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Department of Nutrition, University of Tennessee, Knoxville, TN 37996;
* University of Tennessee College of Veterinary Medicine, Knoxville, TN 37996;
Office of Biological and Environmental Research and
** Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; and
University of California, Los Angeles, CA 90095
3To whom correspondence and reprint requests should be addressed. E-mail: mdhar{at}utk.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mouse chromosome 7 • Atp10c, type IV P-type ATPase • insulin resistance
Human obesity and its related disorders including type 2 diabetes, atherosclerosis, nonalcoholic fatty liver disease (NAFLD),4 and nonalcoholic steatohepatitis are influenced by genetic as well as environmental factors (nutritional and/or hormonal) that can more easily be studied in murine models and then translated into human homologs and phenotypes (1–4). Appropriate mouse models offer many advantages including the availability of inbred strains and genetically altered resources (5). Several polygenic rodent models of obesity and type 2 diabetes have been developed; the underlying genetic factors in these models have been studied by quantitative trait locus (QTL) mapping analysis, and several QTL associated with body weight, body fat, and type 2 diabetes phenotypes have been identified (6,7). These QTL are thought to more closely mimic the presumed polygenic inheritance of obesity and type 2 diabetes in humans than do the single-gene rodent obesity models.
At Oak Ridge National Laboratory (ORNL), we generated and maintain a large collection of mouse stocks that carry radiation-induced chromosomal deletions at the pink-eyed dilution (p) locus on mouse chromosome 7 (MMU-7) (8–10). By assessing the body fat of two of the distally extending heterozygous deletions, p23DFiOD and p30PUb, we showed that the p-linked-obesity-locus 1 (plo 1) region, on proximal MMU-7 contains gene(s) affecting body fat in these mice (11). p30PUb is the longest distally extending p deletion, extending distal to p, between ubiquitin protein ligase E3A (Ube3a) and the imprinted gene in the Prader-Willi syndrome region (Ipw). p23DFiOD, on the other hand is shorter and its distal breakpoint maps to the chromosomal region between the genes 
-aminobutyric acid receptor (Gabrb3) and Ube3a (8). Interspecific backcross analysis suggests that p30PUb carries a deletion of <1 cM (9,10). Mice heterozygous for either of the two p deletions have nearly twice the body fat of mice when the deletion is inherited maternally as when it is inherited paternally. Plo 1 overlaps with the critical region of MMU-7 linked to QTL for body weight/fat and type 2 diabetes phenotypes (11,12).
Data suggest that the phenotype of the plo 1 locus may be due to haploinsufficiency for a novel type IV P-type ATPase, Atp10c, mapping between Gabrb3 and Ube3a on MMU-7. Atp10c, a novel phospholipid translocase is the only transcript mapping to this critical region of <1cM. Atp10c has 21 exons; in p23DFiOD heterozygotes, the first two exons including the 5' promoter region of Atp10c are deleted, whereas in the p30PUb heterozygotes, the complete gene of 21 exons and the 5' and 3' flanking regions are deleted (Dhar, M., unpublished data).
We and two other groups have also identified and characterized the sequence of the human ortholog, ATP10C on human chromosome 15q12 (12–14). ATP10C maps to the Angelman Syndrome (AS) critical region. AS arises from the loss of maternal gene expression in human chromosome 15q11-q13, and is most commonly caused by a 4-Mb maternally inherited deletion, with at least 4 additional genetic mechanisms. Most clinical features of AS are neurobehavioral, arising from loss of function of the UBE3A gene that maps 
200 kb proximal to ATP10C (13,14). Human geneticists have identified a class of AS patients with the additional phenotype of an increased BMI (15). Several mouse models of AS including p30PUb also show a mild neurobehavioral phenotype with an adult-onset obesity appearing at 5–6 mo of age (16). There is also a uniparental disomy mouse model of AS with late-onset obesity reported by Cattanach et al., (17). Atp10c/ATP10C is maternally expressed (paternally imprinted) in both mouse and human brains (13,14,18). As described above, our data show that Atp10c is functionally imprinted such that only the maternal allele is expressed and that loss of the maternal allele causes obesity (11). These studies thus suggest an association of Atp10c/ATP10C with some forms of obesity and lipid metabolism disorders in both mice and humans.
In the present report, we describe results of studies initiated to characterize the obesity phenotypes associated with p30PUb mutants and to assess the overall effect of the heterozygous deletion of Atp10c on obesity-related metabolic abnormalities. Experiments to evaluate the response of the mice carrying the heterozygous deletion of Atp10c to the fat content of their diets were undertaken.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weight and adiposity index.
Total body weights were measured every 2 wk and body fat measurements were carried out as described earlier (11). When the mice were 12–16 wk old, food was withdrawn at 1600 h and the mice were killed the next day between 900 and 1200 h. An adiposity index (AI) was calculated for each mouse as the ratio of the sum of the fat pads divided by the weight of the eviscerated carcass (minus the dissected adipose depots). The major organs, liver, spleen, and kidneys were also weighed and the data recorded.
Glucose (GTT) and insulin (ITT) tolerance tests.
GTT and ITT were carried out on 8- to 12-wk-old conscious mice fed the high-fat diets for 4 and 8 wk. For GTT, mice were food deprived for 14–16 h and then a 200 g/L glucose solution was administered i.p. Tail blood glucose was measured at time 0 (before glucose injection) and at 30, 60, 90, and 120 min after injection with the glucometer (One Touch Ultra, Lifescan).
For ITT, mice were food deprived for 2 h and then 0.375 U/kg of Humulin R (Eli Lilly) was injected i.p. Tail blood glucose was measured at time 0 (before insulin injection) and at 30, 45, 60, 90, and 120 min after injection with the glucometer.
Plasma glucose was measured after mice were food deprived for 2 h as the 0 time values in ITT and after being food deprived for 16–18 h as the 0 time values in GTT. The two sets of values were compared.
Plasma insulin, leptin, and lipids. Blood was sampled from the tail of the mice (food-deprived for 16–18 h) after they consumed the 45% fat diet for 4 and 12 wk. Plasma total cholesterol (TC), HDL cholesterol, unesterified cholesterol (UC), and triacylglycerol (TG) concentrations were measured by enzymatic colorimetric assays as described (19). The sum of VLDL and LDL cholesterol was obtained as the difference between TC and HDL cholesterol. Cholesteryl ester (CE) was calculated as the difference between TC and UC.
For plasma insulin and leptin, blood was collected by heart puncture from 12-wk-old mice when they were killed for the body composition analyses described above. Plasma insulin was measured using a rat insulin RIA kit with rat insulin as the standard; leptin was measured using a mouse leptin RIA kit (Linco).
Histological examination of the heart, liver, and pancreas. The heart, liver, and pancreas were fixed in 10% buffered formalin. The hearts were transversely sectioned proximal and distal to the atria. The tissues were routinely processed by paraffin embedding, sectioned (5–8 µm), and stained with hematoxylin and eosin. Sections of the liver were also stained with periodic acid-Schiff (PAS) and PAS diastase for evaluation of cytoplasmic carbohydrate deposition. Routine light microscopy was used to examine the tissues.
Statistical methods. For all of the analyses, female and male mice were tested separately. Data were grouped according to the sex, the diet, and the genotype of the mice used. Statistical analysis was performed using unpaired t tests comparing mutants and controls in each group to determine the effects of the genotype on the variables analyzed (Statview Version 4.5). Differences with P < 0.05 were considered significant. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2–2.5 times the actual weights of the individual fat pads and the AIs and 1–1.5 times the weights of the eviscerated carcasses of both the control lean pxpx mice and the p30PUb pats (Table 1, Fig. 2). However, in the male p30PUb mats, the weights of the individual fat pads, the corresponding AIs, and the weights of the eviscerated carcasses were not higher. Male p30PUb mats fed the 10% fat diet for 8 wk and killed for body fat analysis had 24.5% greater body weight, 1.5–2.0 times the actual weights of the individual fat pads and the Ais, and 1–1.5 times the weights of the eviscerated carcasses, than the p30PUb controls (Table 1).
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Plasma insulin and leptin.
Compared with the controls, both the female (P = 0.004) and male (P = 0.02) p30PUb mats were hyperglycemic after being food deprived for only 2 h, suggesting an abnormal glucose homeostasis. Female p30PUb mats had 2.2 times higher insulin (0.229 ± 0.02 pmol/L, P = 0.04) and 3.0 times higher leptin (26.3 ± 5.70 µg/L, P = 0.01) levels than the lean control mice (insulin = 0.102 ± 0.01 pmol/L, leptin = 8.68 ± 2.90 µg/L). Male p30PUb mats on the other hand, did not differ from controls in their blood insulin and leptin levels. However, the blood insulin and leptin levels of the male p30PUb mats (insulin = 0.379 ± 0.02 pmol/L, leptin = 28.7 ± 10.90 µg/L) that consumed the 10% fat diet were significantly higher than those of the corresponding pxpx controls (insulin = 0.067 ± 0.01 pmol/L, leptin = 2.2 ± 0.75 µg/L).
Glucose tolerance and insulin resistance. The obese p30PUb mats had a significantly higher hyperglycemic response to a given dose of glucose and insulin than either the px/px or p30PUb pats, suggesting that insulin resistance occurs only with maternal inheritance (Figs. 3, 4). This effect was observed after 4 wk of consuming the 45% fat diet and persisted at 8 wk. Similar glucose intolerance and insulin resistance were also observed in the male p30PUb mats fed the 10% fat diet for the same time (Fig. 5).
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Maternal vs. paternal inheritance of p30PUb. Increased adiposity, impaired glucose tolerance, insulin resistance, and fatty liver disease are observed in p30PUb mats but not p30PUb pats. The body weight and adiposity of p30PUb pats were similar to that of the px /px control mice (Figs. 1Aand 2A). Furthermore, after consuming the 45% fat diet for 4 wk, p30PUb pats had normal responses to glucose and insulin tolerance testing (Fig. 3). Histopathological analysis of their livers did not show substantial lipid deposition (Fig. 6C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Glucose homeostasis is tightly controlled by a balance between glucose absorption from the intestine, production by the liver, and uptake and metabolism by the muscle and fat. Thus, insulin resistance may arise due to a defect in insulin signaling in one or all of the three target tissues, i.e., liver, skeletal muscle, and adipose tissue, in which they become less responsive to insulin (20–23). The defect may lie in any of the steps involving insulin secretion and the final glucose disposal or uptake in the peripheral tissues.
The data presented here suggest that the p30PUb heterozygote mutants represent a novel genetic, diet-induced model of insulin resistance characterized by hyperinsulinemia, hyperglycemia, hyperlipidemia, and obesity in association with glucose intolerance. These studies indicated some sex-specific differences. Body weights of female p30PUb mats increased significantly after consumption of the 45% fat diet for 4 wk. Their fat pads and the eviscerated carcasses were also significantly heavier when they were killed at 12 wk compared with the p30PUb pats. Male p30PUb mats fed the 45% fat diet had significantly increased body weights but not adipose tissue or eviscerated carcass weights compared with their control littermates. Although adiposity and lean carcass weight did not increase, male p30PUb mats fed the 45% fat diet tended to have higher values for these measurements compared with controls (P = 0.192–0.122). We hypothesize that this increase in both the control and the mutant male mice was due in part to the effect of the 45% fat diet on this background strain because these measurements were significantly higher when they were fed a 10% fat diet. The mutants are maintained on a C57BL/6J background, in which obesity is diet induced (24). Male C57BL/6J mice tend to be heavier than their female littermates (25). At this age, the effects on the adipose tissue deposition and lean carcass weight from the 45% fat and the mutation are indistinguishable. Potentially, if the male mice were fed the 45% fat diet for a longer period of time, the effect due to the mutation might significantly alter adiposity as seen in the female mice. Similarly, the blood insulin and leptin levels in male p30PUb mats fed the 45% fat diet did not differ from the control mice. However, there were significant differences in the blood insulin and leptin levels in male p30PUb mats fed the 10% fat diet. This can also be attributed to the diet-induced increase in the adipose tissue by the background and the sex of the control mice. It is noteworthy that even though mutant male mice fed a 45% fat diet did not differ from controls in adiposity, glucose and insulin tolerance and hypertriglyceridemia were affected. Even though there are sex differences, the conclusion that maternal inheritance of the deletion of a region of MMU-7 associated with QTL for body weight and type 2 diabetes phenotypes contributes to the above phenotype is unquestionable.
Obesity is strongly associated with insulin resistance manifested by defects in insulin secretion and/or action. The results of the GTT and ITT confirm that the mutant mice are insulin resistant compared with the control littermates. This effect occurred in mice fed the 10 and 45% fat diets; therefore, it is due to the mutation and not the fat content of the diet. The hyperlipidemia in the obese mutant mice was secondary to the development of insulin resistance and obesity; it occurred only when the obese mutants were fed the 45% fat diet for a period of at least 3 mo. This effect was not seen when the mutants were fed the same diet for only 1 mo. Similarly, histopathological analysis showed mild fat infiltration within hepatocytes at 8 wk, which progressed to severe fatty liver disease by 12 wk.
The weight gain and the corresponding increase in adiposity in p30PUb pats did not differ from controls. These data along with the GTT and ITT data show that the phenotypes in p30PUb heterozygotes may be due to the maternal inheritance of the p30PUb deletion.
By positional cloning and molecular analyses, we showed that a murine type IV P-type ATPase, Atp10c and its human ortholog ATP10C, encoding a putative phospholipid translocase, is a prime candidate affecting body fat and/or lipid metabolism in mice and humans (11,12). As reported earlier, the p30PUb deletion encompasses the chromosomal region containing three genes, Gabrb3, Atp10c, and Ube3a, whereas the plo 1 region, critical to the changes in the body weight, contains Atp10c and Ube3a only (8,11). Because Ube3a transgenic mice are not obese, it is reasonable to expect that it is not the candidate for obesity or altered glucose and lipid metabolism disorders associated with p30PUb mats (26,27). Thus, the maternal inheritance of Atp10c in the heterozygous deletion mutant mice may be responsible for the altered glucose tolerance, increased adiposity, and NAFLD after consumption of the diet containing 10 or 45% fat for only 8–16 wk. The heterozygous deletion of the complete Atp10c gene leads to defects in glucose homeostasis, clearly affecting the liver and adipose tissue. Whether this is due solely to Atp10c and whether the liver and adipose tissue are the only two tissues affected must be established.
Atp10c is a putative phospholipid translocase and it is conceivable that Atp10c plays a role in lipid trafficking and maintenance of the phospholipid asymmetry and fluidity of the plasma membrane; thus, loss of Atp10c function by maternal deletion (coupled with paternal silencing) upsets the normal membrane milieu and perturbs glucose and lipid metabolism. The exact biological role, however, is not known. It is important to identify the primary biochemical defects and then determine how these subsequently alter metabolism in the whole animal. In the present study, we established the validity of our mice as a novel model for obesity, type 2 diabetes, and NAFLD. Specific metabolic tests to identify metabolic pathway(s) and the target tissue(s) associated with Atp10c will be initiated. These experiments will give us important information about glucose homeostasis in skeletal muscle, adipose tissue, and liver associated with a heterozygous deletion of a novel ATPase in our mouse model.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK53393–01.
4 Abbreviations used: AI, adiposity index; AS, Angelman syndrome; CE, cholesteryl ester; %E, % energy; Gabrb3, -aminobutyric acid (GABA-A) receptor, subunit ß 3; GTT, glucose tolerance test; Ipw, imprinted gene in the Prader-Willi syndrome region; ITT, insulin tolerance test; mats, mutant pxpl heterozygotes which inherit the deletion p30PUb maternally; MMU-7, mouse chromosome 7; NAFLD, nonalcoholic fatty liver disease; ORNL, Oak Ridge National Laboratory; PAS, periodic acid-Schiff; pats, mutant pxpl heterozygotes which inherit the deletion p30PUb paternally; QTL, quantitative trait loci; TC, total cholesterol; TG, triglyceride; Ube3a, ubiquitin protein ligase E3A; UC, unesterified cholesterol.
Manuscript received 17 August 2003. Initial review completed 8 September 2003. Revision accepted 14 December 2003.
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