Analysis of lines of mice selected for fat content. 3. Flux through the de novo lipid synthesis pathway

The flux through the de novo fatty acid synthesis pathway was estimated in lines of mice which differed substantially in fat content following 26 generations of selection at 10 weeks of age. Previous estimates of lipogenic enzyme activities had indicated an increase in the capacity for lipogenesis in the Fat compared to the Lean line. Therefore the in vivo flux in lipogenesis was measured in both liver and gonadal fat pad (GFP) tissues of males at 5 and 10 weeks of age, using the rate of incorporation of 3H from 3H2O and 14C from acetate and citrate into total lipids. At both ages and in both tissues the Fat line had a higher flux, about 20% increase in the liver and up to three-fold increase (range 1.2- to 3.4-fold) in the GFP. We conclude that direct selection for fatness in mice has resulted in metabolic changes in the rate of de novo fatty acid synthesis, and that the changes are largely detectable before 10 weeks, the age of selection.     

Liver metabolism traits in two rabbit lines divergently selected for intramuscular fat

Intramuscular fat (IMF) has a large effect in the sensory properties of meat because it affects tenderness, juiciness and flavour. A divergent selection experiment for IMF in longissimus dorsi (LD) muscle was performed in rabbits. Since liver is the major site of lipogenesis in rabbits, the objective of this work is to study the liver metabolism in the lines of the divergent selection experiment. Intramuscular fat content, perirenal fat weight, liver weight, liver lipogenic activities and plasma metabolites related to liver metabolism were measured in the eighth generation of selection. Direct response on IMF was 0.34 g/100 g of LD, which represented 2.7 SD of the trait, and selection showed a positive correlated response in the perirenal fat weight. High-IMF line showed greater liver size and greater liver lipogenic activities of enzymes glucose-6-phosphate dehydrogenase and malic enzyme. We did not find differences between lines for fatty acid synthase lipogenic activity. With regard to plasma metabolites, low-IMF line showed greater plasma concentration of triglycerides, cholesterol, bilirubin and alkaline phosphatase than high-IMF line, whereas high-IMF line showed greater albumin and alanine transaminase concentrations than low-IMF line. We did not observe differences between lines for glucose, total protein and plasma concentrations. Phenotypic correlations between fat (IMF and perirenal fat weight) and liver traits showed that liver lipogenesis affects fat deposition in both, muscle and carcass. However, the mechanisms whereby liver lipogenesis affected IMF content remain to be clarified.     

Genetics of Murine Liver and Kidney Arylsulfatase B

Mice from 12 inbred strains were surveyed for variation of kidney and liver arylsulfatase levels. Kidney variation was due to differences in the activity of arylsulfatase B. Twofold higher activities of arylsulfatase B in SWR/J kidney compared to A/HeJ kidney were determined by an autosomal gene which may be identical to the structural gene for arylsulfatase B since the SWR/J enzyme was more heat-stable than the A/HeJ enzyme. C57BL/6J mice possessed two-fold higher liver arylsulfatase levels than did A/HeJ mice. The major portion of this variation could be attributed to differences in arylsulfatase B, and appeared to be inherited in autosomal fashion. Although some evidence supports the existence of a major locus influencing liver arylsulfatase activity, this must be substantiated by further studies. Whatever the nature of the genetic factors involved, they do not appear to involve structural genes since no differences were discernible between the enzymes of the two strains relevant to Km, heat stability, electrophoretic mobility, pH optimum, activation energy, or response to several inhibitors. Furthermore, the rank ordering of strains on the basis of kidney arylsulfatase activity differed markedly from that which pertained to liver activity. Kidney arylsulfatase levels, but not brain or liver arylsulfatase activities, appear subject to androgenic influences.     

Genetic prenatal maternal effects on organ size in mice and their potential contribution to evolution

Two embryo transfer experiments were carried out in order to estimate the magnitude of prenatal maternal effects, independent of postnatal maternal factors, on the growth of internal organs and fat pads in mice. Reciprocal embryo transfers between the inbred mouse strains C3HeB/FeJ and SWR/J yielded three significant findings. First, all traits were not equally influenced by prenatal maternal factors. Genetic prenatal maternal factors, stemming from the genotype of the uterine mother, had a significant effect on testis weight, subcutaneous fat pad weight and epididymal fat pad weight in 21 day old progeny, but they had no effect on cranial capacity, an index of brain size, kidney weight, or liver weight. Prenatal litter size, defined as the sum of live and dead pups at birth, had a significant negative relationship with 21 day testis weight and kidney weight, and a significant positive association with subcutaneous and epididymal fat pad weights. Cranial capacity and liver weight at 21 days postnatally were not influenced by prenatal litter size. Second, the experiments demonstrated that there was ontogenetic variability in the strength of prenatal maternal effects. At 70 days of age, only subcutaneous fat pad weight was significantly influenced by genetic prenatal effects, and prenatal litter size had a significant negative relationship only with subcutaneous fat pad weight and body weight. Third, genetic prenatal effects had a significant influence on the among-trait covariances at 21 days postnatally, but not at 70 days. Because multivariate evolution involves covariances among characters, the latter results suggest that prenatal effects due to the mother's genotype can affect phenotypic evolution of mammals, especially for selection imposed early in life.     

Hormonal and lipogenic and gluconeogenic enzymatic responses in LA/N-corpulent rats

Genetically obese normotensive rats, LA/N-corpulent (cp), were fed ad libitum diets containing either 54% sucrose or cooked corn starch for 12 weeks. Twenty-four rats were used for the study; half were corpulent (cp/cp) and half were lean (cp/+ or +/+). Fasting levels of plasma insulin, glucose, corticosterone, glucagon and growth hormone, and activities of liver and epididymal fat pad glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), and liver and kidney glucose-6-phosphatase (G6Pase), fructose 1,6-diphosphatase (FDPase), and phosphoenolpyruvate carboxykinase (PEPCK) were measured. A significant phenotype effect was observed in insulin, corticosterone, growth hormone, and liver G6PD, ME, FDPase, and kidney PEPCK, G6Pase, FDPase, and epididymal fat pad G6PD and ME (corpulent greater than lean), and glucagon (lean greater than corpulent). Diet effect (sucrose greater than starch) was significant for plasma glucose, liver ME, and kidney G6Pase. Although not significant at the P less than 0.05 level, insulin, corticosterone, liver G6PD and FDPase and kidney FDPase tended to be higher in sucrose-fed rats. This study suggests that the corpulent rat is more lipogenic and gluconeogenic than the lean, and that the hormones responsible are effective in keeping both the lipogenic and gluconeogenic enzyme activity elevated.     

Strain differences among mice in taste psychophysics of sucrose octaacetate

SWR/J inbred mice consistently avoided a 10^–4 M sucroseoctaacetate (SOA) solution in unconditioned two-bottle preferencetests whereas mice of all other inbred strains tested did not(confirming a previous report that used SWR mice of a differentsubline). In a conditioned taste aversion procedure SWR/J miceavoided SOA at concentrations from 10^–3 M to 10^–7M but not at 10^–8 M. Various other inbred strains firstfailed to avoid SOA at concentrations from 10^–3 M to 10^–5M. The major strain difference between SWR and other inbredmice was robust across rearing regimes and when tested withother psychophysical procedures. In single-bottle, free-lickingtests SWR/J mice differed from C57L/J mice in response to SOAfollowing extremely brief exposure to the SOA.The SOA detectionthreshold differences indicated by these psychophysical proceduresare also consistent with differences reported from electrophysiologicalrecordings from glossopharyngeal and chorda tympani nerves inmice of several of the same strains.     

Increased insulin mediated glucose metabolism in fat cells from I versus C57BL mice

The purpose of this study was to determine whether adipocytes from I strain mice, which are characterized by a greater in vivo glucose tolerance than most other strains, had a higher capacity to utilize glucose in response to physiological concentrations of insulin. Using C57BL mice as a control strain, we examined the effect of insulin on glucose metabolism in epididymal and inguinal adipocytes from 2-month-old male mice. Body weight was only slightly less (7%) for the I mice than for the C57BL mice, but fat pad sizes were 60 and 20% less for epididymal and inguinal depots, respectively, in the I mice. Fat cell size was also smaller in epididymal adipocytes from the I mice than from the C57BL mice. Fat cell size of inguinal adipocytes was similar in the two strains. Without insulin the rates of [U-14C]glucose incorporation into CO2 or lipids were twofold higher in cells from the I mice than in those from the C57BL mice. Maximal insulin concentration (2.5 nM) increased glucose metabolism by 140 and 500% in epididymal and inguinal adipose cells, respectively, in the I mice versus 30 and 50% in the C57BL mice. The maximal effect of insulin was reached at a much higher insulin concentration in the I mice than in the C57BL mice. The activity of fatty acid synthetase was four- to sixfold higher in fat cells from I than in those from C57BL mice. These results demonstrate an increased insulin responsiveness of glucose metabolism in fat cells from the I mice related to an increased lipogenic capacity. Furthermore, they show that adipose tissue in mice exhibits significant regional differences in terms of insulin responsiveness of glucose metabolism.     

The effects of diet composition on body fat and hepatic steatosis in an animal (Peromyscus californicus) model of the metabolic syndrome

The objective of this research was to determine body composition, total fat content, fat distribution, and serum leptin concentration in hyperlipidemic (high responder, HR) and normolipidemic (low responder, LR) California mice (Peromyscus californicus). In our initial experiments, we sought to determine whether differences in regional fat storage were associated with hyperlipidemia in this species. To further characterize the hepatic steatosis in the mice, we performed 2 additional experiments by using a diet containing 45% of energy as fat. The body fat content of mice fed a low fat-diet (12.3% energy as fat) was higher than that of mice fed a moderate-fat diet (25.8% energy as fat). Total body fat did not differ between HR and LR mice. There was no significant difference between intraabdominal, gonadal, or inguinal fat pad weights. Liver weights of HR mice fed the moderate-fat diet were higher than those of LR mice fed the same diet, and the moderate-fat diet was associated with nonalcoholic fatty liver (NAFL). Mice fed the 45% diet had higher histologic score for steatosis but very little inflammatory response. Chemical analysis indicated increased lipid in the livers of mice fed the high-fat diet compared with those fed the low-fat diet. HR and LR mice had similar serum leptin concentrations. California mice develop NAFL without excess fat accumulation elsewhere. NAFL was influenced by genetic and dietary factors. These mice may be a naturally occuring model of partial lipodystrophy.     

Differences in lipogenesis in tissues of control and gold-thioglucose obese mice after an isocaloric meal

Lipogenesis was measured in 2 and 5 week gold-thioglucose (GTG) obese mice after a single meal of 0.5 g of standard chow. Compared to control mice the rate of lipogenesis in GTG obese mice, was 4-fold higher in liver and 10-fold higher in white adipose tissue (WAT). In brown adipose tissue (BAT) of GTG-injected mice the lipogenic rate was only 50% of that of controls. These results indicate that the increased lipid synthesis observed in GTG-injected mice is not due solely to hyperphagia and that some other stimuli, such as increased basal insulin levels and/or decreased thermogenesis and insulin resistance in BAT, contribute to the high rates of fat synthesis in this animal model of obesity.     

Analysis of lines of mice selected for fat content. 2. Correlated responses in the activities of enzymes involved in lipogenesis

Estimates of the activities (Vmax) of six enzymes involved in de novo fat synthesis were made in replicated lines of mice differing in fat content. These lines had been selected high and low for 20 generations with three replicates each of Fat, Control and Lean lines and for a further eight generations high and low as an unreplicated line. The activities of ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS), cytoplasmic malate dehydrogenase (MDH), malic enzyme (ME) and pyruvate kinase (PK) were determined in vitro in both liver and gonadal fatpad tissues taken at ages five and ten weeks. The activities of ACL, ACC, FAS and ME were significantly higher in the Fat than the Lean lines, and the differences were more pronounced at the earlier age and in the gonadal fatpad where activities in the Fat lines were higher by factors of 3.5, 2.4, 2.5 and 3.5 respectively. The activity of PK was unchanged in each tissue. MDH activity was significantly lower in adipose tissue in the Fat lines than the Lean lines at age ten weeks but not at age five weeks or in liver tissue. Results from replicates indicated that random genetic drift affected enzyme activities but nevertheless significant changes in activity were associated with the direction of selection. The changes in enzyme activity reported here are similar to those known to be associated with major mutations causing obesity in mice.     

Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats

This study was designed to monitor the developmental changes in insulinemia and lipogenic enzyme activities in both inguinal adipose tissue and liver during suckling (7, 9, 14, and 17 days of age) and weaning (22 and 30 days of age) on to either a low-fat or a high-fat diet in lean (Fa/fa) and obese (fa/fa) rats. Tissues were removed through surgery and genotypes were retrospectively determined. During suckling, there was no difference in liver enzyme activities between the two groups. In contrast, adipose tissue fatty acid synthetase was increased by 50% and citrate cleavage enzyme and malic enzyme by 30% by 9 days of age. By 17 days of age, there was a threefold elevation in these enzyme activities and 6-phosphogluconic dehydrogenase and a twofold increase in glucose-6-phosphate dehydrogenase per inguinal fat pad in fa/fa versus Fa/fa. Consistent with these results, fat pad weight was increased by 20%, 50%, and 100% at 9, 14, and 17 days of age, respectively, in obese as compared to lean pups. However only by 17 days of age could a slight but significant increase in insulin level be detected in obese pups. Enlargement of inguinal fat pad accelerated after weaning on to a low-fat diet and still more after weaning on to a high-fat diet. Weaning on to a low-fat diet elicited an induction of hepatic lipogenic enzymes two or three times greater in fa/fa than in lean pups, while weaning on to a high-fat diet blunted the differences between genotypes. The lipogenic enzyme activities displayed per total inguinal fat were three to ten times greater in obese than in lean pups, regardless of the diet. However, adipose tissue lipogenic enzyme activities were much lower after weaning on to a high-fat than on to a low-fat diet in obese pups. The high-fat diet was as effective as the low-fat diet in triggering hyperinsulinemia in obese pups. The increased adipose tissue capacity for lipogenesis, starting during the suckling period, could play an important etiologic role in the development and maintenance of obesity in the Zucker rat.-Bazin, R., and M. Lavau. Development of hepatic and adipose tissue lipogenic enzymes and insulinemia during suckling and weaning on to a high-fat diet in Zucker rats.     

Analysis of lines of mice selected for fat content. 1. Correlated responses in the activities of NADPH-generating enzymes

Estimates of the activities (Vmax) of four enzymes that generate the coenzyme NADPH, an absolute requirement for tissue fatty-acid synthesis, and of the concentration of NADP plus NADPH were made in lines of mice differing in fat content. These lines had been selected from the same base population for 20 generations, and 3 high, 3 low replicates and 1 unselected control were used. Analyses were performed on liver and gonadal fat pad (GFP) of males at 5 and 10 weeks of age. In both the liver and the GFP, measurable activities of the four enzymes: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (IDH) and malic enzyme (ME) expressed per mg soluble protein were, with minor exceptions, higher in the Fat (F) than in the Lean (L) lines at both ages; the highest ratio being 2.2 for ME in the GFP. The relationships between these measurable activities (Vmax) and in vivo lipogenesis are not however known. When expressed per gram tissue, the ratios for F to L in the GFP were less than 1 in most cases, presumably because of the very different adipocyte numbers and/or sizes between the lines. There were no significant differences between the lines in the concentration of NADP plus NADPH per gram tissue in liver or GFP, suggesting that F lines converted NADP to NADPH faster than L lines. It is predicted that selection on the enzyme activities would be less efficient than direct selection at changing fat content.     

Conjugated Linoleic Acid Does Not Improve Insulin Tolerance in Mice*

Objective: To determine if the addition or removal of dietary conjugated linoleic acid (CLA) would alter insulin tolerances in mice from two genetic lines. Research Methods and Procedures: High metabolic rate (MH) and low metabolic rate (ML) mice were assigned to consume 1) a control diet ad libitum, 2) a control diet at a restricted intake, or 3) a diet containing 1% CLA ad libitum. After 9 weeks, an insulin tolerance test was conducted, and a portion of the mice were killed. All remaining mice consumed the control diet ad libitum. Insulin tolerance tests were conducted 11 and 32 days after the diet change, and mice were killed 3 days after each test. Body fatness, fat pad weights, and serum insulin concentrations of mice were determined at each time‐point. Two follow‐up experiments were also conducted. Results: Restricted mice had insulin sensitivities not different than control mice. CLA‐fed MH mice in experiment 1 were resistant (p < 0.001) to insulin on each day measured. CLA‐fed ML mice were slightly resistant (p = 0.08) to exogenous insulin on day 0 of recovery and not different from control mice on day 11 or 32. Glucose response to insulin in MH mice fed CLA in experiments 2 or 3 did not differ from control mice. Discussion: Mice fed CLA did not have improved insulin tolerances compared with control mice. In some cases, dietary CLA may cause insulin resistance. MH mice seem more sensitive to CLA than ML mice.     

Effect of hyperinsulinemia on acid cholesterol ester hydrolase activity in liver, heart and epididymal fat pad preparations from rats and mice

The effect of insulin on lysosomal acid cholesterol ester hydrolase activity was studied in liver, heart and fat pad preparations from rats and mice. Hyperinsulinemia was induced for a period of 6 days in rats by the subcutaneous administration of exogenous insulin by an osmotic minipump. The effect of more chronic endogenous hyperinsulinemia was studied using genetic strains of diabetic (db/db) mice at 12 weeks of age. Mouse liver and heart preparations were characterized as having an acid pH optimum of 4.5-5 for cholesterol ester hydrolase activity; a smaller but distinct pH optimum could also be observed at pH 7. In contrast, hydrolase activity in mouse fat pad preparations had only one distinct pH optimum of 6.5. Hyperinsulinemia in rats and mice resulted in a significant decrease in acid cholesterol ester hydrolase activity in heart preparations, but had no consistent effect on acid hydrolase activity observed in liver and fat pad preparations. This decrease in lysosomal acid cholesterol ester hydrolase activity in cardiac tissue due to hyperinsulinemia cannot be related to any changes in lipoprotein turnover caused by insulin or diabetes.     

Different sensitivity of inbred mice to hepatocarcinogen ortho-aminoazotoluene may be due to differences in the negative control mechanisms of hepatocyte proliferation

A most convenient model to study mechanisms of live organism response to chemical carcinogens is tumor induction in murine liver by aminoazodyes, in particular by ortho-aminoazotoluene (OAT). We studied both early and late stages of hepatocarcinogenesis on several lines of inbred mice differing in sensibility to OAT. By means of autoradiography, we examined proliferative activity of hepatocytes obtained from the liver of sensitive (A/He, DD, SWR) and resistant to OAT AKR, CC57Br, BALB/c lines of mice, which were injected carcinogen. The level of p53, p21Cip1, bax, mdm2, cyclin G, gadd45 genes expression in the liver of mice of different lines given OAT injection was studied by multiplex PCR method. Carcinogen caused a decrease of hepatocyte proliferative activity induced by partial hypatectomy (PHE), and an increase in p53, p21Cip, bax, mdm2, and cyclin G genes within mice of A/He, DD and SWR lines. Cell fusion experiments on hepatocytes obtained from regenerating murine liver sensitive to A/He line carcinogen and given long-time OAT administrations with resting and proliferating fibroblasts of NIH 3T3 mice revealed no obvious suppression of DNA synthesis in heterokaryons. Unlike, in fusion experiments on serum-stimulated fibroblasts with hepatocytes obtained from the liver of BALB/c line mice also given OAT suppression of DNA synthesis in stimulated fibroblasts in heterokaryons was observed 15 days following PHE. These results enable us to conclude that OAT administrations break negative endogenous mechanisms of hepatocyte proliferation control in the liver of mice sensitive to carcinogenes.     

Liver X receptors regulate de novo lipogenesis in a tissue-specific manner in C57BL/6 female mice

The liver X receptors (LXRs) play a key role in cholesterol and bile acid metabolism but are also important regulators of glucose metabolism. Recently, LXRs have been proposed as a glucose sensor affecting LXR-dependent gene expression. We challenged wild-type (WT) and LXRαβ(-/-) mice with a normal diet (ND) or a high-carbohydrate diet (HCD). Magnetic resonance imaging showed different fat distribution between WT and LXRαβ(-/-) mice. Surprisingly, gonadal (GL) adipocyte volume decreased on HCD compared with ND in WT mice, whereas it slightly increased in LXRαβ(-/-) mice. Interestingly, insulin-stimulated lipogenesis of isolated GL fat cells was reduced on HCD compared with ND in LXRαβ(-/-) mice, whereas no changes were observed in WT mice. Net de novo lipogenesis (DNL) calculated from Vo(2) and Vco(2) was significantly higher in LXRαβ(-/-) than in WT mice on HCD. Histology of HCD-fed livers showed hepatic steatosis in WT mice but not in LXRαβ(-/-) mice. Glucose tolerance was not different between groups, but insulin sensitivity was decreased by the HCD in WT but not in LXRαβ(-/-) mice. Finally, gene expression analysis of adipose tissue showed induced expression of genes involved in DNL in LXRαβ(-/-) mice compared with WT animals as opposed to the liver, where expression of DNL genes was repressed in LXRαβ(-/-) mice. We thus conclude that absence of LXRs stimulates DNL in adipose tissue, but suppresses DNL in the liver, demonstrating opposite roles of LXR in DNL regulation in these two tissues. These results show tissue-specific regulation of LXR activity, a crucial finding for drug development.     

Weight loss in tumour-bearing mice is not associated with changes in resistin gene expression in white adipose tissue.

Resistin, a product of white adipose tissue, is postulated to induce insulin resistance in obesity and regulate adipocyte differentiation. The aim of this study was to examine resistin gene expression in adipose tissue from mice bearing the MAC16 adenocarcinoma, which induces cancer cachexia with marked wasting of adipose tissue and skeletal muscle mass. MAC16-bearing mice lost weight progressively over the period following tumour transplantation, while the weight of control mice remained stable. Leptin mRNA in gonadal fat was 50 % lower in MAC16 mice than in controls (p < 0.05). Plasma insulin concentrations were also significantly lower in the MAC16 group (p < 0.05). However, resistin mRNA level in gonadal fat in MAC16 mice was similar to controls (94 % of controls). Thus, despite severe weight loss and significant falls in leptin expression and insulin concentration, resistin gene expression appears unchanged in white adipose tissue of mice with MAC16 tumour. Maintenance of resistin production may help inhibit the formation of new adipocytes in cancer cachexia.