Hepatic oleate regulates one-carbon metabolism during high carbohydrate feeding.

Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.

Hepatic Oleate Regulates Insulin-like Growth Factor-Binding Protein 1 Partially through the mTORC1-FGF21 Axis during High-Carbohydrate Feeding.

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the rate-liming step of monounsaturated fatty acid biosynthesis and is a key regulator of systemic glucose metabolism. Mice harboring either a global (GKO) or liver-specific deletion (LKO) of Scd1 display enhanced insulin signaling and whole-body glucose uptake. Additionally, GKO and LKO mice are protected from high-carbohydrate diet-induced obesity. Given that high-carbohydrate diets can lead to chronic metabolic diseases such as obesity, diabetes, and hepatic steatosis, it is critical to understand how Scd1 deficiency confers metabolically beneficial phenotypes. Here we show that insulin-like growth factor-binding protein 1 (IGFBP1), a hepatokine that has been reported to enhance insulin signaling, is significantly elevated in the liver and plasma of GKO and LKO mice fed a low-fat high-carbohydrate diet. We also observed that the expression of hepatic Igfbp1 is regulated by oleic acid (18:1n9), a product of SCD1, through the mTORC1-FGF21 axis both in vivo and in vitro.

Hepatic stearoyl-CoA desaturase-1 deficiency induces fibrosis and hepatocellular carcinoma-related gene activation under a high carbohydrate low fat diet.

Stearoyl-CoA desaturase-1 (SCD1) is a pivotal enzyme in lipogenesis, which catalyzes the synthesis of monounsaturated fatty acids (MUFA) from saturated fatty acids, whose ablation downregulates lipid synthesis, preventing steatosis and obesity. Yet deletion of SCD1 promotes hepatic inflammation and endoplasmic reticulum stress, raising the question of whether hepatic SCD1 deficiency promotes further liver damage, including fibrosis. To delineate whether SCD1 deficiency predisposes the liver to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), we employed in vivo SCD1 deficient global and liver-specific mouse models fed a high carbohydrate low-fat diet and in vitro established AML12 mouse cells. The absence of liver SCD1 remarkably increased the saturation of liver lipid species, as indicated by lipidomic analysis, and led to hepatic fibrosis. Consistently, SCD1 deficiency promoted hepatic gene expression related to fibrosis, cirrhosis, and HCC. Deletion of SCD1 increased the circulating levels of Osteopontin, known to be increased in fibrosis, and alpha-fetoprotein, often used as an early marker and a prognostic marker for patients with HCC. De novo lipogenesis or dietary supplementation of oleate, an SCD1-generated MUFA, restored the gene expression related to fibrosis, cirrhosis, and HCC. Although SCD1 deficient mice are protected against obesity and fatty liver, our results show that MUFA deprivation results in liver injury, including fibrosis, thus providing novel insights between MUFA insufficiency and pathways leading to fibrosis, cirrhosis, and HCC under lean non-steatotic conditions.

Diet Preference, Feed Efficiency and Expression of the Sodium-Dependent Glucose Transporter Isoform 1 and Sweet Taste Receptors in the Jejunum of Lambs Supplemented with Different Flavours.

This study investigated the effect of dietary flavour supplements on the preference, feed efficiency and expression of the sweet taste receptor family 1 members 2 and 3 (T1R2 + T1R3), and sodium-glucose linked transporter 1 (SGLT1) genes in the lambs' small intestines. Eight, five-month-old, Israeli crossbred Assaf lambs were offered 16 different non-nutritive commercial flavours in rolled barley and ground corn. Capsicum and sucram were the most preferred non-aroma flavours (p = 0.020), while milky (p < 0.001) was the most preferred powder-aroma flavour. For the metabolic and relative gene expression study, eight lambs were randomly assigned to either sucram, capsicum, a mix containing sucram and capsicum at 1:1 ratio or no flavour for control in a 4 × 2 cross-over design. The total collection of urine (females only), faeces and refusals was carried out, and T1R2, T1R3 and SGLT1 relative gene expression evaluated from the proximal jejunum biopsies. Flavour had no significant effect on the feed intake (p = 0.934), but capsicum increased the average daily weight gain per metabolic body weight (p = 0.049). The T1R3 gene was expressed highest in the mix treatment (1.7; p = 0.005). Collectively, our findings indicate that flavours can be used to motivate feed acceptance and improve the weight gain in lambs.

Metabolic Effects of Vitamin B1 Therapy under Overnutrition and Undernutrition Conditions in Sheep.

As a precursor for a universal metabolic coenzyme, vitamin B1, also known as thiamine, is a vital nutrient in all living organisms. We previously found that high-dose thiamine therapy prevents overnutrition-induced hepatic steatosis in sheep by enhancing oxidative catabolism. Based on this capacity, we hypothesized that thiamine might also reduce whole-body fat and weight. To test it, we investigated the effects of high-dose thiamine treatment in sheep under overnutrition and calorie-restricted undernutrition to respectively induce positive energy balance (PEB) and negative energy balance (NEB). Eighteen mature ewes were randomly assigned to three treatment groups (n = 6 each). The control group (CG) was administered daily with subcutaneous saline, whereas the T5 and T10 groups were administered daily with equivoque of saline containing 5 mg/kg and 10 mg/kg of thiamine, respectively. Bodyweight and blood biochemistry were measured twice a week for a period of 22 days under PEB and for a consecutive 30 days under NEB. Surprisingly, despite the strong effect of thiamine on liver fat, no effect on body weight or blood glucose was detectable. Thiamine did, however, increase plasma concentration of non-esterified fatty acids (NEFA) during NEB (575.5 ± 26.7, 657.6 ± 29.9 and 704.9 ± 26.1 µEqL-1 for CG, T5, and T10, respectively: p < 0.05), thereby favoring utilization of fatty acids versus carbohydrates as a source of energy. Thiamine increased serum creatinine concentrations (p < 0.05), which paralleled a trending increase in urea (p = 0.09). This may indicate an increase in muscle metabolism by thiamine. Reduction of fat content by thiamine appears more specific to the liver than to adipose tissue. Additional studies are needed to evaluate the potential implications of high-dose vitamin B1 therapy in muscle metabolism.

High-dose vitamin B1 therapy prevents the development of experimental fatty liver driven by overnutrition.

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.

Hyperglycemia-stimulating diet induces liver steatosis in sheep.

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 < 0.0001). As a result, the HC lambs developed systemic- (HOMA-IR of 7.3 vs. 3.1; P < 0.0001), and adipose- (ADIPO-IR of 342.7 vs. 74.4; P < 0.0001) insulin resistance, significant adiposity (P < 0.0001), and higher plasma triglycerides (P < 0.05). Circulating leukocytes in the HC lambs had higher mRNA expression levels of the proinflammatory markers CCL2 (P < 0.01) and TNF-alpha (P < 0.04), and IL1B trended higher (P < 0.1). Remarkably, lambs on the HC diet developed substantial liver steatosis (mean fat content of 8.1 vs. 5.3% in the LC group; P < 0.0001) with a higher histological steatosis score (2.1 vs. 0.4; P < 0.0002). Hepatic steatosis was most-strongly associated with blood glucose and insulin levels but negatively correlated with circulating fatty acids-indicating a more significant contribution from hepatic DNL than from adipose lipolysis. Sheep may prove an attractive large-animal model of fatty liver and metabolic comorbidities resulting from excess carbohydrate-based energy early in life.

Improved Folch Method for Liver-Fat Quantification.

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.

Intravenous Infusions of Glycerol Versus Propylene Glycol for the Regulation of Negative Energy Balance in Sheep: A Randomized Trial.

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.