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Adipose tissue is the primary site of energy storage, playing important roles in health. While adipose research largely focuses on obesity, fat also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety and total energy homeostasis. SMAD2/3 proteins are downstream mediators of activin signaling, which regulate critical preadipocyte and mature adipocyte functions. Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity. The direct contributions of Smad2 and Smad3 in adipose tissues, however, are unknown. Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 on diet-induced adiposity using Smad2 or Smad3 "floxed" mice intercrossed with Adiponectin-Cre mice. Additionally, we examined visceral and subcutaneous preadipocyte differentiation efficiency in vitro. Almost all wild type subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A suppressed differentiation of preadipocytes from both depots. Smad2 conditional knockout (Smad2cKO) mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3 conditional knockout (Smad3cKO) male mice displayed a trend of reduced body weight on high-fat diet. On both diets, Smad3cKO mice displayed an adipose depot-selective phenotype, with a significant reduction in subcutaneous fat mass but not visceral fat mass. Our data suggest that Smad3 is an important contributor to the maintenance of subcutaneous white adipose tissue in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation.

Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by ω-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this work, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in hepatic DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid homeostasis. The Abcd3 KO mouse presents with increased hepatic long-chain DCAs, increased urine medium-chain DCAs, lipodystrophy, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial fatty acid β-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-13C6]-glucose (i.p.), enrichments of 13C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of 13C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of 13C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of 13C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or – as an additional astrocytic fuel – it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of 13C was reduced in the interictal state. Triheptanoin restored the correlation between 13C enrichments of pyruvate relative to most of the TCA cycle intermediates in “epileptic” mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

Peroxisomes metabolize a specific subset of fatty acids, which include dicarboxylic fatty acids (DCAs) generated by ω-oxidation. Data obtained in vitro suggest that the peroxisomal transporter ABCD3 (also known as PMP70) mediates the transport of DCAs into the peroxisome, but in vivo evidence to support this role is lacking. In this study, we studied an Abcd3 KO mouse model generated by CRISPR-Cas9 technology using targeted and untargeted metabolomics, histology, immunoblotting, and stable isotope tracing technology. We show that ABCD3 functions in DCA metabolism and uncover a novel role for this peroxisomal transporter in lipid metabolic homeostasis. The Abcd3 KO mouse presents with lipodystrophy, increased circulating free fatty acids, decreased ketone bodies, enhanced hepatic cholesterol synthesis and decreased hepatic de novo lipogenesis. Moreover, our study suggests that DCAs are metabolized by mitochondrial β-oxidation when ABCD3 is not functional, reflecting the importance of the metabolic compartmentalization and communication between peroxisomes and mitochondria. In summary, this study provides data on the role of the peroxisomal transporter ABCD3 in hepatic lipid homeostasis and DCA metabolism, and the consequences of peroxisomal dysfunction for the liver.

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins (EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Phosphoenolpyruvate carboxykinase (Pck1) is a metabolic enzyme that is integral to the gluconeogenic and glyceroneogenic pathways. However, Pck1's role in macrophage metabolism and function is unknown. Using stable isotopomer MS analysis in a mouse model with a myeloid cell–specific Pck1 deletion, we show here that this deletion increases the proinflammatory phenotype in macrophages. Incubation of LPS-stimulated bone marrow–derived macrophages (BMDM) with [U-13C]glucose revealed reduced 13C labeling of citrate and malate and increased 13C labeling of lactate in Pck1-deleted bone marrow–derived macrophages. We also found that the Pck1 deletion in the myeloid cells increases reactive oxygen species (ROS). Of note, this altered macrophage metabolism increased expression of the M1 cytokines TNFα, IL-1β, and IL-6. We therefore conclude that Pck1 contributes to M1 polarization in macrophages. Our findings provide important insights into the factors determining the macrophage inflammatory response and indicate that Pck1 activity contributes to metabolic reprogramming and polarization in macrophages.

Read the highlighted article ‘Effects of a dietary ketone ester on hippocampal glycolytic and tricarboxylic acid cycle intermediates and amino acids in a 3xTgAD mouse model of Alzheimer's disease’ on page 195.

There is mounting evidence that oxidative glucose metabolism is impaired in epilepsy and recent work has further characterized the metabolic mechanisms involved. In healthy people eating a traditional diet, including carbohydrates, fats and protein, the major energy substrate in brain is glucose. Cytosolic glucose metabolism generates small amounts of energy, but oxidative glucose metabolism in the mitochondria generates most ATP, in addition to biosynthetic precursors in cells. Energy is crucial for the brain to signal "normally," while loss of energy can contribute to seizure generation by destabilizing membrane potentials and signaling in the chronic epileptic brain. Here we summarize the known biochemical mechanisms that contribute to the disturbance in oxidative glucose metabolism in epilepsy, including decreases in glucose transport, reduced activity of particular steps in the oxidative metabolism of glucose such as pyruvate dehydrogenase activity, and increased anaplerotic need. This knowledge justifies the use of alternative brain fuels as sources of energy, such as ketones, TCA cycle intermediates and precursors as well as even medium chain fatty acids and triheptanoin.

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules ((2)H- or (13)C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of (13)C labeled glucose into specific CAC metabolite pools.

Recent work suggests that diet affects brain metabolism thereby impacting cognitive function. Our objective was to determine if a western diet altered brain metabolism, increased blood-brain barrier (BBB) transport and inflammation, and induced cognitive impairment in C57BL/6 (WT) mice and low-density lipoprotein receptor null (LDLr -/-) mice, a model of hyperlipidemia and cognitive decline. We show that a western diet and LDLr -/- moderately influence cognitive processes as assessed by Y-maze and radial arm water maze. Also, western diet significantly increased BBB transport, as well as microvessel factor VIII in LDLr -/- and microglia IBA1 staining in WT, both indicators of activation and neuroinflammation. Interestingly, LDLr -/- mice had a significant increase in 18F- fluorodeoxyglucose uptake irrespective of diet and brain 1H-magnetic resonance spectroscopy showed increased lactate and lipid moieties. Metabolic assessments of whole mouse brain by GC/MS and LC/MS/MS showed that a western diet altered brain TCA cycle and β-oxidation intermediates, levels of amino acids, and complex lipid levels and elevated proinflammatory lipid mediators. Our study reveals that the western diet has multiple impacts on brain metabolism, physiology, and altered cognitive function that likely manifest via multiple cellular pathways.

Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxia-inducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis., Clear cell renal cancers (ccRCC) display elevated intracellular lipid storage. Here the authors show that such lipid accumulation is due to the repression of carnitine palmitoyltransferase 1A (CPT1A) enzyme that impairs fatty acid (FA) transport into the mitochondrion resulting in reduced FA beta oxidation.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

Our previous work in perfused rat livers has demonstrated that 4-hydroxynonenal (HNE) is catabolized predominantly via beta oxidation. Therefore, we hypothesized that perturbations of beta oxidation, such as diet-altered fatty acid oxidation activity, could lead to changes in HNE levels. To test our hypothesis, we (i) developed a simple and sensitive GC/MS method combined with mass isotopomer analysis to measure HNE and HNE analogs, 4-oxononenal (ONE) and 1,4-dihydroxynonene (DHN), and (ii) investigated the effects of four diets (standard, low fat, ketogenic, and high fat mix diets) on HNE, ONE, and DHN concentrations in rat livers. Our results showed that livers from rats fed ketogenic diet or high fat mix diet had high ω-6 polyunsaturated fatty acid concentrations and markers of oxidative stress. However, high concentrations of HNE (1.6 ± 0.5 nmol/g) and ONE (0.9 ± 0.2 nmol/g) were only found in livers from rats fed the high fat mix diet. Livers from rats fed the ketogenic diet had low HNE (0.8 ± 0.1 nmol/g) and ONE (0.4 ± 0.07 nmol/g), similar to rats fed the standard diet. A possible explanation is that the predominant pathway of HNE catabolism (i.e. beta oxidation) is activated in the liver by the ketogenic diet. This is consistent with a 10 fold decrease in malonyl-CoA in livers from rats fed a ketogenic diet compared to a standard diet. The accelerated catabolism of HNE lowers HNE and HNE analog concentrations in livers from rats fed the ketogenic diet. On the other hand, rats fed the high fat mix diet had high rates of lipid synthesis and low rates of fatty acid oxidation, resulting in the slowing down of the catabolic disposal of HNE and HNE analogs. Thus, decreased HNE catabolism by a high fat mix diet induces high concentrations of HNE and HNE analogs. The results of the present work suggested a potential causal relationship to metabolic syndrome induced by western diets (i.e. high fat mix), as well as the effects of the ketogenic diet on the catabolism of lipid peroxidation products in liver.

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Phenylbutyrate is used in humans for treating inborn errors of ureagenesis, certain forms of cancer, cystic fibrosis and thalassemia. The known metabolism of phenylbutyrate leads to phenylacetylglutamine, which is excreted in urine. We have identified phenylbutyrylglutamine as a new metabolite of phenylbutyrate in human plasma and urine. We describe the synthesis of phenylbutyrylglutamine and its assay by gas chromatography/mass spectrometry as a tert-butyldimethylsilyl or methyl derivative, using standards of [2H5]phenylbutyrylglutamine and phenylpropionylglutamine. After administration of phenylbutyrate to normal humans, the cumulative urinary excretion of phenylacetate, phenylbutyrate, phenylacetylglutamine and phenylbutyrylglutamine amounts to about half of the dose of phenylbutyrate. Thus, additional metabolites of phenylbutyrate are yet to be identified. Copyright © 2002 John Wiley & Sons, Ltd.

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.

Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via β-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.

The mechanistic link of ketosis to neuroprotection under certain pathological conditions continues to be explored. We investigated whether chronic ketosis induced by ketogenic diet results in the partitioning of ketone bodies toward oxidative metabolism in brain. We hypothesized that diet-induced ketosis results in increased shunting of ketone bodies toward citric acid cycle and amino acids with decreased carbon shunting from glucose. Rats were fed standard (STD) or ketogenic (KG) diets for 3.5 weeks and then infused with [U-(13) C]glucose or [U-(13) C]acetoacetate tracers. Concentrations and (13) C-labeling pattern of citric acid cycle intermediates and amino acids were analyzed from brain homogenates using stable isotopomer mass spectrometry analysis. The contribution of [U-(13) C]glucose to acetyl-CoA and amino acids decreased by ~ 30% in the KG group versus STD, whereas [U-(13) C]acetoacetate contributions were more than two-fold higher. The concentration of GABA remained constant across groups; however, the (13) C labeling of GABA was markedly increased in the KG group infused with [U-(13) C]acetoacetate compared to STD. This study reveals that there is a significant contribution of ketone bodies to oxidative metabolism and GABA in diet-induced ketosis. We propose that this represents a fundamental mechanism of neuroprotection under pathological conditions.

We measured the mRNA and protein expression of the key gluconeogenic enzymes in human liver biopsy specimens and found that only hepatic pyruvate carboxylase protein levels related strongly with glycemia. We assessed the role of pyruvate carboxylase in regulating glucose and lipid metabolism in rats through a loss-of-function approach using a specific antisense oligonucleotide (ASO) to decrease expression predominantly in liver and adipose tissue. Pyruvate carboxylase ASO reduced plasma glucose concentrations and the rate of endogenous glucose production in vivo. Interestingly, pyruvate carboxylase ASO also reduced adiposity, plasma lipid concentrations, and hepatic steatosis in high fat–fed rats and improved hepatic insulin sensitivity. Pyruvate carboxylase ASO had similar effects in Zucker Diabetic Fatty rats. Pyruvate carboxylase ASO did not alter de novo fatty acid synthesis, lipolysis, or hepatocyte fatty acid oxidation. In contrast, the lipid phenotype was attributed to a decrease in hepatic and adipose glycerol synthesis, which is important for fatty acid esterification when dietary fat is in excess. Tissue-specific inhibition of pyruvate carboxylase is a potential therapeutic approach for nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes.

Researchers view analysis of the citric acid cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolismin blood-perfused brain tissue,which considers the astrocyteneuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data.

Mitochondrial dysfunction has been increasingly shown as a critical process that makes certain areas of the brain more susceptible not only to neurological disease but also to aging. Quantitative histochemistry is a series of procedures for measuring select metabolites in discrete regions of the brain, as they exist in vivo. The development of this method has been useful in establishing energy imbalance following ischemia but more recently has become useful in studying those processes related to the mitochondria which make the brain more susceptible to a variety of neurological insults. The relatively inexpensive cost to assay a given brain metabolite makes this methodology useful in the interpretation of molecular and biochemical responses in terms of the condition of the tissue following a neurological insult.

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1alpha and HIF-1beta subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1alpha, is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1alpha hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1alpha accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1alpha hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1alpha accumulation during hypoxia. Regulation of HIF-1alpha by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.