Pro-survival Phenotype of HIF-1α: Neuroprotection Through Inflammatory Mechanisms.

Hypoxia-inducible factor 1 (HIF-1) is a major player in the oxygen sensor system as well as a transcription factor. HIF-1 is also associated in the pathogenesis of many brain diseases including Alzheimer's disease (AD), epilepsy and stroke. HIF-1 regulates the expression of many genes such as those involved in glycolysis, erythropoiesis, angiogenesis and proliferation in hypoxic condition. Despite several studies, the mechanism through which HIF-1 confers neuroprotection remains unclear, one of them is modulating metabolic profiles and inflammatory pathways. Characterization of the neuroprotective role of HIF-1 may be through its stabilization and the regulation of target genes that aid in the early adaptation to the oxidative stressors. It is interesting to note that mounting data from recent years point to an additional crucial regulatory role for hypoxia-inducible factors (HIFs) in inflammation. HIFs in immune cells regulate the production of glycolytic energy as well as innate immunity, pro-inflammatory gene expression, and mediates activation of pro-survival pathways. The present review highlights the contribution of HIF-1 to neuroprotection where inflammation is the crucial factor in the pathogenesis contributing to neural death. The potential mechanisms that contribute to neuroprotection as a result of the downstream targets of HIF-1α are discussed. Such mechanisms include those mediated through IL-10, an anti-inflammatory molecule involved in activating pro-survival signaling mechanisms via AKT/ERK and JAK/STAT pathways.

SMAD2 and SMAD3 differentially regulate adiposity and the growth of subcutaneous white adipose tissue.

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.

Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis but the underlying mechanism remains unclear. In this study we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischaemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin10 (IL10). Therefore we hypothesised that ketosis-stabilised HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, eight-week-old rats were fed either the standard chow diet (SD) or the ketogenic (KG) diet for 4 weeks before ischaemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet fed group.

Acute Hyperglycemia Exacerbates Hemorrhagic Transformation after Embolic Stroke and Reperfusion with tPA: A Possible Role of TXNIP-NLRP3 Inflammasome.

OBJECTIVES: Acute hyperglycemia (HG) exacerbates reperfusion injury after stroke. Our recent studies showed that acute HG upregulates thioredoxin-interacting protein (TXNIP) expression, which in turn induces inflammation and neurovascular damage in a suture model of ischemic stroke. The aim of the present study was to investigate the effect of acute HG on TXNIP-associated neurovascular damage, in a more clinically relevant murine model of embolic stroke and intravenous tissue plasminogen activator (IV-tPA) reperfusion. MATERIALS AND METHODS: HG was induced in adult male mice, by intraperitoneal injection of 20% glucose. This was followed by embolic middle cerebral artery occlusion (eMCAO), with or without IV-tPA (10 mg/kg) given 3 h post embolization. Brain infarction, edema, hemoglobin content, expression of matrix metalloproteinase (MMP-9), vascular endothelial growth factor A (VEGFA), tight junction proteins (claudin-5, occluding, and zonula occludens-1), TXNIP, and NOD-like receptor protein3 (NLRP3)-inflammasome activation were evaluated at 24 h after eMCAO. RESULTS: HG alone significantly increased TXNIP in the brain after eMCAO, and this was associated with exacerbated hemorrhagic transformation (HT; as measured by hemoglobin content). IV-tPA in HG conditions showed a trend to decrease infarct volume, but worsened HT after eMCAO, suggesting that HG reduces the therapeutic efficacy of IV-tPA. Further, HG and tPA-reperfusion did not show significant differences in expression of MMP-9, VEGFA, junction proteins, and NLRP3 inflammasome activation between the groups. CONCLUSION: The current findings suggest a potential role for TXNIP in the occurrence of HT in hyperglycemic conditions following eMCAO. Further studies are needed to understand the precise role of vascular TXNIP on HG/tPA-induced neurovascular damage after stroke.

Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1.

Macrophages (MΦs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MΦ activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MΦs (BMDM) from Slc2a1M-/- mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MΦ lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1M-/- BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1M-/- BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MΦs of lean Slc2a1M-/- mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/- mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1M-/- BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MΦ were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MΦ function in chronic diseases.

Chronic Ketosis Modulates HIF1α-Mediated Inflammatory Response in Rat Brain.

Hypoxia inducible factor alpha (HIF1α) is associated with neuroprotection conferred by diet-induced ketosis, but the underlying mechanism remains unclear. In this study, we use a ketogenic diet in rodents to induce a metabolic state of chronic ketosis, as measured by elevated blood ketone bodies. Chronic ketosis correlates with neuroprotection in both aged and following focal cerebral ischemia and reperfusion (via middle cerebral artery occlusion, MCAO) in mouse and rat models. Ketone bodies are known to be used efficiently by the brain, and metabolism of ketone bodies is associated with increased cytosolic succinate levels that inhibits prolyl hydroxylases allowing HIF1α to accumulate. Ketosis also regulates inflammatory pathways, and HIF1α is reported to be essential for gene expression of interleukin 10 (IL10). Therefore, we hypothesized that ketosis-stabilized HIF1α modulates the expression of inflammatory cytokines orchestrating neuroprotection. To test changes in cytokine levels in rodent brain, 8-week-rats were fed either the standard chow diet (SD) or the KG diet for 4 weeks before ischemia experiments (MCAO) were performed and the brain tissues were collected. Consistent with our hypothesis, immunoblotting analysis shows IL10 levels were significantly higher in KG diet rat brain compared to SD, whereas the TNFα and IL6 levels were significantly lower in the brains of KG diet-fed group.

Overcompensation of CoA Trapping by Di(2-ethylhexyl) Phthalate (DEHP) Metabolites in Livers of Wistar Rats.

Di(2-ethylhexyl) phthalate (DEHP) is commonly used as a plasticizer in various industrial and household plastic products, ensuring widespread human exposures. Its routine detection in human bio-fluids and the propensity of its monoester metabolite to activate peroxisome proliferator activated receptor-α (PPARα) and perturb lipid metabolism implicate it as a metabolic disrupter. In this study we evaluated the effects of DEHP exposure on hepatic levels of free CoA and various CoA esters, while also confirming the metabolic activation to CoA esters and partial ß-oxidation of a DEHP metabolite (2-ethyhexanol). Male Wistar rats were exposed via diet to 2% (w/w) DEHP for fourteen-days, following which hepatic levels of free CoA and various CoA esters were identified using liquid chromatography-mass spectrometry. DEHP exposed rats showed significantly elevated free CoA and increased levels of physiological, DEHP-derived and unidentified CoA esters. The physiological CoA ester of malonyl-CoA and DEHP-derived CoA ester of 3-keto-2-ethylhexanoyl-CoA were the most highly elevated, at eighteen- and ninety eight-times respectively. We also detected sixteen unidentified CoA esters which may be derivative of DEHP metabolism or induction of other intermediary metabolism metabolites. Our results demonstrate that DEHP is a metabolic disrupter which affects production and sequestration of CoA, an essential cofactor of oxidative and biosynthetic reactions.

Macrophages with a deletion of the phosphoenolpyruvate carboxykinase 1 (Pck1) gene have a more proinflammatory phenotype.

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-1ß, 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.

Disruption of brain-derived neurotrophic factor production from individual promoters generates distinct body composition phenotypes in mice.

Brain-derived neurotrophic factor (BDNF) is a key neuropeptide in the central regulation of energy balance. The Bdnf gene contains nine promoters, each producing specific mRNA transcripts that encode a common protein. We sought to assess the phenotypic outcomes of disrupting BDNF production from individual Bdnf promoters. Mice with an intact coding region but selective disruption of BDNF production from Bdnf promoters I, II, IV, or VI (Bdnf-e1-/-, -e2-/-, -e4-/-, and -e6-/-) were created by inserting an enhanced green fluorescent protein-STOP cassette upstream of the targeted promoter splice donor site. Body composition was measured by MRI weekly from age 4 to 22 wk. Energy expenditure was measured by indirect calorimetry at 18 wk. Food intake was measured in Bdnf-e1-/- and Bdnf-e2-/- mice, and pair feeding was conducted. Weight gain, lean mass, fat mass, and percent fat of Bdnf-e1-/- and Bdnf-e2-/- mice (both sexes) were significantly increased compared with wild-type littermates. For Bdnf-e4-/- and Bdnf-e6-/- mice, obesity was not observed with either chow or high-fat diet. Food intake was increased in Bdnf-e1-/- and Bdnf-e2-/- mice, and pair feeding prevented obesity. Mutant and wild-type littermates for each strain (both sexes) had similar total energy expenditure after adjustment for body composition. These findings suggest that the obesity phenotype observed in Bdnf-e1-/- and Bdnf-e2-/- mice is attributable to hyperphagia and not altered energy expenditure. Our findings show that disruption of BDNF from specific promoters leads to distinct body composition effects, with disruption from promoters I or II, but not IV or VI, inducing obesity.

Post-resuscitation Arterial Blood Pressure on Survival and Change of Capillary Density Following Cardiac Arrest and Resuscitation in Rats.

Transient global brain ischemia, induced by cardiac arrest and resuscitation, results in reperfusion injury leading to delayed selective neuronal cell loss and post-resuscitation mortality. This study determined the effects of post-resuscitation hypotension and hypothermia on long-term survival following cardiac arrest and resuscitation. The capillary density was also determined. Based on the mean arterial blood pressure (MABP) at 1 h of recovery, the normotension group (MABP 80-120 mmHg) and hypotension group (MABP <80 mmHg) were defined. The overall survival was determined at 4 days of recovery. Brain microvascular density was assessed using immunohistochemistry of the glucose transporter, GLUT-1. The pre-arrest MABP was similar in each group; at 1 h after resuscitation, the MABP in the normotension groups was about 80% of their pre-arrest values; the hypotension group had a significantly lower MABP compared to the normotension group. The overall survival rate was lower in the hypotension group compared to the normotension group (36%, 4/11 vs. 67%, 14/21) under the normothermic condition. Brain blood flow in the hypotension group was lower (33% decrease) compared to the normotension group at 1-h post-resuscitation. Compared to the pre-arrest baseline, the capillary density was significantly increased at 14 days of recovery (355 ± 42 vs. 469 ± 50, number/mm2) in the cortex. The capillary density in hippocampus was also increased at 4-30 days following cardiac arrest and resuscitation. Our results suggest that rats able to maintain their post-resuscitation blood pressure at normotension, had higher brain blood flow during the early recovery phase, and improved survival outcome following cardiac arrest and resuscitation. In addition, cardiac arrest and resuscitation induced angiogenesis in brain in the first month of recovery.

Novel ketone body therapy for managing Alzheimer's disease: An Editorial Highlight for 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.

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.

Diet-Induced Ketosis Protects Against Focal Cerebral Ischemia in Mouse.

Over the past decade we have consistently shown that ketosis is neuroprotective against ischemic insults in rats. We reported that diet-induced ketotic rats had a significant reduction in infarct volume when subjected to middle cerebral artery occlusion (MCAO), and improved survival and recovery after cardiac arrest and resuscitation. The neuroprotective mechanisms of ketosis (via ketogenic diet; KG) include (i) ketones are alternate energy substrates that can restore energy balance when glucose metabolism is deficient and (ii) ketones modulate cell-signalling pathways that are cytoprotective. We investigated the effects of diet-induced ketosis following transient focal cerebral ischemia in mice. The correlation between levels of ketosis and hypoxic inducible factor-1alpha (HIF-1α), AKT (also known as protein kinase B or PKB) and 5' AMP-activated protein kinase (AMPK) were determined. Mice were fed with KG diet or standard lab-chow (STD) diet for 4 weeks. For the MCAO group, mice underwent 60 min of MCAO and total brain infarct volumes were evaluated 48 h after reperfusion. In a separate group of mice, brain tissue metabolites, levels of HIF-1α, phosphorylated AKT (pAKT), and AMPK were measured. After feeding a KG diet, levels of blood ketone bodies (beta-hydroxyburyrate, BHB) were increased. There was a proportional decrease in infarct volumes with increased blood BHB levels (KG vs STD; 4.2 ± 0.6 vs 7.8 ± 2.2 mm3, mean ± SEM). A positive correlation was also observed with HIF-1α and pAKT relative to blood BHB levels. Our results showed that chronic ketosis can be induced in mice by KG diet and was neuroprotective against focal cerebral ischemia in a concentration dependent manner. Potential mechanisms include upregulation of cytoprotective pathways such as those associated with HIF-1α, pAKT and AMPK.

Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

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.

Aging Effect on Post-recovery Hypofusion and Mortality Following Cardiac Arrest and Resuscitation in Rats.

In this study we investigated the effect of aging on brain blood flow following transient global ischemia. Male Fisher rats (6 and 24 months old) underwent cardiac arrest (15 min) and resuscitation. Regional brain (cortex, hippocampus, brainstem and cerebellum) blood flow was measured in non-arrested rats and 1-h recovery rats using [14C] iodoantipyrene (IAP) autoradiography; the 4-day survival rate was determined in the two age groups. The pre-arrest baseline blood flows were similar in cortex, brainstem and cerebellum between the 6-month and the 24-month old rats; however, the baseline blood flow in hippocampus was significantly lower in the 24-month old group. At 1 h following cardiac arrest and resuscitation, both 6-month and 24-month groups had significantly lower blood flows in all regions than the pre-arrest baseline values; compared to the 6-month old group, the blood flow was significantly lower (about 40% lower) in all regions in the 24-month old group. The 4-day survival rate for the 6-month old rats was 50% (3/6) whereas none of the 24-month old rats (0/10) survived for 4 days. The data suggest that there is an increased vulnerability to brain ischemic-reperfusion injury in the aged rats; the degree of post-recovery hypoperfusion may contribute to the high mortality in the aged rats following cardiac arrest and resuscitation.

Protective Effect of Dl-3-n-Butylphthalide on Recovery from Cardiac Arrest and Resuscitation in Rats.

In this study we investigated the effect of Dl-3-n-butylphthalide (NBP), a clinically used drug for stroke patients in China, on the recovery following cardiac arrest and resuscitation in rats. Male Wistar rats (3-month old) underwent cardiac arrest (12 min) and resuscitation. Rats were randomly assigned to the following groups: sham non-arrested group, vehicle group (vehicle-treated, 7 days before cardiac arrest and 4 days post-resuscitation), NBP pre-treated group (NBP-treated, 7 days before cardiac arrest), and NBP post-treated group (NBP-treated, 4 days post-resuscitation). Overall survival rates and hippocampal neuronal counts were determined in each group at 4 days post-resuscitation. Results showed that NBP pre-treated group (80 %) and NBP post-treated group (86 %) had significantly higher survival rates compared to that of the vehicle group (50 %). At 4 days of recovery, only about 20 % of hippocampal neurons were preserved in the vehicle group compared to the sham non-arrested group. The hippocampal CA1 cell counts in the NBP pre-treated group and NBP post-treated group were significantly higher than the counts in the vehicle group, about 50-60 % of the counts of non-arrested rats. The data suggest that NBP has both preventive and therapeutic effect on improving outcome following cardiac arrest and resuscitation, and NBP might be a potential early phase treatment for patients recovered from cardiac arrest and resuscitation.

Catabolism of (2E)-4-hydroxy-2-nonenal via ω- and ω-1-oxidation stimulated by ketogenic diet.

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 ß-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.

Sterol regulatory element-binding protein-1 (SREBP-1) is required to regulate glycogen synthesis and gluconeogenic gene expression in mouse liver.

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.

Decreased carbon shunting from glucose toward oxidative metabolism in diet-induced ketotic rat brain.

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.

Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel ß-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA. The latter γ-diketo CoA ester undergoes two parallel cyclization processes. One process yields a mixture of tautomers, i.e., cyclopentenyl- and cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the ε-nitrogen of lysine but without carbons from lysine. The cyclic CoA esters were identified in rat livers perfused with levulinate and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Lastly, 3,6-diketoheptanoyl-CoA, like 2,5-diketohexane, pyrrolates free lysine and, presumably, lysine residues from proteins. This may represent a new pathway for protein pyrrolation. The cyclic CoA esters and related pyrrolation processes may play a role in the toxic effects of 4-hydroxypentanoate.

Contribution of brain glucose and ketone bodies to oxidative metabolism.

UNLABELLED: Ketone bodies are an alternative energy substrate to glucose in brain. Under conditions of oxidative stress, we hypothesize that ketosis stabilizes glucose metabolism by partitioning glucose away from oxidative metabolism towards ketone body oxidation. In this study we assessed oxidative metabolism in ketotic rat brain using stable isotope mass spectrometry analysis. The contribution of glucose and ketone bodies to oxidative metabolism was studied in cortical brain homogenates isolated from anesthetized ketotic rats. To induce chronic ketosis, rats were fed either a ketogenic (high-fat, carbohydrate restricted) or standard rodent chow for 3 weeks and then infused intravenously with tracers of [U-(13)C] glucose or [U-(13)C] acetoacetate for 60 min. The measured percent contribution of glucose or ketone bodies to oxidative metabolism was analyzed by measuring the (13)C-label incorporation into acetyl-CoA. Using mass spectrometry (gas-chromatography; GC-MS, and liquid-chromatography; LCMS) and isotopomer analysis, the fractional amount of substrate oxidation was measured as the M + 2 enrichment (%) of acetyl-CoA relative to the achieved enrichment of the infused precursors, [U-(13)C]glucose or [U-(13)C] acetoacetate. RESULTS: the percent contribution of glucose oxidation in cortical brain in rats fed the ketogenic diet was 71.2 ± 16.8 (mean% ± SD) compared to the standard chow, 89.0 ± 14.6. Acetoacetate oxidation was significantly higher with ketosis compared to standard chow, 41.7 ± 9.4 vs. 21.9 ± 10.6. These data confer the high oxidative capacity for glucose irrespective of ketotic or non-ketotic states. With ketosis induced by 3 weeks of diet, cortical brain utilizes twice as much acetoacetate compared to non-ketosis.