Hepatoprotective effects of tetrahydropalmatine against NAFLD through autophagy activation and lipid metabolic reprogramming via the AMPK-mTOR-Sirt1 axis.

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BackgroundNonalcoholic fatty liver disease (NAFLD) is becoming a global epidemic without effective treatment currently available. NAFLD is characterized by an increase in hepatic de novo lipogenesis (DNL) and inadequate compensatory enhancement in fatty acid oxidation (FAO), which disturbs lipid homeostasis. In NAFLD, lipid metabolism relies heavily on metabolic reprogramming. Moreover, lipid metabolism plays an essential role in switching between lipogenesis and FAO, which is beneficial for the anti-NAFLD therapy. Our recent study demonstrated that the phytochemical tetrahydropalmatine (THP) has positive efficacy in hepatocellular carcinoma (HCC). However, it remains unclear whether the therapeutic benefits of THP are primarily due to delaying the progression of hepatic steatosis to HCC. PurposeThis work aimed to systemically evaluate the pharmacological functions and underlying mechanisms of THP in NAFLD using both in vitro and in vivo models. MethodsNAFLD models were established using high-fat diet (HFD)-fed mice in vivo and palmitic acid- and oleic acid-challenged hepatocytes in vitro. Metabonomics analysis concomitant with biochemical indices and computational biology assays were performed comprehensively to reveal the key link between the treatment of NAFLD and the AMPK-SREBP-1c-Sirt1 signaling axis. ResultsHepatic metabolomics analysis revealed that THP altered lipid metabolism by enhancing FAO and inhibiting glycolysis, tricarboxylic acid cycle, and urea cycle in HFD-fed mice. Analysis of gene expression showed that THP profoundly suppressed hepatic DNL and promoted FAO. THP supplementation not only significantly decreased body/liver weight gain and serum indices but also ameliorated hepatic steatosis. Simultaneously, impaired lipotoxicity was observed in vivo and in vitro after THP supplementation, protecting against steatosis-driven injury. Metabolic phenotype assays showed that THP promoted switching from glycolysis inhibition to FAO enhancement in steatotic cells, resulting in reprogramming lipid metabolism. Mechanistically, THP accelerated lipid oxidation by activating AMPK-SREBP-1c-Sirt1 axis signaling. Applying molecular docking combined with surface plasmon resonance and cellular thermal shift assay target engagement, as well as siRNA assays, AMPKα was confirmed as a direct molecular target of THP. ConclusionIn summary, THP ameliorates hepatic steatosis in NAFLD by switching lipid metabolism via the AMPK-SREBP-1c-Sirt1 pathway. This work provides an attractive phytochemical component for therapy against hepatic steatosis in NAFLD.

IntroductionThis paper reviews the regulatory effects of hydrogen sulfide (H2S) in non-alcoholic fatty liver disease (NAFLD). The research background indicates that NAFLD has become one of the most common chronic liver diseases worldwide, with a complex pathogenesis involving insulin resistance and lipid metabolism disorders. Hydrogen sulfide, as an important gaseous signaling molecule, exerts protective effects in NAFLD through multiple pathways, including antioxidant, anti-inflammatory, lipid metabolism regulation, apoptosis inhibition, and insulin resistance improvement. This article provides a detailed summary of the research overview of NAFLD, the biological characteristics of hydrogen sulfide, its mechanisms of action in NAFLD, empirical studies, and evaluates and prospects current research, aiming to provide a new theoretical basis and experimental support for the treatment of NAFLD.PurposeThis literature review aims to systematically synthesize the regulatory roles of hydrogen sulfide (H2S) in non-alcoholic fatty liver disease (NAFLD). It provides a comprehensive overview of NAFLD research, the biological characteristics of H2S, the mechanisms underlying H2S’s action in NAFLD, and relevant empirical studies. Additionally, the review evaluates the current state of research and prospects future directions, with the goal of offering novel theoretical foundations and experimental support for NAFLD treatment.Patients and MethodsIt adopts a comprehensive literature analysis approach. Analyze literature on NAFLD pathogenesis, H2S (synthesis/function), and NAFLD treatments (in vitro, animal models, clinical studies).ResultsNAFLD links to insulin resistance/inflammation; H2S protects against NAFLD by mitigating inflammation, oxidative stress, and lipid accumulation, while improving insulin sensitivity and inhibiting apoptosis. However, its precise mechanisms, stage-specific effects, and therapeutic safety require further clinical validation.ConclusionHydrogen sulfide (H2S), a gaseous signal, exerts multi-mechanistic protective effects in NAFLD. Current limitations include unclear signaling mechanisms and lack of targeted delivery systems. Future research should focus on stage-specific mechanisms, optimized H2S donors, and combination therapies for NAFLD treatment.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract How Mycobacterium tuberculosis (Mtb) rewires macrophage energy metabolism to facilitate survival is poorly characterized. Here, we used extracellular flux analysis to simultaneously measure the rates of glycolysis and respiration in real time. Mtb infection induced a quiescent energy phenotype in human monocyte-derived macrophages and decelerated flux through glycolysis and the TCA cycle. In contrast, infection with the vaccine strain, M. bovis BCG, or dead Mtb induced glycolytic phenotypes with greater flux. Furthermore, Mtb reduced the mitochondrial dependency on glucose and increased the mitochondrial dependency on fatty acids, shifting this dependency from endogenous fatty acids in uninfected cells to exogenous fatty acids in infected macrophages. We demonstrate how quantifiable bioenergetic parameters of the host can be used to accurately measure and track disease, which will enable rapid quantifiable assessment of drug and vaccine efficacy. Our findings uncover new paradigms for understanding the bioenergetic basis of host metabolic reprogramming by Mtb. https://doi.org/10.7554/eLife.39169.001 Introduction Mechanisms underlying the pathogenesis induced by Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), are poorly understood, and increasing evidence suggests that Mtb subverts the host’s immune response to establish a persistent infection (Cambier et al., 2014; Hmama et al., 2015; Józefowski et al., 2008). Crucial to the success of the immune system to control microbial infection is the metabolic plasticity of immune cells to activate antimicrobial mechanisms in macrophages and activate T cells in response to microbial invasion. Precise coordination between diverse metabolic pathways underlies this plasticity (Ganeshan and Chawla, 2014; Loftus and Finlay, 2016; Mathis and Shoelson, 2011), which is disrupted by pathogenic bacteria. Hence, host-directed therapies are increasingly considered for adjunctive treatment of tuberculosis (Guler and Brombacher, 2015; Mahon and Hafner, 2015; Wallis and Hafner, 2015). Studies suggest that Mtb pathogenicity is reinforced with participation of metabolic pathways from the host, including evidence suggesting that Mtb adaptation to the host environment requires catabolism of host-derived lipids (Daniel et al., 2011; Muñoz-Elías and McKinney, 2005; Pandey and Sassetti, 2008; Rohde et al., 2012; Lee et al., 2013). This is assumed to be induced through Mtb regulating metabolic thresholds of the host macrophage (Mehrotra et al., 2014). Recent studies suggested that there is a shift from oxidative phosphorylation towards glycolysis in macrophages infected with an avirulent strain (H37Ra) or dead γ-irradiated Mtb (Gleeson et al., 2016), and in Mtb (H37Rv)-infected mouse lungs using transcriptomic profiling and confocal imaging (Shi et al., 2015). Lachmandas et al. (Lachmandas et al., 2016) demonstrated that the switch to aerobic glycolysis observed in human peripheral blood mononuclear cells stimulated with dead Mtb lysate is TLR2-dependent, and is mediated in part through the AKT-mTOR (mammalian target of rapamycin) pathway. While this evidence supports the conclusion that dead Mtb reprograms host energy metabolism, the actual underlying mechanisms with live virulent Mtb infection enabling it to persist in humans remain elusive. Furthermore, the metabolic health of the Mtb-infected cell is poorly defined as there is a lack of knowledge on exactly what metabolic health comprises, and what should be measured. Thus, development of a technological advance to address these gaps in our knowledge is expected to uncover the fundamental role of host energy metabolism in allowing Mtb to persist for decades without causing disease. Aberrant cellular bioenergetics have been associated with, and are often the cause of, diseases such as diabetes, cancer, neurodegeneration, and cardiac disease. The dysfunctional energy metabolism in these diseases has been successfully investigated using extracellular flux (XF) analysis (Devarajan et al., 2011; Hill et al., 2009; Salabei et al., 2016; Wu et al., 2007; Lee et al., 2017; Cronin-Furman et al., 2013). XF analysis monitors the rate of oxygen consumed by cells (oxygen consumption rate, OCR) and the release of protons from the cells into the extracellular medium (extracellular acidification rate, ECAR) non-invasively in real time (Figure 1A). Measurements of cellular respiration and acidification form the foundation of our understanding of bioenergetics because cells use two main pathways to produce ATP, namely oxidative phosphorylation (OXPHOS) and glycolysis. This technology is largely unexplored in the field of bacterial pathogenesis, with a few studies focused on Helicobacter pylori infections (Hammond et al., 2015; Saha et al., 2010), but studies on live virulent Mtb pathogenesis are lacking. Figure 1 Download asset Open asset Schematic illustration of cellular metabolism pathways and XF assays used to analyze metabolic pathways. (A) The XF measures oxygen consumption rate (OCR) of the cell, which is mostly consumed at complex IV of the electron transport chain (ETC) in the mitochondria, and extracellular acidification rate (ECAR), which is generated from lactic acid produced from pyruvate, the end-product of glycolysis, and carbonic acid produced from CO2 released during the TCA cycle. Assays performed on the XF include: (B) mitochondrial respiration test, (C) extracellular acidification test, (D) glycolytic rate assay, (E) mitochondrial fuel test, (F) fatty acid oxidation assay and (G) real-time ATP rate assay. Oligo, oligomycin; FCCP, cyanide-4-[trifluoromethoxy]phenylhydrazone; AntiA and Rot, antimycin A and rotenone; 2-DG, 2-Deoxyglucose; G-6-P, glucose-6-phosphate; G-3-P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; α-KG, α-ketoglutarate; OAA, oxaloacetate. https://doi.org/10.7554/eLife.39169.002 In this study, we used extracellular flux analysis to explore the modulation of the energy metabolism of differentiated THP-1 macrophages and human monocyte derived macrophages (hMDM) infected with live virulent Mtb, the slow-growing non-pathogenic vaccine strain, M. bovis BCG (BCG) and dead-Mtb. We examined how mycobacterial burden affects OXPHOS and the glycolysis of macrophages, we investigated ATP production by glycolysis and OXPHOS during mycobacterial infection, and assessed the capacity, dependency and flexibility of mitochondria on glucose, glutamine or fatty acids during infection. Lastly, we confirmed our findings with [U-13C]glucose stable isotope tracing experiments. By adapting a real-time, non-invasive bioenergetic platform to study the bioenergetics of the Mtb-infected host cell, we have generated new knowledge that may contribute towards a better understanding of Mtb persistence and development of novel approaches for host-directed therapeutic interventions. Results Mtb infection depresses the rate of mitochondrial respiration in macrophages Mitochondria are regarded as the energy factory of the cell that generates ATP through OXPHOS. It is reasonable to expect that on infection with Mtb, host energy metabolism is rewired, which has implications for understanding how Mtb causes disease. To examine the effect of mycobacterial infection on host OXPHOS, we made use of an extracellular flux analyzer (XF, Agilent Seahorse, Santa Clara, CA) and the mitochondrial respiration test (Nicholls et al., 2010) to determine key respiratory parameters in mammalian cells. These include basal respiration (Basal Resp), which is the initial OCR measured before addition of any inhibitors minus the non-mitochondrial respiration; ATP-linked respiration (ATP-linked OCR), which is determined after addition of oligomycin that inhibits ATP synthase (Complex V) and thus approximates the respiration required to drive ATP synthesis; proton leak, which is the difference between the ATP-linked OCR and the non-mitochondrial respiration; maximal respiration (Max Resp), which is induced after addition of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) that uncouples ATP synthesis from electron transport; spare respiratory capacity (SRC), which is the difference between maximal respiration and basal respiration; and non-mitochondrial respiration (Non-Mito Resp), which is the OCR after addition of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor (Figure 1B). SRC is an important parameter that reflects the ability of the cell to increase respiration to increase the supply of ATP in scenarios when the energy demand exceeds supply under conditions of stress or increased work load. Several lines of evidence preclude any contribution of the infecting Mtb to the measured OCR of infected macrophages. Firstly, we have previously demonstrated that 106 Mtb consumed 10–20 pmoles O2/min (Lamprecht et al., 2016) in contrast to 100–200 pmoles O2/min consumed by 80 000 to 100 000 macrophages under the basal conditions measured in this study. Thus, at a multiplicity of infection (MOI) of 1, 105 Mtb would result in a negligible contribution (<1 pmoles O2/min) to the OCR of the uninfected macrophages. Secondly, this basal OCR of Mtb was measured in media favourable for Mtb respiration and growth, whereas the intracellular macrophage environment is not conducive to ‘healthy’ Mtb respiration. Thirdly, Mtb infection at MOI of 1 and 2.5 progressively decreases the basal respiration (OCR) of both THP-1 macrophages and hMDMs (Figure 2—figure supplement 1) relative to that of uninfected macrophages. Should Mtb contribute to basal respiration (OCR), we should see an increase in OCR with increasing number of Mtb infecting the macrophages. Fourthly, the growth media of the infected macrophages after the time of infection and treatment was removed and the cells were washed in the XF assay medium before the XF assay, to remove most extracellular mycobacteria. When the washes of the macrophages were plated out on 7H11 agar plates, less than 200 CFU were obtained per well from the washes of the infected hMDMs (MOI 5), and less than 100 CFU per well from the infected THP-1 cells (MOI 5). To demonstrate that these extracellular mycobacteria do not contribute to the OCR readings of the infected macrophages, the final wash was transferred to a separate XF cell culture microplate and a separate mitochondrial respiration assay was performed on any extracellular bacteria present in the washes. The OCR and ECAR readings obtained were below 0 pmol/min and at 0 mpH/min, respectively, and the extracellular bacteria did not respond to the sequential injections of oligomycin, FCCP and rotenone and antimycin A (Figure 2—figure supplement 2A–D). Thus, the infecting mycobacteria do not contribute to the measured OCR of the infected macrophages under our conditions. It is not possible to ensure that every cell will be infected in in vitro infections, thus the percentage of uninfected cells will contribute to the resulting XF profiles. We infected the macrophages with Mtb-green fluorescent protein reporter strain (Mtb-GFP) and used bright-field and fluorescence microscopy to determine the percentage of cells that were infected. We found that there was an increase in the percentage of infected cells with an increase in MOI of both the THP-1 cells and the hMDMs (Figure 2—figure supplement 2E–G). Although the percentage of uninfected cells will contribute to the readout of the XF profiles, previous studies have demonstrated that lipids shed by intracellular mycobacteria, such as TDM and PIM2, spread via the endocytic network throughout the macrophage, and via exocytic vesicles to neighboring uninfected cells (Beatty et al., 2000; Xu et al., 1994) and can elicit the production of proinflammatory cytokines (Rhoades et al., 2003). Consequently, the bioenergetic metabolism of the ‘by-stander’ uninfected cells will also be modulated. Thus, the XF profiles are providing collective data of a mixed population of macrophages. Overall, our data demonstrated that strain pathogenicity and burden have distinct effects on virtually all respiratory parameters. Figure 2A and B shows that infection of THP-1 macrophages with Mtb or BCG significantly decreased the respiratory parameters: Basal Resp, ATP-linked OCR, proton leak and Max Resp (and SRC in the case of Mtb), and increased Non-Mito Resp. Similar patterns were observed at lower MOIs of 1 and 2.5, but to lesser degrees (Figure 2—figure supplement 1A–D). Infection with the dead Mtb only significantly reduced the respiratory parameters at MOIs of 2.5 and 5 (Figure 2—figure supplement 1C–D and Figure 2A–B). Figure 2 with 3 supplements see all Download asset Open asset Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI. Respiratory profiles (OCR) and respiratory parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and ∆Dead Mtb (heat-killed Mtb) at MOIs of 5 for 24 h. Refer to Figure 2—figure supplement 1 for profiles of lower MOIs. After obtaining basal respiration, cells were subjected to oligomycin (Oligo, 1.5 µM), which inhibits ATP synthase and demonstrates the mitochondrial ATP-linked OCR, followed by FCCP (cyanide-4-[trifluoromethoxy]phenylhydrazone), which uncouples mitochondrial respiration and maximizes OCR (1 µM for THP-1 and hMDMs), and finally antimycin A and rotenone (AntiA and Rot), which inhibit complex III and I in the ETC, respectively, and shut down respiration (0.5 µM of each for THP-1; 2.5 µM of each for hMDMs). Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. https://doi.org/10.7554/eLife.39169.003 Mtb infection of hMDMs dramatically reduced the respiratory parameters of the macrophage, while significantly increasing Non-Mito Resp (Figure 2C–D). Smaller reductions in the respiratory parameters were observed at lower Mtb MOIs of 1 and 2.5 with an increase in Non-Mito Resp (Figure 2—figure supplement 1E–H). Notably, contrary to Mtb, BCG infection increased the Max Resp and SRC of the hMDMs at all MOIs investigated. At a MOI of 5, BCG decreased the Basal Resp, ATP-linked OCR and proton leak, while increasing the Non-Mito Resp (Figure 2C–D). At lower MOIs, BCG had little effect on the other respiratory parameters (Figure 2—figure supplement 1E–H). Dead Mtb did not affect the respiratory parameters of the hMDMs at MOIs of 1 and 5, but a MOI of 2.5 increased the Max Resp and SRC of the macrophages as in the BCG infection (Figure 2C–D and Figure 2—figure supplement 1E–H). In Mtb-infected macrophages, the oxidative burst (via NADPH oxidase, which consumes O2) induced by a combination of infection, uncoupling with FCCP and inhibition of the ETC after treatment with antimycin A and rotenone increases the OCR above the initial OCR before treatment with oligomycin (Figure 2A and C). This results in the calculated basal respiration having a negative value (Figure 2B and D). As we have used standard equations to calculate the basal respiration (Nicholls et al., 2010), we propose that when the non-mitochondrial respiration is greater than the initial OCR readings before the addition of any inhibitors, an additional mitochondrial respiration assay should be performed without the addition of the FCCP to determine the non-mitochondrial respiration, to obtain the values of basal respiration and proton leak. Using this format, the true (positive) values for basal respiration and proton leak are obtained (Figure 2—figure supplement 3). The basal respiration of both Mtb and BCG infections at a MOI of 2.5 and 5 were less than that of uninfected THP-1 cells, and the proton leak of BCG was less than that of the uninfected THP-1 cells (Figure 2—figure supplement 3A–D). The non-mitochondrial respiration was increased in both of these infections at a MOI of 5. Similar patterns were observed with the Mtb- and BCG-infected hMDMs at a MOI of 5 (Figure 2—figure supplement 3E,F). In sum, there are profound contrasting respiratory differences among Mtb, BCG and dead Mtb infection of the macrophages. In particular, Max Resp, SRC and Non-Mito Resp are strongly influenced by the mycobacterial strain, burden and macrophage type. Mtb infection of hMDMs decreases Max Resp and SRC in contrast to BCG increasing Max Resp and SRC, and both strains increase Non-Mito Resp. SRC has consequences on how the macrophage responds to environmental stresses such as nutrient availability, redox state and changes in pH. Thus, an increase in the SRC of hMDMs following infection with potential vaccine candidates may aid identification of promising candidates. Strikingly, dead Mtb infection still alters the bioenergetic metabolism of the macrophage, in particular that of the THP-1 cells. This has implications for pharmacological killing of Mtb, as killing intracellular Mtb will not fully restore the macrophage’s bioenergetic metabolism to that of the uninfected macrophage. However, pharmacological killing will improve the bioenergetic profile of the live Mtb-infected macrophages, in particular, the ATP-linked Resp and the Non-Mito Resp. Therefore, improvements in these parameters of the infected macrophages can be used as indicators of effective pharmacological killing of Mtb during screening of potential anti-TB drug leads in Mtb-infected macrophages. Mtb infection reduces the extracellular acidification rate of the macrophage Glycolysis is the second pathway used to supply ATP for the energy requirements of the cell, in addition to anabolic intermediates. Here, we measured the glycolytic parameters of mycobacterial infected cells, including the glucose metabolism extracellular acidification rate after addition of glucose; the maximal glycolytic capacity (Gly capacity) following inhibition of OXPHOS ATP synthesis with oligomycin; and the non-glycolytic extracellular acidification measured after treatment of the mycobacterial infected cells with 2-deoxyglucose (2-DG), an inhibitor of hexokinase II, which catalyzes the first step of glycolysis (Figure 1C). The difference between the extracellular acidification rate of glucose metabolism rate and the maximal glycolytic capacity of the cells defines the spare glycolytic reserve. Mtb strikingly decreased the glycolytic parameters of both types of macrophages after 24 h (Figure 3). In THP-1 cells, all the mycobacterial strains reduced the glycolytic parameters at MOIs of 5 (Figure 3A–B) and 2.5 (Figure 3—figure supplement 1C,D), with dead Mtb having the least effects. At a MOI of 1, Mtb decreased the glycolytic parameters, BCG increased the glycolytic parameters and dead Mtb had no effect (Figure 3—figure supplement 1A–B). Figure 3 with 1 supplement see all Download asset Open asset Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI. ECAR profiles and glycolytic parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and dead Mtb at MOI of 5 for 24 h. Refer to Figure 3—figure supplement 1 for profiles at lower MOIs. After obtaining non-glycolytic acidification, glucose (Glc, 10 mM) was added to the cells, followed by oligomycin (1.5 µM), which inhibits ATP synthase inducing maximal glycolysis to compensate for loss of mitochondrial generated ATP, and finally 2-deoxyglucose (2-DG, 100 mM) to inhibit glycolysis and demonstrate that the prior acidification was generated by glycolysis. Profiles and glycolytic parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. https://doi.org/10.7554/eLife.39169.007 In hMDMs, Mtb at a MOI of 2.5 had little effect and at a MOI of 1 increased glucose metabolism extracellular acidification. (Figure 3—figure supplement 1E–H). Contrary to THP-1 cells, BCG and dead Mtb infection of hMDMs increased glucose metabolism acidification and the glycolytic capacity at all MOIs investigated. Increases in the non-glycolytic acidification were observed in the BCG and dead Mtb infections, probably as a result of the carbonic acid produced from CO2 generated by the tricarboxylic acid cycle (TCA). These results underscore the different modulations of dead and live Mtb on macrophage bioenergetics. In sum, marked glycolytic differences were observed between the virulent and non-virulent infections, with Mtb infection significantly reducing glucose metabolism extracellular acidification in the macrophages. BCG and dead-Mtb infections induced contrasting effects dependent on macrophage cell type, with a decrease in THP-1 glucose metabolism extracellular acidification versus an increase in hMDM extracellular acidification. Mtb infection shifts the bioenergetic phenotype of the macrophage towards quiescence To determine how mycobacterial infection shifts the energy metabolism of the macrophage, basal OCR was plotted as a function of ECAR to form a bioenergetic phenogram that the energy phenotypes of the macrophages. The energy phenotype of cells can be as glycolytic or quiescent (Figure Figure Download asset Open asset demonstrate that increasing MOI of Mtb shifts macrophages towards quiescent energy Basal OCR and ECAR from the respiratory assay (Figure before addition of oligomycin were plotted to of THP-1 cells and hMDMs infected with Mtb, BCG and ∆Dead Mtb at MOIs of 1, 2.5 and 5. Data are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. In THP-1 cells and hMDMs, infection with Mtb the most shift from an phenotype towards that of a phenotype with increasing MOI (Figure and D). In contrast, BCG and dead Mtb infections of THP-1 cells 2.5 and induced shifts towards quiescence (Figure and whereas in hMDMs, only BCG infection at a MOI of 5 a shift towards Dead Mtb did not affect the OCR of the hMDMs but decreased the ECAR at lower MOIs (Figure the differences between live and dead Mtb. In sum, our data demonstrate that Mtb infection shifts the energy phenotypes of human macrophages towards a metabolic quiescent Mtb decreases the glycolytic proton rate of macrophages studies using Mtb (Lachmandas et al., 2016), Mtb and (Gleeson et al., 2016), or profiling (Shi et al., to the that Mtb aerobic glycolysis for ATP as the In XF acidification of the extracellular as measured by is not for glycolysis as the mitochondrial TCA CO2 that is in the extracellular medium and to the acidification of the extracellular medium et al., In the glycolytic rate assay and inhibition of mitochondrial respiration after addition of rotenone and antimycin A of the contribution of the mitochondrial respiration to the rate of proton (Figure of the mitochondrial proton rate from the proton rate the glycolytic proton rate (Figure supplement 1A–D). To the is added to inhibit glycolytic acidification (Figure glycolysis to the ability of the cell to increase glycolysis after OXPHOS has been with rotenone and antimycin Figure 5 with 1 supplement see all Download asset Open asset Mtb infection reduces the glycolytic proton rate of macrophages. (A) Extracellular acidification can be by both and protons produced from pyruvate, the final of glycolysis, in addition to carbonic acid generated from CO2 from oxidation in the proton rate the glycolytic to be from the mitochondrial (Figure supplement 1A–D). Basal and glycolytic of THP-1 cells and hMDMs infected with Mtb, BCG and ∆Dead Mtb at MOI of 5 for h. Refer to Figure supplement for profiles at lower MOIs. basal of ECAR and OCR, to determine basal glycolytic rotenone and antimycin A were added to determine This was followed by addition of to ensure that the observed was by glycolysis. Profiles and are representative of two independent experiments. Data shown are the mean ± SD (n = Student’s t test relative to the uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. rate of ATP production was calculated as the of glycolytic ATP rate to and ATP rate that was from the ATP-linked OCR, a of of ATP in (F) THP-1 cells and (G) hMDM cells infected with Mtb, BCG or ∆Dead Mtb at MOI for h. Refer to Figure supplement for contribution of glycolysis and OXPHOS to the rate of ATP are SD (n = 6 biological replicates). Student’s t #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. The proton rates in Figure are the calculated values of the glycolytic without the acidification contribution from mitochondrial respiration. Basal glycolysis and glycolysis of the macrophages, which is induced when mitochondrial ATP synthase is the cell to use glycolysis to the ATP are as is that Mtb significantly reduces the basal and glycolytic rates of THP-1 cells (MOI 2.5 and 5, Figure Figure supplement 1E–H). In contrast, BCG and dead Mtb infections increased the basal glycolytic rates of THP-1 cells at all MOIs Furthermore, glycolysis was increased in BCG-infected cells at all MOIs, and in dead Mtb infections at a MOI of differences were observed between the live and dead Mtb In hMDMs, Mtb (MOI decreased both the glycolytic rate and the glycolytic rate significantly (Figure but lower MOIs (1 and did not affect the glycolytic rates of hMDMs (Figure supplement BCG infection increased both the glycolytic rate and the glycolytic rates at all MOIs, whereas dead Mtb had no effect (Figure Figure supplement In sum, profound differences were observed in the of infections with virulent versus non-virulent mycobacterial Mtb decreases the glycolytic in human macrophages at mycobacterial whereas BCG increases the glycolytic However, dead Mtb infection increases the in THP-1 cells and has no effect in When we the which is considered a of the glycolytic rate and with ECAR data of the infected macrophages, we found that the observed were dependent on the infecting mycobacterial To the reduced basal and of Mtb-infected THP-1 cells (Figure Figure supplement the observed decreased glycolytic ECAR and glycolytic capacity (Figure Figure 3—figure supplement 1A–D). In contrast, distinct differences were observed in the BCG infection of the THP-1 cells, increases in both basal and (Figure Figure supplement were to reductions in the glucose metabolism ECAR and glycolytic capacity (Figure Figure 3—figure supplement These were not observed in the BCG infection of hMDMs, increased basal and (Figure Figure supplement were by increased glucose metabolism ECAR and glycolytic capacity (Figure Figure 3—figure supplement 1E–H). However, between and ECAR were observed with dead Mtb infection of THP-1 cells with increased basal (Figure Figure supplement to or no reductions in glycolytic ECAR (Figure Figure 3—figure supplement 1A–D). in hMDMs, the changes in basal and in Mtb infection 1 and the

Foods, herbs, and phytoconstituents in alleviating metabolic dysfunction-associated fatty liver disease: A review

Methylation of 4-aminobutyrate aminotransferase (ABAT) by dnmy3b regulates chondrocytes metabolism and the development of osteoarthritis (OA)

Our previous studies have reported the effect of swietenine (a major bioactive component of Swietenia macrophylla seeds) in reversing and potentiating the effect of metformin in hyperglycemia and hyperlipidaemia in diabetic rats. Moreover, we reported that the anti-inflammatory effect of swietenine is mediated via the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). This study evaluated the effect of swietenine and its mechanisms in nonalcoholic fatty liver disease (NAFLD) in high-fat diet/streptozotocin-induced diabetic mice. The effect was assessed by determining blood biochemical parameters (glucose, cholesterol, triglycerides, alanine transaminase (ALT), asparate transaminase (AST), alkaline phosphatase (ALP), glutathione (GSH), total antioxidant capacity (TAC), and malondialdehyde (MDA)) and liver biochemical parameters (liver index, cholesterol, and triglycerides). Hepatic lipid accumulation (initial causative factor in NAFLD) was determined by oil-O-red staining. Gene expression (qPCR) and immunohistochemical studies were performed to elucidate the mechanism of swietenine’s effect in NAFLD. The critical regulators (genes and proteins) involved in lipogenesis (ACLY, ACC1, FASN, SREBP1c, and ChREBPβ) and oxidative stress (Nrf2, NQO-1 and HO-1) pathways were determined. In mice fed with a high-fat diet followed by streptozotocin injection, the liver cholesterol, triglycerides, and lipids were elevated. These increases were reversed by the oral administration of swietenine, 80 mg/kg body weight, on alternate days for eight weeks. Gene expression and immunohistochemical studies showed that swietenine reversed the elevated levels of crucial enzymes of lipogenesis (ACLY, ACC1 and FASN) and their master transcription factors (SREBP1c and ChREBPβ). Furthermore, swietenine activated the Nrf2 antioxidant defense mechanism, as evidenced by the upregulated levels of Nrf2, NQO-1, and HO-1. It is concluded that swietenine shows beneficial effects in diabetes-induced NAFLD via inhibiting lipogenesis and activating the Nrf2 pathway.

Introduction: Non-alcoholic fatty liver disease (NAFLD) has gradually become the primary cause of fatty liver disease. Betel nuts have been used to treat gastrointestinal diseases. Methods: In the present study, we analyzed the pathology, serology, gut flora, and metabolites in a rat model of NAFLD, with and without betel nut alkaloid treatment, using an integrated approach involving pathology, serological testing, 16S rRNA gene sequencing, and ultra-performance liquid chromatography-mass spectrometry metabolomics. Results: Two rats were used for model validation. Thirty SD rats were included and divided into the normal group (C group), NAFLD model group (M group), low-dose group, medium-dose group (T group), and high-dose group with intraperitoneal injection of arecoline. The expression of blood lipids was significantly downregulated at all three arecoline concentrations (p < 0.05). Alpha-diversity analysis of the intestinal flora showed significant differences among the three groups, with a significant reduction in population diversity in the M group and a recovery of population diversity after arecoline treatment. At the phylum level, the relative abundance of Firmicutes was significantly higher in the T group and Proteobacteria in the M group. The KEGG metabolic pathways included polyketide sugar unit biosynthesis and hypertrophic cardiomyopathy. Thirty-three significantly different metabolites were identified among the groups. Significantly different metabolites between groups T and M included indolepyruvate, 2-deoxystreptamine, sakuranetin, glycyl-leucine, and riboflavin. The KEGG metabolic pathway suggested a potential role for arachidonic acid metabolism, serotonergic synapses, neuroactive ligand-receptor interactions, tyrosine metabolism, and regiomelanin. Vitamin digestion and absorption, as well as regulation of lipolysis in adipocytes, were the main metabolic pathways that distinguished the T vs. M groups. PGE2 is involved in several metabolic pathways. Correlation analysis showed that 29 bacterial species were significantly associated with PGE2 levels in the M and T groups. Vagococcus, Lawsonia, Christensenella, unidentified Erysipelotrichaceae, unidentified Coriobacteriaceae, and five other bacterial groups are unique in the PGE2 metabolic pathway regulated by arecoline. Discussion: Arecoline has lipid-lowering effects and may exert therapeutic effects in NAFLD through intestinal metabolites and intestinal flora, as well as through the Butyricicoccus/Christensenella/Coriobacteriaceae-COX2/PGE2 pathway. Thus, arecoline may represent a potential drug or target for NAFLD treatment.

Introduction: Out-of-hospital cardiac arrest (OHCA) is associated with severe metabolic and acid-base disturbances, driven in part by mitochondrial dysfunction. Impaired mitochondrial function leads to lactic acidosis and impaired oxygen extraction. Ubiquinol and alpha-lipoic acid are important components of mitochondrial aerobic respiration. ALA functions as an important cofactor for multiple enzyme complexes in the Krebs cycle, while Coenzyme Q10 is a key electron carrier in the mitochondrial electron transport chain. In this study, we measured the cellular oxygen consumption rates (OCR) and the effects of in vitro Coenzyme Q10 and alpha-lipoic acid administration on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) from OHCA patients. Hypothesis: Administration of Coenzyme Q10 and ALA improves cellular oxygen consumption parameters in patients post-OHCA. Methods: Blood samples were collected from a cohort of patients post-OHCA and healthy controls. Cellular OCR parameters were measured in PBMCs pre- and post treatment with Coenzyme Q10 or ALA. The mitochondrial respiration was measured using an XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Seahorse Bioscience). A linear mixed-effects model was used to compare the OCR parameters between controls and post-OHCA patients and to estimate the treatment effect of Coenzyme Q10 and ALA, including a group-by-treatment interaction term. Results: A total of 53 patients with OHCA and 53 controls were included. Basal (4.88 [IQR:3.24, 7.76] vs. 6.97 [IQR:4.40, 9.86] pmol/min/µg protein, p=0.039) and maximal OCR (12.32 [IQR:7.06, 31.08] vs 29.2 [IQR:19.4, 40.42], p=&lt;0.0001) were significantly lower in the OHCA patients compared to the healthy controls. Basal (33%, p&lt;0.001) and maximal (47%, p=0.03) OCR increased after Coenzyme Q10 treatment in OHCA patients but not in healthy controls (Table 1). Basal (135%,p&lt;0.001) and maximal (89%, p=0.03) OCR also increased after ALA treatment in OHCA patients but not in healthy controls (Table 2). Conclusion: Mitochondrial metabolic profiles of OHCA patients demonstrated lower baseline cellular oxygen consumption when compared to healthy controls.Mitochondrial respiration increased significantly in PBMCs from cardiac arrest patients when treated with Coenzyme Q10 and ALA. These results suggest that Coenzyme Q10 and ALA could potentially have therapeutic benefits in cardiac arrest via metabolic effects on mitochondrial cellular respiration.

The interaction between nonalcoholic fatty liver disease (NAFLD) and chronic hepatitis B infection (CBI) was unclear. We aimed to investigate the association between NAFLD and CBI and the effect of NAFLD on response to antiviral therapy in pediatric population. All children aged 0-18years with liver biopsy-proven NAFLD, CBI, and co-existing NAFLD and CBI were consecutively collected. Children with co-existing CBI and NAFLD were considered as cases and n:m matched with simple NAFLD and simple CBI patients in the same cohort, respectively. In longitude study, the role of NAFLD in antiviral response was further analyzed in children with CBI who received antiviral treatment. Logistic or Cox regression models were used appropriately for analysis. 765 subjects were finally enrolled with 62 co-existing patients, 560 CBI patients, and 143 NAFLD patients. Multivariate analysis showed that HBV DNA level was negatively associated with NAFLD in CBI children (OR 0.376, 95% CI 0.173-0.818). Conversely, the severity of steatosis and levels of serum lipid profile were found to be inversely associated with CBI in NAFLD subjects. Then, in longitude study, we found that HBsAg loss at 96weeks of antiviral treatment was independently associated with NAFLD (aHR 3.245, 95% CI 1.288-8.176). An inverse association between CBI and NAFLD reciprocally existed in pediatric population. In longitude study, HBsAg loss was associated with NAFLD at week 96 of antiviral therapy.

Mechanism investigation of anti-NAFLD of Shugan Yipi Granule based on network pharmacology analysis and experimental verification

Introduction: Lactic acidosis and impaired oxygen extraction due to mitochondrial dysfunction are common post-arrest. Thiamine, a cofactor for pyruvate dehydrogenase, is necessary for aerobic metabolism. In two randomized controlled trials (RCTs) testing the effect of thiamine vs. placebo in out-of-hospital and in-hospital post-arrest patients (NCT03450707 and NCT02974257), no relationship was found between thiamine treatment and the primary outcome of change in lactate over 24 hours. Cellular oxygen consumption rates (OCRs) were measured in a subset of patients at baseline. Maximal and spare OCRs measure the capacity of mitochondria to increase cellular respiration from their basal state when stimulated, and may identify patients likely to benefit from thiamine. We conducted a post-hoc analysis of the two RCTs to evaluate the primary outcome in subgroups defined by baseline OCRs. Hypothesis: Patients with higher maximal and spare OCRs at baseline are more likely to benefit from thiamine treatment, as indicated by lower lactate levels. Methods: Basal, maximal and spare OCRs, collected at enrollment, were measured in peripheral blood mononuclear cells using an XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Seahorse Bioscience). Lactates (at 6, 12, and 24 hours) were log-transformed and analyzed using a linear mixed model controlling for baseline lactate. In patients who expired &lt;24 hours, lactate was imputed using the last available value with a 20% increase. Subgroups were defined by baseline OCR values above or below the median. The primary outcome was lactate at 24 hours, presented as a geometric mean difference (GMD). Results: A total of 74 patients had baseline OCR measurements. There was no statistically significant difference in lactate at 24 hours between placebo and thiamine in all patients (p=0.179) or in the low OCR (below median) groups. In patients with baseline maximum or spare OCRs above the median, lactate levels were significantly lower in those receiving thiamine (24 hour GMDs of 0.61 (95% CI: 0.45-0.82; p=0.001), and 0.53 (95% CI: 0.38-0.74; p&lt;0.001), respectively; Figure 1). Conclusion: In a post-hoc analysis of two RCTs, thiamine significantly improved the primary outcome in the subgroup of patients with higher spare and maximal oxygen consumption rates at baseline. Baseline OCRs could potentially identify post-arrest patients likely to benefit from thiamine.

The metabolic syndrome is often accompanied by liver disease, specifically nonalcoholic fatty liver disease (NAFLD). NAFLD is a clinicopathological entity characterized by accumulation of fat in the liver in the setting of minimal to no alcohol intake. It represents a spectrum of liver disease ranging from steatosis to advanced fibrosis or cirrhosis and can lead to hepatocellular carcinoma. The causes of NAFLD are multifactorial and include genetic predisposition and environmental factors including overnutrition. NAFLD is not a defining characteristic of the metabolic syndrome, but these conditions frequently coexist, especially in the setting of obesity. The prevalence of NAFLD in pediatrics has not been clearly determined, but this is considered the most common pediatric liver disease. An important challenge is that NAFLD is typically asymptomatic and is usually not diagnosed unless the clinician specifically evaluates the patient for it. The diagnosis of NAFLD is established through a liver biopsy. There are pathogenetic links between NAFLD and the metabolic syndrome, but the precise pathogeneses of these conditions remain to be fully elucidated. The effects of NAFLD on the natural history of the metabolic syndrome, and vice versa, are not known. However, it is possible that these conditions may exacerbate one another. Further research is needed to answer important questions regarding pediatric NAFLD and its relationship to the metabolic syndrome. What is clear is that given the high prevalence of overweight and obesity, clinicians need to be well informed about the risk of liver disease in the patient with metabolic syndrome.

PurposeThe rs641738 C>T in membrane-bound O-acyltransferase domain-containing protein 7 (MBOAT7) is implicated, along with the rs738409 C>G polymorphism in patatin-like phospholipase domain-containing protein 3 (PNPLA3), in nonalcoholic fatty liver disease (NAFLD). The association of these polymorphisms and NAFLD are investigated in Hispanic children with obesity.MethodsObese children with and without NAFLD were enrolled at a pediatric tertiary care health system and genotyped for MBOAT7 rs641738 C>T and PNPLA3 rs738409 C>G. NAFLD was characterized by the ultrasonographic presence of hepatic steatosis along with persistently elevated liver enzymes. Genetic variants and demographic and biochemical data were analyzed for the effects on NAFLD.ResultsAmong 126 enrolled subjects, 84 in the case group had NAFLD and 42 in the control group did not. The two groups had similar demographic distribution. NAFLD was associated with abnormal liver enzymes and elevated triglycerides and cholesterol (p<0.05). Children with NAFLD had higher percentage of PNPLA3 GG genotype at 70.2% versus 31.0% in non-NAFLD, and lower MBOAT7 TT genotype at 4.8% versus 16.7% in non-NAFLD (p<0.05). PNPLA3 rs738409 C>G had an additive effect in NAFLD; however, MBOAT7 rs641738 C>T had no effects alone or synergistically with PNPLA3 polymorphism. NAFLD risk increased 3.7-fold in subjects carrying PNPLA3 GG genotype and decreased in MBOAT7 TT genotype.ConclusionIn Hispanic children with obesity, PNPLA3 rs738409 C>G polymorphism increased the risk for NAFLD. The role of MBOAT7 rs641738 variant in NAFLD is less evident.

Background The association between nonalcoholic fatty liver disease (NAFLD) and Helicobacter pylori (H. pylori) is controversial. We conducted a retrospective study to clarify the seroprevalence of H. pylori infection and the relationship between NAFLD and H. pylori infection in north urban Chinese. Methods The retrospective study was performed at Aerospace Center Hospital in Beijing. All subjects in this study were a healthy population who underwent health examinations at the hospital between 2012 and 2015. A logistic regression model was used to calculate the association between NAFLD and H. pylori infection. Age, gender, underlying diseases, and metabolic syndrome (MS) were adjusted. Effects of NAFLD on H. pylori infection in a different age, gender, and number of MS characteristic subgroups were analyzed. Results There were 7803 (43.4%) subjects with H. pylori infection, 3726 (20.7%) with mild NAFLD, 730 (4.1%) with moderate NAFLD, and 369 (2.1%) with severe NAFLD among 17971 subjects. H. pylori infection was related to the seroprevalence of any level of NAFLD, including mild, moderate, and severe NAFLD (OR = 1.607, 95% CI: 1.487-1.736; OR = 1.770, 95% CI: 1.519-2.063; and OR = 2.120, 95% CI: 1.714-2.526, respectively). The results of subgroup analysis showed that the risk of incident NAFLD from H. pylori infection had significant interactions by subjects with or without MS characteristics. Moreover, as the number of MS characteristics in patients with a fatty liver increased, the risk of H. pylori infection also increased. Conclusions NAFLD may be associated with H. pylori infection in a Chinese population. Younger, male NAFLD patients and those meeting more characteristics of MS were more likely to have H. pylori infection.