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Abstract Hepatic steatosis is strongly associated with chronic liver disease and systemic metabolic disorder. Adipose lipolysis is a recognized principal source of intrahepatic fat in various metabolic disorders, including non-alcoholic fatty liver disease. We hypothesized that, in the premorbid state, hepatic de novo lipogenesis (DNL) driven by excess carbohydrates abundance might play a more significant role. We employed a novel nutritional model in sheep of two distinct carbohydrates abundances. During 4 months of the dietary treatment, lambs were monitored for metabolic and terminal liver parameters. Lambs grown on the high-calorie (HC) diet were consistently more hyperglycemic and hyperinsulinemic than lambs grown on the lower-calorie (LC) diet (P

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders. This article has an associated First Person interview with the joint first authors of the paper.

Fatty liver represents a significant metabolic pathology of excess intrahepatic fat in domestic animals and humans. Quantification of hepatic-fat content is therefore essential for diagnosis and investigation of liver and metabolic disease. However, the reproducibility of hepatic steatosis analysis is often low due to subjective and technical factors. We hypothesized that improvement in tissue-lipids extraction efficiency would contribute to the accuracy and precision of liver-fat determination. To test it, we investigated the effect of standardized tissue sonication on liver-fat quantification by the Folch method in sheep. Liver samples from grownup lambs of lean (n = 16) and fatty (n = 15) livers, and from pregnant ewes (n = 6) who died from pregnancy toxemia (PT), were used for hepatic-fat content determination with or without tissue sonication. In the grown lambs, an average hepatic-fat content of 6.6% was determined in sonicated compared to 5.1% in non-sonicated specimens (P = 0.0002). Similarly, in ewes with PT, an average of 12.5% was determined with sonication compared to 10.8% without it (P = 0.0006), and the reproducibility was higher with sonication (CV of 3.1 vs. 6.1%, respectively). Thus, tissue sonication improved the efficiency of liver-lipids extraction and was significant to the accuracy and precision of hepatic-fat determination. Enzymatic quantification of triglycerides was moderately correlated with the results obtained gravimetrically (r = 0.632, P < 0.005). The reported data provide reliable reference values for pregnancy toxemic sheep. The significant improvement in liver-fat quantification observed with the reported revised protocol is likely applicable to most mammals and humans.

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.

The necrotrophic fungus Botrytis cinerea, is considered a major cause of postharvest losses in a wide range of crops. The common fungal extracellular membrane protein (CFEM), containing a conserved eight-cysteine pattern, was found exclusively in fungi. Previous studies in phytopathogenic fungi have demonstrated the role of membrane-bound and secreted CFEM-containing proteins in different aspects of fungal virulence. However, non-G protein-coupled receptor (non-GPCR) membrane CFEM proteins have not been studied yet in phytopathogenic fungi. In the present study, we have identified a non-GPCR membrane-bound CFEM-containing protein, Bcin07g03260, in the B. cinerea genome, and generated deletion mutants, ΔCFEM-Bcin07g03260, to study its potential role in physiology and virulence. Three independent ΔCFEM-Bcin07g03260 mutants showed significantly reduced progression of a necrotic lesion on tomato (Solanum lycopersicum) leaves. Further analysis of the mutants revealed significant reduction (approximately 20–30%) in conidial germination and consequent germ tube elongation compared with the WT. Our data complements a previous study of secreted ΔCFEM1 mutants of B. cinerea that showed reduced progression of necrotic lesions on leaves, without effect on germination. Considering various functions identified for CFEM proteins in fungal virulence, our work illustrates a potential new role for a non-GPCR membrane CFEM in pathogenic fungi to control virulence in the fungus B. cinerea.

Negative energy balance (NEB) is a state of insufficient dietary-energy consumption, characterized by the breakdown of adipose fat to meet the physiological energy expenditure. Extensive NEB, as common in high-yielding transitioning ruminants, drives significant metabolic disturbance and pathologies such as pregnancy toxemia and ketosis. Strategies to minimize the severity of NEB include the use of energy-dense feed supplements, like glycerol and propylene glycol (PG), or IV glucose infusion during severe hypoglycemia. PG and glycerol have been studied mainly by oral or ruminal administration, which exposes them to substantial metabolism in the digestive system. To investigate their direct benefits to mitigating NEB, we intravenously infused them into sheep induced into NEB by feed restriction. Sixteen 5-month-old ewe lambs at NEB were IV-treated with 170 mL isotonic saline containing 15% glycerol or 15% PG. Both PG and glycerol effectively reduced hyperketonemia by 57% and 61%, and inhibited adipose lipolysis by 73.6% and 73.3%, respectively. Surprisingly, only glycerol was glucogenic (p < 0.0001) and insulinotropic (p < 0.0075), while PG was primarily utilized for production of lactate (p < 0.0001). Tissue-damage biomarkers indicated hemolytic activity for PG. This study revealed glycerol as a superior IV treatment for effective relief of NEB. Since it carries no risk of glucose overloading, glycerol IV infusion may also have clinical advantages over glucose for treatment of pregnancy toxemia and ketosis.