Reciprocal Hemizygosity Analysis of Mouse Hepatic Lipase Reveals Influence on Obesity

Objectives: We previously demonstrated coincident quantitative trait loci (QTLs) for percentage body fat, plasma hepatic lipase (HL) activity, and plasma cholesterol on mouse chromosome 7. In the present study, we investigated whether hepatic lipase (Lipc) is an obesity gene, whether Lipc interacts with an unknown gene on chromosome 7, and how HL activity is linked to the chromosome 7 locus. Research Methods and Procedures: BSB mice are a model of complex obesity due to interactions among genes from C57BL/6J and Mus spretus (SPRET) in (C57BL/6J × SPRET) × C57BL/6J backcross mice. Five crosses tested the impact on obesity of combinations of inactive (knockout) and wild‐type Lipc alleles from C57BL/6J or SPRET in a reciprocal hemizygosity analysis. Results: The combined data from this allelic series suggest that Lipc alleles, and not alleles from a gene linked to Lipc, influence obesity. No interaction between Lipc and chromosome 7 was demonstrated. We confirmed the chromosome 7 QTLs for obesity, HL activity, and cholesterol. Because obesity and HL activity are not consistently associated in the BSB model, linkage of HL activity to chromosome 7 is not secondary to obesity per se. We also report, for the first time to our knowledge, a QTL in mammals for food intake. Discussion: This use of reciprocal hemizygosity analysis in mammals, which, to our knowledge, is the first reported, reveals its power to detect previously unknown effects of Lipc on obesity.     

A novel mouse Chromosome 2 congenic strain with obesity phenotypes

Linkage studies have identified many chromosomal regions containing obesity genes in mice. However, only a few of these quantitative trait loci (QTLs) have been used to guide the production of congenic mouse strains that retain obesity phenotypes. We seek to identify chromosomal regions containing obesity genes in the BSB model of spontaneous obesity because the BSB model is a multigenic obesity model. Previous studies identified QTLs on Chromosomes (Chrs) 2, 6, 7,12, and 15. BSB mice are made by backcross of lean C57BL/6J × Mus spretus. F₁s were backcrossed to C57BL/6J mice to produce BSB progeny. We have constructed a new BSB cross and produced congenic mice with obesity phenotypes by marker-directed selection called B6.S–D2Mit194–D2Mit311. We found a highly significant QTL for percentage body lipid on Chr 2 just proximal to the Agouti locus. Chr 2 congenics were constructed to determine whether the main effects would be detectable. We observed highly significant linkage of the Chr 2 congenic containing Agouti and containing markers distal to D2Mit311 and proximal to D2Mit194. Thus, this congenic contains approximately 14.6 cM or 30 Mb (about 1.1% of the spretus mouse genome) and several hundred genes. The obesity phenotype of the QTL is retained in the congenic. The congenic can now be used to model the genetic and physiological basis for a relatively simple, perhaps monogenic, obesity.     

Sensitivity to dietary obesity linked to a locus on Chromosome 15 in a CAST/Ei × C57BL/6J F2 intercross

Details of a new model of diet-dependent polygenic obesity are presented. CAST/Ei (Mus m. castaneus) mice remain lean after 12 weeks on a high-fat (32 kcal% fat) diet, while C57BL/6J mice become obese. The genes responsible for the obesity segregate in an F₂ population derived from an intercross between CAST/Ei and C57BL/6J mice. Quantitative trait analysis, with simple sequence length polymorphisms (SSLPs) at loci previously linked to rodent obesities, identified a quantitative trait locus (QTL) on Chromosome (Chr) 15, accounting for approximately 9% of the variance in adiposity and 14% of the variance in mesenteric depot size. This locus appears to be at the same location as the dietary obesity-3 (Do3) locus controlling body fat content, which was previously identified in an F₂ population derived from an SWR/J × AKR/J cross. This is also at the same location as the multigenic obesity-4 (Mob4) locus found in BSB mice, which display spontaneous polygenic obesity. Suggestive linkage also was found at loci close to the single gene mutations A ^y on Chr 2 and tub on Chr 7. Received 15 January 1996 / Accepted 12 May 1996     

Epistatic interaction between two nonstructural loci on chromosomes 7 and 3 influences hepatic lipase activity in BSB mice

BSB mice exhibit a wide range of obesity despite being produced by a backcross of lean C57BL/6J (B) x lean Mus spretus (SPRET/Pt) F1 animals x B. Previous linkage studies identified a quantitative trait locus (QTL) on mouse chromosome 7 with coincident peaks for hepatic lipase activity, obesity, and plasma cholesterol. However, these mice were not analyzed for gene x gene epistasis. Hepatic lipase activity is correlated with obesity and plasma cholesterol levels. In this study, we identified QTLs for plasma hepatic lipase activity with three statistical mapping methods: maximum likelihood interval mapping, Bayesian nonepistatic mapping, and Bayesian epistatic mapping. Bayesian epistatic mapping detected not only the QTL on chromosome 7 but also an additional QTL on chromosome 3, which has a weak main effect but a strong interaction with chromosome 7. SPRET/Pt alleles of the QTL on each chromosome promote hepatic lipase activity. The proportion of phenotypic variance explained by the epistatic effect is higher than that explained by the main effect of the QTL on chromosome 7.     

Gnpat does not play an essential role in systemic iron homeostasis in murine model

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat^−/−), Gnpat/Hfe double-knockout (Gnpat^−/−Hfe^−/− or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpat^fl/fl;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat^+/+) and Gnpat^−/− mice, between Hfe^−/− and DKO mice, or between Gnpat^fl/fl and Gnpat^fl/fl;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat^−/− and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe^−/− mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe^−/− mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe^−/− mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.     

BSB: A New Mouse Model of Multigenic Obesity

We report here a new mouse model of multigenic obesity. Backcross progeny ((C57BL/6J x Mus spretus)F1 x C57BL/6J), designated as BSB mice, range from 1% to 50% body fat. Since both parental strains are relatively lean, the wide range of the phenotype in the BSB mice indicates the involvement of multiple genes to produce obesity. Obesity in BSB mice results from increases in both intraabdominal and subcutaneous fat and is associated with hyperinsulinemia, hyperglycemia, and hyperlipidemia. Female and male BSB mice do not differ in the degree of obesity obtained. Stimulated plasma corticosterone levels are reduced in obese male and female mice. The development of appropriate genetic markers and statistical methods have made it feasible to analyze quantitative polygenic traits in animal models by employing F2 or backcross progeny. Thus, this BSB model is uniquely suited to the genetic analysis of the multifactorial quantitative trait of obesity and its associated phenotypes. (OBESITY RESEARCH 1993;1:271–280)     

Extensive changes in innate immune gene expression in obese Göttingen minipigs do not lead to changes in concentrations of circulating cytokines and acute phase proteins

The usefulness of Göttingen minipigs as models for obesity and obesity‐related pathologies is well established. The low‐grade inflammation associated with obesity involves a range of innate immune factors; however, to our knowledge, the impact of obesity on innate immune factor expression has not been studied in Göttingen minipigs. Therefore, we studied the expression of innate immune genes in liver and adipose tissues as well as serum concentrations of cytokines and acute phase proteins in obese vs. lean Göttingen minipigs. In the liver, of 35 investigated genes, the expression of nine was significantly different in obese pigs (three up‐regulated, six down‐regulated). Of 33 genes in adipose tissues, obesity was associated with changed expression of 12 genes in the visceral adipose tissue (VAT) (three up‐regulated), 11 in the abdominal retroperitoneal adipose tissue (RPAT) (seven of these up‐regulated) and eight in the subcutaneous adipose tissue (SAT) from the neck (five of which were up‐regulated). Obesity‐associated expression changes were observed for three genes in all adipose tissues, namely chemokine (C‐C motif) ligand 3‐like 1 (up‐regulated), CD200 molecule (down‐regulated) and interleukin 1 receptor antagonist (up‐regulated) with interleukin 1 receptor antagonist being the most highly regulated gene in both VAT and RPAT. Looking at patterns of expression across the three types of adipose tissues, obesity was associated with an increased number of acute phase proteins differentially expressed between adipose tissues and a decreased tissue‐specific expression of cytokines and chemokines. In contrast to obese humans, no changes in serum concentrations of haptoglobin, C‐reactive protein, serum amyloid A, tumor necrosis factor‐α and interleukin 6 were found in obese Göttingen minipigs.     

Role of adiponectin and inflammation in insulin resistance of Mc3r and Mc4r knockout mice

Objective: To investigate the involvement of hypoadiponectinemia and inflammation in coupling obesity to insulin resistance in melanocortin‐3 receptor and melanocortin‐4 receptor knockout (KO) mice (Mc3/4rKO). Research Methods and Procedures: Sera and tissue were collected from 6‐month‐old Mc3rKO, Mc4rKO, and wild‐type C57BL6J litter mates maintained on low‐fat diet or exposed to high‐fat diet (HFD) for 1 or 3 months. Inflammation was assessed by both real‐time polymerase chain reaction analysis of macrophage‐specific gene expression and immunohistochemistry. Results: Mc4rKO exhibited hypoadiponectinemia, exacerbated by HFD and obesity, previously reported in murine models of obesity. Mc4r deficiency was also associated with high levels of macrophage infiltration of adipose tissue, again exacerbated by HFD. In contrast, Mc3rKO exhibited normal serum adiponectin levels, irrespective of diet or obesity, and a delayed inflammatory response to HFD relative to Mc4rKO. Discussion: Our findings suggest that severe insulin resistance of Mc4rKO fed a HFD, as reported in other models of obesity such as leptin‐deficient (Lep^ob/Lep^ob) and KK‐A^y mice, is linked to reduced serum adiponectin and high levels of inflammation in adipose tissue. Conversely, maintenance of normal serum adiponectin may be a factor in the relatively mild insulin‐resistant phenotype of severely obese Mc3rKO. Mc3rKO are, thus, a unique mouse model where obesity is not associated with reduced serum adiponectin levels. A delay in macrophage infiltration of adipose tissue of Mc3rKO during exposure to HFD may also be a factor contributing to the mild insulin resistance in this model.     

Inflammation Correlates With Markers of T‐Cell Subsets Including Regulatory T Cells in Adipose Tissue From Obese Patients

Accumulation of cytotoxic and T‐helper (T_h)1 cells together with a loss of regulatory T cells in gonadal adipose tissue was recently shown to contribute to obesity‐induced adipose tissue inflammation and insulin resistance in mice. Human data on T‐cell populations in obese adipose tissue and their potential functional relevance are very limited. We aimed to investigate abundance and proportion of T‐lymphocyte sub‐populations in human adipose tissue in obesity and potential correlations with anthropometric data, insulin resistance, and systemic and adipose tissue inflammation. Therefore, we analyzed expression of marker genes specific for pan‐T cells and T‐cell subsets in visceral and subcutaneous adipose tissue from highly obese patients (BMI >40 kg/m², n = 20) and lean to overweight control subjects matched for age and sex (BMI <30 kg/m²; n = 20). All T‐cell markers were significantly upregulated in obese adipose tissue and correlated with adipose tissue inflammation. Proportions of cytotoxic T cells and T_h1 cells were unchanged, whereas those of regulatory T cells and T_h2 were increased in visceral adipose tissue from obese compared to control subjects. Systemic and adipose tissue inflammation positively correlated with the visceral adipose abundance of cytotoxic T cells and T_h1 cells but also regulatory T cells within the obese group. Therefore, this study confirms a potential role of T cells in human obesity‐driven inflammation but does not support a loss of protective regulatory T cells to contribute to adipose tissue inflammation in obese patients as suggested by recent animal studies.     

High-Fat Diet-Induced Adiposity,Adipose Inflammation,Hepatic Steatosis and Hyperinsulinemia in Outbred CD-1 Mice

High-fat diet (HFD) has been applied to a variety of inbred mouse strains to induce obesity and obesity related metabolic complications. In this study, we determined HFD induced development of metabolic disorders on outbred female CD-1 mice in a time dependent manner. Compared to mice on regular chow, HFD-fed CD-1 mice gradually gained more fat mass and consequently exhibited accelerated body weight gain, which was associated with adipocyte hypertrophy and up-regulated expression of adipose inflammatory chemokines and cytokines such as Mcp-1 and Tnf-α. Increased fat accumulation in white adipose tissue subsequently led to ectopic fat deposition in brown adipose tissue, giving rise to whitening of brown adipose tissue without altering plasma level of triglyceride. Ectopic fat deposition was also observed in the liver, which was associated with elevated expression of key genes involved in hepatic lipid sequestration, including Ppar-γ2, Cd36 and Mgat1. Notably, adipose chronic inflammation and ectopic lipid deposition in the liver and brown fat were accompanied by glucose intolerance and insulin resistance, which was correlated with hyperinsulinemia and pancreatic islet hypertrophy. Collectively, these results demonstrate sequentially the events that HFD induces physiological changes leading to metabolic disorders in an outbred mouse model more closely resembling heterogeneity of the human population.     

Mapping of obesity QTLs in a cross between mouse lines divergently selected on fat content

A genome-wide quantitative trait locus (QTL) analysis was performed in a polygenic obesity mouse model resulting from a long-term selection experiment. The parental lines were outbred lines divergently selected for 53 generations for high-fat (fat, F line) or low-fat (lean, L line) percentage (fat%) that differed fivefold in fat% at 14 weeks of age. An F₂ population of 436 mice was used for the QTL analysis with 71 markers distributed across the genome. The analysis revealed significant QTLs Fob1 (for F-line obesity QTL 1), Fob2, Fob3, and Fob4, on Chromosomes (Chrs) 2, 12, 15, and X, respectively. None of these QTLs map to regions of known single gene obesity mutations (Lep ^ob , Lepr ^db , Cpe ^fat , A ^y , tub), though they map to regions of previously described obesity QTLs and candidate genes. The effects of Fob1, Fob3, Fob4 were additive, and that of Fob2 was dominant. Fob2 also showed a significant female-specific effect. Fob1, Fob2, Fob3, and Fob4 explained 4.9%, 19.5%, 14.4%, and 7.3% of the F₂ phenotypic variance for fat%, respectively. This study identified four loci that contributed to the response to divergent selection and control a significant proportion of the difference in obesity between the F and L lines. Received: 28 May 1999 / Accepted: 31 August 1999