Sarcopenia in alcohol-related liver disease (ALD) is of high clinical significance, but there are no effective treatments. Hyaluronan 35 kDa (HA35), a glycosaminoglycan polymer, modulates responses to Toll-like receptor 4 (TLR4), a lipopolysaccharide receptor, to improve hepatic and macrophage function in ALD. We evaluated skeletal muscle responses to HA35 in preclinical models of ALD. Responses to ethanol, lipopolysaccharide (LPS), and HA35 were studied in differentiated murine C2C12/human-induced pluripotent stem cell (hiPSC)-derived myotubes, and wild-type/HA receptor CD44 knockout (CD44-/-) mouse models of ALD (mALD). Signaling molecules and measures of protein homeostasis (proteostasis) were quantified by immunoblots, mitochondrial oxidative function was determined by high-sensitivity respirofluorometry, membrane elasticity by atomic force microscopy, and muscle contractile responses to electrical stimulation ex vivo were quantified. Multiomics, weighted gene co-expression network, and image analyses were also performed. Ethanol caused a sarcopenic phenotype in myotubes and mALD (lower myotube/muscle fiber diameter, muscle mass), less protein synthesis, impaired mTORC1 signaling, and higher autophagy markers and unaltered membrane elasticity. Expressions of LPS (TLR2, TLR4) and HA (CD44) receptors were upregulated by ethanol. In myotubes, LPS treatment caused a sarcopenic phenotype at lower concentrations in ethanol-treated myotubes. HA35 reversed ethanol/LPS-induced lower protein synthesis, impaired mTORC1 signaling, and mitochondrial complex I function in myotubes/muscle tissue. Muscle concentrations of HA fragments were lower in mALD, but beneficial responses to HA35 occurred without restoring HA concentrations, and HA35 was not beneficial in CD44-/- mALD. Sarcopenia, signaling perturbations, and mitochondrial oxidative dysfunction in mALD are reversed by HA35, allowing for rapid clinical translation (ongoing clinicaltrials.gov, NCT05018481).NEW & NOTEWORTHY Sarcopenia in alcohol-related liver disease (ALD) is frequent and contributes to adverse clinical outcomes with limited treatment options. Hyaluronans (glycosaminoglycan polymers) of specific fragment sizes (30-40 kDa) have anti-inflammatory and tissue-protective properties. In an array of preclinical models of ALD, we demonstrate that HA35 reverses signaling, mitochondrial oxidative function, and phenotypic perturbations associated with sarcopenia in ALD. These effects are mediated without restoring the low muscle tissue concentrations of HA in ALD.

Exercise modulates multiple physiological systems, including skeletal muscle and the gut microbiome (GMB). Ammonia, a microbiome-derived cytotoxic metabolite, causes cellular hyperammonemic stress (HAS) in chronic diseases. We investigated the impact of voluntary wheel running (VWR) on GMB during HAS in a mouse model. Male C57BL/6J mice were randomized to treatment with either ammonium acetate (AmAc) (2.5 mmol/kg/day) or vehicle for 6 weeks. Stool 16S rRNA sequencing was performed at baseline, pre-intervention, and post-intervention. GMB diversity, taxa-level abundance, and correlation analyses were performed. Overall GMB composition remained stable between baseline and pre-intervention across groups (r > 0.57; P < 0.001). Following interventions, VWR or usual activity (UA), alpha-diversity was highest in AmAc-treated, specifically AmAc-VWR, mice. Eubacterium xylanophilum was reduced in AmAc-UA vs other groups (P < 0.05). Akkermansia abundance declined over time in UA mice, but in AmAc-VWR mice, this depletion was reversed (P = 0.002). Clostridium sensu stricto 1 and Eubacterium ventriosum were increased in AmAc-VWR mice (P < 0.05). Correlation analysis revealed high stability in PBS-UA (r = 0.667; P < 0.001), moderate restructuring in AmAc-VWR (r = 0.566; P < 0.001), and PBS-VWR (r = 0.385; P = 0.0099). HAS-induced GMB instability, with loss of beneficial taxa, including short-chain fatty acid-producing bacteria, was partially ameliorated by VWR. Exercise-mediated GMB modulation may be a strategy to mitigate HAS-induced complications in chronic diseases.IMPORTANCEVoluntary exercise is recommended in chronic diseases to improve outcomes, but biological responses in disease are not well characterized. Perturbations in the metabolism of ammonia, a microbiome-generated toxin, occur in chronic diseases that can be compounded by muscle-generated ammonia during exercise. Exercise-induced molecular responses are adversely affected by hyperammonemic stress of chronic diseases, including liver cirrhosis. We investigated gut microbiome changes during voluntary wheel running, which replicates human endurance exercise in a preclinical mouse model of hyperammonemia. Adverse impacts of Hyperammonemic stress included a reduction in short-chain fatty acid producers that were reversed by voluntary wheel running. Our data lay the foundation for future studies on how endurance-type exercise promotes a favorable gut microbial composition and strategies to use exercise as a regulator of hyperammonemic stress via targeting the gut microbiome.

Supplementary material for American Journal of Physiology-Endocrinology and Metabolism article: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

AIM:Voluntary exercise improves clinical outcomes in healthy subjects, but increased muscle ammoniagenesis may limit beneficial responses during hyperammonaemia in chronic diseases. Responses to 4-weeks voluntary wheel running (VWR) were compared with usual activity (UA) to determine if hyperammonaemia alters VWR responses and if VWR alters muscle responses to hyperammonaemia. METHODS:Eight- to 10-week-old male C57BL/6J mice were treated with 6 weeks of subcutaneous infusion of 2.5 mmol kg-1 day-1 ammonium acetate (AmAc) or vehicle (PBS) via an osmotic pump. Two weeks after the start of infusion, mice were assigned to the intervention (VWR or UA). Wheel runs were measured, and weekly average rotations, distance, and circadian patterns were analysed. Indirect calorimetry was performed pre- and post-intervention. Mice were euthanized 4 weeks after the start of VWR/UA, and organs (including muscles) were harvested, weighed, and muscle histomorphometry performed for fibre diameter/type. Protein synthesis by ex vivo puromycin incorporation, autophagy markers, expression of signalling proteins (mTORC1 pathway, eukaryotic initiation factor-2-α phosphorylation), and ammonia disposal enzymes were quantified by immunoblots. Mitochondrial oxidative function was measured by high-sensitivity respirofluorometry using substrate, uncoupler, inhibitor, and titration protocols. Fluorometric assays were done for ammonia measurements. RESULTS:Gastrocnemius muscle mass (p < 0.01), muscle fibre area (p < 0.01), and grip strength were lower in AmAc-UA than in PBS-UA mice and higher with VWR than UA in AmAc mice (p < 0.001). Expression of electron transport chain proteins and some components of mitochondrial oxidative function were less (p < 0.05 or less) in AmAc-UA than PBS-UA, and these perturbations were reversed in the AmAc-VWR mice (p < 0.05 or less). Global muscle protein synthesis (p < 0.05) and components of the mTORC1 pathway expression (p < 0.05) were higher, while myostatin expression was lower with VWR than UA in AmAc mice (p < 0.05). Expression of autophagy markers P62 and LC3-II was not different with VWR or UA in AmAc mice, while Beclin1 was higher in VWR compared with UA, regardless of treatment group (p < 0.001). Expression of muscle ammonia disposal pathway enzymes, including glutamate dehydrogenase and pyrroline-5-carboxylate synthase, was higher (p ≤ 0.05) in AmAc-UA versus PBS-UA and increased in only PBS-VWR mice (p < 0.05). CONCLUSION:VWR reverses hyperammonaemia-induced sarcopenia, protein synthesis/autophagy signalling perturbations, and mitochondrial oxidative dysfunction. Muscle mass, grip strength, signalling, and mitochondrial responses to VWR were not affected by hyperammonaemia. Increased expression of enzymes involved in the ammonia disposal pathway in skeletal muscle may be an adaptive response to hyperammonaemia. These data provide the rationale for exercise programmes in chronic diseases, including cirrhosis, even with hyperammonaemia.

Supplementary material for American Journal of Physiology-Endocrinology and Metabolism article: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

Co-presentation of anti GBM and anti MPO antibodies is considered to be a rare phenomena. When patients present both types of antibodies, the condition is referred to as “double positive” for anti-GBM and ANCA associated antibodies which can present clinically as rapidly progressive glomerulonephritis [RPGN], often associated with alveolar bleeding and renal manifestations which constitutes a medical emergency associated with increased morbidity and mortality. In current clinical practice, the diagnosis is obtained through serological tests that detect the presence of antibodies and kidney biopsy to identify their specific histopathological changes. We highlight the role of clinical Biochemistry laboratory and specifically LIA technique in playing a crucial role in identifying the disease thus providing timely intervention to aid better patient outcome.

The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26MTA1; Ptenf/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future.

Co-presentation of anti GBM and anti MPO antibodies is considered to be a rare phenomena. When patients present both types of antibodies, the condition is referred to as "double positive" for anti-GBM and ANCA associated antibodies which can present clinically as rapidly progressive glomerulonephritis [RPGN], often associated with alveolar bleeding and renal manifestations which constitutes a medical emergency associated with increased morbidity and mortality. In current clinical practice, the diagnosis is obtained through serological tests that detect the presence of antibodies and kidney biopsy to identify their specific histopathological changes. We highlight the role of clinical Biochemistry laboratory and specifically LIA technique in playing a crucial role in identifying the disease thus providing timely intervention to aid better patient outcome.

Perturbations in the metabolism of ammonia, a cytotoxic endogenous metabolite, occur in a number of chronic diseases, with consequent hyperammonemia. Increased skeletal muscle ammonia uptake causes metabolic, molecular, and phenotype alterations including cataplerosis of (loss of tricarboxylic acid cycle (TCA) cycle intermediate) α-ketoglutarate (αKG), mitochondrial oxidative dysfunction, and senescence-associated molecular phenotype (SAMP). L-Isoleucine (Ile) is an essential, branched-chain amino acid (BCAA) that simultaneously provides acetyl-CoA as an oxidative substrate and succinyl-CoA for anaplerosis (providing TCA cycle intermediates). Our multiomics analyses in myotubes and skeletal muscle from hyperammonemic mice and human patients with cirrhosis showed perturbations in BCAA transporters and catabolism. We, therefore, determined if Ile reverses hyperammonemia-induced impaired mitochondrial oxidative function and SAMP. Studies were performed in differentiated murine C2C12 myotubes that were early passage, late passage (senescent), or those depleted of LAT1/SLC7A5 and human induced pluripotent stem cell-derived myotubes (hiPSCM). Ile reverses hyperammonemia-induced reduction in the maximum respiratory capacity, complex I, II, and III functions in early passage murine myotubes and hiPSCM. Consistently, low ATP content and impaired global protein synthesis (high energy requiring cellular process) during hyperammonemia are reversed by Ile in murine myotubes and hiPSCM. Lower abundance of critical regulators of protein synthesis in mTORC1 signaling, and increased phosphorylation of eukaryotic initiation factor 2α are also reversed by Ile. Genetic depletion studies showed that Ile responses are independent of the amino acid transporter LAT1/SLC7A5. Our studies show that Ile reverses the hyperammonemia-induced impaired mitochondrial oxidative function, cataplerosis, and SAMP in a LAT1/SLC7A5 transporter-independent manner.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG–MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG–MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.

Scope: Resistance of castrate-resistant prostate cancer (CRPC) to enzalutamide (Enz) involves the expression of constitutively active androgen receptor splice variant (AR-V7). In addition to altered AR pathways, CRPC is characterized by "non-AR-driven" signaling, which includes an overexpression of metastasis-associated protein 1 (MTA1). Combining natural compounds with anticancer drugs may enhance drug effectiveness while reducing adverse effects. In this study, the in vitro and in vivo anticancer effects of Gnetin C (GnC) alone and in combination with Enz against CRPC are examined.Methods and results: The effects of GnC alone and in combination with Enz are assessed by cell viability, clonogenic survival, cell migration, and AR and MTA1 expression using 22Rv1 cells. The tumor growth in vivo is assessed by bioluminescent imaging, western blots, RT-PCR, and IHC. GnC alone and in combined treatment inhibit cell viability, clonogenic survival and migration, and AR and MTA1 expression in 22Rv1 cells. The underlying AR- and MTA1-targeted anticancer mechanisms of treatments in vivo involve inhibition of proliferation and angiogenesis, and induction of apoptosis.Conclusion: The findings demonstrate that GnC alone and GnC combined with Enz effectively inhibits AR- and MTA1-promoted tumor-progression in advanced CRPC, which indicates its potential as a novel therapeutic approach for CRPC.