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Background and purpose Overexpression or aberrant activation of the T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes gene expression and growth of solid tumours, implying that TOPK would be a rational target in developing novel anticancer drugs. Acetylshikonin, a diterpenoid compound isolated from Lithospermum erythrorhizon root, exerts a range of biological activities. Here we have investigated whether acetylshikonin, by acting as an inhibitor of TOPK, can attenuate the proliferation of colorectal cancer cells and the growth of patient-derived tumours, in vitro and in vivo. Experimental approach Targets of acetylshikonin, were identified using kinase profiling analysis, kinetic/binding assay, and computational docking analysis and knock-down techniques. Effects of acetylshikonin on colorectal cancer growth and the underlying mechanisms were evaluated in cell proliferation assays, propidium iodide and annexin-V staining analyses and western blots. Patient-derived tumour xenografts in mice (PDX) and immunohistochemistry were used to assess anti-tumour effects of acetylshikonin. Key results Acetylshikonin directly inhibited TOPK activity, interacting with the ATP-binding pocket of TOPK. Acetylshikonin suppressed cell proliferation by inducing cell cycle arrest at the G1 phase, stimulated apoptosis, and increased the expression of apoptotic biomarkers in colorectal cancer cell lines. Mechanistically, acetylshikonin diminished the phosphorylation and activation of TOPK signalling. Furthermore, acetylshikonin decreased the volume of PDX tumours and reduced the expression of TOPK signalling pathway in xenograft tumours. Conclusion and implications Acetylshikonin suppressed growth of colorectal cancer cells by attenuating TOPK signalling. Targeted inhibition of TOPK by acetylshikonin might be a promising new approach to the treatment of colorectal cancer.

[Image: see text] Intracellular nucleotides and acyl-CoAs are metabolites that are central to the regulation of energy metabolism. They set the cellular energy charge and redox state, act as allosteric regulators, modulate signaling and transcription factors, and thermodynamically activate substrates for oxidation or biosynthesis. Unfortunately, no method exists to simultaneously quantify these biomolecules in tissue extracts. A simple method was developed using ion-pairing reversed-phase high-performance liquid chromatography–electrospray-ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to simultaneously quantify adenine nucleotides (AMP, ADP, and ATP), pyridine dinucleotides (NAD(+) and NADH), and short-chain acyl-CoAs (acetyl, malonyl, succinyl, and propionyl). Quantitative analysis of these molecules in mouse liver was achieved using stable-isotope-labeled internal standards. The method was extensively validated by determining the linearity, accuracy, repeatability, and assay stability. Biological responsiveness was confirmed in assays of liver tissue with variable durations of ischemia, which had substantial effects on tissue energy charge and redox state. We conclude that the method provides a simple, fast, and reliable approach to the simultaneous analysis of nucleotides and short-chain acyl-CoAs.

De novo lipogenesis (DNL) is disrupted in a wide range of human disease. Thus, quantification of DNL may provide insight into mechanisms and guide interventions if it can be performed rapidly and noninvasively. DNL flux is commonly measured by 2H incorporation into fatty acids following deuterated water (2H2O) administration. However, the sensitivity of this approach is limited by the natural abundance of 13C, which masks detection of 2H by mass spectrometry. Here we report that high-resolution Orbitrap gas-chromatography mass-spectrometry resolves 2H and 13C fatty acid mass isotopomers, allowing DNL to be quantified using lower 2H2O doses and shorter experimental periods than previously possible. Serial measurements over 24-hrs in mice detects the nocturnal activation of DNL and matches a 3H-water method in mice with genetic activation of DNL. Most importantly, DNL is detected in overnight-fasted humans in less than an hour and is responsive to feeding during a 4-h study. Thus, 2H specific MS provides the ability to study DNL in settings that are currently impractical., Fat synthesis is necessary for normal physiology, but its dysregulation contributes to the pathology of many diseases. Here, the authors report a high-resolution mass spectrometry approach that quantifies fat synthesis flux in humans and mice following a brief and low dose of deuterated water.

Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, 1H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed.

BackgroundSkin cancer is one of the most commonly diagnosed cancers worldwide. The 5-year survival rate of the most aggressive late-stage skin cancer ranges between 20 and 30%. Thus, the discovery and investigation of novel target therapeutic agents that can effectively treat skin cancer is of the utmost importance. The T-lymphokine-activated killer cell-originated protein kinase (TOPK), which belongs to the serine-threonine kinase class of the mitogen-activated protein kinase kinase (MAPKK) family, is highly expressed and activated in skin cancer. The present study investigates the role of 3-deoxysappanchalcone (3-DSC), a plant-derived functional TOPK inhibitor, in suppressing skin cancer cell growth.PurposeIn the context of skin cancer prevention and therapy, we clarify the effect and mechanism of 3-DSC on different types of skin cancer and solar-simulated light (SSL)-induced skin hyperplasia.MethodsIn an in vitro study, western blotting and in vitro kinase assays were utilized to determine the protein expression of TOPK and its activity, respectively. Pull-down assay with 3-DSC and TOPK (wild-type and T42A/N172 mutation) was performed to confirm the direct interaction between T42A/N172 amino acid sites of TOPK and 3-DSC. Cell proliferation and anchorage-independent cell growth assays were utilized to determine the effect of 3-DSC on cell growth. In an in vivo study, the thickness of skin and tumor size were measured in the acute SSL-induced inflammation mouse model or SK-MEL-2 cell-derived xenografts mouse model treated with 3-DSC. Immunohistochemistry analysis of tumors isolated from SK-MEL-2 cell-derived xenografts was performed to determine whether cell-based results observed upon 3-DSC treatment could be recapitulated in vivo.Results3-DSC is able to inhibit cell proliferation in skin cancer cells in an anchorage-dependent and anchorage-independent manner by regulation of TOPK and its related signaling pathway in vitro. We also found that application of 3-DSC reduced acute SSL-induced murine skin hyperplasia. Additionally, we observed that 3-DSC decreased SK-MEL-2 cell-derived xenograft tumor growth through attenuating phosphorylation of TOPK and its downstream effectors including ERK, RSK, and c-Jun.ConclusionsOur results suggest that 3-DSC may function in a chemopreventive and chemotherapeutic capacity by protecting against UV-induced skin hyperplasia and inhibiting tumor cell growth by attenuating TOPK signaling, respectively.

Carbohydrates and lipids are primary energy sources in mammals; thus, their oxidation and synthesis are carefully controlled. The tricarboxylic acid (TCA) cycle is necessary to oxidize these substrates, and in liver it also supports their synthesis. However, its role in regulating these processes, particularly de novo lipogenesis (DNL) is underappreciated. We report that activation of hepatic gluconeogenesis (GNG) is required to trigger the canonical lipid and carbohydrate response to fasting in mice. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes cataplerotic flux from the TCA cycle and is required for GNG from lactate, pyruvate and amino acids. Knockdown (KD) or knockout (KO) of PEPCK caused the massive accumulation of TCA cycle intermediates and KO mice developed fatty liver after an overnight fast. Lipidomic profiling of liver suggested increased lipid desaturation and elongation with loss of PEPCK. 2H NMR analysis of liver lipids following 2H2O administration demonstrated elevated fasting DNL in KO mice, which was similar to fed WT mice. In addition, fasting ketogenesis, estimated by infusions of [U-13C]BHB and [3,4-13C]AcAc, was decreased in KO mice. Despite an impaired fasting response, the expression of genes related to control of fat oxidation and DNL were not decreased, suggesting that loss of regulation was mediated by a metabolic mechanism. Inhibition of GNG produced a reduced mitochondrial redox state which limited oxidative pathways and decreased acetyl-CoA concentration. Inhibition of GNG also decreased fasting ATP requirements, resulting in a high energy charge which suppressed activation of AMPK, its canonical suppression of acetyl-CoA carboxylase and caused increased malonyl-CoA during fasting. Hence, PEPCK mediated cataplerosis and GNG during fasting is not only an important source of glucose, it is also necessary to shift cellular redox and energetics to states necessary to suppress lipogenesis and maintain ketogenesis during fasting. Disclosure S. Deja: None. J. Duarte: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. G. Vale: None. J. Young: Board Member; Self; Metalytics. Consultant; Self; Pfizer Inc. J. Browning: None. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184, P41EB015908); Robert A. Welch Foundation (I-1804)

Liver triglyceride accumulation associated with insulin resistance is partially mediated by elevated de novo lipogenesis (DNL); thus, limiting this pathway is a promising therapeutic strategy for NAFLD. Targeted suppression of acetyl-CoA carboxylase (ACC) activity depletes malonyl-CoA resulting in lower DNL and a simultaneous increase in fat oxidation due to activation of CPT-1. Recently developed ACC1/2 liver specific double KO mice (LDKO) are protected from hepatic steatosis and closely recapitulate pharmacological inhibition of ACC1/2 in humans with NAFLD. However, hepatic ACC inhibition causes surprising elevations in circulating triglyceride and glucose concentrations. LDKO mice had worsened glucose tolerance, which was most consequential during a HFD and especially prominent during fasting. Glucose labeling from 2H2O and [U-13C3] lactate was higher in LDKO hepatocytes, an effect that was dependent on the availability of long chain fatty acids, suggesting that loss of malonyl-CoA and activation of CPT-1 promote gluconeogenesis (GNG). Ketogenesis was increased in LDKO hepatocytes and showed limited response to insulin. Acetyl-CoA was elevated in the fed state, as previously reported, but was normal during fasting when hyperglycemia was most prominent. LDKO mice exhibited massive accumulation of liver citrate and a high fasted hepatic energy charge, which are both known to activate GNG. Hence, despite improved hepatic lipid profile and insulin sensitivity, LDKO liver activates GNG. The resulting hyperinsulinemia partially ameliorates the glycemia phenotype, but may also induce hepatomegaly through canonical signaling pathways and effects on energy status. Thus, activation of fat oxidation in liver lowers lipid accumulation, but has downstream metabolic effects on energetically dependent pathways, such as GNG, that may promote elevated glucose levels in contexts of reduced insulin action, such as fasting and diet induced obesity. Disclosure S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. C. Kim: None. J. Browning: None. J.D. Horton: Board Member; Self; Pfizer Inc. Consultant; Self; Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Stock/Shareholder; Self; Catabasis Pharmaceuticals. Other Relationship; Self; Regeneron Pharmaceuticals, Sanofi. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184, P41EB015908); Robert A. Welch Foundation (I-1804)

Hepatic lipid synthesis and Vldl production plays a major role in whole body lipid homeostasis. Insulin imposed regulation of liver metabolism is in large part executed on a transcriptional level. Several published studies focused on the effect of hepatic insulin resistance and the role of mTORC1 in liver lipid metabolism, sometimes with conflicting results. Here we used mouse genetic models with short-term liver specific removal of the insulin receptor (complete signaling pathway inactivation), Tsc1 (mTORC1 activation), Raptor (mTORC1 inactivation), Pten (simultaneous mTORC1 and Akt activation), and Raptor Pten (simultaneous mTORC1 inactivation and Akt activation) to precisely dissect the role of insulin signaling in the regulation of lipid metabolism. In vivo 2H2O administration, 1H and 2H NMR analyses revealed hepatic de novo fatty acid (FA) and triglyceride (TG) synthesis, export, as well as FA profiles both in liver and plasma. By direct examination of in vivo hepatic de novo lipogenesis (DNL), the direction and magnitude of changes observed in lipogenic gene expression did not correlate well with differences observed in DNL flux. For example, PtenLKO mice had a 3-fold increase in DNL flux with little effect on lipogenic gene expression. Our results suggest that in some contexts other factors, possibly including metabolite availability, TCA cycle metabolism, redox state or energy charge impose stronger control over DNL than regulation of gene expression. We also show that triglyceride synthesis and incorporation of de novo synthesized FA into triglycerides is regulated by mTORC1. While mTORC1 positively regulates export of newly synthesized TG it has a negative effect on export of de novo produced FA. Disclosure B. Kucejova: None. S. Deja: None. J. Duarte: None. X. Fu: None. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184)

Elevated hepatic de novo lipogenesis (DNL) is a key characteristic of patients with Nonalcoholic Fatty Liver Disease (NAFLD). Liver pyruvate carboxylase (PC) may be critical for DNL since PC replenishes oxaloacetate that is converted to citrate as source for acetyl-CoA during DNL. PC may also play a role in triglyceride (TG) synthesis by replenishing oxaloacetate that is used for glyceroneogenesis. Thus, PC is considered a lipogenic enzyme, though most evidence has been derived from non-liver cell culture models and indirect measures in livers of animal models. Liver-specific PC knockout mice (LPCKO) have lower circulating TG on a high fat diet, but fasting liver TG was not improved. To determine whether liver-specific PC is required for DNL and glyceroneogenesis, we examined LPCKO mice using a 1H/2H NMR approach to directly measure flux through DNL and TG-glycerol synthesis. PCf/f and LPCKO mice were exposed to either standard chow or a high sucrose diet (HSD) for 10 days. Ad libitum fed PCf/f and LPCKO mice were then administered D2O overnight. Liver lipids were extracted, and positional isotopomer analysis by 1H/2H NMR was used to determine DNL and TG-glycerol synthesis fluxes. DNL flux was not significantly different between PCf/f and LPCKO mice under standard chow conditions and only tended to be moderately lower in LPCKO mice after a short-term HSD treatment. TG-glycerol synthesis was also similar between PCf/f and LPCKO, regardless of diet. Surprisingly, liver triglyceride levels were modestly higher in standard chow fed LPCKO than PCf/f mice, and no significant differences were found between HSD-fed PCf/f and LPCKO mice. Our initial findings suggest that PC is not required for DNL and TG-glycerol synthesis during the fed state and therefore indicate that alternative pathways may exist to supply of cytosolic acetyl-CoA for lipid synthesis. Disclosure M.R. Inigo: None. S. Deja: None. B. Kucejova: None. J.A. Fletcher: None. X. Fu: None. S.C. Burgess: None. Funding Robert A. Welch Foundation (I-1804)

Genetic variants that increase the risk of fatty liver disease and cirrhosis have recently been identified in the proximity of membrane-bound O-acyltransferase domain-containing 7 (MBOAT7). To elucidate the link between these variants and fatty liver disease, we characterized Mboat7 liver-specific KO mice (Mboat7 LSKO). Chow-fed Mboat7 LSKO mice developed fatty livers and associated liver injury. Lipidomic analysis of liver using MS revealed a pronounced reduction in 20-carbon PUFA content in phosphatidylinositols (PIs) but not in other phospholipids. The change in fatty acid composition of PIs in these mice was associated with a marked increase in de novo lipogenesis because of activation of SREBP-1c, a transcription factor that coordinates the activation of genes encoding enzymes in the fatty acid biosynthesis pathway. Hepatic removal of both SREBP cleavage-activating protein (Scap) and Mboat7 normalized hepatic triglycerides relative to Scap-only hepatic KO, showing that increased SREBP-1c processing is required for Mboat7-induced steatosis. This study reveals a clear relationship between PI fatty acid composition and regulation of hepatic fat synthesis and delineates the mechanism by which mutations in MBOAT7 cause hepatic steatosis., Graphical abstract

Key points Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. Abstract Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

The heart oxidizes fatty acids, carbohydrates, and ketone bodies inside the tricarboxylic acid (TCA) cycle to generate the reducing equivalents needed for ATP production. Competition between these substrates makes it difficult to estimate the extent of pyruvate oxidation. Previously, hyperpolarized pyruvate detected propionate-mediated activation of carbohydrate oxidation, even in the presence of acetate. In this report, the optimal concentration of propionate for the activation of glucose oxidation was measured in mouse hearts perfused in Langendorff mode. This study was performed with a more physiologically relevant perfusate than the previous work. Increasing concentrations of propionate did not cause adverse effects on myocardial metabolism, as evidenced by unchanged O2consumption, TCA cycle flux, and developed pressures. Propionate at 1 mM was sufficient to achieve significant increases in pyruvate dehydrogenase flux (3×), and anaplerosis (6×), as measured by isotopomer analysis. These results further demonstrate the potential of propionate as an aid for the correct estimation of total carbohydrate oxidative capacity in the heart. However, liquid chromotography/mass spectroscopy-based metabolomics detected large changes (~30-fold) in malate and fumarate pool sizes. This observation leads to a key observation regarding mass balance in the TCA cycle; flux through a portion of the cycle can be drastically elevated without changing the O2consumption.

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of (2)H and (13)C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from (2)H and (13)C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1–C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical with (2)H or (13)C NMR.

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the US population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, we studied flux through pathways of hepatic oxidative metabolism and gluconeogenesis using 5 simultaneous stable isotope tracers in ketotic (24-hour-fasted) individuals with a wide range of hepatic triglyceride content levels (0%–52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic acid (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when β-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as calculated hepatic oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is a consequence not of acetyl-CoA production per se, but rather of how acetyl-CoA is further metabolized in liver.

Rats selectively bred for high capacity running (HCR) or low capacity running (LCR) display divergence for intrinsic aerobic capacity and hepatic mitochondrial oxidative capacity, both factors associated with susceptibility for nonalcoholic fatty liver disease. Here, we tested if HCR and LCR rats display differences in susceptibility for hepatic steatosis after 16 wk of high-fat diets (HFD) with either 45% or 60% of kcals from fat. HCR rats were protected against HFD-induced hepatic steatosis, whereas only the 60% HFD induced steatosis in LCR rats, as marked by a doubling of liver triglycerides. Hepatic complete fatty acid oxidation (FAO) and mitochondrial respiratory capacity were all lower in LCR compared with HCR rats. LCR rats also displayed lower hepatic complete and incomplete FAO in the presence of etomoxir, suggesting a reduced role for noncarnitine palmitoyltransferase-1-mediated lipid catabolism in LCR versus HCR rats. Hepatic complete FAO and mitochondrial respiration were largely unaffected by either chronic HFD; however, 60% HFD feeding markedly reduced 2-pyruvate oxidation, a marker of tricarboxylic acid (TCA) cycle flux, and mitochondrial complete FAO only in LCR rats. LCR rats displayed lower levels of hepatic long-chain acylcarnitines than HCR rats but maintained similar levels of hepatic acetyl-carnitine levels, further supporting lower rates of β-oxidation, and TCA cycle flux in LCR than HCR rats. Finally, only LCR rats displayed early reductions in TCA cycle genes after the acute initiation of a HFD. In conclusion, intrinsically high aerobic capacity confers protection against HFD-induced hepatic steatosis through elevated hepatic mitochondrial oxidative capacity.

SummaryDysregulated mitochondrial metabolism during hepatic insulin resistance may contribute to pathophysiologies ranging from elevated glucose production to hepatocellular oxidative stress and inflammation. Given that obesity impairs insulin action but paradoxically activates mTORC1, we tested whether insulin action and mammalian target of rapamycin complex 1 (mTORC1) contribute to altered in vivo hepatic mitochondrial metabolism. Loss of hepatic insulin action for 2 weeks caused increased gluconeogenesis, mitochondrial anaplerosis, tricarboxylic acid (TCA) cycle oxidation, and ketogenesis. However, activation of mTORC1, induced by the loss of hepatic Tsc1, suppressed these fluxes. Only glycogen synthesis was impaired by both loss of insulin receptor and mTORC1 activation. Mice with a double knockout of the insulin receptor and Tsc1 had larger livers, hyperglycemia, severely impaired glycogen storage, and suppressed ketogenesis, as compared to those with loss of the liver insulin receptor alone. Thus, activation of hepatic mTORC1 opposes the catabolic effects of impaired insulin action under some nutritional states.

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.

Hepatic metabolism is critically altered by the pathology of many diseases, including obesity, diabetes and NAFLD. Quantitative estimation of gluconeogenic flux and TCA cycle turnover are clinically useful parameters as they represent major biosynthetic and energetic pathways that are perturbed by these diseases. NMR spectroscopy is an important technique used to study hepatic fluxes since it can precisely distinguish between 2H and 13C metabolic tracers, and provide position-specific enrichment. However, the throughput of NMR for flux analysis is hindered by its low sensitivity, making microscale experiments that have limited biological mass (e.g., primary hepatocytes, tissue biopsies or mouse blood analysis) often infeasible. Thus, we investigate an alternative approach based on GC-MS measurements of glucose mass isotopomers and mathematical modeling of metabolic fluxes. In contrast with NMR, MS approaches are very sensitive, but cannot directly distinguish nuclei or positional enrichment information. Hence, we validated a GC-MS method in isolated perfused mouse liver and in vivo infusions in rats against NMR measurements. A medium-scale metabolic network containing atom transitions for both hydrogen and carbon atoms in the gluconeogenic pathway and TCA cycle was able to deconvolute 2H and 13C labeling information and estimate metabolic fluxes. Although some fluxes were significantly different between the NMR and GC-MS, there was excellent correlation between the two approaches. Finally, we applied GC-MS based simultaneous 2H and 13C approach in primary mouse hepatocytes cultured in 60 mm dishes, an experiment not feasible using standard NMR equipment. This opens future possibilities for drug screening and translational flux studies. Disclosure S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. J. Young: None. S.C. Burgess: None.

Production of glucose by the liver is crucial for maintaining whole-body gluconeogenesis and is dependent on carbon substrates provided by anaplerotic pathways into the TCA cycle. In the liver, the large majority of anaplerotic flux is through the conversion of pyruvate to oxaloacetate by the action of pyruvate carboxylase (PC). To investigate the role of PC in hepatic gluconeogenesis, we produced a liver-specific PC knockout mouse (LPKCO). LPCKO mice have impaired ability to convert pyruvate into glucose. Surprisingly, they display no difference in blood glucose under fed and overnight fasted conditions. LPCKO mice, however, have elevated concentrations of ketone bodies in plasma under both fed and fasted conditions, suggesting a switch from gluconeogenesis to ketogenesis. Additionally, we observed changes in kidney gene expression that suggest a compensatory upregulation of renal gluconeogenesis. Using stable isotope tracers, we analyzed anaplerotic and TCA cycle fluxes in LPCKO livers and found that LPCKO livers are deficient in anaplerotic flux and have a large reduction but not total ablation of hepatic gluconeogenesis. The LPCKO livers also show a significant reduction in TCA cycle turnover and reduced concentrations of several TCA cycle metabolites. On a high-fat diet (HFD), LPCKO mice gain slightly less weight than their non-transgenic littermates. After 12 weeks of HFD the LPCKO mice have significantly lower blood glucose and plasma insulin compared to non-transgenic littermates. When subjected to a glucose tolerance test, HFD-fed LPCKO mice show significantly better glucose tolerance than non-transgenic littermates. Overall, we demonstrate that flux through hepatic PC is required for anaplerosis and TCA cycle function in the liver and that restricting hepatic gluconeogenesis and TCA cycle turnover may help to protect against the deleterious metabolic effects of high fat diet. Disclosure D. Cappel: None. S. Deja: None. X. Fu: None. S.C. Burgess: None.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme that is highly expressed in the liver and kidney but is also expressed at lower levels in a variety of other tissues where it may play adjunct roles in fatty acid esterification, amino acid metabolism, and/or TCA cycle function. PEPCK is expressed in the enterocytes of the small intestine, but it is unclear whether it supports a gluconeogenic rate sufficient to affect glucose homeostasis. To examine potential roles of intestinal PEPCK, we generated an intestinal PEPCK knockout mouse. Deletion of intestinal PEPCK ablated ex vivo gluconeogenesis but did not significantly affect glycemia in chow, high-fat diet, or streptozotocin-treated mice. In contrast, postprandial triglyceride secretion from the intestine was attenuated in vivo, consistent with a role in fatty acid esterification. Intestinal amino acid profiles and13C tracer appearance into these pools were significantly altered, indicating abnormal amino acid trafficking through the enterocyte. The data suggest that the predominant role of PEPCK in the small intestine of mice is not gluconeogenesis but rather to support nutrient processing, particularly with regard to lipids and amino acids.NEW & NOTEWORTHY The small intestine expresses gluconeogenic enzymes for unknown reasons. In addition to glucose synthesis, the nascent steps of this pathway can be used to support amino acid and lipid metabolisms. When phosphoenolpyruvate carboxykinase, an essential gluconeogenic enzyme, is knocked out of the small intestine of mice, glycemia is unaffected, but mice inefficiently absorb dietary lipid, have abnormal amino acid profiles, and inefficiently catabolize glutamine. Therefore, the initial steps of intestinal gluconeogenesis are used for processing dietary triglycerides and metabolizing amino acids but are not essential for maintaining blood glucose levels.

SummaryGluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D.

Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.

The manner in which insulin resistance impinges on hepatic mitochondrial function is complex. Although liver insulin resistance is associated with respiratory dysfunction, the effect on fat oxidation remains controversial, and biosynthetic pathways that traverse mitochondria are actually increased. The tricarboxylic acid (TCA) cycle is the site of terminal fat oxidation, chief source of electrons for respiration, and a metabolic progenitor of gluconeogenesis. Therefore, we tested whether insulin resistance promotes hepatic TCA cycle flux in mice progressing to insulin resistance and fatty liver on a high-fat diet (HFD) for 32 weeks using standard biomolecular and in vivo (2)H/(13)C tracer methods. Relative mitochondrial content increased, but respiratory efficiency declined by 32 weeks of HFD. Fasting ketogenesis became unresponsive to feeding or insulin clamp, indicating blunted but constitutively active mitochondrial β-oxidation. Impaired insulin signaling was marked by elevated in vivo gluconeogenesis and anaplerotic and oxidative TCA cycle flux. The induction of TCA cycle function corresponded to the development of mitochondrial respiratory dysfunction, hepatic oxidative stress, and inflammation. Thus, the hepatic TCA cycle appears to enable mitochondrial dysfunction during insulin resistance by increasing electron deposition into an inefficient respiratory chain prone to reactive oxygen species production and by providing mitochondria-derived substrate for elevated gluconeogenesis.

The three water-soluble chitosan derivatives bearing double functional groups were synthesized with 2,3-epoxypropyltrimethylammonium chloride and benzaldehyde as modifiers through formation of Schiff base, reduction, N-methylation and O-quaternization. The synthesized chitosan derivatives were applied to cotton fabrics together with citric acid (CA) as the crosslinking agent to evaluate their use as durable antimicrobial textile finishing agents. The optimal finishing conditions were investigated and antibacterial activities of finished fabrics were tested with “Shake-Flask Test”. The fabrics finished showed strong antimicrobial activities and fairly good durability. The antibacterial efficiency of S. aureus and E. coli was more than 99% and 96% respectively, when crosslinked with CA (14%, o.w.f) through a two-bath process. The order of antimicrobial activities of the chitosan derivatives was O-quaternized-N,N-biethyl-N-benzyl ammonium chitosans chloride (O-QCTS-DEBn) ≥ O-quaternized-N-chitosan Schiff bases (O-QCTSS) > O-quaternized-N-benzyl-chitosans (O-QCTS-Bn). The antibacterial activity of using O-QCTSS finishing fabric was still more than 75% after 20 consecutive home launderings.

Hepatic ketogenesis provides a vital systemic fuel during fasting because ketone bodies are oxidized by most peripheral tissues and, unlike glucose, can be synthesized from fatty acids via mitochondrial β-oxidation. Since dysfunctional mitochondrial fat oxidation may be a cofactor in insulin-resistant tissue, the objective of this study was to determine whether diet-induced insulin resistance in mice results in impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis. Ketone turnover (μmol/min) in the conscious and unrestrained mouse was responsive to induction and diminution of hepatic fat oxidation, as indicated by an eightfold rise during the fed (0.50+/−0.1)-to-fasted (3.8+/−0.2) transition and a dramatic blunting of fasting ketone turnover in PPARα−/−mice (1.0+/−0.1). C57BL/6 mice made obese and insulin resistant by high-fat feeding for 8 wk had normal expression of genes that regulate hepatic fat oxidation, whereas 16 wk on the diet induced expression of these genes and stimulated the function of hepatic mitochondrial fat oxidation, as indicated by a 40% induction of fasting ketogenesis and a twofold rise in short-chain acylcarnitines. Together, these findings indicate a progressive adaptation of hepatic ketogenesis during high-fat feeding, resulting in increased hepatic fat oxidation after 16 wk of a high-fat diet. We conclude that mitochondrial fat oxidation is stimulated rather than impaired during the initiation of hepatic insulin resistance in mice.

Sucrose fatty acid esters were synthesized by the transesterification of sucrose with aliphatic esters under ultrasound irradiation in good yield (⩾73%). The optimum reaction conditions for the transesterification reaction include a molar ratio of sucrose to fatty acid ethyl ester of 2:1 and the use of a 13% mol anhydrous K2CO3 catalyst. The optimum reaction temperature was set at 70 °C, the optimum reaction time was 2 h, and the optimum reaction pressure was 11 kPa. The reaction had excellent monoester selectivity. The proportion of monoester (6-monoester + 6′-monoester) in the purified products was up to 92–95% via flash column chromatography over silica gel, the ratios of 6-monoester/6′-monoester are 2.1–2.7, and the sucrose monoesters were identified by HPLC–MS, NMR and IR.

Pyruvate transport across the inner mitochondrial membrane is believed to be a prerequisite step for gluconeogenesis in hepatocytes, which is important for maintenance of normoglycemia during prolonged food deprivation, but also contributes to hyperglycemia in diabetes. To determine the requirement for mitochondrial pyruvate import in gluconeogenesis, mice with liver-specific deletion of mitochondrial pyruvate carrier 2 (LS-Mpc2−/−) were generated. Loss of MPC2 impaired, but did not completely abolish, hepatocyte pyruvate metabolism, labelled pyruvate conversion to TCA cycle intermediates and glucose, and glucose production from pyruvate. Unbiased metabolomic analyses of livers from fasted LS-Mpc2−/− mice suggested that alterations in amino acid metabolism, including pyruvate-alanine cycling, might compensate for loss of MPC2. Indeed, inhibition of pyruvate-alanine transamination further reduced mitochondrial pyruvate metabolism and glucose production by LS-Mpc2−/− hepatocytes. These data demonstrate an important role for MPC2 in controlling hepatic gluconeogenesis and illuminate a compensatory mechanism for circumventing a block in mitochondrial pyruvate import.

Textured Ca modified (Pb,La)TiO 3 (PLCT) films were deposited on Pt/Ti/SiO 2 /Si substrates using a metal-organic decomposition (MOD) process. The microstructure of the PLCT thin film was investigated by X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Electric properties were measured using the Pt/PLCT/Pt capacitor structure. The PLCT films exhibit good ferroelectric and dielectric properties.

Textured calcium modified (Pb,La)TiO3 (PLCT) films were deposited on Pt/Ti/SiO2/Si substrates by using a metal-organic decomposition (MOD) process. The PLCT films exhibit good ferroelectric properties, a very low leakage current and a sharp PLCT/Pt interface. The (100) texture of the PLCT film is growth-controlled; the (100) oriented grains grow preferentially so as to minimize the surface energy. Particularly, the (100) preferred orientation is easy to form in the PLCT film with a layered structure for which the substrate almost does not affect the nucleation and growth of the film.

Pb(Zr0.48Ti0.52)O-3 thin films at 20% excess Pb were synthesized on Pt/Ti/SiO2/Si(100) substrates at different annealing temperatures by a metal-organic decomposition process. The microstructure of the PZT films was investigated by x-ray diffraction and atomic force microscopy. The composition of the films was characterized by Rutherford Backscattering Spectroscopy (RBS). These results showed that The PZT films have perovskite phase coexisted with PbO2 phase. The PbO2 phase mainly was formed by excess Pb which congregate at boundaries of crystalline grains during the annealing process and may be absorbed part of oxygen ion at normal sites, thus leading to an increase of oxygen vacancies in the PZT film. PbO2 phase and oxygen vacancies act as pinning centres, which has an effect on the ferroelectric domain switching. This eventually resulted in an increase of fatigue rate in PZT films. (C) 2001 Kluwer Academic Publishers.

In this paper we studied the processes of blistering and exfoliation on the surface of crystal silicon, the evolution of defects/strains in the crystal silicon caused by hydrogen and helium coimplantation during annealing, and the formation of platelets and bubbles in the crystal. It is shown that H+ and He+ coimplantation produces a synergistic effect, which greatly decreases the total implantation dose, compared with either just H+ or He+ implantation. We also present the effect of coimplantation and analyse the different roles of H and He in the process of exfoliation during annealing. It seems that the essential role of hydrogen is to interact chemically with the defects in the silicon and create H-stabilized platelets, while the role of helium is to effuse into these platelets and exert a pressure on the inner surface of these platelets. The damage caused by coimplantation is lower than by hydrogen implantation (at the dose that exfoliation requires).

Zr-rich lead zirconate titanate thin films with highly preferential (1 0 0) orientation, PZT80/20 and PZT85/15, and randomly oriented PZT70/30 thin films have been successfully prepared on (1 1 1)Pt-coated Si substrates using the sol–gel process with rapid thermal annealing (RTA) at 700°C for 200 s. The results of X-ray diffraction, including rocking curve measurements, suggest that PZT80/20 and PZT85/15 have a single (1 0 0) orientation with the rocking curve FWHM of around 0.5°. The dielectric properties of each film have been measured as well. The results show that highly (1 0 0)-oriented PZT85/15 films exhibit higher remnant polarization (2Pr=41.4 μC/cm2) and higher coercive field (2Ec=111 kV/cm) than randomly oriented PZT70/30 films (2Pr=33.4 μC/cm2 and 2Ec=106 kV/cm, respectively).

Single crystal silicon samples were implanted with H + ions at an energy of 30 KeV, and a dose of 4×10 16 /cm 2 . These samples were annealed in the temperature range from 100°C to 500°C. Four-crystal X-ray diffractometer (FCXRD), cross-sectional transmission electron microscopy (XTEM), Rutherford backscattering spectroscopy and channeling (RBS/C) were used to characterize the distribution and magnitude of the strain and extended defects in the samples. Elastic recoil detection analysis (ERDA) was used to measure the redistribution of hydrogen in Si samples during annealing. Strain distributions in the samples were simulated from the rocking curves by computer. The results help to understand the evolution of the strain and defects during annealing in the high dose H + -implanted silicon samples.

Highly (100)-oriented lanthanum- and calcium-modified PbTiO 3 (PLCT68/17/15 and PLCT80/10/10) ferroelectric thin films were successfully prepared on Pt/Ti/SiO 2 /Si substrates by a simple sol–gel spin-on process. The effects of thin film composition, film thickness and annealing temperature on the (100) orientation were reported. Highly (100)-oriented PLCT80/10/10 and PLCT68/17/15 thin films with pure perovskite could be obtained with around 0.3 and 0.45 μm thicknesses, respectively, when annealed at 600°C for 1 h. The films' crystallization and orientation were studied using the techniques of X-ray diffraction (XRD) and atomic force microscopy (AFM).

The oxidation behavior of a new type of wrought Ni–Fe–Cr–Alsuperalloy has been investigated systematically in the temperature range of1100 to 1300°C. Results are compared with those of alloy 214, Inconel600, and GH 3030. It is shown that the oxidation resistance of the newsuperalloy is excellent and much better than that of the comparisonalloys. Scanning electron microscopy (SEM), electron probe microanalysis(EPMA), and X-ray diffraction (XRD) experiments reveal that the excellentoxidation resistance of the new superalloy is due to the formation of adense, stable and continuous Al2O3 and Cr2O3 oxide layer at hightemperatures. Differential thermal analysis (DTA) shows that the formationof Cr2O3 and Al2O3 oxide layers on the new superalloy reaches a maximum at1060 and 1356°C, respectively. The Cr2O3 layer peels off easily, and thesingle dense Al2O3 layer remains, giving good oxidation resistance attemperatures higher than 1150°C. In addition, the new superalloypossesses high mechanical strength at high temperatures. On-site testsshowed that the new superalloy has ideal oxidation resistance and can beused at high temperatures up to 1300°C in various oxidizing andcorrosion atmospheres, such as those containing SO2, CO2 etc., for longperiods.

A simple nebulized spray pyrolysis method has been applied to prepare MgO thin films with (200) preferential orientation on (100) Si substrates by using magnesium acetate as starting reactants. The experimental results revealed that the substrates temperature and the oxygen flow played important roles in the formation of (200) oriented MgO film. The surface morphology and some properties of the prepared MgO thin films were examined by atomic force microscopy, transmission electron microscopy, X-ray diffraction and spread resistance processing technique.

MgO thin films have been prepared on Si substrates by a novel and simple sol-gel method using magnesium nitrate and collodion as starting material. Solutions consisting of magnesium nitrate in a mixture of collodion and ethanol were spin-coated onto silicon substrates. It was found that collodion was a necessary component to form stable sols and the crystallization and structures were clearly dependent on the amount of the collodion and the annealing temperature. The MgO thin films with good crystallization were obtained after annealing at 800°C. Meanwhile, the microstructure of the MgO films was examined by transmission electron microscopy and atomic force microscopy.

PGC-1β plays pleiotropic roles in regulating intermediary metabolism and has been shown to regulate both catabolic and anabolic processes in liver. We sought to evaluate the effects of PGC-1β on liver energy metabolism by generating mice with postnatal, liver-specific deletion of PGC-1β (LS-PGC-1β(-/-) mice). LS-PGC-1β(-/-) mice were outwardly normal, but exhibited a significant increase in hepatic triglyceride content at 6 weeks of age. Hepatic steatosis was due, at least in part, to impaired capacity for fatty acid oxidation and marked mitochondrial dysfunction. Mitochondrial DNA content and the expression of genes encoding multiple steps in mitochondrial fatty acid oxidation and oxidative phosphorylation pathways were significantly diminished in LS-PGC-1β(-/-) mice. Liquid chromatography mass spectrometry-based analyses also revealed that acetylcarnitine and butyrylcarnitine levels were depleted whereas palmitoylcarnitine content was increased in LS-PGC-1β(-/-) liver, which is consistent with attenuated rates of fatty acid oxidation. Interestingly, loss of PGC-1β also significantly impaired inducible expression of glycolytic and lipogenic enzymes that occurs with high carbohydrate diet refeeding after a prolonged fast. These results suggest that PGC-1β plays dual roles in regulating hepatic fatty acid metabolism by controlling the expression of programs of genes involved in both fatty acid oxidation and de novo fatty acid synthesis.

In this paper, Ca modified (Pb, La)TiO3 (PLCT) films were deposited on Pt coated silicon substrates using metal-organic decomposition (MOD) process. The preferred orientations polycrystalline films were varied with heating treatment temperature. The PLCT films exhibit good ferroelectric and dielectric properties at room temperature. The large area PLCT thin films with (100) preferred orientation may be application for future non-volatile memory devices (NVFRAM's) and dynamic random access memories (DRAM's).

Summary The hepatic TCA cycle supports oxidative and biosynthetic metabolism. This dual responsibility requires anaplerotic pathways, such as pyruvate carboxylase (PC), to generate TCA cycle intermediates necessary for biosynthesis without disrupting oxidative metabolism. Liver-specific PC knockout (LPCKO) mice were created to test the role of anaplerotic flux in liver metabolism. LPCKO mice have impaired hepatic anaplerosis, diminution of TCA cycle intermediates, suppressed gluconeogenesis, reduced TCA cycle flux, and a compensatory increase in ketogenesis and renal gluconeogenesis. Loss of PC depleted aspartate and compromised urea cycle function, causing elevated urea cycle intermediates and hyperammonemia. Loss of PC prevented diet-induced hyperglycemia and insulin resistance but depleted NADPH and glutathione, which exacerbated oxidative stress and correlated with elevated liver inflammation. Thus, despite catalyzing the synthesis of intermediates also produced by other anaplerotic pathways, PC is specifically necessary for maintaining oxidation, biosynthesis, and pathways distal to the TCA cycle, such as antioxidant defenses.