Control of immunity via nutritional interventions.

Nutrition affects all physiological processes including those linked to the development and function of our immune system. Here, we discuss recent evidence and emerging concepts supporting the idea that our newfound relationship with nutrition in industrialized countries has fundamentally altered the way in which our immune system is wired. This will be examined through the lens of studies showing that mild or transient reductions in dietary intake can enhance protective immunity while also limiting aberrant inflammatory responses. We will further discuss how trade-offs and priorities begin to emerge in the context of severe nutritional stress. In those settings, specific immunological functions are heightened to re-enforce processes and tissue sites most critical to survival. Altogether, these examples will emphasize the profound influence nutrition has over the immune system and highlight how a mechanistic exploration of this cross talk could ultimately lead to the design of novel therapeutic approaches that prevent and treat disease.

Remodelling of the translatome controls diet and its impact on tumorigenesis.

Fasting is associated with a range of health benefits1-6. How fasting signals elicit changes in the proteome to establish metabolic programmes remains poorly understood. Here we show that hepatocytes selectively remodel the translatome while global translation is paradoxically downregulated during fasting7,8. We discover that phosphorylation of eukaryotic translation initiation factor 4E (P-eIF4E) is induced during fasting. We show that P-eIF4E is responsible for controlling the translation of genes involved in lipid catabolism and the production of ketone bodies. Inhibiting P-eIF4E impairs ketogenesis in response to fasting and a ketogenic diet. P-eIF4E regulates those messenger RNAs through a specific translation regulatory element within their 5' untranslated regions (5' UTRs). Our findings reveal a new signalling property of fatty acids, which are elevated during fasting. We found that fatty acids bind and induce AMP-activated protein kinase (AMPK) kinase activity that in turn enhances the phosphorylation of MAP kinase-interacting protein kinase (MNK), the kinase that phosphorylates eIF4E. The AMPK-MNK-eIF4E axis controls ketogenesis, revealing a new lipid-mediated kinase signalling pathway that links ketogenesis to translation control. Certain types of cancer use ketone bodies as an energy source9,10 that may rely on P-eIF4E. Our findings reveal that on a ketogenic diet, treatment with eFT508 (also known as tomivosertib; a P-eIF4E inhibitor) restrains pancreatic tumour growth. Thus, our findings unveil a new fatty acid-induced signalling pathway that activates selective translation, which underlies ketogenesis and provides a tailored diet intervention therapy for cancer.

Metabolic epilepsies amenable to ketogenic therapies: Indications, contraindications, and underlying mechanisms.

Metabolic epilepsies arise in the context of rare inborn errors of metabolism (IEM), notably glucose transporter type 1 deficiency syndrome, succinic semialdehyde dehydrogenase deficiency, pyruvate dehydrogenase complex deficiency, nonketotic hyperglycinemia, and mitochondrial cytopathies. A common feature of these disorders is impaired bioenergetics, which through incompletely defined mechanisms result in a wide spectrum of neurological symptoms, such as epileptic seizures, developmental delay, and movement disorders. The ketogenic diet (KD) has been successfully utilized to treat such conditions to varying degrees. While the mechanisms underlying the clinical efficacy of the KD in IEM remain unclear, it is likely that the proposed heterogeneous targets influenced by the KD work in concert to rectify or ameliorate the downstream negative consequences of genetic mutations affecting key metabolic enzymes and substrates-such as oxidative stress and cell death. These beneficial effects can be broadly grouped into restoration of impaired bioenergetics and synaptic dysfunction, improved redox homeostasis, anti-inflammatory, and epigenetic activity. Hence, it is conceivable that the KD might prove useful in other metabolic disorders that present with epileptic seizures. At the same time, however, there are notable contraindications to KD use, such as fatty acid oxidation disorders. Clearly, more research is needed to better characterize those metabolic epilepsies that would be amenable to ketogenic therapies, both experimentally and clinically. In the end, the expanded knowledge base will be critical to designing metabolism-based treatments that can afford greater clinical efficacy and tolerability compared to current KD approaches, and improved long-term outcomes for patients.

Suppression of enteroendocrine cell glucagon-like peptide (GLP)-1 release by fat-induced small intestinal ketogenesis: a mechanism targeted by Roux-en-Y gastric bypass surgery but not by preoperative very-low-calorie diet.

OBJECTIVE: Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms. DESIGN: Jejunal mucosa biopsies from patients undergoing RYGB surgery were retrieved before and after very-low calorie diet, at time of surgery and 6 months postoperatively. Samples were analysed by global protein expression analysis and Western blotting. Biological functionality of these findings was explored in mice and enteroendocrine cells (EECs) primary mouse jejunal cell cultures. RESULTS: The most prominent change found after RYGB was decreased jejunal expression of the rate-limiting ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGCS), corroborated by decreased ketone body levels. In mice, prolonged high-fat feeding induced the expression of mHMGCS and functional ketogenesis in jejunum. The effect of ketone bodies on gut peptide secretion in EECs showed a ∼40% inhibition of GLP-1 release compared with baseline. CONCLUSION: Intestinal ketogenesis is induced by high-fat diet and inhibited by RYGB surgery. In cell culture, ketone bodies inhibited GLP-1 release from EECs. Thus, we suggest that this may be a mechanism by which RYGB can remove the inhibitory effect of ketone bodies on EECs, thereby restituting the responsiveness of EECs resulting in increased meal-stimulated levels of GLP-1 after surgery.

Hepatocyte-specific Sirt6 deficiency impairs ketogenesis.

Sirt6 is an NADH (NAD+)-dependent deacetylase with a critical role in hepatic lipid metabolism. Ketogenesis is controlled by a signaling network of hepatic lipid metabolism. However, how Sirt6 functions in ketogenesis remains unclear. Here, we demonstrated that Sirt6 functions as a mediator of ketogenesis in response to a fasting and ketogenic diet (KD). The KD-fed hepatocyte-specific Sirt6 deficiency (HKO) mice exhibited impaired ketogenesis, which was due to enhanced Fsp27 (fat-specific induction of protein 27), a protein known to regulate lipid metabolism. In contrast, overexpression of Sirt6 in mouse primary hepatocytes promoted ketogenesis. Mechanistically, Sirt6 repressed Fsp27ß expression by interacting with Crebh (cAMP response element-binding protein H) and preventing its recruitment to the Fsp27ß gene promoter. The KD-fed HKO mice also showed exacerbated hepatic steatosis and inflammation. Finally, Fsp27 silencing rescued hypoketonemia and other metabolic phenotypes in KD-fed HKO mice. Our data suggest that the Sirt6-Crebh-Fsp27 axis is pivotal for hepatic lipid metabolism and inflammation. Sirt6 may be a pharmacological target to remedy metabolic diseases.

Effect of withholding early parenteral nutrition in PICU on ketogenesis as potential mediator of its outcome benefit.

BACKGROUND: In critically ill children, omitting early use of parenteral nutrition (late-PN versus early-PN) reduced infections, accelerated weaning from mechanical ventilation, and shortened PICU stay. We hypothesized that fasting-induced ketogenesis mediates these benefits. METHODS: In a secondary analysis of the PEPaNIC RCT (N = 1440), the impact of late-PN versus early-PN on plasma 3-hydroxybutyrate (3HB), and on blood glucose, plasma insulin, and glucagon as key ketogenesis regulators, was determined for 96 matched patients staying ≥ 5 days in PICU, and the day of maximal 3HB-effect, if any, was identified. Subsequently, in the total study population, plasma 3HB and late-PN-affected ketogenesis regulators were measured on that average day of maximal 3HB effect. Multivariable Cox proportional hazard and logistic regression analyses were performed adjusting for randomization and baseline risk factors. Whether any potential mediator role for 3HB was direct or indirect was assessed by further adjusting for ketogenesis regulators. RESULTS: In the matched cohort (n = 96), late-PN versus early-PN increased plasma 3HB throughout PICU days 1-5 (P < 0.0001), maximally on PICU day 2. Also, blood glucose (P < 0.001) and plasma insulin (P < 0.0001), but not glucagon, were affected. In the total cohort (n = 1142 with available plasma), late-PN increased plasma 3HB on PICU day 2 (day 1 for shorter stayers) from (median [IQR]) 0.04 [0.04-0.04] mmol/L to 0.75 [0.04-2.03] mmol/L (P < 0.0001). The 3HB effect of late-PN statistically explained its impact on weaning from mechanical ventilation (P = 0.0002) and on time to live PICU discharge (P = 0.004). Further adjustment for regulators of ketogenesis did not alter these findings. CONCLUSION: Withholding early-PN in critically ill children significantly increased plasma 3HB, a direct effect that statistically mediated an important part of its outcome benefit.

Role of ketone signaling in the hepatic response to fasting.

Ketosis is a metabolic adaptation to fasting, nonalcoholic fatty liver disease (NAFLD), and prolonged exercise. ß-OH butyrate acts as a transcriptional regulator and at G protein-coupled receptors to modulate cellular signaling pathways in a hormone-like manner. While physiological ketosis is often adaptive, chronic hyperketonemia may contribute to the metabolic dysfunction of NAFLD. To understand how ß-OH butyrate signaling affects hepatic metabolism, we compared the hepatic fasting response in control and 3-hydroxy-3-methylglutaryl-CoA synthase II (HMGCS2) knockdown mice that are unable to elevate ß-OH butyrate production. To establish that rescue of ketone metabolic/endocrine signaling would restore the normal hepatic fasting response, we gave intraperitoneal injections of ß-OH butyrate (5.7 mmol/kg) to HMGCS2 knockdown and control mice every 2 h for the final 9 h of a 16-h fast. In hypoketonemic, HMGCS2 knockdown mice, fasting more robustly increased mRNA expression of uncoupling protein 2 (UCP2), a protein critical for supporting fatty acid oxidation and ketogenesis. In turn, exogenous ß-OH butyrate administration to HMGCS2 knockdown mice decreased fasting UCP2 mRNA expression to that observed in control mice. Also supporting feedback at the transcriptional level, ß-OH butyrate lowered the fasting-induced expression of HMGCS2 mRNA in control mice. ß-OH butyrate also regulates the glycemic response to fasting. The fast-induced fall in serum glucose was absent in HMGCS2 knockdown mice but was restored by ß-OH butyrate administration. These data propose that endogenous ß-OH butyrate signaling transcriptionally regulates hepatic fatty acid oxidation and ketogenesis, while modulating glucose tolerance. NEW & NOTEWORTHY Ketogenesis regulates whole body glucose metabolism and ß-OH butyrate produced by the liver feeds back to inhibit hepatic ß-oxidation and ketogenesis during fasting.

Liraglutide acutely suppresses glucagon, lipolysis and ketogenesis in type 1 diabetes.

In view of the occurrence of diabetic ketoacidosis associated with the use of sodium-glucose transport protein-2 inhibitors in patients with type 1 diabetes (T1DM) and the relative absence of this complication in patients treated with liraglutide in spite of reductions in insulin doses, we investigated the effect of liraglutide on ketogenesis. Twenty-six patients with inadequately controlled T1DM were randomly divided into 2 groups of 13 patients each. After an overnight fast, patients were injected, subcutaneously, with either liraglutide 1.8 mg or with placebo. They were maintained on their basal insulin infusion and were followed up in our clinical research unit for 5 hours. The patients injected with placebo maintained their glucose and glucagon concentrations without an increase, but there was a significant increase in free fatty acids (FFA), acetoacetate and ß-hydoxybutyrate concentrations. In contrast, liraglutide significantly reduced the increase in FFA, and totally prevented the increase in acetoacetate and ß-hydroxybutyrate concentrations while suppressing glucagon and ghrelin concentrations. Thus, a single dose of liraglutide is acutely inhibitory to ketogenesis.

Combination of aerobic exercise and an arginine, alanine, and phenylalanine mixture increases fat mobilization and ketone body synthesis.

During exercise, blood levels of several hormones increase acutely. We hypothesized that consumption of a specific combination of amino acids (arginine, alanine, and phenylalanine; A-mix) may be involved in secretion of glucagon, and when combined with exercise may promote fat catabolism. Ten healthy male volunteers were randomized in a crossover study to ingest either A-mix (3 g/dose) or placebo (3 g of dextrin/dose). Thirty minutes after ingesting, each condition subsequently performed workload trials on a cycle ergometer at 50% of maximal oxygen consumption for 1 h. After oral intake of A-mix, the concentrations of plasma ketone bodies and adrenalin during and post-exercise were significantly increased. The area under the curve for glycerol and glucagon was significantly increased in the post-exercise by A-mix administration. These results suggest that pre-exercise ingestion of A-mix causes a shift of energy source from carbohydrate to fat combustion by increasing secretion of adrenalin and glucagon.

The hypophagic response to heat stress is not mediated by GPR109A or peripheral ß-OH butyrate.

Rising temperatures resulting from climate change will increase the incidence of heat stress, negatively impacting the labor force and food animal production. Heat stress elevates circulating ß-OH butyrate, which induces vasodilation through GPR109a. Interestingly, both heat stress and intraperitoneal ß-OH butyrate administration induce hypophagia. Thus, we aimed to investigate the role of ß-OH butyrate in heat stress hypophagia in mice. We found that niacin, a ß-OH butyrate mimetic that cannot be oxidized to generate ATP, also reduces food intake. Interestingly, the depression in food intake as a result of 8-h intraperitoneal niacin or 48-h heat exposure did not result from changes in hypothalamic expression of orexigenic or anorexigenic signals (AgRP, NPY, or POMC). Genetically eliminating GPR109a expression did not prevent the hypophagic response to heat exposure, intraperitoneal ß-OH butyrate (5.7 mmol/kg), or niacin (0.8 mmol/kg). Hepatic vagotomy eliminated the hypophagic response to ß-OH butyrate and niacin but did not affect the hypophagic response to heat exposure. We subsequently hypothesized that the hypophagic response to heat stress may depend on direct effects of ß-OH butyrate at the central nervous system: ß-OH butyrate induced hormonal changes (hyperinsulinemia, hypercorticosteronemia, and hyperleptinemia), or gene expression changes. To test these possibilities, we blocked expression of hepatic hydroxyl methyl glutaryl CoA synthase II (HMGCS2) to prevent hepatic ß-OH butyrate synthesis. Mice that lack HMGCS2 maintain a hypophagic response to heat stress. Herein, we establish that the hypophagia of heat stress is independent of GPR109a, the hepatic vagus afferent nerve, and hepatic ketone body synthesis.

The tuberous sclerosis complex model Eker (TSC2+/-) rat exhibits hyperglycemia and hyperketonemia due to decreased glycolysis in the liver.

Tuberous sclerosis complex (TSC) presents as benign tumors that affect the brain, kidneys, lungs and skin. The inactivation of TSC2 gene, through loss of heterozygosity is responsible for tumor development in TSC. Since TSC patients are carriers of heterozygous a TSC2; mutation, to reveal the risk factors which these patients carry prior to tumor development is important. In this experiment, Eker rat which carry a mutation in this TSC2 gene were analyzed for their metabolic changes. Wild-type (TSC2+/+) and heterozygous mutant TSC2 (TSC2+/-) Eker rats were raised for 100 days. As a result, the Eker rats were found to exhibit hyperglycemia and hyperketonemia. However the high ketone body production in the liver was observed without accompanying increased levels of plasma free fatty acids or insulin. Further, production of the ketone body ß-hydroxybutyrate was inhibited due to the low NADH/NAD(+) ratio resulting from the restraint on glycolysis, which was followed by inhibition of the malate-aspartate shuttle and TCA cycle. Therefore, we conclude that glycolysis is restrained in the livers of TSC2 heterozygous mutant rats, and these defects lead to abnormal production of acetoacetate.

mTORC1 controls fasting-induced ketogenesis and its modulation by ageing.

The multi-component mechanistic target of rapamycin complex 1 (mTORC1) kinase is the central node of a mammalian pathway that coordinates cell growth with the availability of nutrients, energy and growth factors. Progress has been made in the identification of mTORC1 pathway components and in understanding their functions in cells, but there is relatively little known about the role of the pathway in vivo. Specifically, we have little knowledge regarding the role mTOCR1 has in liver physiology. In fasted animals, the liver performs numerous functions that maintain whole-body homeostasis, including the production of ketone bodies for peripheral tissues to use as energy sources. Here we show that mTORC1 controls ketogenesis in mice in response to fasting. We find that liver-specific loss of TSC1 (tuberous sclerosis 1), an mTORC1 inhibitor, leads to a fasting-resistant increase in liver size, and to a pronounced defect in ketone body production and ketogenic gene expression on fasting. The loss of raptor (regulatory associated protein of mTOR, complex 1) an essential mTORC1 component, has the opposite effects. In addition, we find that the inhibition of mTORC1 is required for the fasting-induced activation of PPARα (peroxisome proliferator activated receptor α), the master transcriptional activator of ketogenic genes, and that suppression of NCoR1 (nuclear receptor co-repressor 1), a co-repressor of PPARα, reactivates ketogenesis in cells and livers with hyperactive mTORC1 signalling. Like livers with activated mTORC1, livers from aged mice have a defect in ketogenesis, which correlates with an increase in mTORC1 signalling. Moreover, we show that the suppressive effects of mTORC1 activation and ageing on PPARα activity and ketone production are not additive, and that mTORC1 inhibition is sufficient to prevent the ageing-induced defect in ketogenesis. Thus, our findings reveal that mTORC1 is a key regulator of PPARα function and hepatic ketogenesis and suggest a role for mTORC1 activity in promoting the ageing of the liver.

Migraine improvement during short lasting ketogenesis: a proof-of-concept study.

BACKGROUND AND PURPOSE: Ketogenesis is a physiological phenomenon due to starvation or a ketogenic diet (KD), a drastic restricted carbohydrate dietary regimen that induces lipid metabolism and ketone body synthesis. Two patients whose migraines disappeared only during, and not outside, cycles of very-low-calorie KD performed to reduce their weight were recently observed. To confirm our observation, in a dietitian clinical setting two parallel groups of migraineurs, one receiving a 1-month very-low-calorie KD prescription followed by a 5-month standard low-calorie diet (SD) and the other a 6-month SD, were followed. METHODS: Ninety-six overweight female migraineurs were enrolled in a diet clinic and blindly received a KD (n = 45) or an SD (n = 51) prescription. Mean monthly attack frequency, number of days with headaches and tablet intake were assessed before and 1, 2, 3 and 6 months after diet initiation. RESULTS: In the KD group, the baseline attack frequency (2.9 attacks per month), number of days with headaches (5.11 days per month) and tablet intake (4.91 doses per month) were significantly reduced after the first month of diet (respectively 0.71, 0.91, 0.51; overall, KD versus baseline, P < 0.0001). During the transition period (first versus second month), the KD group showed a transient worsening of each clinical headache variable (respectively 2.60, 3.60, 3.07), despite being improved compared with baseline, with continuous improvement up to month 6 (respectively 2.16, 2.78, 3.71). In the SD group, significant decreases in the number of days with headaches and tablet intake were observed only from month 3 (P < 0.0001), and in attack frequency at month 6 (P < 0.0001). CONCLUSIONS: The underlying mechanisms of KD efficacy could be related to its ability to enhance mitochondrial energy metabolism and counteract neural inflammation.

Ketone body metabolism and its defects.

Acetoacetate (AcAc) and 3-hydroxybutyrate (3HB), the two main ketone bodies of humans, are important vectors of energy transport from the liver to extrahepatic tissues, especially during fasting, when glucose supply is low. Blood total ketone body (TKB) levels should be evaluated in the context of clinical history, such as fasting time and ketogenic stresses. Blood TKB should also be evaluated in parallel with blood glucose and free fatty acids (FFA). The FFA/TKB ratio is especially useful for evaluation of ketone body metabolism. Defects in ketogenesis include mitochondrial HMG-CoA synthase (mHS) deficiency and HMG-CoA lyase (HL) deficiency. mHS deficiency should be considered in non-ketotic hypoglycemia if a fatty acid beta-oxidation defect is suspected, but cannot be confirmed. Patients with HL deficiency can develop hypoglycemic crises and neurological symptoms even in adolescents and adults. Succinyl-CoA-3-oxoacid CoA transferase (SCOT) deficiency and beta-ketothiolase (T2) deficiency are two defects in ketolysis. Permanent ketosis is pathognomonic for SCOT deficiency. However, patients with "mild" SCOT mutations may have nonketotic periods. T2-deficient patients with "mild" mutations may have normal blood acylcarnitine profiles even in ketoacidotic crises. T2 deficient patients cannot be detected in a reliable manner by newborn screening using acylcarnitines. We review recent data on clinical presentation, metabolite profiles and the course of these diseases in adults, including in pregnancy.

S6 kinase 2 deficiency enhances ketone body production and increases peroxisome proliferator-activated receptor alpha activity in the liver.

UNLABELLED: Nutrient homeostasis is tightly regulated by the balance between energy production and utilization. During fasting, production of ketone bodies as an alternative energy source is critical to maintain nutrient homeostasis. An important component in the nutrient-sensitive signaling pathway is S6 kinase 2 (S6K2), a downstream effector of mammalian target of rapamycin. Here, we show that mice lacking S6K2 exhibit elevated levels of ketone bodies and enhanced peroxisome proliferator-activated receptor alpha (PPARα) activity upon nutrient availability. Consistent with this, knockdown of S6K2 increases the transcriptional activity of PPARα. S6K2 suppresses PPARα by associating with its corepressor, nuclear receptor corepressor 1 (NCoR1), and by inducing the recruitment of NCoR1 to the nucleus. Moreover, ob/ob mice, a genetic model of obesity, have markedly elevated S6K2 activity, and S6K2 was strongly associated with NCoR1 in the nucleus of liver cells. CONCLUSION: Our findings suggest that S6K2 regulates hepatic energy homeostasis by repressing PPARα activity and point to its potential relevance for therapeutic strategies designed to modulate S6K2 activity as a treatment for deregulated ketone body production.

Positional cloning of the combined hyperlipidemia gene Hyplip1.

Familial combined hyperlipidemia (FCHL, MIM-144250) is a common, multifactorial and heterogeneous dyslipidemia predisposing to premature coronary artery disease and characterized by elevated plasma triglycerides, cholesterol, or both. We identified a mutant mouse strain, HcB-19/Dem (HcB-19), that shares features with FCHL, including hypertriglyceridemia, hypercholesterolemia, elevated plasma apolipoprotein B and increased secretion of triglyceride-rich lipoproteins. The hyperlipidemia results from spontaneous mutation at a locus, Hyplip1, on distal mouse chromosome 3 in a region syntenic with a 1q21-q23 FCHL locus identified in Finnish, German, Chinese and US families. We fine-mapped Hyplip1 to roughly 160 kb, constructed a BAC contig and sequenced overlapping BACs to identify 13 candidate genes. We found substantially decreased mRNA expression for thioredoxin interacting protein (Txnip). Sequencing of the critical region revealed a Txnip nonsense mutation in HcB-19 that is absent in its normolipidemic parental strains. Txnip encodes a cytoplasmic protein that binds and inhibits thioredoxin, a major regulator of cellular redox state. The mutant mice have decreased CO2 production but increased ketone body synthesis, suggesting that altered redox status down-regulates the citric-acid cycle, sparing fatty acids for triglyceride and ketone body production. These results reveal a new pathway of potential clinical significance that contributes to plasma lipid metabolism.

Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver.

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 ß-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.

Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

Mitochondrial 2,4-dienoyl-CoA reductase deficiency in mice results in severe hypoglycemia with stress intolerance and unimpaired ketogenesis.

The mitochondrial beta-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids, we created a DECR-deficient mouse line. In Decr(-/-) mice, the mitochondrial beta-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr(-/-) mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C(18:2)), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr(-/-) mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal beta-oxidation and microsomal omega-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1alpha and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.