Sol-moiety: Discovery of a water-soluble prodrug technology for enhanced oral bioavailability of insoluble therapeutics.

Though conceptually attractive, the use of water-soluble prodrug technology to enhance oral bioavailability of highly insoluble small molecule therapeutics has not been widely adopted. In large part, this is due to the rapid enzymatic or chemical hydrolysis of prodrugs within the gastrointestinal tract, resulting in drug precipitation and no overall improvement in oral bioavailability relative to standard formulation strategies. We reasoned that an optimal water-soluble prodrug could be attained if the rate of prodrug hydrolysis were reduced to favor drug absorption rather than drug precipitation. In doing so, the rate of hydrolysis provides a pharmacokinetic control point for drug delivery. Herein, we report the discovery of a water-soluble promoiety (Sol-moiety) technology to optimize the oral bioavailability of highly insoluble small molecule therapeutics, possessing various functional groups, without the need for sophisticated, often toxic, lipid or organic solvent-based formulations. The power of the technology is demonstrated with marked pharmacokinetic improvement of the commercial drugs enzalutamide, vemurafenib, and paclitaxel. This led to a successful efficacy study of a water-soluble orally administered prodrug of paclitaxel in a mouse pancreatic tumor model.

Gut Microbiota-Derived Trimethylamine N-Oxide Contributes to Abdominal Aortic Aneurysm Through Inflammatory and Apoptotic Mechanisms.

BACKGROUND: Large-scale human and mechanistic mouse studies indicate a strong relationship between the microbiome-dependent metabolite trimethylamine N-oxide (TMAO) and several cardiometabolic diseases. This study aims to investigate the role of TMAO in the pathogenesis of abdominal aortic aneurysm (AAA) and target its parent microbes as a potential pharmacological intervention. METHODS: TMAO and choline metabolites were examined in plasma samples, with associated clinical data, from 2 independent patient cohorts (N=2129 total). Mice were fed a high-choline diet and underwent 2 murine AAA models, angiotensin II infusion in low-density lipoprotein receptor-deficient (Ldlr-/-) mice or topical porcine pancreatic elastase in C57BL/6J mice. Gut microbial production of TMAO was inhibited through broad-spectrum antibiotics, targeted inhibition of the gut microbial choline TMA lyase (CutC/D) with fluoromethylcholine, or the use of mice genetically deficient in flavin monooxygenase 3 (Fmo3-/-). Finally, RNA sequencing of in vitro human vascular smooth muscle cells and in vivo mouse aortas was used to investigate how TMAO affects AAA. RESULTS: Elevated TMAO was associated with increased AAA incidence and growth in both patient cohorts studied. Dietary choline supplementation augmented plasma TMAO and aortic diameter in both mouse models of AAA, which was suppressed with poorly absorbed oral broad-spectrum antibiotics. Treatment with fluoromethylcholine ablated TMAO production, attenuated choline-augmented aneurysm initiation, and halted progression of an established aneurysm model. In addition, Fmo3-/- mice had reduced plasma TMAO and aortic diameters and were protected from AAA rupture compared with wild-type mice. RNA sequencing and functional analyses revealed choline supplementation in mice or TMAO treatment of human vascular smooth muscle cells-augmented gene pathways associated with the endoplasmic reticulum stress response, specifically the endoplasmic reticulum stress kinase PERK. CONCLUSIONS: These results define a role for gut microbiota-generated TMAO in AAA formation through upregulation of endoplasmic reticulum stress-related pathways in the aortic wall. In addition, inhibition of microbiome-derived TMAO may serve as a novel therapeutic approach for AAA treatment where none currently exist.

Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

Gut microbial trimethylamine is elevated in alcohol-associated hepatitis and contributes to ethanol-induced liver injury in mice.

There is mounting evidence that microbes residing in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite TMA is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

IL-1 induces mitochondrial translocation of IRAK2 to suppress oxidative metabolism in adipocytes.

Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R-MyD88-IRAK2-PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.

Obesity-linked suppression of membrane-bound O-acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease.

Recent studies have identified a genetic variant rs641738 near two genes encoding membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4) that associate with increased risk of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol-related cirrhosis, and liver fibrosis in those infected with viral hepatitis (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017). Based on hepatic expression quantitative trait loci analysis, it has been suggested that MBOAT7 loss of function promotes liver disease progression (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017), but this has never been formally tested. Here we show that Mboat7 loss, but not Tmc4, in mice is sufficient to promote the progression of NAFLD in the setting of high fat diet. Mboat7 loss of function is associated with accumulation of its substrate lysophosphatidylinositol (LPI) lipids, and direct administration of LPI promotes hepatic inflammatory and fibrotic transcriptional changes in an Mboat7-dependent manner. These studies reveal a novel role for MBOAT7-driven acylation of LPI lipids in suppressing the progression of NAFLD.

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

Loss of HDAC6 alters gut microbiota and worsens obesity.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (Treg) cells. We asked whether mice that lack HDAC6 and have potent suppressive Treg cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4+ and CD8+ T-cell activation yet maintained the enhanced Treg cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.

Postprandial gut microbiota-driven choline metabolism links dietary cues to adipose tissue dysfunction.

The human body is an integrated circuit between microbial symbionts and our Homo sapien genome, which communicate bi-directionally to maintain homeostasis within the human meta-organism. There is now strong evidence that microbes resident in the human intestine can directly contribute to the pathogenesis of obesity and associated cardiometabolic disorders. In fact, gut microbes represent a filter of our greatest environmental exposure - the foods we consume. It is now clear that we each experience a given meal differently, based on our unique gut microbial communities. Biologically active gut microbe-derived metabolites, such as short chain fatty acids, secondary bile acids, and trimethylamine-N-oxide (TMAO), are now uniquely recognized as contributors to obesity and related cardiometabolic disorders. However, mechanistic insights into how microbe-derived metabolites promote obesity are largely unknown. Recent work has demonstrated that the meta-organismal production of the bacterial co-metabolite TMAO is linked to suppression of beiging of white adipose tissue in mice and humans. Furthermore, the TMAO pathway is becoming an increasingly attractive therapeutic target in obesity-associated diseases such as type 2 diabetes, kidney failure, and cardiovascular disease. In this commentary we discuss recent findings linking the TMAO pathway to obesity-associated disorders, and provide additional insights into potential mechanisms driving this microbe-host interaction.

Δ-5 Fatty Acid Desaturase FADS1 Impacts Metabolic Disease by Balancing Proinflammatory and Proresolving Lipid Mediators.

OBJECTIVE: Human genetic variants near the FADS (fatty acid desaturase) gene cluster (FADS1-2-3) are strongly associated with cardiometabolic traits including dyslipidemia, fatty liver, type 2 diabetes mellitus, and coronary artery disease. However, mechanisms underlying these genetic associations are unclear. APPROACH AND RESULTS: Here, we specifically investigated the physiological role of the Δ-5 desaturase FADS1 in regulating diet-induced cardiometabolic phenotypes by treating hyperlipidemic LDLR (low-density lipoprotein receptor)-null mice with antisense oligonucleotides targeting the selective knockdown of Fads1. Fads1 knockdown resulted in striking reorganization of both ω-6 and ω-3 polyunsaturated fatty acid levels and their associated proinflammatory and proresolving lipid mediators in a highly diet-specific manner. Loss of Fads1 activity promoted hepatic inflammation and atherosclerosis, yet was associated with suppression of hepatic lipogenesis. Fads1 knockdown in isolated macrophages promoted classic M1 activation, whereas suppressing alternative M2 activation programs, and also altered systemic and tissue inflammatory responses in vivo. Finally, the ability of Fads1 to reciprocally regulate lipogenesis and inflammation may rely in part on its role as an effector of liver X receptor signaling. CONCLUSIONS: These results position Fads1 as an underappreciated regulator of inflammation initiation and resolution, and suggest that endogenously synthesized arachidonic acid and eicosapentaenoic acid are key determinates of inflammatory disease progression and liver X receptor signaling.

Akt3 inhibits adipogenesis and protects from diet-induced obesity via WNK1/SGK1 signaling.

Three Akt isoforms, encoded by 3 separate genes, are expressed in mammals. While the roles of Akt1 and Akt2 in metabolism are well established, it is not yet known whether Akt3 plays a role in metabolic diseases. We now report that Akt3 protects mice from high-fat diet-induced obesity by suppressing an alternative pathway of adipogenesis via with no lysine protein kinase-1 (WNK1) and serum/glucocorticoid-inducible kinase 1 (SGK1). We demonstrate that Akt3 specifically phosphorylates WNK1 at T58 and promotes its degradation via the ubiquitin-proteasome pathway. A lack of Akt3 in adipocytes increases the WNK1 protein level, leading to activation of SGK1. SGK1, in turn, promotes adipogenesis by phosphorylating and inhibiting transcription factor FOXO1 and, subsequently, activating the transcription of PPARγ in adipocytes. Akt3-deficient mice have an increased number of adipocytes and, when fed a high-fat diet, display increased weight gain, white adipose tissue expansion, and impaired glucose homeostasis. Pharmacological blockade of SGK1 in high-fat diet-fed Akt3-deficient mice suppressed adipogenesis, prevented excessive weight gain and adiposity, and ameliorated metabolic parameters. Thus, Akt3/WNK1/SGK1 represents a potentially novel signaling pathway controlling the development of obesity.

The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue.

Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.

Regulation of Hepatic Triacylglycerol Metabolism by CGI-58 Does Not Require ATGL Co-activation.

Adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) are critical regulators of triacylglycerol (TAG) turnover. CGI-58 is thought to regulate TAG mobilization by stimulating the enzymatic activity of ATGL. However, it is not known whether this coactivation function of CGI-58 occurs in vivo. Moreover, the phenotype of human CGI-58 mutations suggests ATGL-independent functions. Through direct comparison of mice with single or double deficiency of CGI-58 and ATGL, we show here that CGI-58 knockdown causes hepatic steatosis in both the presence and absence of ATGL. CGI-58 also regulates hepatic diacylglycerol (DAG) and inflammation in an ATGL-independent manner. Interestingly, ATGL deficiency, but not CGI-58 deficiency, results in suppression of the hepatic and adipose de novo lipogenic program. Collectively, these findings show that CGI-58 regulates hepatic neutral lipid storage and inflammation in the genetic absence of ATGL, demonstrating that mechanisms driving TAG lipolysis in hepatocytes differ significantly from those in adipocytes.

Emerging roles of flavin monooxygenase 3 in cholesterol metabolism and atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis and associated cardiovascular disease still remain the largest cause of mortality worldwide. Several recent studies have discovered that metabolism of common nutrients by gut microbes can produce a proatherogenic metabolite called trimethylamine-N-oxide (TMAO). The goal of this review is to discuss emerging evidence that the hepatic enzyme that generates TMAO, flavin monooxygenase 3 (FMO3), plays a regulatory role in maintaining whole body cholesterol balance and atherosclerosis development. RECENT FINDINGS: Several independent studies have recently uncovered a link between either FMO3 itself or its enzymatic product TMAO with atherosclerosis and hepatic insulin resistance. These recent studies show that inhibition of FMO3 stimulates macrophage reverse cholesterol transport and protects against atherosclerosis in mice. SUMMARY: A growing body of work demonstrates that nutrients present in high-fat foods (phosphatidylcholine, choline and L-carnitine) can be metabolized by the gut microbial enzymes to generate trimethylamine, which is then further metabolized by the host enzyme FMO3 to produce proatherogenic TMAO. Here, we discuss emerging evidence that the TMAO-producing enzyme FMO3 is centrally involved in the pathogenesis of atherosclerosis by regulating cholesterol metabolism and insulin resistance, and how these new insights provide exciting new avenues for cardiovascular disease therapies.

The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance.

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Cardiomyocyte-specific deficiency of ketone body metabolism promotes accelerated pathological remodeling.

OBJECTIVE: Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined. METHODS: Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed. RESULTS: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals. CONCLUSIONS: These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.

Role of choline deficiency in the Fatty liver phenotype of mice fed a low protein, very low carbohydrate ketogenic diet.

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet - weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction - were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.

Ketone body metabolism and cardiovascular disease.

Ketone bodies are metabolized through evolutionarily conserved pathways that support bioenergetic homeostasis, particularly in brain, heart, and skeletal muscle when carbohydrates are in short supply. The metabolism of ketone bodies interfaces with the tricarboxylic acid cycle, ß-oxidation of fatty acids, de novo lipogenesis, sterol biosynthesis, glucose metabolism, the mitochondrial electron transport chain, hormonal signaling, intracellular signal transduction pathways, and the microbiome. Here we review the mechanisms through which ketone bodies are metabolized and how their signals are transmitted. We focus on the roles this metabolic pathway may play in cardiovascular disease states, the bioenergetic benefits of myocardial ketone body oxidation, and prospective interactions among ketone body metabolism, obesity, metabolic syndrome, and atherosclerosis. Ketone body metabolism is noninvasively quantifiable in humans and is responsive to nutritional interventions. Therefore, further investigation of this pathway in disease models and in humans may ultimately yield tailored diagnostic strategies and therapies for specific pathological states.

Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation.

During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT), encoded by nuclear Oxct1] is required for oxidation of ketone bodies and that germline SCOT-knockout (KO) mice die within 48 h of birth because of hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. Although transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and, while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.