Acetate dependence of tumors.

Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.

Reciprocal regulation of cardiac ß-oxidation and pyruvate dehydrogenase by insulin.

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.

Nonalcoholic Fatty Liver Disease and Cardiovascular Risk: A Scientific Statement From the American Heart Association.

Nonalcoholic fatty liver disease (NAFLD) is an increasingly common condition that is believed to affect >25% of adults worldwide. Unless specific testing is done to identify NAFLD, the condition is typically silent until advanced and potentially irreversible liver impairment occurs. For this reason, the majority of patients with NAFLD are unaware of having this serious condition. Hepatic complications from NAFLD include nonalcoholic steatohepatitis, hepatic cirrhosis, and hepatocellular carcinoma. In addition to these serious complications, NAFLD is a risk factor for atherosclerotic cardiovascular disease, which is the principal cause of death in patients with NAFLD. Accordingly, the purpose of this scientific statement is to review the underlying risk factors and pathophysiology of NAFLD, the associations with atherosclerotic cardiovascular disease, diagnostic and screening strategies, and potential interventions.

CB1Rs in VMH neurons regulate glucose homeostasis but not body weight.

Cannabinoid 1 receptor (CB1R) inverse agonists reduce body weight and improve several parameters of glucose homeostasis. However, these drugs have also been associated with deleterious side effects. CB1R expression is widespread in the brain and in peripheral tissues, but whether specific sites of expression can mediate the beneficial metabolic effects of CB1R drugs, while avoiding the untoward side effects, remains unclear. Evidence suggests inverse agonists may act on key sites within the central nervous system to improve metabolism. The ventromedial hypothalamus (VMH) is a critical node regulating energy balance and glucose homeostasis. To determine the contributions of CB1Rs expressed in VMH neurons in regulating metabolic homeostasis, we generated mice lacking CB1Rs in the VMH. We found that the deletion of CB1Rs in the VMH did not affect body weight in chow- and high-fat diet-fed male and female mice. We also found that deletion of CB1Rs in the VMH did not alter weight loss responses induced by the CB1R inverse agonist SR141716. However, we did find that CB1Rs of the VMH regulate parameters of glucose homeostasis independent of body weight in diet-induced obese male mice.NEW & NOTEWORTHY Cannabinoid 1 receptors (CB1Rs) regulate metabolic homeostasis, and CB1R inverse agonists reduce body weight and improve parameters of glucose metabolism. However, the cell populations expressing CB1Rs that regulate metabolic homeostasis remain unclear. CB1Rs are highly expressed in the ventromedial hypothalamic nucleus (VMH), which is a crucial node that regulates metabolism. With CRISPR/Cas9, we generated mice lacking CB1Rs specifically in VMH neurons and found that CB1Rs in VMH neurons are essential for the regulation of glucose metabolism independent of body weight regulation.

A Highly Durable RNAi Therapeutic Inhibitor of PCSK9.

BACKGROUND: Inclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. METHODS: In this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. RESULTS: The most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84). CONCLUSIONS: In this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442 .).

Loss of astrocyte cholesterol synthesis disrupts neuronal function and alters whole-body metabolism.

Cholesterol is important for normal brain function. The brain synthesizes its own cholesterol, presumably in astrocytes. We have previously shown that diabetes results in decreased brain cholesterol synthesis by a reduction in sterol regulatory element-binding protein 2 (SREBP2)-regulated transcription. Here we show that coculture of control astrocytes with neurons enhances neurite outgrowth, and this is reduced with SREBP2 knockdown astrocytes. In vivo, mice with knockout of SREBP2 in astrocytes have impaired brain development and behavioral and motor defects. These mice also have altered energy balance, altered body composition, and a shift in metabolism toward carbohydrate oxidation driven by increased glucose oxidation by the brain. Thus, SREBP2-mediated cholesterol synthesis in astrocytes plays an important role in brain and neuronal development and function, and altered brain cholesterol synthesis may contribute to the interaction between metabolic diseases, such as diabetes and altered brain function.

Interplay between ChREBP and SREBP-1c coordinates postprandial glycolysis and lipogenesis in livers of mice.

Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp-/- mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp-/- livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp-/- mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin's induction of lipogenic genes, ChREBP mediates glucose's induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present.

Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel.

AIMS: To appraise the clinical and genetic evidence that low-density lipoproteins (LDLs) cause atherosclerotic cardiovascular disease (ASCVD). METHODS AND RESULTS: We assessed whether the association between LDL and ASCVD fulfils the criteria for causality by evaluating the totality of evidence from genetic studies, prospective epidemiologic cohort studies, Mendelian randomization studies, and randomized trials of LDL-lowering therapies. In clinical studies, plasma LDL burden is usually estimated by determination of plasma LDL cholesterol level (LDL-C). Rare genetic mutations that cause reduced LDL receptor function lead to markedly higher LDL-C and a dose-dependent increase in the risk of ASCVD, whereas rare variants leading to lower LDL-C are associated with a correspondingly lower risk of ASCVD. Separate meta-analyses of over 200 prospective cohort studies, Mendelian randomization studies, and randomized trials including more than 2 million participants with over 20 million person-years of follow-up and over 150 000 cardiovascular events demonstrate a remarkably consistent dose-dependent log-linear association between the absolute magnitude of exposure of the vasculature to LDL-C and the risk of ASCVD; and this effect appears to increase with increasing duration of exposure to LDL-C. Both the naturally randomized genetic studies and the randomized intervention trials consistently demonstrate that any mechanism of lowering plasma LDL particle concentration should reduce the risk of ASCVD events proportional to the absolute reduction in LDL-C and the cumulative duration of exposure to lower LDL-C, provided that the achieved reduction in LDL-C is concordant with the reduction in LDL particle number and that there are no competing deleterious off-target effects. CONCLUSION: Consistent evidence from numerous and multiple different types of clinical and genetic studies unequivocally establishes that LDL causes ASCVD.

Inhibition of PCSK9 does not improve lipopolysaccharide-induced mortality in mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that targets LDL receptors (LDLRs) for degradation in liver. Blocking the interaction of PCSK9 with the LDLR potently reduces plasma LDL cholesterol levels and cardiovascular events. Recently, it has been suggested that inhibition of PCSK9 might also improve outcomes in mice and humans with sepsis, possibly by increasing LDLR-mediated clearance of endotoxins. Sepsis is a complication of a severe microbial infection that has shared pathways with lipid metabolism. Here, we tested whether anti-PCSK9 antibodies prevent death from lipopolysaccharide (LPS)-induced endotoxemia. Mice were administered PCSK9 antibodies prior to, or shortly after, injecting LPS. In both scenarios, the administration of PCSK9 antibodies did not alter endotoxemia-induced mortality. Afterward, we determined whether the complete absence of PCSK9 improved endotoxemia-induced mortality in mice with the germ-line deletion of Pcsk9 Similarly, PCSK9 knockout mice were not protected from LPS-induced death. To determine whether low LDLR expression increased LPS-induced mortality, Ldlr-/- mice and PCSK9 transgenic mice were studied after injection of LPS. Endotoxemia-induced mortality was not altered in either mouse model. In a human cohort, we observed no correlation between plasma inflammation markers with total cholesterol levels, LDL cholesterol, and PCSK9. Combined, our data demonstrate that PCSK9 inhibition provides no protection from LPS-induced mortality in mice.

Mogat1 deletion does not ameliorate hepatic steatosis in lipodystrophic (Agpat2-/-) or obese (ob/ob) mice.

Reducing triacylglycerol (TAG) in the liver continues to pose a challenge in states of nonalcoholic hepatic steatosis. MonoacylglycerolO-acyltransferase (MOGAT) enzymes convert monoacylglycerol (MAG) to diacylglycerol, a precursor for TAG synthesis, and are involved in a major pathway of TAG synthesis in selected tissues, such as small intestine. MOGAT1 possesses MGAT activity in in vitro assays, but its physiological function in TAG metabolism is unknown. Recent studies suggest a role for MOGAT1 in hepatic steatosis in lipodystrophic [1-acylglycerol-3-phosphateO-acyltransferase (Agpat)2(-/-)] and obese (ob/ob) mice. To test this, we deletedMogat1in theAgpat2(-/-)andob/obgenetic background to generateMogat1(-/-);Agpat2(-/-)andMogat1(-/-);ob/obdouble knockout (DKO) mice. Here we report that, despite the absence ofMogat1in either DKO mouse model, we did not find any decrease in liver TAG by 16 weeks of age. Additionally, there were no measureable changes in plasma glucose (diabetes) and insulin resistance. Our data indicate a minimal role, if any, of MOGAT1 in liver TAG synthesis, and that TAG synthesis in steatosis associated with lipodystrophy and obesity is independent of MOGAT1. Our findings suggest that MOGAT1 likely has an alternative function in vivo.

Hepatic gluconeogenesis is enhanced by phosphatidic acid which remains uninhibited by insulin in lipodystrophic Agpat2-/- mice.

In this study we examined the role of phosphatidic acid (PA) in hepatic glucose production (HGP) and development of hepatic insulin resistance in mice that lack 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2). Liver lysophosphatidic acid and PA levels were increased ∼2- and ∼5-fold, respectively, in male Agpat2(-/-) mice compared with wild type mice. In the absence of AGPAT2, the liver can synthesize PAs by activating diacylglycerol kinase or phospholipase D, both of which were elevated in the livers of Agpat2(-/-) mice. We found that PAs C16:0/18:1 and C18:1/20:4 enhanced HGP in primary WT hepatocytes, an effect that was further enhanced in primary hepatocytes from Agpat2(-/-) mice. Lysophosphatidic acids C16:0 and C18:1 failed to increase HGP in primary hepatocytes. The activation of HGP was accompanied by an up-regulation of the key gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This activation was suppressed by insulin in the WT primary hepatocytes but not in the Agpat2(-/-) primary hepatocytes. Thus, the lack of normal insulin signaling in Agpat2(-/-) livers allows unrestricted PA-induced gluconeogenesis significantly contributing to the development of hyperglycemia in these mice.

Temporal regulation of nuclear factor one occupancy by calcineurin/NFAT governs a voltage-sensitive developmental switch in late maturing neurons.

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

Leptin ameliorates insulin resistance and hepatic steatosis in Agpat2-/- lipodystrophic mice independent of hepatocyte leptin receptors.

Leptin is essential for energy homeostasis and regulation of food intake. Patients with congenital generalized lipodystrophy (CGL) due to mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) and the CGL murine model (Agpat2(-/-) mice) both have severe insulin resistance, diabetes mellitus, hepatic steatosis, and low plasma leptin levels. In this study, we show that continuous leptin treatment of Agpat2(-/-) mice for 28 days reduced plasma insulin and glucose levels and normalized hepatic steatosis and hypertriglyceridemia. Leptin also partially, but significantly, reversed the low plasma thyroxine and high corticosterone levels found in Agpat2(-/-) mice. Levels of carbohydrate response element binding protein (ChREBP) were reduced, whereas lipogenic gene expression were increased in the livers of Agpat2(-/-) mice, suggesting that deregulated ChREBP contributed to the development of fatty livers in these mice and that this transcription factor is a target of leptin's beneficial metabolic action. Leptin administration did not change hepatic fatty acid oxidation enzymes mRNA levels in Agpat2(-/-) mice. The selective deletion of leptin receptors only in hepatocytes did not prevent the positive metabolic actions of leptin in Agpat2(-/-) mice, supporting the notion that the majority of metabolic actions of leptin are dependent on its action in nonhepatocyte cells and/or the central nervous system.

Deletion of ELOVL6 blocks the synthesis of oleic acid but does not prevent the development of fatty liver or insulin resistance.

Elongation of very long chain fatty acid-like family member 6 (ELOVL6) is a fatty acyl elongase that performs the initial and rate-limiting condensing reaction required for microsomal elongation of long-chain fatty acids. Our previous in vitro studies suggested that ELOVL6 elongated long-chain saturated fatty acids and monounsaturated fatty acids with chain lengths of 12 to 16 carbons. Here, we describe the generation and phenotypic characterization of Elovl6(-/-) mice. As predicted from the in vitro studies, livers from Elovl6(-/-) mice accumulated palmitic (C16:0) and palmitoleic (C16:1, n-7) fatty acids and contained significantly less stearic (C18:0) and oleic (C18:1, n-9) acids, confirming that ELOVL6 is the only enzyme capable of elongating palmitate (C16:0). Unexpectedly, Elovl6(-/-) mice produced vaccenic acid (C18:1, n-7), the elongated product of palmitoleate (C16:1, n-7), suggesting that palmitoleate (C16:1, n-7) to vaccenate (C18:1, n-7) elongation was not specific to ELOVL6. The only detected consequence of deleting Elovl6(-/-) in mice was that their livers accumulated significantly more triglycerides than wild-type mice when fed a fat-free/high-carbohydrate diet. When mice were fed a high-fat diet or ELOVL6 was deleted in ob/ob mice, the absence of ELOVL6 did not alter the development of obesity, fatty liver, hyperglycemia, or hyperinsulinemia. Combined, these results suggest that palmitoleic (C16:1, n-7) and vaccenic (C18:1, n-7) acids can largely replace the roles of oleic acid (C18:1, n-9) in vivo and that the deletion of ELOVL6 does not protect mice from the development of hepatic steatosis or insulin resistance.

A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice.

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.

Regulated regeneration of adipose tissue in lipodystrophic Agpat2-null mice partially ameliorates hepatic steatosis.

Both humans and mice with congenital generalized lipodystrophy due to AGPAT2 deficiency develop diabetes mellitus, insulin resistance, and hepatic steatosis, which have been attributed to the near total loss of adipose tissue (AT). Here, we show that regulated AT regeneration in doxycycline (dox)-fed Tg-AT-hAGPAT2;mAgpat2-/- mice partially ameliorates hepatic steatosis at 12 weeks of age and causes reduced expression of genes involved in hepatic de novo lipogenesis despite partial (∼30-50%) AT regeneration compared to that in wild-type mice. Compared to chow-fed Tg-AT-hAGPAT2;mAgpat2-/- mice, those fed dox diet had markedly reduced serum insulin levels, suggesting an improvement in insulin resistance. Interestingly, the fasting plasma glucose levels in dox-fed Tg-AT-hAGPAT2;mAgpat2-/- mice were no different than those in chow-fed wild-type mice. Indirect calorimetry revealed normalization in the energy balance of dox-fed Tg-AT-hAGPAT2;mAgpat2-/- mice compared to that in chow-fed mice. This study's findings suggest that partial AT regeneration in lipodystrophic mice can ameliorate metabolic derangements.