Nicotinamide N-methyltransferase upregulation contributes to palmitate-elicited peroxisome proliferator-activated receptor transactivation in hepatocytes.

Peroxisome proliferator-activated receptor γ (PPARγ) plays a pivotal role in regulating lipid metabolism and hepatic PPARγ transactivation contributes to fatty liver development. Fatty acids (FAs) are well-known endogenous ligands for PPARγ. Palmitate, a 16-C saturated FA (SFA) and the most abundant SFA in human circulation, is a strong inducer of hepatic lipotoxicity, a central pathogenic factor for various fatty liver diseases. In this study, using both alpha mouse liver 12 (AML12) and primary mouse hepatocytes, we investigated the effects of palmitate on hepatic PPARγ transactivation and underlying mechanisms, as well as the role of PPARγ transactivation in palmitate-induced hepatic lipotoxicity, all of which remain ambiguous currently. Our data revealed that palmitate exposure was concomitant with both PPARγ transactivation and upregulation of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing the degradation of nicotinamide, the predominant precursor for cellular NAD+ biosynthesis. Importantly, we discovered that PPARγ transactivation by palmitate was blunted by NNMT inhibition, suggesting that NNMT upregulation plays a mechanistic role in PPARγ transactivation. Further investigations uncovered that palmitate exposure is associated with intracellular NAD+ decline and NAD+ replenishment with NAD+-enhancing agents, nicotinamide and nicotinamide riboside, obstructed palmitate-induced PPARγ transactivation, implying that cellular NAD+ decline resulted from NNMT upregulation represents a potential mechanism behind palmitate-elicited PPARγ transactivation. At last, our data showed that the PPARγ transactivation marginally ameliorated palmitate-induced intracellular triacylglycerol accumulation and cell death. Collectively, our data provided the first-line evidence supporting that NNMT upregulation plays a mechanistic role in palmitate-elicited PPARγ transactivation, potentially through reducing cellular NAD+ contents.NEW & NOTEWORTHY Hepatic PPARγ transactivation contributes to fatty liver development. Saturated fatty acids (SFAs) induce hepatic lipotoxicity. Here, we investigated whether and how palmitate, the most abundant SFA in the human blood, affects PPARγ transactivation in hepatocytes. We reported for the first time that upregulation of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing the degradation of nicotinamide, the predominant precursor for cellular NAD+ biosynthesis, plays a mechanistic role in regulating palmitate-elicited PPARγ transactivation through reducing intracellular NAD+ contents.

ATF4-mediated CD36 upregulation contributes to palmitate-induced lipotoxicity in hepatocytes.

Hepatic lipotoxicity plays a central role in the pathogenesis of nonalcoholic fatty liver disease; however, the underlying mechanisms remain elusive. Here, using both cultured hepatocytes (AML-12 cells and primary mouse hepatocytes) and the liver-specific gene knockout mice, we investigated the mechanisms underlying palmitate-elicited upregulation of CD36, a class B scavenger receptor mediating long-chain fatty acids uptake, and its role in palmitate-induced hepatolipotoxicity. We found that palmitate upregulates hepatic CD36 expression. Despite being a well-established target gene of PPARγ transactivation, our data demonstrated that the palmitate-induced CD36 upregulation in hepatocytes is in fact PPARγ-independent. We previously reported that the activation of ATF4, one of three canonical pathways activated upon endoplasmic reticulum (ER) stress induction, contributes to palmitate-triggered lipotoxicity in hepatocytes. In this study, our data revealed for the first time that ATF4 plays a critical role in mediating hepatic CD36 expression. Genetic inhibition of ATF4 attenuated CD36 upregulation induced by either palmitate or ER stress inducer tunicamycin in hepatocytes. In mice, tunicamycin upregulates liver CD36 expression, whereas hepatocyte-specific ATF4 knockout mice manifest lower hepatic CD36 expression when compared with control animals. Furthermore, we demonstrated that CD36 upregulation upon palmitate exposure represents a feedforward mechanism in that siRNA knockdown of CD36 in hepatocytes blunted ATF4 activation induced by both palmitate and tunicamycin. Finally, we confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation. Collectively, our data demonstrate that CD36 upregulation by ATF4 activation contributes to palmitate-induced hepatic lipotoxicity.NEW & NOTEWORTHY We provided the initial evidence that ATF4 is a principal transcription factor mediating hepatic CD36 expression in that both palmitate- and ER stress-elicited CD36 upregulation was blunted by ATF4 gene knockdown in hepatocytes, and hepatocyte-specific ATF4 knockout mice manifested lower hepatic CD36 expression. We further confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation in response to exogenous palmitate exposure.

ATF4 activation promotes hepatic mitochondrial dysfunction by repressing NRF1-TFAM signalling in alcoholic steatohepatitis.

OBJECTIVE: Mitochondrial dysfunction plays a dominant role in the pathogenesis of alcoholic liver disease (ALD); however, the underlying mechanisms remain to be fully understood. We previously found that hepatic activating transcription factor 4 (ATF4) activation was associated with mitochondrial dysfunction in ALD. This study aimed to investigate the function and mechanism of ATF4 in alcohol-induced hepatic mitochondrial dysfunction. DESIGN: ATF4 activation was detected in the livers of patients with severe alcoholic hepatitis (AH). The role of ATF4 and mitochondrial transcription factor A (TFAM) in alcohol-induced liver damage was determined in hepatocyte-specific ATF4 knockout mice and liver-specific TFAM overexpression mice, respectively. RESULTS: Hepatic PERK-eIF2α-ATF4 ER stress signalling was upregulated in patients with AH. Hepatocyte-specific ablation of ATF4 in mice ameliorated alcohol-induced steatohepatitis. ATF4 ablation also attenuated alcohol-impaired mitochondrial biogenesis and respiratory function along with the restoration of TFAM. Cell studies confirmed that TFAM expression was negatively regulated by ATF4. TFAM silencing in hepatoma cells abrogated the protective effects of ATF4 knockdown on ethanol-mediated mitochondrial dysfunction and cell death. Moreover, hepatocyte-specific TFAM overexpression in mice attenuated alcohol-induced mitochondrial dysfunction and liver damage. Mechanistic studies revealed that ATF4 repressed the transcription activity of nuclear respiratory factor 1 (NRF1), a key regulator of TFAM, through binding to its promoter region. Clinical relevance among ATF4 activation, NRF1-TFAM pathway disruption and mitochondrial dysfunction was validated in the livers of patients with AH. CONCLUSION: This study demonstrates that hepatic ATF4 plays a pathological role in alcohol-induced mitochondrial dysfunction and liver injury by disrupting the NRF1-TFAM pathway.

Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

Defined as the dysfunction and/or cell death caused by toxic lipids accumulation in hepatocytes, hepatic lipotoxicity plays a pathological role in nonalcoholic fatty liver disease. The cellular and molecular mechanisms underlying lipotoxicity remain to be elucidated. In this study, using AML12 cells, a nontransformed murine hepatocyte cell line, exposed to palmitate (a 16-C saturated fatty acid) as an experimental model, we investigated the role and mechanisms of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing nicotinamide methylation and degradation, in hepatic lipotoxicity. We initially identified activating transcription factor 4 (ATF4) as a major transcription factor for hepatic NNMT expression. Here, we demonstrated that palmitate upregulates NNMT expression via activating ATF4 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent mechanism in that mTORC1 inhibition by both Torin1 and rapamycin attenuated ATF4 activation and NNMT upregulation. We further demonstrated that the mTORC1-dependent ATF4 activation is an integral signaling event of unfolded protein response (UPR) as both ATF4 activation and NNMT upregulation by tunicamycin, a well-documented endoplasmic reticulum (ER) stress inducer, are blunted when hepatocytes were pretreated with Torin1. Importantly, our data uncovered that NNMT upregulation contributes to palmitate-induced hepatotoxicity as NNMT inhibition, via either pharmacological (NNMT inhibitors) or genetic approach (siRNA transfection), provided protection against palmitate lipotoxicity. Our further mechanistic exploration identified protein kinase A (PKA) activation to contribute, at least, partially to the protective effect of NNMT inhibition against lipotoxicity. Collectively, our data demonstrated that NNMT upregulation by the mTORC1-ATF4 pathway activation contributes to the development of lipotoxicity in hepatocytes.

Inositol-requiring enzyme 1α links palmitate-induced mTOR activation and lipotoxicity in hepatocytes.

Hepatic lipotoxicity, hepatocyte dysfunction/cell death induced by saturated fatty acids (SFA), plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms remain unclear. Palmitate is the most abundant SFA in the circulation. In this study, via a small-scale screening of chemical inhibitors using AML12 hepatocytes, we identified mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) to be a culprit in palmitate-induced cell death in hepatocytes in that mTOR inhibition is protective against palmitate-induced cell death. The protective effect of mTORC1 inhibition is independent of autophagy induction, as autophagy inhibition failed to ablate the mTORC1 inhibitor-conferred protection. We have previously reported that the endonuclease activity of inositol-requiring enzyme 1α (IRE1α), one of three canonical signaling pathways of endoplasmic reticulum (ER) stress, was implicated in palmitate-induced cell death in hepatocytes. The continuous mechanistic investigation in this study uncovered that IRE1α is a downstream target of mTORC1 activation upon palmitate exposure and the inhibition of either its endonuclease activity or kinase activity protects against the lipotoxic effect of palmitate. Our research further revealed that protein palmitoylation is potentially involved in palmitate-induced mTORC1 activation and lipotoxicity in hepatocytes. 2-Bromopalmitate, a protein palmitoylation inhibitor, ameliorated palmitate-triggered mTORC1 activation, concomitant with the protection of lipotoxicity in hepatocytes. Collectively, our data have identified that mTORC1 and ER stress are coordinately implicated in hepatocyte cell death in response to palmitate exposure and suggest that this pathway may potentially serve as a therapeutic target for the treatment of NAFLD as well as other metabolic disorders involving lipotoxicity.

ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver development.

BACKGROUND & AIMS: N-nicotinamide methyltransferase (NNMT) is emerging as an important enzyme in the regulation of metabolism. NNMT is highly expressed in the liver. However, the exact regulatory mechanism(s) underlying NNMT expression remains unclear and its potential involvement in alcohol-related liver disease (ALD) is completely unknown. METHODS: Both traditional Lieber-De Carli and the NIAAA mouse models of ALD were employed. A small-scale chemical screening assay and a chromatin immunoprecipitation assay were performed. NNMT inhibition was achieved via both genetic (adenoviral short hairpin RNA delivery) and pharmacological approaches. RESULTS: Chronic alcohol consumption induces endoplasmic reticulum (ER) stress and upregulates NNMT expression in the liver. ER stress inducers upregulated NNMT expression in both AML12 hepatocytes and mice. PERK-ATF4 pathway activation is the main contributor to ER stress-mediated NNMT upregulation in the liver. Alcohol consumption fails to upregulate NNMT in liver-specific Atf4 knockout mice. Both adenoviral NNMT knockdown and NNMT inhibitor administration prevented fatty liver development in response to chronic alcohol feeding; this was also associated with the downregulation of an array of genes involved in de novo lipogenesis, including Srebf1, Acaca, Acacb and Fasn. Further investigations revealed that activation of the lipogenic pathway by NNMT was independent of its NAD+-enhancing action; however, increased cellular NAD+, resulting from NNMT inhibition, was associated with marked liver AMPK activation. CONCLUSIONS: ER stress, specifically PERK-ATF4 pathway activation, is mechanistically involved in hepatic NNMT upregulation in response to chronic alcohol exposure. Overexpression of NNMT in the liver plays an important role in the pathogenesis of ALD. LAY SUMMARY: In this study, we show that nicotinamide methyltransferase (NNMT) - the enzyme that catalyzes nicotinamide degradation - is a pathological regulator of alcohol-related fatty liver development. NNMT inhibition protects against alcohol-induced fatty liver development and is associated with suppressed de novo lipogenic activity and enhanced AMPK activation. Thus, our data suggest that NNMT may be a potential therapeutic target for the treatment of alcohol-related liver disease.

Random vibration-driven continuous-wave CRDS system for calibration-free gas concentration measurement.

Random vibrations were employed to pick up each monochromatic component in a continuous-wave cavity ringdown spectroscopy (CRDS) system using a bichromatic laser source. Light frequencies were selected within flat portions of an absorption profile to suppress the jitter in laser frequency during measurements. An interference effect caused by cavity length variations was suppressed by optimizing the initial fit point for each ringdown transient. The difference in exponential decay rates of two frequencies determined the gas mole fraction, and no calibration of empty cavity losses was necessary. Experiments on varying humidity were conducted, and the results agreed with the readings of a commercial hygrometer.

The TLR4-IRE1α pathway activation contributes to palmitate-elicited lipotoxicity in hepatocytes.

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non-alcoholic fatty liver disease (NAFLD); however, the exact mechanism(s) remain to be clearly elucidated. Toll-like receptor (TLR) 4 plays a fundamental role in activating the innate immune system. Intriguingly, hepatocytes express TLR4 and machinery for TLR4 signalling pathway. That liver-specific TLR4 knockout mice are protective against diet-induced NAFLD suggests that hepatocyte TLR4 signalling pathway plays an important role in NAFLD pathogenesis. Herein, using cultured hepatocytes, we sought to directly examine the role of TLR4 signalling pathway in palmitate-elicited hepatotoxicity and to elucidate underlying mechanism(s). Our data reveal that palmitate exposure up-regulates TLR4 expression at both mRNA and protein levels in hepatocytes, which are associated with NF-κB activation. The inhibition of TLR4 signalling pathway through both pharmacological and genetic approaches abolished palmitate-induced cell death, suggesting that TLR4 signalling pathway activation contributes to palmitate-induced hepatotoxicity. Mechanistic investigations demonstrate that inositol-requiring enzyme 1α (IRE1α), one of three major signal transduction pathways activated during endoplasmic reticulum (ER) stress, is the downstream target of palmitate-elicited TLR4 activation and mechanistically implicated in TLR4 activation-triggered cell death in response to palmitate exposure. Collectively, our data identify that the TLR4-IRE1α pathway activation contributes to palmitate-elicited lipotoxicity in hepatocytes. Our findings suggest that targeting TLR4-IRE1α pathway can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism.

Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Attenuated Hepatic Triglyceride Accumulation in Alcoholic Fatty Liver Disease in Mice.

Poly(ADP-ribose) polymerase (PARP) is an NAD-consuming enzyme and its specific role in the pathogenesis of alcoholic fatty liver disease (AFLD) remains elusive. In this study, we applied PJ34 [N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-2-acetamide hydrochloride] to inhibit hepatic PARP activity to examine the corresponding pathologic alteration in AFLD in mice and the underlying molecular mechanism. We found that PJ34 decreased the intracellular triglyceride (TG) content in hepatocytes. Moreover, PJ34 suppressed the gene expression of diglyceride acyltransferases DGAT1 and DGAT2 and elevated intracellular NAD+ levels in hepatocytes. These mechanistic observations were validated in alcohol-fed mice injected with PJ34 intraperitoneally. Our results indicate that the PJ34 injection attenuated hepatic TG accumulation in alcohol-fed mice. Furthermore, PJ34 injection lowered the gene expression of hepatic sterol regulatory element binding protein 1c, DGAT1, and DGAT2, whereas PJ34 injection augmented hepatic NAD+ levels in alcohol-fed mice. Finally, nicotinamide riboside supplementation alleviated hepatic TG accumulation in alcohol-fed mice. These data indicate that applying PARP-specific inhibitor PJ34 by intraperitoneal injection attenuated hepatic NAD+ depletion and TG accumulation in alcohol-fed mice and may be a potential candidate for use in AFLD therapy.

Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity.

Lipotoxicity induced by saturated fatty acids (SFAs) plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanisms remain to be fully elucidated. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide-dependent deacetylase located primarily inside mitochondria. In this study, we demonstrated that an SFA-rich high-fat diet (HFD) was more detrimental to the liver than an isocaloric unsaturated HFD rich in fatty acids. Unexpectedly, SIRT3 expression and activity were significantly elevated in the livers of mice exposed to the SFA-rich HFD. Using cultured HepG2 and AML-12 hepatocytes, we demonstrated that unlike monounsaturated fatty acids, SFAs up-regulate SIRT3 expression and activity. SIRT3 overexpression renders both the liver and hepatocytes susceptible to palmitate-induced cell death, which can be alleviated by SIRT3 small interfering RNA (siRNA) transfection. In contrast, SIRT3 suppression protects hepatocytes from palmitate cytotoxicity. Further studies revealed that SIRT3 acts as a negative regulator of autophagy, thereby enhancing the susceptibility of hepatocytes to SFA-induced cytotoxicity. Mechanistic investigations revealed that SIRT3 overexpression causes manganese superoxide dismutase deacetylation and activation, which depleted intracellular superoxide contents, leading to adenosine monophosphate-activated protein kinase (AMPK) inhibition and mammalian target of rapamycin C1 activation, resulting in autophagy suppression. In contrast, SIRT3 siRNA gene silencing enhanced autophagy flux. A similar result was observed in the liver tissue of SIRT3 knockout mice. CONCLUSION: Our data indicate that SIRT3 is a negative regulator of autophagy whose activation by SFAs contributes to lipotoxicity in hepatocytes and suggest that restraining SIRT3 overactivation can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism. (Hepatology 2017;66:936-952).

Residual vibration control based on a global search method in a high-speed white light scanning interferometer.

To achieve high-speed measurements using white light scanning interferometers, the scanning devices used need to have high feedback gain in closed-loop operations. However, flexure hinges induce a residual vibration that can cause a misidentification of the fringe order. The reduction of this residual vibration is crucial because the highly nonlinear distortions in interferograms lead to clearly incorrect measured profiles. Input shaping can be used to control the amplitude of the residual vibration. The conventional method uses continuous wavelet transform (CWT) to estimate parameters of the scanning device. Our proposed method extracts equivalent modal parameters using a global search algorithm. Due to its simplicity, ease of implementation, and response speed, this global search method outperforms CWT. The delay time is shortened by searching, because fewer modes are needed for the shaper. The effectiveness of the method has been confirmed by the agreement between simulated shaped responses and experimental displacement information from the capacitive sensor inside the scanning device, and the intensity profiles of the interferometer have been greatly improved. An experiment measuring the surface of a silicon wafer is also presented. The method is shown to be effective at improving the intensity profiles and recovering accurate surface topography. Finally, frequency localizations are found to be almost stable with different proportional gains, but their energy distributions change.

Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.

tert-Butylhydroquinone (tBHQ) protects hepatocytes against lipotoxicity via inducing autophagy independently of Nrf2 activation.

Saturated fatty acids (SFAs) induce hepatocyte cell death, wherein oxidative stress is mechanistically involved. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator of cellular antioxidant defense enzymes. Therefore, Nrf2 activation is regarded as an effective strategy against oxidative stress-triggered cellular damage. In this study, tert-butylhydroquinone (tBHQ), a widely used Nrf2 activator, was initially employed to investigate the potential protective role of Nrf2 activation in SFA-induced hepatoxicity. As expected, SFA-induced hepatocyte cell death was prevented by tBHQ in both AML-12 mouse hepatocytes and HepG2 human hepatoma cells. However, the protective effect of tBHQ is Nrf2-independent, because the siRNA-mediated Nrf2 silencing did not abrogate tBHQ-conferred protection. Alternatively, our results revealed that autophagy activation was critically involved in the protective effect of tBHQ on lipotoxicity. tBHQ induced autophagy activation and autophagy inhibitors abolished tBHQ's protection. The induction of autophagy by tBHQ exposure was demonstrated by the increased accumulation of LC3 puncta, LC3-II conversion, and autophagic flux (LC3-II conversion in the presence of proteolysis inhibitors). Subsequent mechanistic investigation discovered that tBHQ exposure activated AMP-activated protein kinase (AMPK) and siRNA-mediated AMPK gene silencing abolished tBHQ-induced autophagy activation, indicating that AMPK is critically involved in tBHQ-triggered autophagy induction. Furthermore, our study provided evidence that tBHQ-induced autophagy activation is required for its Nrf2-activating property. Collectively, our data uncover a novel mechanism for tBHQ in protecting hepatocytes against SFA-induced lipotoxicity. tBHQ-triggered autophagy induction contributes not only to its hepatoprotective effect, but also to its Nrf2-activating property.

Nuclear factor (erythroid-derived 2)-like 2 activation-induced hepatic very-low-density lipoprotein receptor overexpression in response to oxidative stress contributes to alcoholic liver disease in mice.

UNLABELLED: Chronic alcohol consumption leads to hypertriglyceridemia, which is positively associated with alcoholic liver disease (ALD). However, whether and how it contributes to the development of fatty liver and liver injury are largely unknown. In this study we demonstrate that chronic alcohol exposure differently regulates the expression of very-low-density lipoprotein receptor (VLDLR) in adipose tissue and the liver. Whereas adipose tissue VLDLR is significantly down-regulated, its hepatic expression is dramatically increased after chronic alcohol feeding. While HepG2 cells stably overexpressing VLDLR manifests increased intracellular triglyceride accumulation, VLDLR-deficient mice are protective against fatty liver and liver injury after chronic alcohol exposure. Mechanistic investigations using both in vitro and in vivo systems reveal that oxidative stress-induced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation plays a critical role in alcohol-induced VLDLR up-regulation in hepatocytes, but not in adipocytes. Oxidative stress enhances VLDLR gene expression and protein abundance in primary hepatocytes, concomitant with the Nrf2 activation. Conversely, Nrf2 gene silencing abrogates oxidative stress-induced VLDLR up-regulation in the liver, but not in adipose tissue. In mice, alcohol exposure induces hepatic oxidative stress and Nrf2 activation. Supplementation of N-acetylcysteine alleviates fatty liver and liver injury induced by chronic alcohol exposure, which is associated with suppressed Nrf2 activation and attenuated VLDLR increase in the liver. Furthermore, in comparison to wild-type counterparts, Nrf2-deficient mice demonstrate attenuated hepatic VLDLR expression increase in response to chronic alcohol exposure. CONCLUSION: Chronic alcohol consumption differently alters VLDLR expression in adipose tissue and the liver. Oxidative stress-induced Nrf2 activation is mechanistically involved in VLDLR overexpression in hepatocytes in response to chronic alcohol consumption. Hepatic VLDLR overexpression plays an important role in the pathogenesis of ALD.

Gut-derived ammonia contributes to alcohol-related fatty liver development via facilitating ethanol metabolism and provoking ATF4-dependent de novo lipogenesis activation.

BACKGROUND & AIMS: Dysbiosis contributes to alcohol-associated liver disease (ALD); however, the precise mechanisms remain elusive. Given the critical role of the gut microbiota in ammonia production, we herein aim to investigate whether and how gut-derived ammonia contributes to ALD. METHODS: Blood samples were collected from human subjects with/without alcohol drinking. Mice were exposed to the Lieber-DeCarli isocaloric control or ethanol-containing diets with and without rifaximin (a nonabsorbable antibiotic clinically used for lowering gut ammonia production) supplementation for five weeks. Both in vitro (NH4Cl exposure of AML12 hepatocytes) and in vivo (urease administration for 5 days in mice) hyperammonemia models were employed. RNA sequencing and fecal amplicon sequencing were performed. Ammonia and triglyceride concentrations were measured. The gene and protein expression of enzymes involved in multiple pathways were measured. RESULTS: Chronic alcohol consumption causes hyperammonemia in both mice and human subjects. In healthy livers and hepatocytes, ammonia exposure upregulates the expression of urea cycle genes, elevates hepatic de novo lipogenesis (DNL), and increases fat accumulation. Intriguingly, ammonia promotes ethanol catabolism and acetyl-CoA formation, which, together with ammonia, synergistically facilitates intracellular fat accumulation in hepatocytes. Mechanistic investigations uncovered that ATF4 activation, as a result of ER stress induction and general control nonderepressible 2 activation, plays a central role in ammonia-provoked DNL elevation. Rifaximin ameliorates ALD pathologies in mice, concomitant with blunted hepatic ER stress induction, ATF4 activation, and DNL activation. CONCLUSIONS: An overproduction of ammonia by gut microbiota, synergistically interacting with ethanol, is a significant contributor to ALD pathologies.

The Notch-PDGFRß axis suppresses brown adipocyte progenitor differentiation in early post-natal mice.

De novo brown adipogenesis holds potential in combating the epidemics of obesity and diabetes. However, the identity of brown adipocyte progenitor cells (APCs) and their regulation have not been extensively explored. Here, through in vivo lineage tracing and mouse modeling, we observed that platelet-derived growth factor receptor beta (PDGFRß)+ pericytes give rise to developmental brown adipocytes but not to those in adult homeostasis. By contrast, T-box 18 (TBX18)+ pericytes contribute to brown adipogenesis throughout both developmental and adult stages, though in a depot-specific manner. Mechanistically, Notch inhibition in PDGFRß+ pericytes promotes brown adipogenesis by downregulating PDGFRß. Furthermore, inhibition of Notch signaling in PDGFRß+ pericytes mitigates high-fat, high-sucrose (HFHS)-induced glucose and metabolic impairment in mice during their development and juvenile phases. Collectively, these findings show that the Notch/PDGFRß axis negatively regulates developmental brown adipogenesis, and its repression promotes brown adipose tissue expansion and improves metabolic health.

Estrogen counteracts age-related decline in beige adipogenesis through the NAMPT-regulated ER stress response.

Thermogenic beige adipocytes are recognized as potential therapeutic targets for combating metabolic diseases. However, the metabolic advantages that they offer are compromised with aging. Here we show that treating mice with estrogen (E2), a hormone that decreases with age, can counteract the age-related decline in beige adipogenesis when exposed to cold temperature while concurrently enhancing energy expenditure and improving glucose tolerance in mice. Mechanistically, we found that nicotinamide phosphoribosyl transferase (NAMPT) plays a pivotal role in facilitating the formation of E2-induced beige adipocytes, which subsequently suppresses the onset of age-related endoplasmic reticulum (ER) stress. Furthermore, we found that targeting NAMPT signaling, either genetically or pharmacologically, can restore the formation of beige adipocytes by increasing the number of perivascular adipocyte progenitor cells. Conversely, the absence of NAMPT signaling prevents this process. Together, our findings shed light on the mechanisms regulating the age-dependent impairment of beige adipocyte formation and underscore the E2-NAMPT-controlled ER stress pathway as a key regulator of this process.

Inhibition of NF-κB activation by 4-hydroxynonenal contributes to liver injury in a mouse model of alcoholic liver disease.

Long-term alcohol exposure sensitizes hepatocytes to tumor necrosis factor-α (TNF) cytotoxicity. 4-Hydroxynonenal (4-HNE) is one of the most abundant and reactive lipid peroxides. Increased hepatic 4-HNE contents present in both human alcoholics and alcohol-fed animals. In the present study, we investigated the effects of intracellular 4-HNE accumulation on TNF-induced hepatotoxicity and its potential implication in the pathogenesis of alcoholic liver disease. Male C57BL/6 mice were fed an ethanol-containing or a control diet for 5 weeks. Long-term alcohol exposure increased hepatic 4-HNE and TNF levels. Cell culture studies revealed that 4-HNE, at nontoxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-κB transactivity. Further investigation demonstrated that 4-HNE prevented TNF-induced inhibitor of κBα phosphorylation without affecting upstream IκB kinase activity. An immunoprecipitation assay revealed that increased 4-HNE content was associated with increased formation of 4-HNE-inhibitor of κBα adduction in both 4-HNE-treated hepatocytes and in the livers of alcohol-fed mice. Prevention of intracellular 4-HNE accumulation by bezafibrate, a peroxisome proliferator-activated receptor-α agonist, protected hepatocytes from TNF killing via NF-κB activation. Supplementation of N-acetylcysteine, a glutathione precursor, conferred a protective effect on alcohol-induced liver injury in mice, was associated with decreased hepatic 4-HNE formation, and improved hepatic NF-κB activity. In conclusion, increased 4-HNE accumulation represents a potent and clinically relevant sensitizer to TNF-induced hepatotoxicity. These data support the notion that removal of intracellular 4-HNE can serve as a potential therapeutic option for alcoholic liver disease.

Differential roles of insulin receptor in adipocyte progenitor cells in mice.

The development of white adipose tissue (WAT) occurs during distinct embryonic and postnatal stages, and it is subsequently maintained throughout life. However, the specific mediators and mechanisms responsible for WAT development during different phases remain unclear. In this study, we investigate the role of the insulin receptor (IR) in regulating adipogenesis and adipocyte function within adipocyte progenitor cells (APCs) during WAT development and homeostasis. We use two in vivo adipose lineage tracking and deletion systems to delete IR either in embryonic APCs or adult APCs, respectively, to explore the specific requirements of IR during WAT development and WAT homeostasis in mice. Our data suggest that IR expression in APCs may not be essential for adult adipocyte differentiation but appears to be crucial for adipose tissue development. We reveal a surprising divergent role of IR in APCs during WAT development and homeostasis.