Myostatin Expression,Lymphocyte Population,and Potential Cytokine Production Correlate with Predisposition to High-Fat Diet Induced Obesity in Mice

A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO) resistant (SWR/J) and susceptible (C57BL/6) mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ) potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4⁺/CD44^hi and CD8⁺/CD44^hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.     

Methionine-Restricted C57BL/6J Mice Are Resistant to Diet-Induced Obesity and Insulin Resistance but Have Low Bone Density

Dietary methionine restriction (MR) extends lifespan, an effect associated with reduction of body weight gain, and improvement of insulin sensitivity in mice and rats as a result of metabolic adaptations in liver, adipose tissue and skeletal muscle. To test whether MR confers resistance to adiposity and insulin resistance, C57BL/6J mice were fed a high fat diet (HFD) containing either 0.86% methionine (control fed; CF) or 0.12% methionine (methionine-restricted; MR). MR mice on HFD had lower body weight gain despite increased food intake and absorption efficiency compared to their CF counterparts. MR mice on HFD were more glucose tolerant and insulin sensitive with reduced accumulation of hepatic triglycerides. In plasma, MR mice on HFD had higher levels of adiponectin and FGF21 while leptin and IGF-1 levels were reduced. Hepatic gene expression showed the downregulation of Scd1 while Pparg, Atgl, Cd36, Jak2 and Fgf21 were upregulated in MR mice on HFD. Restriction of growth rate in MR mice on HFD was also associated with lower bone mass and increased plasma levels of the collagen degradation marker C-terminal telopeptide of type 1 collagen (CTX-1). It is concluded that MR mice on HFD are metabolically healthy compared to CF mice on HFD but have decreased bone mass. These effects could be associated with the observed increase in FGF21 levels.     

Insulin facilitates monocyte migration: a possible link to tissue inflammation in insulin-resistance

Mononuclear cells (MNCs) are the primary cell type involved in the pro-inflammatory state of obesity-linked insulin-resistance, and atherosclerosis. Increased serum levels of MMP-9 are reported in insulin-resistant type 2 diabetic patients. Here we demonstrate insulin facilitating human monocytic THP-1 cell chemotaxis via prolonged Erk1/2-dependent induction of MMP-9. In vivo, significantly increased serum levels of MMP-9 were found in obesity-induced hyperinsulinemic C57BL/J6 mice, which were diminished by treatment with the anti-diabetic PPARγ-ligand pioglitazone. In line with this, pioglitazone inhibited Erk1/2-phosphorylation and subsequent insulin-dependent MMP-9 synthesis in THP-1 cells. Thus, insulin increases MMP-9 gelatinolytic activity in monocytic cells, which results in accelerated chemotaxis. Hyperinsulinemia is associated with enhanced MMP-9 serum levels, potentially facilitating monocyte migration to and infiltration of adipose tissue and the arterial wall, thereby contributing to the increased cardiovascular risk in obese, hyperinsulinemic patients.     

Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model

Background

Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.

Methodology

C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.

Principal Findings/Conclusions

A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.     

BVT.2733, a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, attenuates obesity and inflammation in diet-induced obese mice

Background

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice.

Methodology/Principal Findings

C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro.

Conclusions/Significance

These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease.     

Involvement of the GHSR in the developmental programming and metabolic disturbances induced by maternal undernutrition

The mismatch between maternal undernutrition and adequate nutrition after birth increases the risk of developing metabolic diseases. We aimed to investigate whether the hyperghrelinemia during maternal undernourishment rewires the hypothalamic development of the offspring and contributes to the conversion to an obese phenotype when fed a high-fat diet (HFD). Pregnant C57BL/6 J, wild type (WT) and ghrelin receptor (GHSR)^−/− mice were assigned to either a normal nourished (NN) group, or an undernutrition (UN) (30% food restricted) group. All pups were fostered by NN Swiss mice. After weaning, pups were fed a normal diet, followed by a HFD from week 9. Plasma ghrelin levels peaked at postnatal day 15 (P15) in both C57BL/6 J UN and NN pups. Hypothalamic Ghsr mRNA expression was upregulated at P15 in UN pups compared to NN pups and inhibited agouti-related peptide (AgRP) projections. Adequate lactation increased body weight of UN WT but not of GHSR^−/− pups compared to NN littermates. After weaning with a HFD, body weight and food intake was higher in WT UN pups but lower in GHSR^−/− UN pups than in NN controls. The GHSR prevented a decrease in ambulatory activity and oxygen consumption in UN offspring during ad libitum feeding. Maternal undernutrition triggers developmental changes in the hypothalamus in utero which were further affected by adequate feeding after birth during the postnatal period by affecting GHSR signaling. The GHSR contributes to the hyperphagia and the increase in body weight when maternal undernutrition is followed by an obesity prone life environment.     

Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity,Impairs PARP-1 Degradation,and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2^-/- deficient mice and MMP-2^+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2^+/+ mice, MMP-2 null (MMP-2^-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2^+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.     

A High-Fat Diet Delays Age-Related Hearing Loss Progression in C57BL/6J Mice

Objective

Age-related hearing loss (AHL), or presbycusis, is the most common sensory disorder among the elderly. We used C57BL/6J mice as an AHL model to determine a possible association between AHL and a high-fat diet (HFD).

Methods

Forty C57BL/6J mice were randomly assigned to a control or HFD group. Each group was divided into the following subgroups: 1-, 3-, 5- and 12-month groups (HFD, n = 5/subgroup; control, n = 5/subgroup). Nine CBA/N-slc mice were also used as a 12-month control (n = 5) or 12-month HFD (n = 4) group. The mice were fed a HFD or normal (control) diet throughout this study. Hearing function was evaluated at 1, 3, 5 and 12 months using auditory evoked brainstem responses (ABRs). Spiral ganglion cells (SGCs) were also counted.

Results

The elevation of ABR thresholds (at 4 and 32 kHz) at 3 and 5 months was significantly suppressed in the HFD group compared with the control groups for C57BL/6J mice. After 12 months, the elevation of ABR thresholds was significantly suppressed in the HFD group at all frequencies for C57BL/6J mice. In contrast, CBA/N-slc mice displayed opposite outcomes, as ABR thresholds at all frequencies at 12 months were significantly elevated in the HFD group compared with the control group. For the C57BL/6J mice at 12 months, SGC numbers significantly decreased in all parts of the cochleae in the control group compared with the HFD groups. In contrast, for the CBA/N-slc mice, SGC numbers significantly decreased, particularly in the upper parts of the cochleae in the HFD group compared with the control groups.

Conclusions

The elevation in ABR thresholds and SGC loss associated with aging in the HFD-fed C57BL/6J mice were significantly suppressed compared with those in the normal diet-fed mice. These results suggest that HFD delays AHL progression in the C57B/6J mice.     

Macronutrient diet selection in thirteen mouse strains

The strain distribution for macronutrient diet selection was described in 13 mouse strains (AKR/J, NZB/B1NJ, C57BL/6J, C57BL/6ByJ, DBA/2J, SPRET/Ei, CD-1, SJL/J, SWR/J, 129/J, BALB/cByJ, CAST/Ei, and A/J) with the use of a self-selection protocol in which separate carbohydrate, fat, and protein diets were simultaneously available for 26-30 days. Relative to carbohydrate, nine strains consumed significantly more calories from the fat diet; two strains consumed more calories from carbohydrate than from fat (BALB/cByJ, CAST/Ei). Diet selection by SWR/J mice was variable over time, resulting in a lack of preference. One strain (A/J) failed to adapt to the diet paradigm due to inadequate protein intake. Comparisons of proportional fat intake across strains revealed that fat selection/consumption ranged from 26 to 83% of total energy. AKR/J, NZB/B1NJ, and C67BL/6J mice self-selected the highest proportion of dietary fat, whereas the CAST/Ei and BALB/cByJ strains chose the lowest. Finally, epididymal fat depot weight was correlated with fat consumption. There were significant positive correlations in AKR/J and C57BL/6J mice, which are highly sensitive to dietary obesity. However, absolute fat intake was inversely correlated with epididymal fat in two of the lean strains: SWR/J and CAST/Ei. We hypothesize that the SWR/J and CAST/Ei strains are highly sensitive to a negative feedback signal generated by increasing body fat, but the AKR/J and C67BL/6J mice are not. The variation in dietary fat selection across inbred strains provides a tool for dissecting the complex genetics of this trait.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

High Fat Diet-Induced Gut Microbiota Exacerbates Inflammation and Obesity in Mice via the TLR4 Signaling Pathway

Background & Aims

While it is widely accepted that obesity is associated with low-grade systemic inflammation, the molecular origin of the inflammation remains unknown. Here, we investigated the effect of endotoxin-induced inflammation via TLR4 signaling pathway at both systemic and intestinal levels in response to a high-fat diet.

Methods

C57BL/6J and TLR4-deficient C57BL/10ScNJ mice were maintained on a low-fat (10 kcal % fat) diet (LFD) or a high–fat (60 kcal % fat) diet (HFD) for 8 weeks.

Results

HFD induced macrophage infiltration and inflammation in the adipose tissue, as well as an increase in the circulating proinflammatory cytokines. HFD increased both plasma and fecal endotoxin levels and resulted in dysregulation of the gut microbiota by increasing the Firmicutes to Bacteriodetes ratio. HFD induced the growth of Enterobecteriaceae and the production of endotoxin in vitro. Furthermore, HFD induced colonic inflammation, including the increased expression of proinflammatory cytokines, the induction of Toll-like receptor 4 (TLR4), iNOS, COX-2, and the activation of NF-κB in the colon. HFD reduced the expression of tight junction-associated proteins claudin-1 and occludin in the colon. HFD mice demonstrated higher levels of Akt and FOXO3 phosphorylation in the colon compared to the LFD mice. While the body weight of HFD-fed mice was significantly increased in both TLR4-deficient and wild type mice, the epididymal fat weight and plasma endotoxin level of HFD-fed TLR4-deficient mice were 69% and 18% of HFD-fed wild type mice, respectively. Furthermore, HFD did not increase the proinflammatory cytokine levels in TLR4-deficient mice.

Conclusions

HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the gut microbiota composition and increasing its intestinal permeability through the induction of TLR4, thereby accelerating obesity.     

Divergent effects of leptin in mice susceptible or resistant to obesity.

Consumption of a high-fat diet decreases hypothalamic neuropeptide Y (NPY) and increases proopiomelanocortin (POMC) and brown adipose uncoupling protein (UCP)-1 mRNA in obesity-resistant SWR/J but not obesity-prone C57Bl/6J mice. Although leptin was elevated in both strains in response to a high-fat diet, its role in the development of diet-induced obesity has remained unclear since insulin and other factors that affect similar tissue targets are altered. Thus, we administered recombinant leptin by subcutaneous infusion to chow-fed mice to mimic the changes in plasma leptin across its broad physiologic range. We observed strain differences in responsiveness to reduced and elevated leptin levels. A reduction in leptin during fasting evoked a greater response in C57Bl/6J mice by decreasing energy expenditure and thyroxin, increasing corticosterone and stimulating food intake and weight gain during refeeding. However, C57Bl/6J mice were less responsive to an increase in leptin in the fed state. Conversely, the leptin-mediated response to fasting was blunted in SWR/J mice, whereas an increase in leptin profoundly reduced food intake and body weight in SWR/J mice fed ad libitum. Sensitivity to fasting in C57Bl/6J mice was associated with higher hypothalamic NPY mRNA and reduced POMC and UCP-1 mRNA expression, while the robust response to high leptin levels in SWR/J mice was associated with suppression of NPY mRNA. These results indicate that differences in leptin responsiveness between strains might occur centrally or peripherally, leading to alteration in the patterns of food intake, thermogenesis and energy storage.     

Corneal and stomach expression of aldehyde dehydrogenases: from fish to mammals

We have studied the distribution of the ALDH3A1, ALDH1A1 and ALDH2 proteins in the cornea and stomach of several animal species, including mammals (C57BL/6J and SWR/J mice, rat and pig), birds (chicken and turkey), amphibians (frog) and fish (trout and zebrafish). High ALDH3A1 protein levels and catalytic activities were detected in C57BL/6J mouse, rat and pig. We found complete absence of the ALDH3A1 protein in SWR/J mice, which carry the Aldh3a1^c allele characterized by four amino acid substitutions (G88R, I154N, H305R and I352V) and lack of enzymatic activity. This indicates that the SWR/J mouse strain is a natural gene knockout model for ALDH3A1. Traces of ALDH3A1 were detected in rabbit, whereas expression was absent from chicken, turkey, frog, trout, and zebrafish. Interestingly, significant levels of the cytosolic ALDH1A1 and mitochondrial ALDH2 proteins were detected by immunoblot analysis in all examined species that are deficient in ALDH3A1 expression. In contrast, no ALDH1A1 or ALDH2 protein was detected in the species expressing ALDH3A1. It can, therefore, be concluded that corneal expression of ALDH3A1 or ALDH1A1/ALDH2 occurs in a taxon-specific manner, supporting the protective role of these ALDHs in cornea against the UV-induced oxidative damage.     

Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes.     

Accelerated Fatty Acid Oxidation in Muscle Averts Fasting-induced Hepatic Steatosis in SJL/J Mice

The accumulation of triglycerides (TG) in the liver, designated hepatic steatosis, is characteristically associated with obesity and insulin resistance, but it can also develop after fasting. Here, we show that fasting-induced hepatic steatosis is under genetic control in inbred mice. After a 24-h fast, C57BL/6J mice and SJL/J mice both lost more than 20% of body weight and ∼60% of total body TG. In C57BL/6J mice, TG accumulated in liver, producing frank steatosis. In striking contrast, SJL/J mice failed to accumulate any hepatic TG even though they lost nearly as much adipose tissue mass as the C57BL/6J mice. Mice from five other inbred strains developed fasting-induced steatosis like the C57BL/6J mice. Measurements of the uptake of free fatty acids (FA) in vivo and in vitro demonstrated that SJL/J mice were protected from steatosis because their heart and skeletal muscle took up and oxidized twice as much FA as compared with C57BL/6J mice. As a result of this muscle diversion, serum-free FA and ketone bodies rose much less after fasting in SJL/J mice as compared with C57BL/6J mice. When livers of SJL/J and C57BL/6J mice were perfused with similar concentrations of FA, the livers took up and esterified similar amounts. We conclude that SJL/J mice express one or more variant genes that lead to enhanced FA uptake and oxidation in muscle, thereby sparing the liver from FA overload in the fasting state.Liver and adipose tissue coordinate metabolic responses to oscillations in nutrient availability (1, 2). In the postprandial state, the liver secretes triglycerides (TG)⁴ into the blood in very low-density lipoproteins (VLDL). In adipose tissue, lipoprotein lipase hydrolyzes the TG, producing fatty acids (FA) and monoglycerides that enter fat cells for reesterification and storage as TG (1). The activity of adipose tissue lipoprotein lipase is enhanced by the postprandial rise in insulin. At the same time, insulin inhibits lipolysis of stored TG in fat cells, assuring that the TG will be retained in the cells (3).Under fasting conditions, insulin falls and the inhibitory effect of insulin on adipose tissue lipolysis is diminished. The released FA enters the blood and is used as an energy source in liver, heart, and skeletal muscle. In the liver, excess FA are either re-esterified into TG for intracellular storage or oxidized and secreted as ketone bodies, which become the main energy source for the brain. In skeletal muscle during fasting, FA are oxidized to CO₂ (1, 2).We (4–6) and others (7) previously reported that livers of mice accumulate large amounts of TG after fasting for 6–24 h. In the current study, we screened 7 strains of inbred mice to study the genetic control of fasting-induced hepatic TG accumulation. Mice from 6 of 7 strains exhibited fasting-induced fatty liver. In the unique mouse strain (SJL/J), hepatic TG failed to accumulate after a 24-h fast even though the SJL/J mice lost amounts of body weight and adipose tissue that were similar to those of the other 6 strains. To trace the mechanism for the difference in hepatic TG accumulation, we conducted extensive comparisons of SJL/J mice and C57BL/6J mice. We provide evidence that mice from both strains release comparable amounts of FA from adipose tissue into blood after fasting. In the SJL/J mice, the bulk of these FA are taken up by muscle and oxidized. In C57BL/6J mice, FA uptake in muscle is comparatively low, and the excess FA are taken up by the liver where they are converted to TG. Thus, genetic control of muscle FA uptake determines the level of hepatic TG accumulation in fasted mice.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

A mouse model for nonalcoholic steatohepatitis

Background

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of the growing metabolic syndrome epidemic that could progress to cirrhosis. Animal models adequately mimicking this condition in humans are scanty.

Aim

The objective of our study was to investigate whether high-fat diets (HFD) with adequate methionine and choline levels can induce pathophysiological features typical of human NASH in C57BL/6J mice.

Methods

Forty C57BL/6J mice, divided into control and high-fat (HF) groups, were fed low-fat diet and HFD, ad libitum respectively for 20 weeks. At the end of 20 weeks, animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. Adipose tissue depots were collected and liver samples were processed for histological examination.

Results

High-fat feeding led to increased triglyceride accumulation in the liver (8.9 μmol/100 mg liver tissue vs. 2.6 μmol/100 mg for control) and induced histopathological features of human NASH including hepatic steatosis, ballooning inflammation and fibrosis. Expressions of proteins and chemokines predominant in NASH including collagens I, III and IV and platelet derived growth factor (PDGF) A and B were significantly higher in animals fed the HFD. Liver enzymes alanine transaminase, aspartate transaminase and alkaline phosphatase were significantly (P<.05) elevated in the HF group compared to controls. Mice on HFD also developed hyperglycemia, hyperinsulinemia, hypoadiponectinemia along with elevated tumor necrosis factor α, resistin, leptin, free fatty acids, transforming growth factor β and malondialdehyde levels that characterize NASH in humans.

Conclusion

Long-term HF feeding with adequate methionine and choline can induce many of the pathophysiological features typical of human NASH in C57BL/6J mice.     

Resistance to Diet-Induced Obesity and Associated Metabolic Perturbations in Haploinsufficient Monocarboxylate Transporter 1 Mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 ^+/− mice developed normally. However, when fed high fat diet (HFD), MCT1 ^+/− mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 ^+/+ mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 ^+/− mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O₂ consumption and CO₂ production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 ^+/+ mice when fed HFD, were reduced in MCT1 ^+/− mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 ^+/+ mice under high fat diet was prevented in the liver of MCT1 ^+/− mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.     

Comparative biochemistry of murine arylsulfatase B

Arylsulfatase B was purified 4500-fold from liver and kidney of C57BL/6J mice. Hepatic and renal arysulfatase B are apparently determined by a single structural locus; however, posttranslational modification introduces inter- and intratissue microheterogeneity. Partially purified enzyme from C57BL/6J, A/J, C3H/HeJ, and SWR/J mice has similar catalytic properties. The 4500-fold-purified arylsulfatase B from SWR/J and C3H/HeJ mice was more thermostable than that from C57BL/6J and A/J mice, strongly suggesting that the thermostability difference reflects an alteration of the primary structure of the enzyme. Thermal stability of arylsulfatase B was pH dependent and markedly influenced by buffer anion. Variation of thermostability did not appear accountable for the observed activity variation among these strains; however, this possibility cannot be rigorously excluded by presently available data. Thirty-five murine strains were found to possess the As-1 ^a allele (thermostable enzyme), while As-1 ^b was largely restricted to A and C57 strains.This research was supported by PHS Biomedical Sciences Research Support Grant RR-07030.