Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.     

Coffee and caffeine improve insulin sensitivity and glucose tolerance in C57BL/6J mice fed a high-fat diet

We have previously demonstrated that coffee and caffeine ameliorated hyperglycemia in spontaneously diabetic KK-A(y) mice. This present study evaluates the antidiabetic effects of coffee and caffeine on high-fat-diet-induced impaired glucose tolerance in C57BL/6J mice. C57BL/6J mice fed a high-fat diet were given regular drinking water (control group), or a 2.5-fold-diluted coffee or caffeine solution (200 mg/L) for 17 weeks. The ingestion of coffee or caffeine improved glucose tolerance, insulin sensitivity, and hyperinsulinemia when compared with mice in the control group. The adipose tissue mRNA levels of inflammatory adipocytokines (MCP-1 and IL-6) and the liver mRNA levels of genes related to fatty acid synthesis were lower in the coffee and caffeine groups than those in the control group. These results suggest that coffee and caffeine exerted an ameliorative effect on high-fat-diet-induced impaired glucose tolerance by improving insulin sensitivity. This effect might be attributable in part to the reduction of inflammatory adipocytokine expression.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Fucoxanthin-rich seaweed extract suppresses body weight gain and improves lipid metabolism in high-fat-fed C57BL/6J mice

An ethanol extract of fucoxanthin-rich seaweed was examined for its effectiveness as a nutraceutical for body fat-lowering agent and for an antiobese effect based on mode of actions in C57BL/6J mice. Animals were randomized to receive a semi-purified high-fat diet (20% dietary fat, 10% corn oil and 10% lard) supplemented with 0.2% conjugated linoleic acid (CLA) as the positive control, 1.43% or 5.72% fucoxanthin-rich seaweed ethanol extract (Fx-SEE), equivalent to 0.05% or 0.2% dietary fucoxanthin for six weeks. Results showed that supplementation with both doses of Fx-SEE significantly reduced body and abdominal white adipose tissue (WAT) weights, plasma and hepatic triglyceride (TG), and/or cholesterol concentrations compared to the high-fat control group. Activities of adipocytic fatty acid (FA) synthesis, hepatic FA and TG synthesis, and cholesterol–regulating enzyme were also lowered by Fx-SEE supplement. Concentrations of plasma high-density lipoprotein-cholesterol, fecal TG and cholesterol, as well as FA oxidation enzyme activity and UCP1 mRNA expression in epididymal WAT were significantly higher in the Fx-SEE groups than in the high-fat control group. CLA treatment reduced the body weight gain and plasma TG concentration. Overall, these results indicate that Fx-SEE affects the plasma and hepatic lipid profile, fecal lipids and body fat mass, and alters hepatic cholesterol metabolism, FA synthesis and lipid absorption.     

Substrain specific behavioral responses in male C57BL/6N and C57BL/6J mice to a shortened 21-hour day and high-fat diet

ABSTRACT

Altered circadian rhythms have negative consequences on health and behavior. Emerging evidence suggests genetics influences the physiological and behavioral responses to circadian disruption. We investigated the effects of a 21 h day (T = 21 cycle), with high-fat diet consumption, on locomotor activity, explorative behaviors, and health in male C57BL/6J and C57BL/6N mice. Mice were exposed to either a T = 24 or T = 21 cycle and given standard rodent chow (RC) or a 60% high-fat diet (HFD) followed by behavioral assays and physiological measures. We uncovered numerous strain differences within the behavioral and physiological assays, mainly that C57BL/6J mice exhibit reduced susceptibility to the obesogenic effects of (HFD) and anxiety-like behavior as well as increased circadian and novelty-induced locomotor activity compared to C57BL/6N mice. There were also substrain-specific differences in behavioral responses to the T = 21 cycle, including exploratory behaviors and circadian locomotor activity. Under the 21-h day, mice consuming RC displayed entrainment, while mice exposed to HFD exhibited a lengthening of activity rhythms. In the open-field and light-dark box, mice exposed to the T = 21 cycle had increased novelty-induced locomotor activity with no further effects of diet, suggesting daylength may affect mood-related behaviors. These results indicate that different circadian cycles impact metabolic and behavioral responses depending on genetic background, and despite circadian entrainment.     

High-fat feeding rapidly induces obesity and lipid derangements in C57BL/6N mice

C57BL/6N (B6N) is becoming the standard background for genetic manipulation of the mouse genome. The B6N, whose genome is very closely related to the reference C57BL/6J genome, is versatile in a wide range of phenotyping and experimental settings and large repositories of B6N ES cells have been developed. Here, we present a series of studies showing the baseline characteristics of B6N fed a high-fat diet (HFD) for up to 12 weeks. We show that HFD-fed B6N mice show increased weight gain, fat mass, and hypercholesterolemia compared to control diet-fed mice. In addition, HFD-fed B6N mice display a rapid onset of lipid accumulation in the liver with both macro- and microvacuolation, which became more severe with increasing duration of HFD. Our results suggest that the B6N mouse strain is a versatile background for studying diet-induced metabolic syndrome and may also represent a model for early nonalcoholic fatty liver disease.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Scallop protein with endogenous high taurine and glycine content prevents high-fat,high-sucrose-induced obesity and improves plasma lipid profile in male C57BL/6J mice

High-protein diets induce alterations in metabolism that may prevent diet-induced obesity. However, little is known as to whether different protein sources consumed at normal levels may affect diet-induced obesity and associated co-morbidities. We fed obesity-prone male C57BL/6J mice high-fat, high-sucrose diets with protein sources of increasing endogenous taurine content, i.e., chicken, cod, crab and scallop, for 6 weeks. The energy intake was lower in crab and scallop-fed mice than in chicken and cod-fed mice, but only scallop-fed mice gained less body and fat mass. Liver mass was reduced in scallop-fed mice, but otherwise no changes in lean body mass were observed between the groups. Feed efficiency and apparent nitrogen digestibility were reduced in scallop-fed mice suggesting alterations in energy utilization and metabolism. Overnight fasted plasma triacylglyceride, non-esterified fatty acids, glycerol and hydroxy-butyrate levels were significantly reduced, indicating reduced lipid mobilization in scallop-fed mice. The plasma HDL-to-total-cholesterol ratio was higher, suggesting increased reverse cholesterol transport or cholesterol clearance in scallop-fed mice in both fasted and non-fasted states. Dietary intake of taurine and glycine correlated negatively with body mass gain and total fat mass, while intake of all other amino acids correlated positively. Furthermore taurine and glycine intake correlated positively with improved plasma lipid profile, i.e., lower levels of plasma lipids and higher HDL-to-total-cholesterol ratio. In conclusion, dietary scallop protein completely prevents high-fat, high-sucrose-induced obesity whilst maintaining lean body mass and improving the plasma lipid profile in male C57BL/6J mice.     

C57BL/6J and A/J mice fed a high-fat diet delineate components of metabolic syndrome

Objective: The aim of this study was to assess the suitability of A/J and C57BL/6J mice of both sexes as models of some components of the human metabolic syndrome (MetS) under nutritional conditions more comparable with the actual worldwide diet responsible for the increased incidence of the MetS. Research Methods: We fed large cohorts (n = 515) of two strains of mice, A/J and the C57BL/6J, and of both sexes a high‐fat diet (HFD; 60% fat) that, in contrast with most previous reports using saturated fats, was enriched in mono‐ and polyunsaturated fatty acids, thus more closely mimicking most Western diets, or a control diet (10% fat), for 20 weeks. Results: In sharp contrast to previous reports, weight gain and hyperleptinemia were similar in both strains and sexes. Hyperinsulinemia, glucose tolerance, insulin resistance, and hypercholesterolemia were observed, although with important differences between strains and sexes. A/J males displayed severely impaired glucose tolerance and insulin resistance. However, in contrast with C57BL6/J mice, which displayed overt type 2 diabetes, A/J mice of both sexes remained normoglycemic. Discussion: With important differences in magnitude and time course, the phenotypic and metabolic characteristics of both strains and both sexes on this HFD demonstrate that these models are very useful for identifying the mechanisms underlying progression or resistance to subsequent type 2 diabetes.     

Garcinia cambogia extract ameliorates visceral adiposity in C57BL/6J mice fed on a high-fat diet

The aim of present study is to evaluate the effects of Garcinia cambogia on the mRNA levels of the various genes involved in adipogenesis, as well as on body weight gain, visceral fat accumulation, and other biochemical markers of obesity in obesity-prone C57BL/6J mice. Consumption of the Garcinia cambogia extract effectively lowered the body weight gain, visceral fat accumulation, blood and hepatic lipid concentrations, and plasma insulin and leptin levels in a high-fat diet (HFD)-induced obesity mouse model. The Garcinia cambogia extract reversed the HFD-induced changes in the expression pattern of such epididymal adipose tissue genes as adipocyte protein aP2 (aP2), sterol regulatory element-binding factor 1c (SREBP1c), peroxisome proliferator-activated receptor gamma2 (PPARgamma2), and CCAT/enhancer-binding protein alpha (C/EBPalpha). These findings suggest that the Garcinia cambogia extract ameliorated HFD-induced obesity, probably by modulating multiple genes associated with adipogenesis, such as aP2, SREBP1c, PPARgamma2, and C/EBPalpha in the visceral fat tissue of mice.     

Intake compensates for resting metabolic rate variation in female C57BL/6J mice fed high-fat diets

Objective: The literature is divided over whether variation in resting metabolic rate (RMR) is related to subsequent obesity. We set out to see whether the effect of RMR on weight gain in mice could be revealed with high‐fat feeding. Research Methods and Procedures: Female C57BL/6J mice received a low‐ (10 kcal%fat n = 47), medium‐ (45 kcal%fat n = 50), or high‐fat diet (60 kcal%fat n = 50) for 12 weeks. Pre‐treatment RMR was measured by indirect calorimetry. Body composition was estimated using DXA before and after treatment. Results: Mice on the high‐fat diet gained 39% of body mass, whereas control animals gained 3.5%. There was no interaction between RMR and dietary type on weight gain, and there was no association between weight gain and RMR for any of the treatments. RMR accounted for 2.4% of the variation in pre‐treatment food intake corrected for initial body mass; however, the gradient of this relationship indicated that variations in RMR were, on average, compensated for by adjustments in food intake. Discussion: Individual variations in RMR did not predispose mice to weight gain independent of the dietary treatment. Deviations from the relationship between RMR and food intake were not associated with weight gain. This suggests that variations in energy expenditure, caused by RMR and physical activity, are closely linked to dietary intake, and, therefore, well compensated. Individual variations in the strength of this association may underpin individual variability in the responses to diet.     

Aging and glucose homeostasis in C57BL/6J male mice

Age-dependent changes in glucose homeostasis were assessed in specific pathogen-free C57BL/6J male mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucose-stimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired beta cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse.     

Genistein and daidzein modulate hepatic glucose and lipid regulating enzyme activities in C57BL/KsJ-db/db mice

This study examines whether anti-diabetic effects of genistein and daidzein are mediated by hepatic glucose and lipid regulating enzyme activities in type 2 diabetic animals. Male C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and age-matched non-diabetic littermates (db/+) were used in this study. The db/db mice were divided into control, genistein (0.02%, w/w) and daidzein (0.02%, w/w) groups. The blood glucose and HbA(1c) levels were significantly lower in the genistein and daidzein groups than in the control group, while glucose tolerance only was significantly improved in the genistein-supplemented group. The plasma insulin and C-peptide levels did not differ significantly between groups, yet the glucagon level was lower in the genistein and daidzein groups compared to that in the control db/db or db/+ group. The genistein and daidzein supplements increased the insulin/glucagon ratio in the type 2 diabetic animals. While the hepatic glucokinase activity was significantly lower in the db/db control group, the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were significantly higher in the control group compared to the db/+ group. Interestingly, these hepatic glucose metabolizing enzyme activities were reversed by the genistein and daidzein supplementation in db/db mice compared to the control group. The hepatic fatty acid synthase, beta-oxidation and carnitine palmitoyltransferase activities were all significantly lower in the genistein and daidzein groups than in the control group. The genistein and daidzein supplements also improved the plasma total cholesterol, triglyceride, HDL-cholesterol/total cholesterol, free fatty acid and hepatic triglyceride concentrations in the db/db mice. These results suggest that genistein and daidzein exert anti-diabetic effect in type 2 diabetic conditions by enhancing the glucose and lipid metabolism.     

Reductive effect of body weight in C57BL/6J mice fed a high-fat diet by Saesaengmyung Diet

The Saesaengmyung Diet (SD) is a newly developed dietary product to help control weight. The aim of this study was to evaluate whether SD combined with a high-fat (HF) diet could influence body weight, fat accumulation, and glucose levels in blood. C57BL/6J mice were fed for 8 weeks with a standard diet, an HF diet, and an HF + 10% or HF + 20% SD diet. Body weight was recorded weekly, and plasma levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and glucose were analyzed at the end of the study. Weight increases in the 10% or 20% SD group were significantly less than in the HF diet group (p < 0.05). Plasma total cholesterol level significantly decreased by 33.5% in the 10% SD group and 38.8% in the 20% SD group, but the LDL cholesterol, HDL cholesterol, and glucose levels in the SD groups were not significantly changed. Our findings indicate that SD may be beneficial to overweight individuals in the reduction of weight gain induced by an HF diet.     

Self-mutilation of the penis in C57BL/6N mice

A major cause of male reproductive failure in a C57BL/6N mouse production colony is self-inflicted mutilation of the penis. The extent of the damage ranged from loss of the distal end to loss of the entire penis. From January 1974 to August 1976, 645 adult male mice with mutilated penis were removed from this colony--where the monthly census was 9500 mice, mated 1 male to 4 females using a continuous mating system. On necropsy, it was observed that the substance blocking the urethra in the penile stump resulted in urine retention, grossly distending the urinary bladder. Proteus mirabilis and other bacteria isolated from the urethral plugs were considered secondary invaders.     

Adrenal function and the effect of a high-fat diet on C57BL/6J and C57BL/6J-ob/ob mice.

In C57BL/6J mice and the ob/+ and ob/ob mutants total plasma corticosterone levels were found to be statistically different. In C57BL/6J mice the level was 1.9 +/- 0.2 mug/100 ml plasma, in ob/+ mice 8.6 +/- 1.6 mug/100 ml and in ob/ob mice 13.7 +/- 1.5 mug/100 ml. The percentage of protein-bound corticosterone as well as the free endogenous corticosterone levels were also different. Feeding a high-fat diet to young C57BL/6J and C57BL/6J-ob/ob mice for a period of 4 weeks had no effect upon blood glucose, plasma insulin and plasma corticosterone levels. The significantly higher increase in body weight of the high-fat diet groups of both lines of mice was mainly due to fat cell hypertrophy.     

Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice

The effects of exendin-4 on Sirt1 expression as a mechanism of reducing fatty liver have not been previously reported. Therefore, we investigated whether the beneficial effects of exendin-4 treatment on fatty liver are mediated via Sirt1 in high-fat (HF) diet-induced obese C57BL/6J mice and related cell culture models. Exendin-4 treatment decreased body weight, serum free fatty acid (FA), and triglyceride levels in HF-induced obese C57BL/6J mice. Histological analysis showed that exendin-4 reversed HF-induced hepatic accumulation of lipids and inflammation. Exendin-4 treatment increased mRNA and protein expression of Sirt1 and its downstream factor, AMPK, in vivo and also induced genes associated with FA oxidation and glucose metabolism. In addition, a significant increase in the hepatic expression of Lkb1 and Nampt mRNA was observed in exendin-4-treated groups. We also observed increased expression of phospho-Foxo1 and GLUT2, which are involved in hepatic glucose metabolism. In HepG2 and Huh7 cells, mRNA and protein expressions of GLP-1R were increased by exendin-4 treatment in a dose-dependent manner. Exendin-4 enhanced protein expression of Sirt1 and phospho-AMPKα in HepG2 cells treated with 0.4 mM palmitic acid. We also found that Sirt1 was an upstream regulator of AMPK in hepatocytes. A novel finding of this study was the observation that expression of GLP-1R is proportional to exendin-4 concentration and exendin-4 could attenuate fatty liver through activation of Sirt1.     

Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet

C57BL/6J mice were fed a high-fat, carbohydrate-free diet (HFD) for 9 mo. Approximately 50% of the mice became obese and diabetic (ObD), approximately 10% lean and diabetic (LD), approximately 10% lean and nondiabetic (LnD), and approximately 30% displayed intermediate phenotype. All of the HFD mice were insulin resistant. In the fasted state, whole body glucose clearance was reduced in ObD mice, unchanged in the LD mice, and increased in the LnD mice compared with the normal-chow mice. Because fasted ObD mice were hyperinsulinemic and the lean mice slightly insulinopenic, there was no correlation between insulin levels and increased glucose utilization. In vivo, tissue glucose uptake assessed by 2-[(14)C]deoxyglucose accumulation was reduced in most muscles in the ObD mice but increased in the LnD mice compared with the values of the control mice. In the LD mice, the glucose uptake rates were reduced in extensor digitorum longus (EDL) and total hindlimb but increased in soleus, diaphragm, and heart. When assessed in vitro, glucose utilization rates in the absence and presence of insulin were similar in diaphragm, soleus, and EDL muscles isolated from all groups of mice. Thus, in genetically homogenous mice, HFD feeding lead to different metabolic adaptations. Whereas all of the mice became insulin resistant, this was associated, in obese mice, with decreased glucose clearance and hyperinsulinemia and, in lean mice, with increased glucose clearance in the presence of mild insulinopenia. Therefore, increased glucose clearance in lean mice could not be explained by increased insulin level, indicating that other in vivo mechanisms are triggered to control muscle glucose utilization. These adaptive mechanisms could participate in the protection against development of obesity.