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

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

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

Hepatic TAG secretion in mice fed very high fat, low protein, very low carbohydrate diets.

<p>Mice from each dietary group (6 weeks each diet) were fasted for 18 h. Blood was collected prior to (0 h) and after intraperitoneal injection of tyloxapol. (<b>A</b>) Serum TAG concentration and (<b>B</b>) areas under the curve (AUC), <i>n</i>=5 mice/group. Data are presented as means±SEM. <i>a</i>, significantly different compared to chow, <i>p</i>≤0.001 by 1-way ANOVA with Tukey’s post hoc testing.</p

Metabolic parameters of mice maintained on very high fat, low protein, very low carbohydrate diets.

<p>(<b>A</b>) Body weight responses to 6 weeks of maintenance on the experimental paste diets, compared to chow controls. n=10-15 mice/group. a, p≤0.001; <i>b</i>, p≤0.001; <i>c</i>, p≤0.01. See end of this legend for description of the individual comparisons depicted by each letter. (<b>B</b>) Caloric consumption of diet, normalized per mouse, between weeks two and four of the 6 weeks of maintenance on the diets. n=5-10 mice/group. a, p≤0.001; <i>c</i>, p≤0.05. (<b>C</b>) Caloric consumption of diet, normalized per gram of body weight (BW). n=5-10 mice/group. a, p≤0.001; b, p≤0.01. (<b>D</b>) Percent adiposity after 6 weeks on each of the diets. n=5-10 mice/group. a, p≤0.01; <i>c</i>, p≤0.05. For all panels, data are presented as means±SEM. <i>a</i>, significantly different compared to chow; <i>b</i>, significant difference attributable to decrease in protein content (from 10% kcal to 5% kcal) at a fixed choline content; <i>c</i>, significant difference attributable to restriction in choline content (from 5.3 g/kg to 0.3 g/kg) at a fixed protein content; by 1-way ANOVA with Tukey’s post hoc testing.</p

Intrahepatic triglyceride content and hepatic histopathology in mice fed very high fat, low protein, very low carbohydrate diets.

<p>Hepatic histology in mice maintained for 6 weeks on (<b>A</b>–<b>C</b>) standard chow; (<b>D</b>–<b>F</b>) LP/C⁺ diet, which caused very small amounts of mixed large and small droplet steatosis in hepatocytes restricted to zone 2 (a representative example of zone 2 is displayed in panel <b>F</b>), and no inflammation; (<b>G</b>–<b>I</b>) LP/C^- diet, which caused mixed large and small droplet macrovesicular steatosis in a zone 2 distribution (a representative example of zone 2 is displayed in panel <b>I</b>); (<b>J</b>–<b>L</b>) VLP/C⁺ diet, which exhibited small lipid droplets only at higher power; (<b>M</b>–<b>O</b>) VLP/C^- diet, which caused diffuse steatosis that is predominantly small and microvesicular with some macrovesicular droplets. Numerous clusters of inflammatory cells, some of which are likely associated with necrotic hepatocytes, were observed. Livers of mice fed both VLP/C⁺ (<b>P</b>) and VLP/C^- (<b>Q</b>) exhibit inflammatory foci (arrows). (<b>R</b>) Only in livers from VLP/C^--fed were mitotic figures observed (arrow). Scale bars: (<b>A, B, D, E, G, H, J, K, M, N</b>, lower power images taken with standard light microscopy, original magnification at 10X or 20X), 100 μm; (<b>C, F, I, L, O</b>, higher power images taken with confocal microscopy, original magnification at 80X), 10 μm; (<b>P, Q, R</b>, medium power images taken with standard light microscopy, original magnification at 40X), 50 μm.</p

Hepatic macrophage density in mice fed very high fat, low protein, very low carbohydrate diets.

<p>(<b>A</b>) Confocal images of F4/80⁺ macrophages (scale bars, 50 μm) and (<b>B</b>) quantification of F4/80⁺ macrophages normalized to the number of DAPI-stained nuclei from liver sections of mice maintained on the indicated diets for 6 weeks. Data are presented as means±SEM. n=3 mice/group with n=3 20X fields quantified per section/mouse.</p

Respiration studies of hepatic mitochondria isolated from mice fed very low protein and carbohydrate, very high fat diets.

<p>(<b>A</b>) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F₁F₀-ATPase independent condition (state 4), and uncoupled condition in hepatic mitochondria isolated from chow-fed, VLP/C⁺-fed, and VLP/C^--fed (for 6 weeks) mice using palmitoyl-L-carnitine and malate as substrates. <i>n</i>=4 mice/group. (<b>B</b>) Relative respiratory ratios of basal leak (state 2/state 3), respiratory control (RCR, state 3/state 4), and coupling control (CCR, state 4/uncoupled), derived from panel A. (<b>C</b>) Respiration rates in states 2-4 and while uncoupled in hepatic mitochondria isolated from chow-, VLP/C⁺, and VLP/C^--fed mice that respired using the Complex II-electron donor substrate succinate in the presence of rotenone (Complex I activity inhibitor). n=9 mice/group. (<b>D</b>) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel C. (<b>E</b>) Respiration rates in states 2-4 in hepatic mitochondria isolated from chow-, VLP/C⁺, and VLP/C^--fed mice that respired using the Complex III-donor substrate duroquinol plus rotenone. n=9 mice/group. (<b>F</b>) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel E. (<b>G</b>) Respiration rates in states 2-4 in hepatic mitochondria isolated from chow-, VLP/C⁺, and VLP/C^--fed mice that respired using the Complex IV-donor substrate combination TMPD/ascorbate, plus rotenone. n=9 mice/group. (<b>H</b>) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel G. Data are presented as means±SEM. *<i>p</i>≤0.05; **<i>p</i>≤0.01 by 1-way ANOVA with Tukey’s post hoc testing.</p