Description of the fasted serum metabolomic signature of lean and obese cats at maintenance and of obese cats under energy restriction.

This study aimed to investigate the serum metabolomic profile of obese and lean cats as well as obese cats before and after energy restriction for weight loss. Thirty cats, 16 obese (body condition score 8 to 9/9) and 14 lean (body condition score 4 to 5/9), were fed a veterinary weight loss food during a 4-week period of weight maintenance (L-MAINT and O-MAINT). The 16 obese cats were then energy restricted by a 60% energy intake reduction with the same food for a 10-week period (O-RESTRICT). Fasted serum metabolites were measured using nuclear magnetic resonance and direct infusion mass spectrometry after the maintenance period for L-MAINT and O-MAINT cats and after the energy restriction period for O-RESTRICT and compared between groups using a two-sided t-test. Obese cats lost 672 g ± 303 g over the 10-week restriction period, representing a weight loss rate of 0.94 ± 0.28% per week. Glycine, l-alanine, l-histidine, l-glutamine, 2-hydroxybutyrate, isobutryric acid, citric acid, creatine, and methanol were greater in O-RESTRICT compared to O-MAINT. There was a greater concentration of long-chain acylcarnitines in O-RESTRICT compared to both O-MAINT and L-MAINT, and greater total amino acids compared to O-MAINT. Glycerol and 3-hydroxybutyric acid were greater in O-MAINT compared to L-MAINT, as were several lysophosphatidylcholines. Thus, energy restriction resulted in increased dispensable amino acids in feline serum which could indicate alterations in amino acid partitioning. An increase in lipolysis was not evident, though greater circulating acylcarnitines were observed, suggesting that fatty acid oxidation rates may have been greater under calorie restriction. More research is needed to elucidate energy metabolism and substrate utilization, specifically fatty acid oxidation and methyl status, during energy restriction in strict carnivorous cats to optimize weight loss.

Dietary choline, but not L-carnitine, increases circulating lipid and lipoprotein concentrations, without affecting body composition, energy expenditure or respiratory quotient in lean and obese male cats during weight maintenance.

Introduction: Due to the involvement in one-carbon metabolism and lipid mobilization, choline and L-carnitine supplementation have been recommended to minimize hepatic lipid accumulation and support fat oxidation, respectively. This study investigated the lipotropic benefits of choline or L-carnitine supplementation in lean and obese cats maintaining body weight (BW). Methods: Lean [n = 9; body condition score (BCS): 4-5/9] and obese (n = 9; BCS: 8-9/9) adult male neutered colony cats were used in a replicated 3 x 3 complete Latin square design. Treatments included choline (378 mg/kg BW0.67), L-carnitine (200 mg/kg BW) and control (no supplement). Treatments were supplemented to the food for 6 weeks each, with a 2-week washout between treatments. Cats were fed once daily to maintenance energy requirements, and BW and BCS were assessed weekly. Fasted blood collection, indirect calorimetry, and dual-energy X-ray absorptiometry occurred at the end of each treatment period. Serum was analyzed for cholesterol (CHOL), high-density lipoprotein CHOL (HDL-C), triglycerides (TAG), non-esterified fatty acids (NEFA), glucose, creatinine (CREAT), urea, alkaline phosphatase (ALP) and alanine aminotransferase (ALT). Very low-density lipoprotein CHOL (VLDL) and low-density lipoprotein CHOL (LDL-C) were calculated. Data were analyzed using proc GLIMMIX, with group and period as random effects, and treatment, body condition, and their interaction as fixed effects, followed by a Tukey's post-hoc test when significance occurred. Results: Cats supplemented choline had lower food intake (P = 0.025). Treatment did not change BW, BCS and body composition (P > 0.05). Obese cats had greater ALP, TAG, and VLDL, and lower HDL-C compared to lean cats (P < 0.05). Choline resulted in greater CHOL, HDL-C, LDL-C and ALT (P < 0.05). L-carnitine resulted in lower CREAT (P = 0.010). Following the post-hoc test, differences between treatment means were not present for ALP (P = 0.042). No differences were found for glucose, urea or NEFA (P > 0.05). Obese cats had a lower fed respiratory quotient (RQ), regardless of treatment (P = 0.045). Treatment did not affect fed or fasted RQ and energy expenditure (P > 0.05). Discussion: Choline appeared to increase circulating lipid and lipoprotein concentrations regardless of body condition, likely through enhanced lipid mobilization and hepatic elimination. Neither dietary choline or L-carnitine altered body composition or energy metabolism in the lean or obese cats, as compared to control.

Skeletal Muscle Consequences of Phosphatidylethanolamine Synthesis Deficiency.

The maintenance of phospholipid homeostasis is increasingly being implicated in metabolic health. Phosphatidylethanolamine (PE) is the most abundant phospholipid on the inner leaflet of cellular membranes, and we have previously shown that mice with a heterozygous ablation of the PE synthesizing enzyme, Pcyt2 (Pcyt2+/-), develop obesity, insulin resistance, and NASH. Skeletal muscle is a major determinant of systemic energy metabolism, making it a key player in metabolic disease development. Both the total PE levels and the ratio of PE to other membrane lipids in skeletal muscle are implicated in insulin resistance; however, the underlying mechanisms and the role of Pcyt2 regulation in this association remain unclear. Here, we show how reduced phospholipid synthesis due to Pcyt2 deficiency causes Pcyt2+/- skeletal muscle dysfunction and metabolic abnormalities. Pcyt2+/- skeletal muscle exhibits damage and degeneration, with skeletal muscle cell vacuolization, disordered sarcomeres, mitochondria ultrastructure irregularities and paucity, inflammation, and fibrosis. There is intramuscular adipose tissue accumulation, and major disturbances in lipid metabolism with impaired FA mobilization and oxidation, elevated lipogenesis, and long-chain fatty acyl-CoA, diacylglycerol, and triacylglycerol accumulation. Pcyt2+/- skeletal muscle exhibits perturbed glucose metabolism with elevated glycogen content, impaired insulin signaling, and reduced glucose uptake. Together, this study lends insight into the critical role of PE homeostasis in skeletal muscle metabolism and health with broad implications on metabolic disease development.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

Serum metabolomic analysis of the dose-response effect of dietary choline in overweight male cats fed at maintenance energy requirements.

Choline participates in methyl group metabolism and has been recognized for its roles in lipid metabolism, hepatic health and muscle function in various species. Data regarding the impacts of choline on feline metabolic pathways are scarce. The present study investigated how choline intake affects the metabolomic profile of overweight cats fed at maintenance energy. Overweight (n = 14; body condition score:6-8/9) male adult cats were supplemented with five doses of choline in a 5x5 Latin Square design. Cats received a daily dose of choline on extruded food (3620 mg choline/kg diet) for three weeks at maintenance energy requirements (130 kcal/kgBW0.4). Doses were based on body weight (BW) and the daily recommended allowance (RA) for choline for adult cats (63 mg/kg BW0.67). Treatment groups included: Control (no additional choline, 1.2 x NRC RA, 77 mg/kg BW0.67), 2 x NRC RA (126 mg/kg BW0.67), 4 x NRC RA (252 mg/kg BW0.67), 6 x RA (378 mg/kg BW0.67), and 8 x NRC RA (504 mg/kg BW0.67). Serum was collected after an overnight fast at the end of each treatment period and analyzed for metabolomic parameters through nuclear magnetic resonance (NMR) spectroscopy and direct infusion mass spectrometry (DI-MS). Data were analyzed using GLIMMIX, with group and period as random effects, and dose as the fixed effect. Choline up to 8 x NRC RA was well-tolerated. Choline at 6 and 8 x NRC RA resulted in greater concentrations of amino acids and one-carbon metabolites (P < 0.05) betaine, dimethylglycine and methionine. Choline at 6 x NRC RA also resulted in greater phosphatidylcholine and sphingomyelin concentrations (P < 0.05). Supplemental dietary choline may be beneficial for maintaining hepatic health in overweight cats, as it may increase hepatic fat mobilization and methyl donor status. Choline may also improve lean muscle mass in cats. More research is needed to quantify how choline impacts body composition.

ER stress-associated transcription factor CREB3 is essential for normal Ca2+, ATP, and ROS homeostasis.

In mammalian cells, mitochondrial respiration produces reactive oxygen species (ROS) such as superoxide (O2-), which is then converted by the SOD1 enzyme into hydrogen peroxide (H2O2), the predominant form of cytosolic ROS. ROS at high levels can be toxic, but below this threshold are important for physiological processes acting as a second messenger similar to Ca2+. Mitochondrial Ca2+ influx from the ER increases ATP and ROS production, while ATP and ROS can regulate Ca2+ homeostasis, leading to an intricate interplay between Ca2+, ROS, and ATP synthesis. The Unfolded Protein Response (UPR) proteins ATF6α and XBP1 contribute to protection from oxidative stress through upregulation of Sod1 and Catalase genes. Here, UPR-associated protein CREB3 is shown to play a role in balancing Ca2+, ROS, and ATP homeostasis. Creb3-deficient mouse embryonic fibroblast cells (MEF-/-) were susceptible to H2O2-induced oxidative stress while having a functioning antioxidant gene expression response compared to MEF+/+. MEF-/- cells also contained elevated basal cytosolic ROS levels, which was attributed to drastically increased basal mitochondrial respiration and spare respiratory capacity relative to MEF+/+. MEF-/- cells also showed an increase in endoplasmic reticulum Ca2+ release and mitochondrial Ca2+ levels hinting at a potential cause for MEF-/- cell mitochondrial dysfunction. These results suggest that CREB3 is essential for maintaining proper Ca2+, ATP, and ROS homeostasis in mammalian cells.

Circulating direct infusion MS and NMR metabolomic profiles of post-gonadectomy kittens with or without additional dietary choline supplementation.

Choline is beneficial for energy metabolism and growth in various species. Choline may work similarly in kittens at risk of obesity. Direct infusion MS (Di-MS) and NMR spectroscopy were used to investigate the metabolomic signatures of kittens supplemented with or without additional dietary choline for 12 weeks. Fifteen intact male kittens consumed a base diet (3310 mg choline/kg DM) to their daily metabolisable energy requirement (DER) over an 11-week acclimation. Kittens were gonadectomised and assigned, based on body weight, to the base diet (CONTROL, n 7) or the base diet with 300 mg/kgBW0·75 additional choline as choline chloride (CHOLINE, n 8) and offered three times their individual energy requirement divided into three meals. At weeks -1 and 12, fasted blood was sampled and serum analysed for 130 metabolites via Di-MS and fifty-one metabolites via NMR spectroscopy. Changes in fasted metabolites were assessed using a repeated-measures GLIMMIX procedure with time and group as fixed effects, and time as a repeated measure. Metabolites of one-carbon metabolism and lipids increased, and medium-chain acyl carnitines decreased from week -1 to 12 for CHOLINE (P < 0·05), but not CONTROL (P > 0·05). Increases in amino acid, biogenic amine and organic compound concentrations were observed in both groups (P < 0·05). The results suggest impacts of dietary choline at greater intakes than currently recommended on one-carbon metabolism and fatty acid oxidation, and these may promote healthy growth in post-gonadectomy kittens.

Bilayer Forming Phospholipids as Targets for Cancer Therapy.

Phospholipids represent a crucial component for the structure of cell membranes. Phosphatidylcholine and phosphatidylethanolamine are two phospholipids that comprise the majority of cell membranes. De novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine occurs via the Kennedy pathway, and perturbations in the regulation of this pathway are linked to a variety of human diseases, including cancer. Altered phosphatidylcholine and phosphatidylethanolamine membrane content, phospholipid metabolite levels, and fatty acid profiles are frequently identified as hallmarks of cancer development and progression. This review summarizes the research on how phospholipid metabolism changes over oncogenic transformation, and how phospholipid profiling can differentiate between human cancer and healthy tissues, with a focus on colorectal cancer, breast cancer, and non-small cell lung cancer. The potential for phospholipids to serve as biomarkers for diagnostics, or as anticancer therapy targets, is also discussed.

Transcription factor CREB3 is a potent regulator of high-fat diet-induced obesity and energy metabolism.

BACKGROUND: The endoplasmic reticulum senses alterations to cellular homeostasis that activates the unfolded protein response (UPR). UPR proteins are known to aid in regulating glucose and lipid metabolism. CREB3 is a UPR-associated transcription factor whose potential role in regulating energy metabolism remains unclear. METHODS: Eight-week-old wild-type (WT) and Creb3+/- mice were placed on control and high-fat diets (HFD) for 8 weeks, and metabolic phenotypes characterized by weekly weighing, indirect calorimetry, body composition scans, glucose tolerance tests, plasma analysis, tissue lipid quantifications and gene/protein expression analysis. RESULTS: HFD weight gain in Creb3+/- males was reduced by 34% (p < 0.0001) and females by 39.5% (p = 0.014) from their WT counterparts. No differences were found in HFD food intake or total fecal lipids between genotypes. Creb3+/- mice had increased energy expenditure and respiratory exchange ratios (p = 0.002) relative to WT. Creb3+/- mice had significant reductions in absolute fat and lean tissue, while Creb3+/- females had significant reductions in body fat% and increased lean% composition (p < 0.0001) compared to WT females. Creb3+/- mice were protected from HFD-induced basal hyperglycemia (males p < 0.0001; females p = 0.0181). Creb3+/- males resisted HFD-induced hepatic lipid accumulation (p = 0.025) and glucose intolerance compared to WT (p < 0.0001) while Creb3+/- females were protected from lipid accumulation in skeletal muscle (p = 0.001). Despite the metabolic differences of Creb3+/- mice on HFD, lipid plasma profiles did not significantly differ from WT. Fasted Creb3+/- mice additionally revealed upregulation of hepatic energy expenditure and gluconeogenic genes such as Pgc-1a and Gr (glucocorticoid receptor) (p < 0.05), respectively. CONCLUSIONS: Reduced expression of CREB3 increased energy expenditure and the respiratory exchange ratio, and protected mice from HFD-induced weight gain, basal hyperglycemia, and sex-specific tissue lipid accumulation. We postulate that CREB3 is a novel key regulator of diet-induced obesity and energy metabolism that warrants further investigation as a potential therapeutic target in metabolic disorders.

Dose-response relationship between dietary choline and serum lipid profile, energy expenditure, and respiratory quotient in overweight adult cats fed at maintenance energy requirements.

Choline is an essential nutrient linked to hepatic lipid metabolism in many animal species, including cats. The current study investigated the serum lipid profiles, serum liver enzymes, respiratory quotients, and energy expenditures of overweight cats fed maintenance diets, in response to graded doses of supplemental dietary choline. Overweight (body condition score [BCS]: ≥6/9) adult male neutered cats (n = 14) were supplemented with five choline chloride doses for 3-wk periods, in a 5 × 5 Latin square design. Doses were based on individual body weight (BW) and the daily recommended allowance (RA) for choline (63 mg/kg BW0.67) according to the National Research Council. Doses were control (no additional choline: 1.2 × RA, 77 mg/kg BW0.67), 2 × RA (126 mg/kg BW0.67), 4 × RA (252 mg/kg BW0.67), 6 × RA (378 mg/kg BW0.67), and 8 × RA (504 mg/kg BW0.67). Choline was top-dressed over the commercial extruded cat food (3,620 mg choline/kg diet), fed once a day at maintenance energy requirements (130 kcal/kgBW0.4). Body weight and BCS were assessed weekly. Fasted blood samples were taken and indirect calorimetry was performed at the end of each 3-wk period. Serum was analyzed for cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, non-esterified fatty acids, glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), and alanine aminotransferase. Very low-density lipoprotein cholesterol (VLDL) and low-density lipoprotein cholesterol were calculated. Data were analyzed via SAS using proc GLIMMIX, with group and period as the random effects, and treatment as the fixed effect. Statistical significance was considered at P < 0.05. Body weight and BCS did not change (P > 0.05). Serum cholesterol, HDL-C, triglycerides, and VLDL increased with 6 × RA (P < 0.05). Serum ALP decreased with 8 × RA (P = 0.004). Choline at 4 × and 6 × RA decreased serum BUN (P = 0.006). Fed or fasted respiratory quotient and energy expenditure did not differ among dietary choline doses (P > 0.05). These results suggest that dietary choline supplementation at 6 × RA may increase hepatic fat mobilization through increased lipoprotein transport and beneficially support hepatic health in overweight cats. Future studies that combine these results with existing knowledge of feline weight loss and hepatic lipidosis are warranted.

Choline is an essential nutrient important for lipid metabolism in the liver of many mammals. In the present study, fourteen overweight cats had their commercial extruded cat food top-dressed with different amounts of choline chloride supplement. The amounts of choline were based on the individual body weights and the published recommended allowance (RA) for dietary choline intake in adult cats. The choline treatments were control (no additional choline added, 1.2 × RA), 2 × RA, 4 × RA, 6 × RA, and 8 × RA. The cats were separated into five groups. Each group received the choline treatments once daily for 3 wk per treatment. Choline at 6 × RA increased serum cholesterol, triglycerides, and lipoproteins. There were no significant differences in respiratory quotient or energy expenditure with choline intake. The results of this study suggest that choline at 6 × RA increases the transport of lipids from the liver. This may be beneficial in supporting liver health in overweight cats. Future studies should investigate supplementing choline to cats undergoing weight loss and those at risk of developing fatty liver.

Dietary choline in gonadectomized kittens improved food intake and body composition but not satiety, serum lipids, or energy expenditure.

Gonadectomy is a major risk factor for feline obesity. The lipotropic effects of choline have demonstrated benefits for growth and carcass composition in livestock. The consumption of supplemental choline on body weight (BW), body composition, lipid metabolism, energy expenditure (EE), and serum satiety hormones were evaluated in 15 gonadectomized male kittens. Kittens were offered a base diet formulated for growth (3310mg choline/kg dry matter [DM]) to daily energy requirements (DER) over an 11-week acclimation. Post-gonadectomy, kittens were assigned to a base diet (CONTROL, n = 7) or choline group (base diet with additional choline at 300mg/kg BW0.75 as a top dress) (CHOLINE, n = 8). For 12-weeks post-neuter, kittens were offered three times their DER over three meals to mimic ad libitum feeding. At week -1 and 12, body composition was assessed using dual energy x-ray absorptiometry (DXA), 24-hour indirect calorimetry was performed for EE and respiratory quotients (RQ), and fasted serum samples were analyzed for lipid compounds and satiety hormones. Daily food intake (FI) and weekly BW were measured. Data was analyzed as a repeated measures of variance (ANCOVA) using the GLIMMIX procedure with time and group as fixed effects. CHOLINE had lower mean daily FI and lower rates of BW accretion (P<0.05) in contrast to CONTROL. All absolute body composition data increased over time for both groups, with lower increases in total tissue mass (P = 0.031) and fat mass (P = 0.005) in CHOLINE. Serum satiety hormones and lipid compounds did not differ (P>0.05) between groups, but both groups experienced a decrease in low-density lipoproteins and increase in high-density lipoproteins (P<0.05). Primary substrate utilization showed lipid use when fasted and use of protein or mixed macronutrients in the fed state. Fed state EE decreased post-gonadectomy (P = 0.004), however, CHOLINE did not affect total EE or RQ. These results suggest that supplemental dietary choline reduces FI, BW, and fat mass and may help to reduce the propensity of weight gain and subsequent obesity in gonadectomized feline populations.

Pcyt2 deficiency causes age-dependant development of nonalcoholic steatohepatitis and insulin resistance that could be attenuated with phosphoethanolamine.

The mechanisms of NASH development in the context of age and genetics are not fully elucidated. This study investigates the age-dependent liver defects during NASH development in mice with heterozygous deletion of Pcyt2 (Pcyt2+/-), the rate limiting enzyme in phosphatidylethanolamine (PE) synthesis. Further, the therapeutic potential of Pcyt2 substrate, phosphoethanolamine (PEtn), is examined. Pcyt2+/- were investigated at 2 and 6-8 months (mo) of age and in addition, 6-mo old Pcyt2+/- with developed NASH were supplemented with PEtn for 8 weeks and glucose and fatty acid metabolism, insulin signaling, and inflammation were examined. Heterozygous ablation of Pcyt2 causes changes in liver metabolic regulators from young age, prior to the development of liver disease which does not occur until adulthood. Only older Pcyt2+/- experiences perturbed glucose and fatty acid metabolism. Older Pcyt2+/- liver develops NASH characterized by increased glucose production, accumulation of TAG and glycogen, and increased inflammation. Supplementation with PEtn reverses Pcyt2+/- steatosis, inflammation, and other aspects of NASH, showing that was directly caused by Pcyt2 deficiency. Pcyt2 deficiency is a novel mechanism of metabolic dysregulation due to reduced membrane ethanolamine phospholipid synthesis, and the metabolite PEtn offers therapeutic potential for NASH reversion.

Phosphatidylethanolamine homeostasis under conditions of impaired CDP-ethanolamine pathway or phosphatidylserine decarboxylation.

Phosphatidylethanolamine is the major inner-membrane lipid in the plasma and mitochondrial membranes. It is synthesized in the endoplasmic reticulum from ethanolamine and diacylglycerol (DAG) by the CDP-ethanolamine pathway and from phosphatidylserine by decarboxylation in the mitochondria. Recently, multiple genetic disorders that impact these pathways have been identified, including hereditary spastic paraplegia 81 and 82, Liberfarb syndrome, and a new type of childhood-onset neurodegeneration-CONATOC. Individuals with these diseases suffer from multisystem disorders mainly affecting neuronal function. This indicates the importance of maintaining proper phospholipid homeostasis when major biosynthetic pathways are impaired. This study summarizes the current knowledge of phosphatidylethanolamine metabolism in order to identify areas of future research that might lead to the development of treatment options.

Serum Lipid, Amino Acid and Acylcarnitine Profiles of Obese Cats Supplemented with Dietary Choline and Fed to Maintenance Energy Requirements.

Obesity is a health concern for domestic cats. Obesity and severe energy restriction predispose cats to feline hepatic lipidosis. As choline is linked to lipid metabolism, we hypothesized that dietary choline supplementation would assist in reducing hepatic fat through increased lipoprotein transport and fatty acid oxidation. Twelve obese cats (body condition score [BCS] ≥ 8/9) were split into two groups. Cats were fed a control (n = 6; 4587 mg choline/kg dry matter [DM]) or a high choline diet (n = 6; 18,957 mg choline/kg DM) for 5 weeks, for adult maintenance. On days 0 and 35, fasted blood was collected, and the body composition was assessed. Serum lipoprotein and biochemistry profiles, plasma amino acids and plasma acylcarnitines were analyzed. The body weight, BCS and body composition were unaffected (p > 0.05). Choline increased the serum cholesterol, triacylglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol and plasma methionine (p < 0.05) and decreased the serum blood urea nitrogen and alkaline phosphatase (p < 0.05). Choline also reduced the plasma acylcarnitine to free carnitine ratio (p = 0.006). Choline may assist in eliminating hepatic fat through increased fat mobilization and enhanced methionine recycling.

Addition of dietary methionine but not dietary taurine or methyl donors/receivers to a grain-free diet increases postprandial homocysteine concentrations in adult dogs.

Grain-based ingredients are replaced in part by pulse ingredients in grain-free pet foods. Pulse ingredients are lower in methionine and cysteine, amino acid (AA) precursors to taurine synthesis in dogs. Although recent work has investigated plasma and whole blood taurine concentrations when feeding grain-free diets, supplementation of a grain-free diet with various nutrients involved in the biosynthesis of taurine has not been evaluated. This study aimed to investigate the effects of supplementing a complete grain-free dry dog food with either methionine (MET), taurine (TAU), or methyl donors (choline) and methyl receivers (creatine and carnitine; CCC) on postprandial AA concentrations. Eight healthy Beagle dogs were fed one of the three treatments or the control grain-free diet (CON) for 7 d in a 4 × 4 Latin square design. On day 7, cephalic catheters were placed and one fasted sample (0 min) and a series of nine post-meal blood samples were collected at 15, 30, 60, 90, 120, 180, 240, 300, and 360 min. Data were analyzed as repeated measures using the PROC GLIMMIX function in SAS (Version 9.4). Dogs fed MET had greater plasma and whole blood methionine concentrations from 30 to 360 min after a meal (P < 0.0001) and greater plasma homocysteine concentrations from 60 to 360 min after a meal (P < 0.0001) compared with dogs fed CON, TAU, and CCC. Dogs fed TAU had greater plasma taurine concentrations over time compared with dogs fed CON (P = 0.02) but were not different than dogs fed MET and CCC (P > 0.05). In addition, most AAs remained significantly elevated at 6 h post-meal compared with fasted samples across all treatments. Supplementation of creatine, carnitine, and choline in grain-free diets may play a role in sparing the methionine requirement without increasing homocysteine concentrations. Supplementing these nutrients could also aid in the treatment of disease that causes metabolic or oxidative stress, including cardiac disease in dogs, but future research is required.

Choline transporter-like proteins 1 and 2 are newly identified plasma membrane and mitochondrial ethanolamine transporters.

The membrane phospholipids phosphatidylcholine and phosphatidylethanolamine (PE) are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. Although multiple transport systems have been established for choline, ethanolamine transport is poorly characterized and there is no single protein assigned a transport function for ethanolamine. The solute carriers 44A (SLC44A) known as choline transporter-like proteins-1 and -2 (CTL1 and CTL2) are choline transporter at the plasma membrane and mitochondria. We report a novel function of CTL1 and CTL2 in ethanolamine transport. Using the lack or the gain of gene function in combination with specific antibodies and transport inhibitors we established two distinct ethanolamine transport systems of a high affinity, mediated by CTL1, and of a low affinity, mediated by CTL2. Both transporters are Na+-independent ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations in the CTL1 gene (M1= SLC44A1ΔAsp517 and M2= SLC44A1ΔSer126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 in M2 cells reduced the ethanolamine transport, the flux through the CDP-ethanolamine Kennedy pathway, and PE synthesis. In contrast, overexpression of CTL1 in M2 cells improved ethanolamine transport and PE synthesis. These data firmly establish that CTL1 and CTL2 are the first identified ethanolamine transporters in whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the Kennedy pathway and intracellular redistribution of ethanolamine.

Dietary intake of amino acids and vitamins compared to NRC requirements in obese cats undergoing energy restriction for weight loss.

BACKGROUND: This study aimed to determine if obese cats undergoing energy restriction for weight loss would meet the National Research Council's (NRC) indispensable amino acid and vitamin recommendations when fed a purpose-formulated diet. Thirty cats were placed into one of two groups; obese (BCS 8 to 9/9; n = 16) and lean (BCS 4 to 5/9; n = 14) and included in a non-randomized retrospective observational study. Cats were fed a veterinary weight loss food during a 4-week period of weight maintenance. Obese cats (O-MAINT) refers to obese cats during this period, L-MAINT to lean cats. After this initial 4-week period, the lean cats finished the study at this time and the 16 obese cats continued and were energy restricted for a 10-week period (O-RESTRICT). Analysis for dietary concentrations of indispensable amino acid and vitamin contents were performed. Daily food intakes were used to determine minimum, maximum and average daily intakes of individual nutrients for all three groups and compared against NRC 2006 minimum requirements (MR), adequate intakes (AI) and recommended allowances (RA) for adult cats. RESULTS: Over 10 weeks, O-RESTRICT cats lost 672 g ± 303 g, representing a weight loss rate of 0.94 ± 0.28% per week. Daily intake of the majority of indispensable amino acids and vitamins was greater than the NRC 2006 recommended allowance (RA per kg ideal body weight ^0.67), except for arginine, choline, crude protein, phenylalanine plus tyrosine and threonine. All O-RESTRICT cats had minimum, average, and maximum arginine intakes less than the NRC AI. Minimum daily intake of choline was below NRC RA for all O-RESTRICT cats and below NRC MR for two. All, except one, O-RESTRICT cats had a maximum and average choline intake below RA. CONCLUSIONS: All cats remained clinically healthy and showed no clinical signs of deficiency. Dietary choline and arginine requirements of obese cats as well as health risks associated with low dietary intake during energy restriction warrant further investigation.

Dietary polyacetylene falcarinol upregulated intestinal heme oxygenase-1 and modified plasma cytokine profile in late phase lipopolysaccharide-induced acute inflammation in CB57BL/6 mice.

Unlike polyphenols, which are widely available in the diet, polyacetylenes are available only from the Apiaceae family vegetables, including carrot, parsnip, fennel, celery, and many herbs (parsley, lovage, etc). The aim of this study was to investigate the hypothesis that polyacetylene falcarinol (FA) reduces intestinal inflammation and examine its similarity of effect to isothiocyanate R-sulforaphane during the late phase of acute inflammation. To this end, 3-month-old male CB57BL/6 mice were fed twice daily for 1 week with 5 mg/kg of FA, sulforaphane, or vehicle before receiving an intraperitoneal injection of 5 mg/kg endotoxin (lipopolysaccharide [LPS]) to induce modest acute inflammation. The expression of intestinal and hepatic heme oxygenase-1 at the mRNA and protein levels, circulating cytokines, as well as intestinal and mesenteric n-6 and n-3 fatty acid lipid mediators was compared 24 hours after LPS administration to examine its effects on the late phase of inflammation. Intestinal nuclear factor (erythroid-derived 2)-like 2 target enzyme heme oxygenase-1 was upregulated 8.42-fold at the mRNA level and 10.7-fold at the protein level by FA-supplemented diet. However, the FA-supplemented diet produced a unique type-2 plasma cytokine skew after LPS treatment. Plasma cytokines interleukin (IL)-4, IL-13, IL-9, and IL-10 were upregulated, reflecting the cytokine profile of reduced type 1 inflammation. A detailed lipidomic analysis of n-6 and n-3 fatty acid pro- and anti-inflammatory pathways in the mesentery and intestinal mucosa showed that FA diet was more similar to the control groups than to other LPS treated groups. In this study, we demonstrated that FA-supplemented diet produced a unique immunomodulatory effect not observed with sulforaphane in late phases of inflammation. These results support the hypothesis that FA may have role as a dietary immunosuppressant in patients with inflammatory gastrointestinal as well as other inflammatory disorders that may be alleviated by increasing consumption of carrot or other FA-containing food sources.