitus (NIDDM) with the GYS1 locus were reported recently in Finnish and Japanese populations. We have performed linkage and association analyses of GYS1 and seven additional DNA markers on 19q with NIDDM, and with parameters of insulin action in the Pima Indians. We have found a significant association of NIDDM with GYS1 genotypes (p = 0.009), and with common GYS1 alleles (p = 0.022) in the Pima Indians. We have performed a detailed comparative analysis of the GYS1 gene, mRNA, and protein product in insulin-sensitive and insulin-resistant Pima Indians. No mutations in GYS1 coding sequences were detected; nor did we find alterations of GYS1 mRNA expression or of its basal enzymatic activity in insulin-resistant Pima Indians. These results contrasted with a 25% reduction of immunoreactive protein in insulin-resistant subjects as detected by Western blotting with an antibody specific for the C-terminal end of GYS1 (t-test p = 0.024; Wilcoxon's rank-sum test, p = 0.04). Because no mutations were detected in the DNA encoding this epitope, the difference in immunoreactivity may reflect post-translational modification(s) of the protein rather than a difference in the gene itself, or it could have occurred by chance. We conclude that our data do not indicate alterations in the GYS1 gene as the cause for the observed association, and that a different locus near GYS1 may be the contributing genetic element.
There is evidence that insulin resistance and obesity are associated with relative increases in the proportion of glycolytic type IIb muscle fibers and decreases in the proportion of oxidative type I fibers. Futhermore, insulin resistance and obesity are associated with the fatty acid (FA) profile of structural membrane lipids. The present study was undertaken to define interrelationships between muscle fiber type and oxidative capacity, muscle membrane FA composition, and insulin action and obesity. Muscle morphology, insulin action, and body fat content were measured in 48 male nondiabetic Pima Indians. Percent body fat (pFAT, determined by hydrodensitometry) correlated negatively with percentage of type I fibers (r = -0.44, P = 0.002) and positively with percentage of type IIb fibers (r = 0.40, P = 0.005). Consistent with this finding, pFAT was also significantly related to oxidative capacity of muscle, as assessed by NADH staining (r = -0.47, P = 0.0007) and citrate synthase (CS) activity (r = -0.43, P = 0.008). Insulin action was correlated with oxidative capacity (CS; r = 0.41, P = 0.01) and weakly correlated with percentage of type IIb fibers (r = -0.29, P = 0.05). In addition, relationships were shown between muscle fiber type and FA composition (e.g., percentage of type I fibers related to n-3 FA; r = 0.37, P = 0.01). Thus leaness and insulin sensitivity are associated with increased oxidative capacity and unsaturation of membranes in skeletal muscle. Present studies support the hypothesis that muscle oxidative capacity and fiber type may play a genetically determined or an environmentally modified role in development of obesity and insulin resistance.
Leptin, a recently discovered protein produced in adipocytes, may be important in the regulation of body energy stores. In humans, leptin is present in the circulation in direct proportion to the amount of body fat. In rodents, insulin seems to regulate the production of leptin, but there is only limited evidence that this occurs in humans. We, therefore, measured plasma leptin concentrations in 13 insulin-sensitive and 13 insulin-resistant Pima Indians at baseline, at the end of 100 min of physiologic hyperinsulinemia (mean plasma insulin = 563 pmol/L) and after a further 100 min of supraphysiologic hyperinsulinemia (mean plasma insulin = 11,910 pmol/L), during a 2-step hyperinsulinemic-euglycemic glucose clamp. At baseline, plasma leptin concentrations were directly related to percent body fat, determined by hydrodensitometry (r = 0.85, P < 0.0001). After adjusting for percent body fat, there were no differences in fasting plasma leptin concentrations between insulin-sensitive and insulin-resistant subjects. Plasma leptin concentrations did not change in response to insulin in either the insulin-sensitive or insulin-resistant subjects. These results suggest that insulin does not acutely regulate plasma leptin concentrations in humans.
The product of the obese (ob) gene, leptin, is a secreted protein that is important in the regulation of body weight. Mice with mutations in the ob gene are obese and diabetic and manifest reduced physical as well as metabolic activity. In this study, we tested the possibility that mutations in the OB gene may contribute to human obesity. We report the isolation and partial sequence of the human OB gene and the screening of 105 obese patients for mutations in the protein coding sequence using the technique of single-strand conformational polymorphism. No coding sequence polymorphism was found, suggesting that mutations in the coding sequence of the OB gene do not constitute a common cause of increased body weight in humans. We also identified a highly polymorphic simple dinucleotide repeat DNA polymorphism in this gene that will be useful for genetic studies.
The homologues of single genes that cause obesity in rodents are suggested as candidate genes for modulation of body composition in humans. Among these genes are the four mouse mutations-diabetes (db), obesity (ob), tubby (tub), and yellow agouti (Ay). Variation in the human counterparts to these genes (OB, DB, TUB, and ASP, respectively) may contribute to human obesity, which is thought to have a substantial genetic component. To initially assess the potential contribution of these genes to human obesity, we examined polymorphic DNA markers that, by virtue of syntenic relationships to appropriate regions of the mouse genome, should be closely linked to the human counterparts of these genes. Using combined data from 716 Pima Indians comprising 217 nuclear families, we have tested a number of polymorphic microsatellite markers (three at DB, two at OB, five at TUB, and three at ASP) for sib-pair linkage to BMI, percentage body fat, resting metabolic rate, 24-h energy expenditure, and 24-h respiratory quotient. No significant linkages were found in an analysis of all sibships or in an analysis restricted to discordant sib pairs.
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