Acetaminophen-Induced Hepatotoxicity in Obesity and Nonalcoholic Fatty Liver Disease: A Critical Review.

The epidemic of obesity, type 2 diabetes and nonalcoholic liver disease (NAFLD) favors drug consumption, which augments the risk of adverse events including liver injury. For more than 30 years, a series of experimental and clinical investigations reported or suggested that the common pain reliever acetaminophen (APAP) could be more hepatotoxic in obesity and related metabolic diseases, at least after an overdose. Nonetheless, several investigations did not reproduce these data. This discrepancy might come from the extent of obesity and steatosis, accumulation of specific lipid species, mitochondrial dysfunction and diabetes-related parameters such as ketonemia and hyperglycemia. Among these factors, some of them seem pivotal for the induction of cytochrome P450 2E1 (CYP2E1), which favors the conversion of APAP to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). In contrast, other factors might explain why obesity and NAFLD are not always associated with more frequent or more severe APAP-induced acute hepatotoxicity, such as increased volume of distribution in the body, higher hepatic glucuronidation and reduced CYP3A4 activity. Accordingly, the occurrence and outcome of APAP-induced liver injury in an obese individual with NAFLD would depend on a delicate balance between metabolic factors that augment the generation of NAPQI and others that can mitigate hepatotoxicity.

Xenobiotic-Induced Aggravation of Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD), which is often linked to obesity, encompasses a large spectrum of hepatic lesions, including simple fatty liver, steatohepatitis, cirrhosis and hepatocellular carcinoma. Besides nutritional and genetic factors, different xenobiotics such as pharmaceuticals and environmental toxicants are suspected to aggravate MAFLD in obese individuals. More specifically, pre-existing fatty liver or steatohepatitis may worsen, or fatty liver may progress faster to steatohepatitis in treated patients, or exposed individuals. The mechanisms whereby xenobiotics can aggravate MAFLD are still poorly understood and are currently under deep investigations. Nevertheless, previous studies pointed to the role of different metabolic pathways and cellular events such as activation of de novo lipogenesis and mitochondrial dysfunction, mostly associated with reactive oxygen species overproduction. This review presents the available data gathered with some prototypic compounds with a focus on corticosteroids and rosiglitazone for pharmaceuticals as well as bisphenol A and perfluorooctanoic acid for endocrine disruptors. Although not typically considered as a xenobiotic, ethanol is also discussed because its abuse has dire consequences on obese liver.

Role of Mitochondrial Cytochrome P450 2E1 in Healthy and Diseased Liver.

Cytochrome P450 2E1 (CYP2E1) is pivotal in hepatotoxicity induced by alcohol abuse and different xenobiotics. In this setting, CYP2E1 generates reactive metabolites inducing oxidative stress, mitochondrial dysfunction and cell death. In addition, this enzyme appears to play a role in the progression of obesity-related fatty liver to nonalcoholic steatohepatitis. Indeed, increased CYP2E1 activity in nonalcoholic fatty liver disease (NAFLD) is deemed to induce reactive oxygen species overproduction, which in turn triggers oxidative stress, necroinflammation and fibrosis. In 1997, Avadhani's group reported for the first time the presence of CYP2E1 in rat liver mitochondria, and subsequent investigations by other groups confirmed that mitochondrial CYP2E1 (mtCYP2E1) could be found in different experimental models. In this review, we first recall the main features of CYP2E1 including its role in the biotransformation of endogenous and exogenous molecules, the regulation of its expression and activity and its involvement in different liver diseases. Then, we present the current knowledge on the physiological role of mtCYP2E1, its contribution to xenobiotic biotransformation as well as the mechanism and regulation of CYP2E1 targeting to mitochondria. Finally, we discuss experimental investigations suggesting that mtCYP2E1 could have a role in alcohol-associated liver disease, xenobiotic-induced hepatotoxicity and NAFLD.

Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanism-based toxicity.

Steatosis is a liver lesion reported with numerous pharmaceuticals. Prior studies showed that severe impairment of mitochondrial fatty acid oxidation (mtFAO) constantly leads to lipid accretion in liver. However, much less is known about the mechanism(s) of drug-induced steatosis in the absence of severe mitochondrial dysfunction, although previous studies suggested the involvement of mild-to-moderate inhibition of mtFAO, increased de novo lipogenesis (DNL), and impairment of very low-density lipoprotein (VLDL) secretion. The objective of our study, mainly carried out in human hepatoma HepaRG cells, was to investigate these 3 mechanisms with 12 drugs able to induce steatosis in human: amiodarone (AMIO, used as positive control), allopurinol (ALLO), D-penicillamine (DPEN), 5-fluorouracil (5FU), indinavir (INDI), indomethacin (INDO), methimazole (METHI), methotrexate (METHO), nifedipine (NIF), rifampicin (RIF), sulindac (SUL), and troglitazone (TRO). Hepatic cells were exposed to drugs for 4 days with concentrations decreasing ATP level by less than 30% as compared to control and not exceeding 100 × Cmax. Among the 12 drugs, AMIO, ALLO, 5FU, INDI, INDO, METHO, RIF, SUL, and TRO induced steatosis in HepaRG cells. AMIO, INDO, and RIF decreased mtFAO. AMIO, INDO, and SUL enhanced DNL. ALLO, 5FU, INDI, INDO, SUL, RIF, and TRO impaired VLDL secretion. These seven drugs reduced the mRNA level of genes playing a major role in VLDL assembly and also induced endoplasmic reticulum (ER) stress. Thus, in the absence of severe mitochondrial dysfunction, drug-induced steatosis can be triggered by different mechanisms, although impairment of VLDL secretion seems more frequently involved, possibly as a consequence of ER stress.

Cytochrome P450 2E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis.

Acetaminophen (APAP) hepatotoxicity is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a highly toxic metabolite generated by cytochrome P450 2E1 (CYP2E1). Thus, pathological conditions increasing CYP2E1 activity can favour APAP-induced liver injury, which is characterized by massive hepatocellular necrosis and secondary sterile inflammation. In a recent work, Wang et al. showed that APAP-induced hepatotoxicity was exacerbated in a murine model of type 1 diabetes induced by the administration of streptozotocin (STZ). Higher hepatotoxicity was in particular associated with a stronger proinflammatory response of the resident macrophages. Although the authors carried out numerous investigations, they did not study hepatic CYP2E1, nor discussed the possible role of this enzyme in the exacerbation of APAP hepatotoxicity. However, numerous investigations reported hepatic CYP2E1 induction in STZ-treated rodents, which could be secondary to insulinopenia and ketosis. This commentary also discusses the role of insulin resistance in CYP2E1 induction observed in obesity and nonalcoholic fatty liver disease. Investigators studying APAP-induced liver injury in the context of insulinopenia or hyperinsulinemia are thus encouraged to consider CYP2E1 as a significant player in the observed phenotypic changes.

Moderate chronic ethanol consumption exerts beneficial effects on nonalcoholic fatty liver in mice fed a high-fat diet: possible role of higher formation of triglycerides enriched in monounsaturated fatty acids.

PURPOSE: Several clinical studies suggested that light-to-moderate alcohol intake could alleviate nonalcoholic fatty liver disease (NAFLD), but the underlying mechanism is still poorly understood. METHODS: Mice fed a high-fat diet (HFD) were submitted or not to moderate ethanol intake for 3 months (ca. 10 g/kg/day) via drinking water. Biochemical, analytical and transcriptomic analyses were performed in serum and liver. RESULTS: Serum ethanol concentrations in ethanol-treated HFD mice comprised between 0.5 and 0.7 g/l throughout the experiment. NAFLD improvement was observed in ethanol-treated HFD mice as assessed by reduced serum transaminase activity. This was associated with less microvesicular and more macrovacuolar steatosis, the absence of apoptotic hepatocytes and a trend towards less fibrosis. Liver lipid analysis showed increased amounts of fatty acids incorporated in triglycerides and phospholipids, reduced proportion of palmitic acid in total lipids and higher desaturation index, thus suggesting enhanced stearoyl-coenzyme A desaturase activity. mRNA expression of several glycolytic and lipogenic enzymes was upregulated. Genome-wide expression profiling and gene set enrichment analysis revealed an overall downregulation of the expression of genes involved in collagen fibril organization and leukocyte chemotaxis and an overall upregulation of the expression of genes involved in oxidative phosphorylation and mitochondrial respiratory chain complex assembly. In addition, mRNA expression of several proteasome subunits was upregulated in ethanol-treated HFD mice. CONCLUSIONS: Moderate chronic ethanol consumption may alleviate NAFLD by several mechanisms including the generation of non-toxic lipid species, reduced expression of profibrotic and proinflammatory genes, restoration of mitochondrial function and possible stimulation of proteasome activity.

Drug-induced liver injury in obesity and nonalcoholic fatty liver disease.

Obesity is commonly associated with nonalcoholic fatty liver (NAFL), a benign condition characterized by hepatic lipid accumulation. However, NAFL can progress in some patients to nonalcoholic steatohepatitis (NASH) and then to severe liver lesions including extensive fibrosis, cirrhosis and hepatocellular carcinoma. The entire spectrum of these hepatic lesions is referred to as nonalcoholic fatty liver disease (NAFLD). The transition of simple fatty liver to NASH seems to be favored by several genetic and environmental factors. Different experimental and clinical investigations showed or suggested that obesity and NAFLD are able to increase the risk of hepatotoxicity of different drugs. Some of these drugs may cause more severe and/or more frequent acute liver injury in obese individuals whereas others may trigger the transition of simple fatty liver to NASH or may worsen hepatic lipid accumulation, necroinflammation and fibrosis. This review presents the available information regarding drugs that may cause a specific risk in the context of obesity and NAFLD. These drugs, which belong to different pharmacological classes, include acetaminophen, halothane, methotrexate, rosiglitazone and tamoxifen. For some of these drugs, experimental investigations confirmed the clinical observations and unveiled different pathophysiological mechanisms which may explain why these pharmaceuticals are particularly hepatotoxic in obesity and NAFLD. Because obese people often take several drugs for the treatment of different obesity-related diseases, there is an urgent need to identify the main pharmaceuticals that may cause acute liver injury on a fatty liver background or that may enhance the risk of severe chronic liver disease.

Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure.

Exposure to xenobiotics could favor the transition of nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis in obese patients. Recently, we showed in different models of NAFL that benzo[a]pyrene (B[a]P) and ethanol coexposure induced a steatohepatitis-like state. One model was HepaRG cells incubated with stearate and oleate for 2 weeks. In the present study, we wished to determine in this model whether mitochondrial dysfunction and reactive oxygen species (ROS) overproduction could be involved in the occurrence of this steatohepatitis-like state. CRISPR/Cas9-modified cells were also used to specify the role of aryl hydrocarbon receptor (AhR), which is potently activated by B[a]P. Thus, nonsteatotic and steatotic HepaRG cells were treated with B[a]P, ethanol, or both molecules for 2 weeks. B[a]P/ethanol coexposure reduced mitochondrial respiratory chain activity, mitochondrial respiration, and mitochondrial DNA levels and induced ROS overproduction in steatotic HepaRG cells. These deleterious effects were less marked or absent in steatotic cells treated with B[a]P alone or ethanol alone and in nonsteatotic cells treated with B[a]P/ethanol. Our study also disclosed that B[a]P/ethanol-induced impairment of mitochondrial respiration was dependent on AhR activation. Hence, mitochondrial dysfunction and ROS generation could explain the occurrence of a steatohepatitis-like state in steatotic HepaRG cells exposed to B[a]P and ethanol.

Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells.

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with nine hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether drug-induced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver. Among the nine drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in nonsteatotic cells. None of the nine drugs decreased mtDNA levels. However, increased mtDNA was observed with five drugs, especially in nonsteatotic cells. The mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), PGC1ß, and AMP-activated protein kinase α-subunit. Hence, drug-induced impairment of mtDNA translation might not be rare, and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload.

Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity.

BACKGROUND: Obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which refers to a large spectrum of hepatic lesions including fatty liver, nonalcoholic steatohepatitis (NASH) and cirrhosis. Different investigations showed or suggested that obesity and NAFLD are able to increase the risk of hepatotoxicity of different drugs. Some of these drugs could induce more frequently an acute hepatitis in obese individuals whereas others could worsen pre-existing NAFLD. AIM: The main objective of the present review was to collect the available information regarding the role of NAFLD as risk factor for drug-induced hepatotoxicity. For this purpose, we performed a data-mining analysis using different queries including drug-induced liver injury (or DILI), drug-induced hepatotoxicity, fatty liver, nonalcoholic fatty liver disease (or NAFLD), steatosis and obesity. The main data from the collected articles are reported in this review and when available, some pathophysiological hypotheses are put forward. RELEVANCE FOR PATIENTS: Drugs that could pose a potential risk in obese patients include compounds belonging to different pharmacological classes such as acetaminophen, halothane, methotrexate, rosiglitazone, stavudine and tamoxifen. For some of these drugs, experimental investigations in obese rodents confirmed the clinical observations and unveiled different pathophysiological mechanisms which could explain why these pharmaceuticals are particularly hepatotoxic in obesity and NAFLD. Other drugs such as pentoxifylline, phenobarbital and omeprazole might also pose a risk but more investigations are required to determine whether this risk is significant or not. Because obese people often take several drugs for the treatment of different obesity-related diseases such as type 2 diabetes, hyperlipidemia and coronary heart disease, it is urgent to identify the main pharmaceuticals that can cause acute hepatitis on a fatty liver background or induce NAFLD worsening.

Bisphenol a induces steatosis in HepaRG cells using a model of perinatal exposure.

Human exposure to bisphenol A (BPA) could favor obesity and related metabolic disorders such as hepatic steatosis. Investigations in rodents have shown that these deleterious effects are observed not only when BPA is administered during the adult life but also with different protocols of perinatal exposure. Whether perinatal BPA exposure could pose a risk in human is currently unknown, and thus appropriate in vitro models could be important to tackle this major issue. Accordingly, we determined whether long-term BPA treatment could induce steatosis in human HepaRG cells by using a protocol mimicking perinatal exposure. To this end, the kinetics of expression of seven proteins differentially expressed during liver development was determined during a 4-week period of cell culture required for proliferation and differentiation. By analogy with data reported in rodents and humans, our results indicated that the period of cell culture around day 15 and day 18 after seeding could be considered as the "natal" period. Consequently, HepaRG cells were treated for 3 weeks with BPA (from 0.2 to 2000 nM), with a treatment starting during the proliferating period. BPA was able to induce steatosis with a nonmonotonic dose response profile, with significant effects on neutral lipids and triglycerides observed for the 2 nM concentration. However, the expression of many enzymes involved in lipid and carbohydrate homeostasis was unchanged in exposed HepaRG cells. The expression of other potential BPA targets and enzymes involved in BPA biotransformation was also determined, giving answers as well as new questions regarding the mechanisms of action of BPA. Hence, HepaRG cells provide a valuable model that can prove useful for the toxicological assessment of endocrine disruptors on hepatic metabolisms, in particular in the developing liver. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1024-1036, 2017.

Chronic and low exposure to a pharmaceutical cocktail induces mitochondrial dysfunction in liver and hyperglycemia: Differential responses between lean and obese mice.

Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.

Mid-infrared fibre evanescent wave spectroscopy of serum allows fingerprinting of the hepatic metabolic status in mice.

Non-alcoholic fatty liver disease is associated with obesity, diabetes, and metabolic syndrome. The detection of systemic metabolic changes associated with alterations in the liver status during non-alcoholic fatty liver disease could improve patient follow-up. The aim of the present study was to evaluate the potential of mid-infrared fibre evanescent wave spectroscopy as a minimum-invasive method for evaluating the liver status during non-alcoholic fatty liver disease. Seventy-five mice were subjected to a control, high-fat or high-fat-high carbohydrate diets. We analysed the serum biochemical parameters and mRNA levels of hepatic genes by quantitative RT-PCR. Steatosis was quantified by image analysis. The mid-infrared spectra were acquired from serum, and then analysed to develop a predictive model of the steatosis level. Animals subjected to enriched diets were obese. Hepatic steatosis was found in all animals. The relationship between the spectroscopy-predicted and observed levels of steatosis, expressed as percentages of the liver biopsy area, was not linear. A transition around 10% steatosis was observed, leading us to consider two distinct predictive models (<10% and >10%) based on two different sets of discriminative spectral variables. The model performance was evaluated using random cross-validation (10%). The hypothesis that additional metabolic changes occur beyond this transition was supported by the fact that it was associated with increased serum ALT levels, and Col1α1 chain mRNA levels. Our data suggest that mid-infrared spectroscopy combined with statistical analysis allows identifying serum mid-infrared signatures that reflect the liver status during non-alcoholic fatty liver disease.

The effect of water suppression on the hepatic lipid quantification, as assessed by the LCModel, in a preclinical and clinical scenario.

OBJECTIVE: To investigate the effect of water suppression on the hepatic lipid quantification, using the LCModel. MATERIALS AND METHODS: MR spectra with and without water suppression were acquired in the liver of mice at 4.7 T and patients at 3 T, and processed with the LCModel. The Cramér-Rao Lower Bound (CRLB) values of the seven lipid resonances were determined to assess the impact of water suppression on hepatic lipid quantification. A paired t test was used for comparison between the CRLBs obtained with and without water suppression. RESULTS: For the preclinical data, in the high (low) fat fraction subset an overall impairment in hepatic lipid quantification, i.e. an increase of CRLBs (no significant change of CRLBs) was observed in spectra acquired with water suppression. For the clinical data, there were no substantial changes in the CRLB with water suppression. Because (1) the water suppression does not overall improve the quantification of the lipid resonances and (2) the MR spectrum without water suppression is always acquired for fat fraction calculation, the optimal data-acquisition strategy for liver MRS is to acquire only the MR spectrum without water suppression. CONCLUSION: For quantification of hepatic lipid resonances, it is advantageous to perform MR spectroscopy without water suppression in a clinical and preclinical scenario (at moderate fields).

Chronic exposure to low doses of pharmaceuticals disturbs the hepatic expression of circadian genes in lean and obese mice.

Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.

Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease.

The worldwide epidemic of obesity and insulin resistance favors nonalcoholic fatty liver disease (NAFLD). Insulin resistance (IR) in the adipose tissue increases lipolysis and the entry of nonesterified fatty acids (NEFAs) in the liver, whereas IR-associated hyperinsulinemia promotes hepatic de novo lipogenesis. However, several hormonal and metabolic adaptations are set up in order to restrain hepatic fat accumulation, such as increased mitochondrial fatty acid oxidation (mtFAO). Unfortunately, these adaptations are usually not sufficient to reduce fat accumulation in liver. Furthermore, enhanced mtFAO without concomitant up-regulation of the mitochondrial respiratory chain (MRC) activity induces reactive oxygen species (ROS) overproduction within different MRC components upstream of cytochrome c oxidase. This event seems to play a significant role in the initiation of oxidative stress and subsequent development of nonalcoholic steatohepatitis (NASH) in some individuals. Experimental investigations also pointed to a progressive reduction of MRC activity during NAFLD, which could impair energy output and aggravate ROS overproduction by the damaged MRC. Hence, developing drugs that further increase mtFAO and restore MRC activity in a coordinated manner could ameliorate steatosis, but also necroinflammation and fibrosis by reducing oxidative stress. In contrast, physicians should be aware that numerous drugs in the current pharmacopoeia are able to induce mitochondrial dysfunction, which could aggravate NAFLD in some patients.

Melanocortin-3 receptors and metabolic homeostasis.

Attenuated activity of the central nervous melanocortin system causes obesity and insulin resistance. Obese rodents treated with melanocortins exhibit improvements in obesity and metabolic homeostasis that are not mutually dependent, suggesting metabolic actions that are independent of weight changes. These responses are generally thought to involve G-protein-coupled receptors expressed in the brain. Melanocortin-4 receptors (MC4Rs) regulate satiety and autonomic nervous system and thyroid function. MC3Rs are expressed in hypothalamic and limbic regions involved in controlling ingestive behaviors and autonomic function. Mc3r-/- mice exhibit increased adiposity and an accelerated diet-induced obesity. While this phenotype is not dependent on hyperphagia, data on the regulation of food intake by MC3Rs are inconsistent. Recent investigations by our laboratory suggest a unique combination of behavioral and metabolic disorders in Mc3r-/- mice. MC3Rs are critical for the expression of the anticipatory response and metabolic homeostasis when food intake occurs outside the normal voluntary rhythms driven by photoperiod. Using a Cre-Lox strategy, we can now investigate MC3Rs expressed in different brain regions and organ systems in the periphery. While focusing on the functions of neural MC3Rs, early results suggest an additional layer of complexity with central and peripheral MC3Rs involved in the defense of body weight.

Differences in early acetaminophen hepatotoxicity between obese ob/ob and db/db mice.

Clinical investigations suggest that hepatotoxicity after acetaminophen (APAP) overdose could be more severe in the context of obesity and nonalcoholic fatty liver disease. The pre-existence of fat accumulation and CYP2E1 induction could be major mechanisms accounting for such hepatic susceptibility. To explore this issue, experiments were performed in obese diabetic ob/ob and db/db mice. Preliminary investigations performed in male and female wild-type, ob/ob, and db/db mice showed a selective increase in hepatic CYP2E1 activity in female db/db mice. However, liver triglycerides in these animals were significantly lower compared with ob/ob mice. Next, APAP (500 mg/kg) was administered in female wild-type, ob/ob, and db/db mice, and investigations were carried out 0.5, 2, 4, and 8 h after APAP intoxication. Liver injury 8 h after APAP intoxication was higher in db/db mice, as assessed by plasma transaminases, liver histology, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. In db/db mice, however, the extent of hepatic glutathione depletion, levels of APAP-protein adducts, c-Jun N-terminal kinase activation, changes in gene expression, and mitochondrial DNA levels were not greater compared with the other genotypes. Furthermore, in the db/db genotype plasma lactate and ß-hydroxybutyrate were not specifically altered, whereas the plasma levels of APAP-glucuronide were intermediary between wild-type and ob/ob mice. Thus, early APAP-induced hepatotoxicity was greater in db/db than ob/ob mice, despite less severe fatty liver and similar basal levels of transaminases. Hepatic CYP2E1 induction could have an important pathogenic role when APAP-induced liver injury occurs in the context of obesity and related metabolic disorders.

Genetic dissection of the functions of the melanocortin-3 receptor, a seven-transmembrane G-protein-coupled receptor, suggests roles for central and peripheral receptors in energy homeostasis.

The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.

Homeostastic and non-homeostatic functions of melanocortin-3 receptors in the control of energy balance and metabolism.

The central nervous melanocortin system is a neural network linking nutrient-sensing systems with hypothalamic, limbic and hindbrain neurons regulating behavior and metabolic homeostasis. Primary melanocortin neurons releasing melanocortin receptor ligands residing in the hypothalamic arcuate nucleus are regulated by nutrient-sensing and metabolic signals. A smaller group of primary neurons releasing melanocortin agonists in the nucleus tractus solitarius in the brainstem are also regulated by signals of metabolic state. Two melanocortin receptors regulate energy homeostasis. Melanocortin-4 receptors regulate satiety and autonomic outputs controlling peripheral metabolism. The functions of melanocortin-3 receptors (MC3R) expressed in hypothalamic and limbic structures are less clear. Here we discuss published data and preliminary observations from our laboratory suggesting that neural MC3R regulate inputs into systems governing the synchronization of rhythms in behavior and metabolism with nutrient intake. Mice subjected to a restricted feeding protocol, where a limited number of calories are presented at a 24h interval, rapidly exhibit bouts of increased wakefulness and activity which anticipate food presentation. The full expression of these responses is dependent on MC3R. Moreover, MC3R knockout mice are unique in exhibiting a dissociation of weight loss from improved glucose homeostasis when subject to a restricted feeding protocol. While mice lacking MC3R fed ad libitum exhibit normal to moderate hyperinsulinemia, when subjected to a restricted protocol they develop hyperglycemia, glucose intolerance, and dyslipidemia. Collectively, our data suggest that the central nervous melanocortin system is a point convergence in the control of energy balance and the expression of rhythms anticipating nutrient intake.