Dietary obesity linked to genetic loci on chromosomes 9 and 15 in a polygenic mouse model.

Loci linked to sensitivity to dietary obesity were identified by Quantitative Trait Locus (QTL) analysis of two mapping populations derived from a cross between AKR/J and SWR/J mice. AKR/J mice are sensitive to dietary obesity when fed a high fat diet while SWR/J mice are resistant. Intercrosses between these strains segregate the phenotype of sensitivity to dietary obesity. Using an F2 mapping population of 931 male mice we found significant linkage with a QTL on chromosome 9 (Likelihood of the Odds [LOD] ratio of 4.85) and another QTL on chromosome 15 (LOD = 3.93). The presence of a QTL on chromosome 15 was confirmed in a separate mapping population of 375 male F1 x SWR/J mice (LOD = 3.82). These two loci are designated dietary obese 2 (Do2) and dietary obese 3 (Do3) for the chromosome 9 and 15 loci, respectively. Both of these chromosomal regions contain candidate genes which may contribute to variation in the phenotype. These loci also exert a significant control over individual adipose depot weights.

Polymorphisms in the taste receptor gene (Tas1r3) region are associated with saccharin preference in 30 mouse strains.

The results of recent studies suggest that the mouse Sac (saccharin preference) locus is identical to the Tas1r3 (taste receptor) gene. The goal of this study was to identify Tas1r3 sequence variants associated with saccharin preference in a large number of inbred mouse strains. Initially, we sequenced approximately 6.7 kb of the Tas1r3 gene and its flanking regions from six inbred mouse strains with high and low saccharin preference, including the strains in which the Sac alleles were described originally (C57BL/6J, Sac(b); DBA/2J, Sac(d)). Of the 89 sequence variants detected among these six strains, eight polymorphic sites were significantly associated with preferences for 1.6 mm saccharin. Next, each of these eight variant sites were genotyped in 24 additional mouse strains. Analysis of the genotype-phenotype associations in all 30 strains showed the strongest association with saccharin preference at three sites: nucleotide (nt) -791 (3 bp insertion/deletion), nt +135 (Ser45Ser), and nt +179 (Ile60Thr). We measured Tas1r3 gene expression, transcript size, and T1R3 immunoreactivity in the taste tissue of two inbred mouse strains with different Tas1r3 haplotypes and saccharin preferences. The results of these experiments suggest that the polymorphisms associated with saccharin preference do not act by blocking gene expression, changing alternative splicing, or interfering with protein translation in taste tissue. The amino acid substitution (Ile60Thr) may influence the ability of the protein to form dimers or bind sweeteners. Here, we present data for future studies directed to experimentally confirm the function of these polymorphisms and highlight some of the difficulties of identifying specific DNA sequence variants that underlie quantitative trait loci.

Ventromedial hypothalamic lesions increase pancreatic sensitivity to streptozotocin in rats.

Rats with electroytic lesions of the ventromedial hypothalamus (VMX rats) and sham-operated controls (SHAM rats) were injected with streptozotocin (STZ) at a dose of 50 mg/kg 48 h after the lesions were made. VMX rats were significantly more sensitive to STZ in that over 70% died within 6 wk, shereas none of the SHAM rats dies. When smaller doses of STZ were given to VMX rats (30--35 mg/kg), a large percentage still died, although the survivors appeared equally as diabetic (in terms of hyperglycemia and hypoinsulinemia) as SHAM rats given a larger dose of STZ. At 25 mg/kg, the surviving VMX rats were more hyperglycemic than matched controls. We suggest that the increased B-cell activity known to occur in VMX animals might be the important factor in the increased sensitivity to STZ observed. We speculate that similar variation in pancreatic B-cell response to an environmental injury may be an important determinant of diabetes susceptibility in man.

Sexual dimorphism of hyperglycemia and glucose tolerance in Wistar fatty rats.

Obese and lean male and female Wistar fatty rats were fed a high-sucrose (68% of calories) diet from 5 to 22 wk of age. Obese males, but not obese females, developed hyperglycemia in the fed state and were more glucose intolerant during an intragastric glucose tolerance test than obese females. Lean Wistar fatty rats did not become hyperglycemic on the sucrose diet. Obese males also showed a smaller insulin response during the glucose tolerance test than did obese females. The Wistar fatty rat is a sexually dimorphic model of non-insulin-dependent diabetes mellitus in which the male but not the female obese rats become diabetic. The diabetic condition and impaired glucose tolerance in the obese male Wistar fatty rat may be related to impaired pancreatic insulin release and peripheral insulin resistance.

Peptides and the control of meal size.

There are now a large number of experiments demonstrating that peripheral administration of exogenous cholecystokinin or its synthetic analogue, CCK-8, reduces meal size in a number of species. The peptide interacts with other factors which influence satiety, and treatments thought to be effective in eliciting secretion of cholecystokinin have predictable effects on meal size. Cholecystokinin is effective in the genetically obese Zucker rat, obese rats with lesions of the ventromedial hypothalamus, and subdiaphragmatically vagotomized rats. Somatostatin and bombesin are also reasonable candidates for satiety factors. Intraperitoneal naloxone reduces meal size in rats, and beta-endorphin injected intraventricularly causes an increase in meal size of 50% over 30 minutes. We conclude that cholecystokinin and bombesin may interact in weight regulation and control of meal time food intake.

A novel ATPase on mouse chromosome 7 is a candidate gene for increased body fat.

A region of mouse chromosome 7, just distal to the pink-eyed (p) dilution locus, contains a gene or genes, which we have named p-locus-associated obesity (plo1), affecting body fat. Mice heterozygous for the most distally extending chromosomal deletions of this region have nearly double the body fat of mice when the deletion is inherited maternally as when it is inherited paternally. We have physically mapped the 1-Mb critical region, which lies between the Gabrb3 and Ube3a/Ipw genes, and DNA sequencing has localized a new member of the third subfamily of P-type ATPases to the minimal region specifying the trait. This gene, which we have called p-locus fat-associated ATPase (pfatp) is differentially expressed in human and mouse tissues with predominant expression in the testis and lower levels of expression in adipose tissue and other organs. We propose this ATPase as the prime candidate for the gene at the plo1 locus modulating body fat content in the mouse. The unusual inheritance pattern of this phenotype suggests either genomic imprinting, known to occur in other local genes (Ube3a, Ipw), or an effect of maternal haploinsufficiency during pregnancy or lactation on body fat in the progeny.

Microsatellite marker panels for use in high-throughput genotyping of mouse crosses.

Several microsatellite genotyping panel sets have been developed that are polymorphic between C57BL/6J and CAST/Ei mice, or C57BL/6J and DBA/2J. One set of markers for each strain pair has an intermarker distance of approximately 20 cM, and a second set has an intermarker distance of 5 cM. The 20-cM set contains 105 markers for C57BL/6J x DBA/2J and 108 for C57BL/6J x CAST/Ei, divided into 13 panels. Each 5-cM set includes 350 markers arranged into 45 panels. A panel contains a number of primer pairs whose fluorescently labeled PCR products can be pooled together and separated on one lane of a polyacrylamide gel. The sets are arranged by the size of the PCR product and by the type of fluorescent dye; 5-cM sets are also arranged by chromosomal region. The 20-cM sets are most useful for full-genome scans, the 5-cM sets are useful for full-genome and/or for region-specific chromosome screens. Both sets were proven as useful tools for speed congenic development, quantitative trait loci (QTL) analysis and physical mapping. These panel sets provide a throughput of 1,536-2,304 mouse genotypes daily per one gel-based system. Whole genome scans of one animal require 13 or 48 gel lanes, with 20 cM or 5 cM density, respectively.

Immunocytochemical detection of insulin in rat hypothalamus and its possible uptake from cerebrospinal fluid.

Insulin-like immunoreactivity (IRI) was detected in the rat hypothalamus, particularly in the paraventricular, periventricular, supraoptic, suprachiasmatic, arcuate, and lateral hypothalamic nuclei. The immunostainable IRI was diffusely distributed in comparison to the neuronal concentrations of immunostainable vasopressin in the periventricular nucleus, or of IRI in islet B cells, suggesting that immunostainable IRI in the hypothalamus is not concentrated in neuronal perikarya. To determine if insulin in cerebrospinal fluid (CSF) may be a source of some insulin in brain tissue, [125I]iodoinsulin was stereotaxically injected into a lateral cerebral ventricle, and the uptake of radioactivity into periventricular hypothalamus was localized by both quantitative autoradiography of paraffin-embedded brain sections and by measuring the radioactivity present in microdissected brain regions. In brains that received lateral ventricular injections of labeled insulin, the concentration of radioactivity in the periventricular region of the hypothalamus, as revealed by autoradiographic grains, was significantly greater than that in the periventricular region of brains that received lateral ventricular injections of labeled insulin mixed with an equimolar excess of an unlabeled peptide (insulin, ribonuclease, or both together). The highest levels of radioactivity detected in both autoradiographic and microdissection procedures were in regions nearest to the third ventricle, suggesting that insulin in the lateral ventricles has access to the periventricular neuropile in the hypothalamus. The staining pattern of immunostainable insulin in the hypothalamus along with the distribution of radioactivity after CSF injection of labeled insulin are consistent with the hypothesis that insulin is taken up into brain from the CSF.

Blunted natriuretic response to an acute sodium load in obese hypertensive dogs.

Several studies support the premise that there is a strong relation between obesity and high blood pressure. Although the mechanism for obesity-related hypertension has not yet been fully elucidated, recent studies have suggested that abnormalities in renal sodium handling may be involved in the pathogenesis of obesity-induced hypertension. The purpose of the present study was to determine the effects of an acute saline load on renal excretory function in dogs with obesity-induced hypertension and in normotensive lean dogs. Experiments were performed in two groups of conscious, chronically instrumented dogs. One group of dogs (obese) was fed a high-fat diet for 5-6 weeks, and the other group (lean) ate a normal diet. The body weight of the obese dog group (26.3 +/- 0.7 kg) was 45% higher than the lean dog group (18.1 +/- 0.3 kg). Mean arterial pressure averaged 126 +/- 2 mm Hg in the obese dogs and 100 +/- 1 mm Hg in the lean dogs. The lean dogs had an average heart rate of 104 +/- 7 beats per minute, whereas the obese dogs averaged 134 +/- 8 beats per minute. Plasma renin activity was also significantly higher in the obese dogs. Both groups of dogs were given 135 meq sodium chloride over 60 minutes via an intravenous infusion of isotonic saline. Sodium and water excretion increased significantly in response to the acute saline load. However, the natriuresis and diuresis was markedly attenuated in the obese hypertensive dogs. During the first 40 minutes of saline loading, the increase in sodium and water excretion was 50-70% lower in the obese hypertensive dogs. The results of the present study indicate that obese hypertensive dogs have a reduced capability to excrete an acute sodium load. This abnormality in renal sodium handling may play a role in the pathogenesis of obesity-induced hypertension.

Dietary fat, genetic predisposition, and obesity: lessons from animal models.

This review focuses on animal studies that examine the role of dietary fat in obesity. It is evident from animal experiments that the percentage of energy derived from fat in the diet is positively correlated with body fat content. With few exceptions, obesity is induced by high-fat diets in monkeys, dogs, pigs, hamsters, squirrels, rats, and mice. The mechanisms responsible for this correlation between body fat and dietary fat content are not clear. It has been proposed that a high-fat diet produces hyperphagia, which is solely responsible for the increased body fat content. However, several studies in various rodent models showed that increased body fat content still results when the hyperphagia is prevented. This suggests that some metabolic effects of high-fat diets, independent of hyperphagia, may also be contributing to the obesity induced by high-fat diets. It is also clear from animal studies that genetic factors significantly modulate the body's response to diets high in fat-derived energy. In contrast with the animal studies, studies in humans that have examined the relation between dietary fat content and body fat are inconclusive. The limitations of cross-sectional studies, the lack of controlled feeding trials, and the importance of genetic variation in response explain the absence of conclusive evidence. The lessons learned from animal models point to dietary fat as one potentially important component in the etiology of human obesity. Additional comprehensive studies are warranted to determine the role of dietary fat in the etiology of human obesity.

Genetics of obesity in humans and animal models.

Human obesity has a significant genetic component which contributes to the risk for this disorder. The application of molecular genetic techniques to identify these genes using a variety of approaches, including information from animal models, will help clarify the role of specific genes in the etiology of human obesity. Identification of these genetic mechanisms is likely to lead to new approaches, both pharmacologic and nonpharmacologic, for the prevention and treatment of this disease.

Bombesin reduces food intake of normal and hypothalamically obese rats and lowers body weight when given chronically.

Bombesin is a peptide hormone reported to reduce meal size when administered in rats. In the first experiment, synthetic bombesin was injected subcutaneously into normal rats and obese rats with lesions of the ventromedial hypothalamus just prior to the presentation of food. A dose-dependent suppression of meal size occurred for both groups, showing that the peptide has this action in obese as well as normal animals. In a second experiment, a conditioned taste aversion was not formed with a dose of bombesin which suppressed meal size by approximately 50% while the animals did develop an aversion with a dose of LiCl reported to reduce meal size equivalently. In a third experiment, rats were placed on a feeding schedule where they received three 30-min meals each day. After weights had stabilized under this paradigm, bombesin was administered just prior to each meal for six days. The bombesin caused a consistent suppression of meal size when the animals were allowed 30-min meals such that the rats lost weight over the six-day period. When this experiment was repeated with 60-min meals apparent tolerance developed to these actions of bombesin.

The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.

The androgen-dependent regulation for the gene encoding the kidney androgen regulated protein (Kap) was examined in transgenic mice expressing luciferase (luc) under the control of the murine Kap promoter. Biophotonic imaging was used to visualize luciferase expression from the kidneys and various organs that was confirmed using luminometer assays. Kap-luc expression was observed at high levels in kidneys, epididymides, testes, and seminal vesicles in male mice, and in kidneys, ovaries, and uterus in female mice. Kap-luc expression was modulated by androgen and anti-androgen treatment in both male and female mice. Male mice were treated daily with the anti-androgenic compounds, cyproterone acetate (50 and 100 mg/kg/day) and flutamide (50 and 100 mg/kg/day), or vehicle for 16 days. Endpoints evaluated included in vivo biophotonic imaging, body weights, organ weights (liver, kidney, testes, epididymides, preputial gland, and seminal vesicles), protein luciferase assays and Western blot analysis. Biophotonic imaging was used to follow Kap-luc expression from each animal throughout the experiment using a sensitive imaging system. These imaging results correlated well with Western blot analysis and traditional endpoints of body and organ weights. Following treatment with anti-androgens, the luciferase signal was found to significantly decrease in the intact male mouse using in vivo biophotonic imaging and correlated with measurements of luciferase activity in homogenized organ extracts. The decrease in epididymal and seminal vesicle weight confirmed the action of the anti-androgens. In vivo imaging documented significant changes in luciferase expression within the first few days of the experiment indicative of the anti-androgenic activity of the drugs. Testosterone treatment significantly increased the Kap-luc bioluminescent signal in female mice. This increased luciferase induction was shown to be inhibited by coadministration of cyproterone (100 mg/kg/day). Our results indicate that biophotonic imaging may provide a useful approach for noninvasively tracking the effects of endocrine disruptors in specific tissues.

A microcomputer-based data acquisition system for continuous recording of feeding and drinking by rats.

A monitoring system to continuously record the daily pattern of drinking and eating of rats is described. This system, based on a North Star microcomputer, can record the amount of food ingested with a temporal resolution of +/- 1.0 second and quantitative accuracy within +/- 5%. Drinking behavior is detected using a drinkometer which also has a temporal resolution of +/- 1.0 second. Data are analyzed by computer to determine absolute amounts of consumption and patterns of intake. The patterns of feeding and drinking recorded by this system are similar to those observed using other monitoring devices.

Infusion of cholecystokinin between meals into free-feeding rats fails to prolong the intermeal interval.

The sulfated octapeptide of cholecystokinin (CCK-8) was infused intraperitoneally into 7 free-feeding male Sprague Dawley rats over a 6-day period. Infusions were given near the end of each free-feeding meal (1.87 microgram/meal/rat), and also during the intermeal interval in gradually increasing doses (0.10-0.63 microgram/5 min/rat). Food intake was continuously monitored and the infusions were controlled by microcomputer. Meal patterns, total food intake, and body weights during drug infusion were compared with data collected during a baseline period when only saline was infused. Meal-contingent CCK-8 infusion produced a significant 29.9% decrease in meal size which persisted throughout the drug-infusion period. Intermeal infusion of CCK-8 failed to prolong the intermeal interval (IMI) but it did initially prevent the compensatory decrease in IMI and increased feeding frequency expected after meal size was reduced. By the last day of drug infusion, total daily food intake recovered to baseline levels due to increased feeding frequency. Body weight was only transiently reduced by CCK-8 infusion. These findings show that tolerance does not develop to the action of CCK-8 to suppress meal size, and the administration of exogenous CCK-8 to free-feeding rats does not persistently prolong the intermeal interval.

Early-onset repeated dieting reduces food intake and body weight but not adiposity in dietary-obese female rats.

As dieting behavior and attempts at weight loss are becoming increasingly common in adolescent girls, we wished to determine whether early-onset repeated dieting influenced the development of obesity and its metabolic correlates. Female rats were fed a high-fat diet and subjected to six cycles of dieting and regain, beginning in the peripubertal period. Although dieted rats weighted less than nondieted high-fat fed controls at the completion of the sixth cycle, body composition analysis revealed that the two groups were equally obese. Cumulative caloric intake was less in dieted rats, suggesting that the pattern of consumption promoted by dieting helped to establish the obesity. Resting metabolic rate did not differ between the two groups. These data suggest that although early-onset repeated dieting may result in reduced body weight, the eventual level of adiposity may be unknowingly elevated, potentially leading to long-term health risks.

Reliability and validity of a macronutrient self-selection paradigm and a food preference questionnaire.

Our laboratory has developed a macronutrient self-selection paradigm (MSSP) designed to vary fat content significantly and systematically with sugar, complex carbohydrates, and protein content in a battery of foods in which fat is commonly consumed in the American diet. We have also developed a food preference questionnaire (FPQ) according to an identical design but using a list of foods mutually exclusive of those presented for selection and intake in the MSSP. Men were tested twice on both instruments, with a 4-week interval between tests. It was determined that the MSSP has strong test-retest reliability for overall fat (r = 0.91) and other macronutrient intake and total caloric intake. In addition, hunger and fullness ratings were reproducible, and fat preferences (r = 0.99) and hedonic responses to foods listed on the FPQ were highly consistent across trials. This study also demonstrated that the MSSP is a valid instrument with respect to the men's reports of habitual intake of fat (r = 0.80) and total carbohydrates on the Block food questionnaire (FQ). In addition, men's fat preferences on the FPQ were validated with respect to overall fat (r = 0.86) and total caloric intake in the MSSP and fat intake (r = 0.83) reported on the Block FQ. The MSSP also has the capability to detect a wide range of fat intake (3.06-50.35% among the present subjects), indicating that this instrument can identify individuals who differ markedly in fat intake or could detect changes in fat preference within subjects. In addition, this paradigm detected a large range of sugar and total caloric intake. It is anticipated that the use of these laboratory tools can enhance our understanding of the relationship between dietary fat intake and obesity.

Race and physical activity: cardiovascular and renal response to sodium loading.

Race and level of physical activity may be important factors affecting cardiovascular responsiveness to acute sodium intake. We examined the effects of a 4-day sodium loading procedure on heart rate (HR), systolic (SBP) and diastolic (DBP) blood pressure, 24-hour electrolyte excretion, and extracellular fluid volume (ECF) in African-American (n = 16) and Caucasian (n = 16) adult males. All subjects were normotensive with either moderately high (> or = 3 day/wk; > or = 20 min/day) or low (< or = 3 day/wk; < or = 20 min/day) physical activity levels. Subjects ingested either placebo or NaCl (0.2 g/kg/day) during two 4-day periods. Sodium loading increased SBP and decreased HR in all high physical activity subjects but not in low physical activity subjects (p < 0.05). Na+ excretion did not differ among groups, although urine volume and K+ excretion were lower, and Na+/K+ excretion ratio was higher in African-American compared to Caucasian subjects (p < 0.05). ECF, as measured by NaBr dilution, was greater in high physical activity subjects compared to low physical activity subjects (p < 0.05). These data suggest that high physical activity levels do not attenuate but rather exaggerate SBP response to a sodium load.

Changes in body composition with conjugated linoleic acid.

Conjugated linoleic acid has been shown to reduce body fat accumulation in several animal models. We have conducted several studies in AKR/J mice showing that CLA reduces body fat accumulation whether animals are fed a high-fat or low-fat diet, with no effect on food intake. One mechanism by which CLA reduces body fat is by increased energy expenditure, which is observed within one week of CLA feeding and is sustained for at least six weeks. The increased energy expenditure is sufficient to account for the decreased fat accumulation. Increased uncoupling protein gene expression does not appear to be involved in the increased energy expenditure. We have observed increased fat oxidation but no decrease in de novo fat biosynthesis with CLA feeding. We have also observed increased liver weights and plasma insulin levels with higher doses of CLA. In all of the studies we have conducted to date we have used a CLA preparation that contains several isomers, primarily c9,t11 and t10,c12. It was assumed that the active form was c9,t11, as CLA was identified as an anticarcinogenic compound from cooked beef, of which the c9,t11 form accounts for 60% to 80% of the CLA. Most of the studies conducted so far must be repeated using the purified isomers in order to determine which isomers are responsible for each of the identified actions of CLA.

Validation of a radiotelemetry system for continuous blood pressure and heart rate monitoring in dogs.

A blood pressure telemetry system with catheter placement in the femoral artery was evaluated over a 119-day period in eight mongrel dogs. Every 3 weeks, the pressures recorded by telemetry were compared with direct, simultaneously recorded blood pressures measured from a femoral artery catheter implanted on the contralateral side. One telemetry device failed within 1 week of implant and was replaced before the beginning of data collection. Continuous blood pressure measures were accurate for a minimum of 17 weeks in all eight dogs. Acute changes in blood pressure and heart rate were not evaluated. Validation by simultaneous recording with a physiological monitor indicated that there was no drift in the telemetry blood pressure or heart rate during the 17-week period. Heart rates recorded by telemetry and a physiological monitor were identical, as were mean arterial pressures. The systolic pressure measured by the physiological monitor was consistently higher than that measured by telemetry. This may have been due to differences in the compliance of the catheter material in the two systems. Two devices failed within approximately 6 months of implant, apparently because of exhaustion of the battery. These findings indicate that this telemetry device can be used for periods of up to 17 weeks without appreciable drift or attenuation of the blood pressure signal. The results also describe a validation procedure for monitoring the accuracy of the telemetry system over extended periods of use. We conclude that this telemetry device can be used for the long-term assessment of blood pressure and heart rate in the dog.