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.

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice.

Non-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ). However, the relevance of hepatocyte PPARγ in NASH associated with obesity is still poorly understood. In this study, hepatocyte PPARγ was knocked out (PpargΔHep) in male and female mice after the development of high-fat diet-induced obesity. The diet-induced obese mice were then maintained on their original diet or switched to a high fat, cholesterol, and fructose (HFCF) diet to induce NASH. Hepatic PPARγ expression was mostly derived from hepatocytes and increased by high fat diets. PpargΔHep reduced HFCF-induced NASH progression without altering steatosis, reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and decreased the area of collagen-stained fibrosis in the liver of HFCF-fed male mice. Moreover, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPARγ-dependent manner. PpargΔHep increased betaine-homocysteine s-methyltransferase expression and reduced homocysteine levels in HFCF-fed male mice. In addition, in a cohort of 102 obese patients undergoing bariatric surgery with liver biopsies, 16 cases were scored with NASH and were associated with increased insulin resistance and hepatic PPARγ expression. Our study shows that hepatocyte PPARγ expression is associated with NASH in mice and humans. In male mice, hepatocyte PPARγ negatively regulates methionine metabolism and contributes to the progression of fibrosis.

Role of hepatic peroxisome proliferator-activated receptor γ in non-alcoholic fatty liver disease.

Peroxisome proliferator-activated receptor γ (PPARγ) belongs to a family of nuclear receptors that could serve as lipid sensors. PPARγ is the target of a group of insulin sensitizers called thiazolidinediones (TZDs) which regulate the expression of genes involved in glucose and lipid metabolism as well as adipokines that regulate metabolic function in other tissues. Non-alcoholic fatty liver disease (NAFLD) has a high prevalence worldwide and is even higher in patients with obesity and insulin resistance. TZD-mediated activation of PPARγ could serve as a good treatment for NAFLD because TZDs have shown anti-fibrogenic and anti-inflammatory effectsin vitro and increase insulin sensitivity in peripheral tissues which improves liver pathology. However, mechanistic studies in mouse models suggest that the activation of PPARγ in hepatocytes might reduce or limit the therapeutic potential of TZD against NAFLD. In this review, we briefly describe the short history of PPAR isoforms, the relevance of their expression in different tissues, as well as the pathogenesis and potential therapeutics for NAFLD. We also discuss some evidence derived from mouse models that could be useful for endocrinologists to assess tissue-specific roles of PPARs, complement reverse endocrinology approaches, and understand the direct role that PPARγ has in hepatocytes and non-parenchymal cells.

Rosiglitazone Requires Hepatocyte PPARγ Expression to Promote Steatosis in Male Mice With Diet-Induced Obesity.

Thiazolidinediones (TZD) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that may reduce hepatic steatosis through their effects in adipose tissue and therefore have been assessed as potential therapies to treat nonalcoholic fatty liver disease (NAFLD) in humans. However, some studies suggest that expression and activation of hepatocyte PPARγ promotes steatosis and that would limit the benefits of TZD as a NAFLD therapy. To further explore this possibility, we examined the impact of short-term rosiglitazone maleate treatment after the development of moderate or severe diet-induced obesity, in both control and adult-onset hepatocyte-specific PPARγ knockout (PpargΔHep) mice. Independent of the level of obesity and hepatic PPARγ expression, the TZD treatment enhanced insulin sensitivity, associated with an increase in white adipose tissue (WAT) fat accumulation, consistent with clinical observations. However, TZD treatment increased hepatic triglyceride content only in control mice with severe obesity. Under these conditions, PpargΔHep reduced diet-induced steatosis and prevented the steatogenic effects of short-term TZD treatment. In these mice, subcutaneous WAT was enlarged and associated with increased levels of adiponectin, while hepatic levels of phosphorylated adenosine 5'-monophosphate-activated protein kinase were also increased. In addition, in mice with severe obesity, the expression of hepatic Cd36, Cidea, Cidec, Fabp4, Fasn, and Scd-1 was increased by TZD in a PPARγ-dependent manner. Taken together, these results demonstrate that hepatocyte PPARγ expression offsets the antisteatogenic actions of TZD in mice with severe obesity. Therefore, in obese and insulin resistant humans, TZD-mediated activation of hepatocyte PPARγ may limit the therapeutic potential of TZD to treat NAFLD.

Hepatocyte-Specific Loss of PPARγ Protects Mice From NASH and Increases the Therapeutic Effects of Rosiglitazone in the Liver.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is commonly observed in patients with type 2 diabetes, and thiazolidinediones (TZD) are considered a potential therapy for NASH. Although TZD increase insulin sensitivity and partially reduce steatosis and alanine aminotransferase, the efficacy of TZD on resolving liver pathology is limited. In fact, TZD may activate peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes and promote steatosis. Therefore, we assessed the role that hepatocyte-specific PPARγ plays in the development of NASH, and how it alters the therapeutic effects of TZD on the liver of mice with diet-induced NASH. METHODS: Hepatocyte-specific PPARγ expression was knocked out in adult mice before and after the development of NASH induced with a high fat, cholesterol, and fructose (HFCF) diet. RESULTS: HFCF diet increased PPARγ expression in hepatocytes, and rosiglitazone further activated PPARγ in hepatocytes of HFCF-fed mice in vivo and in vitro. Hepatocyte-specific loss of PPARγ reduced the progression of HFCF-induced NASH in male mice and increased the benefits derived from the effects of TZD on extrahepatic tissues and non-parenchymal cells. RNAseq and metabolomics indicated that HFCF diet promoted inflammation and fibrogenesis in a hepatocyte PPARγ-dependent manner and was associated with dysregulation of hepatic metabolism. Specifically, hepatocyte-specific loss of PPARγ plays a positive role in the regulation of methionine metabolism, and that could reduce the progression of NASH. CONCLUSIONS: Because of the negative effect of hepatocyte PPARγ in NASH, inhibition of mechanisms promoted by endogenous PPARγ in hepatocytes may represent a novel strategy that increases the efficiency of therapies for NAFLD.

TDP-43 cytoplasmic inclusion formation is disrupted in C9orf72-associated amyotrophic lateral sclerosis/frontotemporal lobar degeneration.

The G4C2 hexanucleotide repeat expansion mutation in the C9orf72 gene is the most common genetic cause underlying both amyotrophic lateral sclerosis and frontotemporal dementia. Pathologically, these two neurodegenerative disorders are linked by the common presence of abnormal phosphorylated TDP-43 neuronal cytoplasmic inclusions. We compared the number and size of phosphorylated TDP-43 inclusions and their morphology in hippocampi from patients dying with sporadic versus C9orf72-related amyotrophic lateral sclerosis with pathologically defined frontotemporal lobar degeneration with phosphorylated TDP-43 inclusions, the pathological substrate of clinical frontotemporal dementia in patients with amyotrophic lateral sclerosis. In sporadic cases, there were numerous consolidated phosphorylated TDP-43 inclusions that were variable in size, whereas inclusions in C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration were quantitatively smaller than those in sporadic cases. Also, C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration homogenized brain contained soluble cytoplasmic TDP-43 that was largely absent in sporadic cases. To better understand these pathological differences, we modelled TDP-43 inclusion formation in fibroblasts derived from sporadic or C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia patients. We found that both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia patient fibroblasts showed impairment in TDP-43 degradation by the proteasome, which may explain increased TDP-43 protein levels found in both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration frontal cortex and hippocampus. Fibroblasts derived from sporadic patients, but not C9orf72 patients, demonstrated the ability to sequester cytoplasmic TDP-43 into aggresomes via microtubule-dependent mechanisms. TDP-43 aggresomes in vitro and TDP-43 neuronal inclusions in vivo were both tightly localized with autophagy markers and, therefore, were likely to function similarly as sites for autophagic degradation. The inability for C9orf72 fibroblasts to form TDP-43 aggresomes, together with the observations that TDP-43 protein was soluble in the cytoplasm and formed smaller inclusions in the C9orf72 brain compared with sporadic disease, suggests a loss of protein quality control response to sequester and degrade TDP-43 in C9orf72-related diseases.

Dysregulation of ErbB Receptor Trafficking and Signaling in Demyelinating Charcot-Marie-Tooth Disease.

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with the majority of cases involving demyelination of peripheral nerves. The pathogenic mechanisms of demyelinating CMT remain unclear, and no effective therapy currently exists for this disease. The discovery that mutations in different genes can cause a similar phenotype of demyelinating peripheral neuropathy raises the possibility that there may be convergent mechanisms leading to demyelinating CMT pathogenesis. Increasing evidence indicates that ErbB receptor-mediated signaling plays a major role in the control of Schwann cell-axon communication and myelination in the peripheral nervous system. Recent studies reveal that several demyelinating CMT-linked proteins are novel regulators of endocytic trafficking and/or phosphoinositide metabolism that may affect ErbB receptor signaling. Emerging data have begun to suggest that dysregulation of ErbB receptor trafficking and signaling in Schwann cells may represent a common pathogenic mechanism in multiple subtypes of demyelinating CMT. In this review, we focus on the roles of ErbB receptor trafficking and signaling in regulation of peripheral nerve myelination and discuss the emerging evidence supporting the potential involvement of altered ErbB receptor trafficking and signaling in demyelinating CMT pathogenesis and the possibility of modulating these trafficking and signaling processes for treating demyelinating peripheral neuropathy.

SIMPLE: A new regulator of endosomal trafficking and signaling in health and disease.

SIMPLE, also known as LITAF, EET1 and PIG7, was originally identified based on its transcriptional upregulation by estrogen, p53, lipopolysaccharide or a microbial cell-wall component. Missense mutations in SIMPLE cause Charcot-Marie-Tooth disease (CMT), and altered SIMPLE expression is associated with cancer, obesity and inflammatory bowel diseases. Despite increasing evidence linking SIMPLE to human diseases, the biological function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Our recent study reveals that SIMPLE is a functional partner of the endosomal sorting complex required for transport (ESCRT) machinery in the regulation of endosome-to-lysosome trafficking and intracellular signaling. Our results indicate that CMT-linked SIMPLE mutants are loss-of-function mutants which act dominantly to impair endosomal trafficking and signaling attenuation. We propose that endosomal trafficking and signaling dysregulation is a key pathogenic mechanism in CMT and other diseases that involve SIMPLE dysfunction.

Motor and sensory neuropathy due to myelin infolding and paranodal damage in a transgenic mouse model of Charcot-Marie-Tooth disease type 1C.

Charcot-Marie-Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt-Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.

Charcot-Marie-Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking.

Mutations in small integral membrane protein of lysosome/late endosome (SIMPLE) cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Here, we report that SIMPLE interacted and colocalized with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functioned with the ESCRT machinery in the control of endosome-to-lysosome trafficking. Our analyses revealed that SIMPLE was required for efficient recruitment of ESCRT components to endosomal membranes and for regulating endosomal trafficking and signaling attenuation of ErbB receptors. We found that the ability of SIMPLE to regulate ErbB trafficking and signaling was impaired by CMT-linked SIMPLE mutations via a loss-of-function, dominant-negative mechanism, resulting in prolonged activation of ERK1/2 signaling. Our findings indicate a function of SIMPLE as a regulator of endosomal trafficking and provide evidence linking dysregulated endosomal trafficking to CMT pathogenesis.

Protein misfolding and clearance in demyelinating peripheral neuropathies: Therapeutic implications.

Peripheral neuropathies such as Charcot-Marie-Tooth disease (CMT) are a group of neurological disorders that affect the peripheral nervous system. Although demyelinating CMT is the most prevalent hereditary peripheral neuropathy, there are currently no effective treatments for patients suffering from this disease. Recent studies by our group and others have provided a link between protein misfolding and demyelinating CMT and indicate that impairment of the proteasome and aggresome-autophagy pathways may contribute to CMT pathogenesis. These studies suggest that targeting protein quality control systems involved in cytoprotection against CMT-associated misfolded proteins could have therapeutic benefits for treating demyelinating CMT.

Mutations associated with Charcot-Marie-Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome-autophagy pathways.

Mutations in SIMPLE cause an autosomal dominant, demyelinating form of peripheral neuropathy termed Charcot-Marie-Tooth disease type 1C (CMT1C), but the pathogenic mechanisms of these mutations remain unknown. Here, we report that SIMPLE is an early endosomal membrane protein that is highly expressed in the peripheral nerves and Schwann cells. Our analysis has identified a transmembrane domain (TMD) embedded within the cysteine-rich (C-rich) region that anchors SIMPLE to the membrane, and suggests that SIMPLE is a post-translationally inserted, C-tail-anchored membrane protein. We found that CMT1C-linked pathogenic mutations are clustered within or around the TMD of SIMPLE and that these mutations cause mislocalization of SIMPLE from the early endosome membrane to the cytosol. The CMT1C-associated SIMPLE mutant proteins are unstable and prone to aggregation, and they are selectively degraded by both the proteasome and aggresome-autophagy pathways. Our findings suggest that SIMPLE mutations cause CMT1C peripheral neuropathy by a combination of loss-of-function and toxic gain-of-function mechanisms, and highlight the importance of both the proteasome and autophagy pathways in the clearance of CMT1C-associated mutant SIMPLE proteins.