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

Human muscle-derived cell populations isolated by differential adhesion rates: Phenotype and contribution to skeletal muscle regeneration in Mdx/SCID mice

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells

High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders

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

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

SummaryAdipose 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

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