Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts.

BACKGROUND: Abnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined. METHODS: Oil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs. RESULTS: Injured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα. CONCLUSIONS: These data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results.

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (ß-HB). Prior work, based on detecting ß-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export ß-HB across the apical membrane. Here, we compare the accuracy of measuring ß-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of ß-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete ß-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming ß-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce ß-HB. Altogether, we substantiate ß-HB secretion in RPE but find that the secretion is equal apically and basally, RPE ß-HB can derive from ketogenic amino acids or fatty acids, and accurate ß-HB assessment requires mass spectrometric analysis.

Restoration of HMGCS2-mediated ketogenesis alleviates tacrolimus-induced hepatic lipid metabolism disorder.

Tacrolimus, one of the macrolide calcineurin inhibitors, is the most frequently used immunosuppressant after transplantation. Long-term administration of tacrolimus leads to dyslipidemia and affects liver lipid metabolism. In this study, we investigated the mode of action and underlying mechanisms of this adverse reaction. Mice were administered tacrolimus (2.5 mg·kg-1·d-1, i.g.) for 10 weeks, then euthanized; the blood samples and liver tissues were collected for analyses. We showed that tacrolimus administration induced significant dyslipidemia and lipid deposition in mouse liver. Dyslipidemia was also observed in heart or kidney transplantation patients treated with tacrolimus. We demonstrated that tacrolimus did not directly induce de novo synthesis of fatty acids, but markedly decreased fatty acid oxidation (FAO) in AML12 cells. Furthermore, we showed that tacrolimus dramatically decreased the expression of HMGCS2, the rate-limiting enzyme of ketogenesis, with decreased ketogenesis in AML12 cells, which was responsible for lipid deposition in normal hepatocytes. Moreover, we revealed that tacrolimus inhibited forkhead box protein O1 (FoxO1) nuclear translocation by promoting FKBP51-FoxO1 complex formation, thus reducing FoxO1 binding to the HMGCS2 promoter and its transcription ability in AML12 cells. The loss of HMGCS2 induced by tacrolimus caused decreased ketogenesis and increased acetyl-CoA accumulation, which promoted mitochondrial protein acetylation, thereby resulting in FAO function inhibition. Liver-specific HMGCS2 overexpression via tail intravenous injection of AAV8-TBG-HMGCS2 construct reversed tacrolimus-induced mitochondrial protein acetylation and FAO inhibition, thus removing the lipid deposition in hepatocytes. Collectively, this study demonstrates a novel mechanism of liver lipid deposition and hyperlipidemia induced by long-term administration of tacrolimus, resulted from the loss of HMGCS2-mediated ketogenesis and subsequent FAO inhibition, providing an alternative target for reversing tacrolimus-induced adverse reaction.

Intestinal Ketogenesis and Permeability.

Consumption of a high-fat diet (HFD) has been suggested as a contributing factor behind increased intestinal permeability in obesity, leading to increased plasma levels of microbial endotoxins and, thereby, increased systemic inflammation. We and others have shown that HFD can induce jejunal expression of the ketogenic rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). HMGCS is activated via the free fatty acid binding nuclear receptor PPAR-α, and it is a key enzyme in ketone body synthesis that was earlier believed to be expressed exclusively in the liver. The function of intestinal ketogenesis is unknown but has been described in suckling rats and mice pups, possibly in order to allow large molecules, such as immunoglobulins, to pass over the intestinal barrier. Therefore, we hypothesized that ketone bodies could regulate intestinal barrier function, e.g., via regulation of tight junction proteins. The primary aim was to compare the effects of HFD that can induce intestinal ketogenesis to an equicaloric carbohydrate diet on inflammatory responses, nutrition sensing, and intestinal permeability in human jejunal mucosa. Fifteen healthy volunteers receiving a 2-week HFD diet compared to a high-carbohydrate diet were compared. Blood samples and mixed meal tests were performed at the end of each dietary period to examine inflammation markers and postprandial endotoxemia. Jejunal biopsies were assessed for protein expression using Western blotting, immunohistochemistry, and morphometric characteristics of tight junctions by electron microscopy. Functional analyses of permeability and ketogenesis were performed in Caco-2 cells, mice, and human enteroids. Ussing chambers were used to analyze permeability. CRP and ALP values were within normal ranges and postprandial endotoxemia levels were low and did not differ between the two diets. The PPARα receptor was ketone body-dependently reduced after HFD. None of the tight junction proteins studied, nor the basal electrical parameters, were different between the two diets. However, the ketone body inhibitor hymeglusin increased resistance in mucosal biopsies. In addition, the tight junction protein claudin-3 was increased by ketone inhibition in human enteroids. The ketone body ß-Hydroxybutyrate (ßHB) did not, however, change the mucosal transition of the large-size molecular FD4-probe or LPS in Caco-2 and mouse experiments. We found that PPARα expression was inhibited by the ketone body ßHB. As PPARα regulates HMGCS expression, the ketone bodies thus exert negative feedback signaling on their own production. Furthermore, ketone bodies were involved in the regulation of permeability on intestinal mucosal cells in vitro and ex vivo. We were not, however, able to reproduce these effects on intestinal permeability in vivo in humans when comparing two weeks of high-fat with high-carbohydrate diet in healthy volunteers. Further, neither the expression of inflammation markers nor the aggregate tight junction proteins were changed. Thus, it seems that not only HFD but also other factors are needed to permit increased intestinal permeability in vivo. This indicates that the healthy gut can adapt to extremes of macro-nutrients and increased levels of intestinally produced ketone bodies, at least during a shorter dietary challenge.

Fasting-induced HMGCS2 expression in the kidney does not contribute to circulating ketones.

Mitochondrial hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme in ketogenesis. The liver expresses high levels of HMGCS2 constitutively as the main ketogenic organ. It has been suggested that the kidney could be ketogenic as HMGCS2 is expressed in the kidney during fasting and diabetic conditions. However, definitive proof of the capacity for the kidney to produce ketones is lacking. We demonstrated that during fasting, HMGCS2 expression is induced in the proximal tubule of the kidney and is peroxisome proliferator activated receptor-α dependent. Mice with kidney-specific Hmgcs2 deletion showed a minor, likely physiologically insignificant, decrease in circulating ketones during fasting. Conversely, liver-specific Hmgcs2 knockout mice exhibited a complete loss of fasting ketosis. Together, these findings indicate that renal HMGCS2 does not significantly contribute to global ketone production and that during fasting, the increase in circulating ketones is solely dependent on hepatic HMGCS2. Proximal tubule HMGCS2 serves functions other than systemic ketone provision.NEW & NOTEWORTHY The mitochondrial enzyme hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) catalyzes the rate-limiting step of ketogenesis. Although the liver constitutively expresses HMGCS2 and is considered the main ketogenic organ, HMGCS2 is induced in the kidney during fasting, leading to the proposal that the kidney contributes to fasting ketosis. We showed kidney HMGCS2 does not contribute to circulating ketones during fasting and cannot compensate for hepatic ketogenic insufficiency.

HMGCS2 Mediation of Ketone Levels Affects Sorafenib Treatment Efficacy in Liver Cancer Cells.

Primary liver cancer is the fifth leading death of cancers in men, and hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases. Sorafenib is a first-line drug for advanced-stage HCC patients. Sorafenib is a multi-target kinase inhibitor that blocks tumor cell proliferation and angiogenesis. Despite sorafenib treatment extending survival, some patients experience side effects, and sorafenib resistance does occur. 3-Hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme for ketogenesis, which synthesizes the ketone bodies, ß-hydroxybutyrate (ß-HB) and acetoacetate (AcAc). ß-HB is the most abundant ketone body which is present in a 4:1 ratio compared to AcAc. Recently, ketone body treatment was found to have therapeutic effects against many cancers by causing metabolic alternations and cancer cell apoptosis. Our previous publication showed that HMGCS2 downregulation-mediated ketone body reduction promoted HCC clinicopathological progression through regulating c-Myc/cyclin D1 and caspase-dependent signaling. However, whether HMGCS2-regulated ketone body production alters the sensitivity of human HCC to sorafenib treatment remains unclear. In this study, we showed that HMGCS2 downregulation enhanced the proliferative ability and attenuated the cytotoxic effects of sorafenib by activating expressions of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-P38, and p-AKT. In contrast, HMGCS2 overexpression decreased cell proliferation and enhanced the cytotoxic effects of sorafenib in HCC cells by inhibiting ERK activation. Furthermore, we showed that knockdown HMGCS2 exhibited the potential migratory ability, as well as decreasing zonula occludens protein (ZO)-1 and increasing c-Myc expression in both sorafenib-treated Huh7 and HepG2 cells. Although HMGCS2 overexpression did not alter the migratory effect, expressions of ZO-1, c-Myc, and N-cadherin decreased in sorafenib-treated HMGCS2-overexpressing HCC cells. Finally, we investigated whether ketone treatment influences sorafenib sensitivity. We showed that ß-HB pretreatment decreased cell proliferation and enhanced antiproliferative effect of sorafenib in both Huh7 and HepG2 cells. In conclusion, this study defined the impacts of HMGCS2 expression and ketone body treatment on influencing the sorafenib sensitivity of liver cancer cells.

SLC38A4 functions as a tumour suppressor in hepatocellular carcinoma through modulating Wnt/ß-catenin/MYC/HMGCS2 axis.

BACKGROUND: Many molecular alterations are shared by embryonic liver development and hepatocellular carcinoma (HCC). Identifying the common molecular events would provide a novel prognostic biomarker and therapeutic target for HCC. METHODS: Expression levels and clinical relevancies of SLC38A4 and HMGCS2 were investigated by qRT-PCR, western blot, TCGA and GEO datasets. The biological roles of SLC38A4 were investigated by functional assays. The downstream signalling pathway of SLC38A4 was investigated by qRT-PCR, western blot, immunofluorescence, luciferase reporter assay, TCGA and GEO datasets. RESULTS: SLC38A4 silencing was identified as an oncofetal molecular event. DNA hypermethylation contributed to the downregulations of Slc38a4/SLC38A4 in the foetal liver and HCC. Low expression of SLC38A4 was associated with poor prognosis of HCC patients. Functional assays demonstrated that SLC38A4 depletion promoted HCC cellular proliferation, stemness and migration, and inhibited HCC cellular apoptosis in vitro, and further repressed HCC tumorigenesis in vivo. HMGCS2 was identified as a critical downstream target of SLC38A4. SLC38A4 increased HMGCS2 expression via upregulating AXIN1 and repressing Wnt/ß-catenin/MYC axis. Functional rescue assays showed that HMGCS2 overexpression reversed the oncogenic roles of SLC38A4 depletion in HCC. CONCLUSIONS: SLC38A4 downregulation was identified as a novel oncofetal event, and SLC38A4 was identified as a novel tumour suppressor in HCC.

Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

Why the diabetic heart is energy inefficient: a ketogenesis and ketolysis perspective.

Lack of glucose uptake compromises metabolic flexibility and reduces energy efficiency in the diabetes mellitus (DM) heart. Although increased use of fatty acid to compensate glucose substrate has been studied, less is known about ketone body metabolism in the DM heart. Ketogenic diet reduces obesity, a risk factor for T2DM. How ketogenic diet affects ketone metabolism in the DM heart remains unclear. At the metabolic level, the DM heart differs from the non-DM heart because of altered metabolic substrate and the T1DM heart differs from the T2DM heart because of insulin levels. How these changes affect ketone body metabolism in the DM heart are poorly understood. Ketogenesis produces ketone bodies by using acetyl-CoA, whereas ketolysis consumes ketone bodies to produce acetyl-CoA, showing their opposite roles in the ketone body metabolism. Cardiac-specific transgenic upregulation of ketogenesis enzyme or knockout of ketolysis enzyme causes metabolic abnormalities leading to cardiac dysfunction. Empirical evidence demonstrates upregulated transcription of ketogenesis enzymes, no change in the levels of ketone body transporters, very high levels of ketone bodies, and reduced expression and activity of ketolysis enzymes in the T1DM heart. Based on these observations, I hypothesize that increased transcription and activity of cardiac ketogenesis enzyme suppresses ketolysis enzyme in the DM heart, which decreases cardiac energy efficiency. The T1DM heart exhibits highly upregulated ketogenesis compared with the T2DM heart because of the lack of insulin, which inhibits ketogenesis enzyme.

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site.

Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, Methanothermococcus thermolithotrophicus HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.

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.

The specific molecular architecture of plant 3-hydroxy-3-methylglutaryl-CoA lyase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.

Overexpression of HMG-CoA synthase promotes Arabidopsis root growth and adversely affects glucosinolate biosynthesis.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the second step of the mevalonate (MVA) pathway. An HMGS inhibitor (F-244) has been reported to retard growth in wheat, tobacco, and Brassica juncea, but the mechanism remains unknown. Although the effects of HMGS on downstream isoprenoid metabolites have been extensively reported, not much is known on how it might affect non-isoprenoid metabolic pathways. Here, the mechanism of F-244-mediated inhibition of primary root growth in Arabidopsis and the relationship between HMGS and non-isoprenoid metabolic pathways were investigated by untargeted SWATH-MS quantitative proteomics, quantitative real-time PCR, and target metabolite analysis. Our results revealed that the inhibition of primary root growth caused by F-244 was a consequence of reduced stigmasterol, auxin, and cytokinin levels. Interestingly, proteomic analyses identified a relationship between HMGS and glucosinolate biosynthesis. Inhibition of HMGS activated glucosinolate biosynthesis, resulting from the induction of glucosinolate biosynthesis-related genes, suppression of sterol biosynthesis-related genes, and reduction in sterol levels. In contrast, HMGS overexpression inhibited glucosinolate biosynthesis, due to down-regulation of glucosinolate biosynthesis-related genes, up-regulation of sterol biosynthesis-related genes, and increase in sterol content. Thus, HMGS might represent a target for the manipulation of glucosinolate biosynthesis, given the regulatory relationship between HMGS in the MVA pathway and glucosinolate biosynthesis.

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.

Crocin ameliorates hepatic steatosis through activation of AMPK signaling in db/db mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity, type 2 diabetes and other metabolic disorders worldwide. Crocin is a carotenoid compound possessing various pharmacological activities. In the present study, we aimed to investigate the effect on fatty liver under diabetic and obese condition and to examine the possible role of AMP-activated protein kinase (AMPK) signaling. METHODS: db/db mice were administrated with crocin and injected with LV-shAMPK or its negative control lentivirus. Metabolic dysfunction, lipogenesis and fatty acid-oxidation in liver were evaluated. RESULTS: In db/db mice, we found that oral administration of crocin significantly upregulated the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in liver. Crocin reduced liver weight, serum levels of alanine aminotransferase, alanine aminotransferase, and liver triglyceride content, and attenuated morphological injury of liver in db/db mice. Crocin inhibited the mRNA expression of lipogenesis-associated genes, including sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor γ, fatty acid synthase, stearoyl-CoA desaturase 1, and diacylglycerol acyltransferase 1, and increased the mRNA expression of genes involved in the regulation of ß-oxidation of fatty acids, including PPARα, acyl-CoA oxidase 1, carnitine palmitoyltransferase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2. Moreover, treatment of crocin resulted in a amelioration of general metabolic disorder, as evidenced by decreased fasting blood glucose, reduced serum levels of insulin, triglyceride, total cholesterol, and non-esterified fatty acid, and improved glucose intolerance. Crocin-induced protective effects against fatty liver and metabolic disorder were significantly blocked by lentivirus-mediated downregulation of AMPK. CONCLUSIONS: The results suggest that crocin can inhibit lipogenesis and promote ß-oxidation of fatty acids through activation of AMPK, leading to improvement of fatty liver and metabolic dysfunction. Therefore, crocin may be a potential promising option for the clinical treatment for NAFLD and associated metabolic diseases.

Fusarisolins A⁻E, Polyketides from the Marine-Derived Fungus Fusarium solani H918.

Five new (fusarisolins A⁻E, 1 to 5) and three known (6 to 8) polyketides were isolated from the marine-derived fungus Fusarium solani H918, along with six known phenolics (9 to 14). Their structures were established by comprehensive spectroscopic data analyses, methoxyphenylacetic acid (MPA) method, chemical conversion, and by comparison with data reported in the literature. Compounds 1 and 2 are the first two naturally occurring 21 carbons polyketides featuring a rare ß- and γ-lactone unit, respectively. All isolates (1 to 14) were evaluated for their inhibitory effects against tea pathogenic fungus Pestalotiopsis theae and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase gene expression. Compound 8 showed potent antifungal activity with an ED50 value of 55 µM, while 1, 8, 13, and 14 significantly inhibited HMG-CoA synthase gene expression.

HMG-CoA synthase 1 is a synthetic lethal partner of BRAFV600E in human cancers.

Contributions of metabolic changes to cancer development and maintenance have received increasing attention in recent years. Although many human cancers share similar metabolic alterations, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Using an RNAi-based screen targeting the majority of the known metabolic proteins, we recently found that oncogenic BRAFV600E up-regulates HMG-CoA lyase (HMGCL), which converts HMG-CoA to acetyl-CoA and a ketone body, acetoacetate, that selectively enhances BRAFV600E-dependent MEK1 activation in human cancer. Here, we identified HMG-CoA synthase 1 (HMGCS1), the upstream ketogenic enzyme of HMGCL, as an additional "synthetic lethal" partner of BRAFV600E Although HMGCS1 expression did not correlate with BRAFV600E mutation in human melanoma cells, HMGCS1 was selectively important for proliferation of BRAFV600E-positive melanoma and colon cancer cells but not control cells harboring active N/KRAS mutants, and stable knockdown of HMGCS1 only attenuated colony formation and tumor growth potential of BRAFV600E melanoma cells. Moreover, cytosolic HMGCS1 that co-localized with HMGCL and BRAFV600E was more important than the mitochondrial HMGCS2 isoform in BRAFV600E-expressing cancer cells in terms of acetoacetate production. Interestingly, HMGCL knockdown did not affect HMGCS1 expression levels, whereas HMGCS1 knockdown caused a compensating increase in HMGCL protein level because of attenuated protein degradation. However, this increase did not reverse the reduced ketogenesis in HMGCS1 knockdown cells. Mechanistically, HMGCS1 inhibition decreased intracellular acetoacetate levels, leading to reduced BRAFV600E-MEK1 binding and consequent MEK1 activation. We conclude that the ketogenic HMGCS1-HMGCL-acetoacetate axis may represent a promising therapeutic target for managing BRAFV600E-positive human cancers.

Involvement of nutrients and nutritional mediators in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2) catalyses the first step of ketogenesis and is critical in various metabolic conditions. Several nutrient molecules were able to differentially modulate HMGCS2 expression levels. Docosahexaenoic acid (DHA, C22:6, n-3), eicosapentaenoic acid (EPA, C20:5, n-3), arachidonic acid (AA, C20:4, n-6), and glucose increased HMGCS2 mRNA and protein levels in HepG2 hepatoma cells, while fructose decreased them. The effect of n-6 AA resulted significantly higher than that of n-3 PUFA, but when combined all these molecules were far less efficient. Insulin reduced HMGCS2 mRNA and protein levels in HepG2 cells, even when treated with PUFA and monosaccharides. Several nuclear receptors and transcription factors are involved in HMGCS2 expression regulation. While peroxysome proliferator activated receptor α (PPAR-α) agonist WY14643 increased HMGCS2 expression, this treatment was unable to affect PUFA-mediated regulation of HMGCS2 expression. Forkhead box O1 (FoxO1) inhibitor AS1842856 reduced HMGCS2 expression and suppressed induction promoted by fatty acids. Cells treatment with liver X receptor alpha (LXRα) agonist T0901317 reduced HMGCS2 mRNA, indicating a role for this transcription factor as suppressor of HMGCS2 gene. Previous observations already indicated HMGCS2 expression as possible nutrition status reference: our results show that several nutrients as well as specific nutritional related hormonal conditions are able to affect significantly HMGCS2 gene expression, indicating a relevant role for PUFA, which are mostly derived from nutritional intake. These insights into mechanisms of its regulation, specifically through nutrients commonly associated with disease risk, indicate HMGCS2 expression as possible reference marker of metabolic and nutritional status.