General Control Nonderepressible 2 (GCN2) Kinase Protects Oligodendrocytes and White Matter during Branched-Chain Amino Acid Deficiency in Mice

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk−/− and GBDK pups. Brains from Bdk−/− pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies

GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment

The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone fibroblast growth factor 21 (FGF21) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF21 and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF21 and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis

Regulation of carbohydrate metabolism by exogenous glucagon in lactating cows

Forty early lactation Holstein cows were assigned to four groups of a 2 x 2 factorial design with two nutritional backgrounds (normal and ketosis-susceptible) and two levels of glucagon (0 and 10 mg/d from 21 to 35 days postpartum) to study the regulation of glucose metabolism. In normal cows, plasma glucagon and glucose concentrations increased by about 6-fold and 10 mg/dl, respectively, during glucagon infusions, but plasma nonesterified fatty acids, [beta]-hydroxybutyrate, and urea nitrogen did not changed. Liver glycogen decreased at d 2 of glucagon infusions, but it was restored by d 7 and increased to 169% of baseline at 3 d after cessation of infusions. Milk and milk protein yield, but not milk lactose yield, decreased during glucagon infusions. Concentrations of phosphoenolpyruvate carboxykinase (PEPCK) mRNA decreased during wk 1 of glucagon infusions, when endogenous insulin secretion increased. In susceptible cows, which had fatty liver and less plasma insulin, glucagon infusions increased plasma glucagon and glucose about 8-fold and 17 mg/dl, respectively, but affected neither PEPCK mRNA nor insulin. Concentrations of PEPCK mRNA were greater overall in susceptible controls than in normal controls;In another experiment with mid-lactation cows, 3.5-h infusions of glucagon at 15 mg/d increased plasma insulin and glucose, but decreased plasma nonesterified fatty acids. PEPCK mRNA decreased 41%, but pyruvate carboxylase mRNA increased 50% by the end of infusions, and fructose 1,6 hisphosphatase mRNA did not change. Liver glycogen decreased an actual value of 2.1%, and two thirds of the decrease occurred during the first 0.75 h of glucagon infusions. The results indicate that glucagon infusions cause an initial net glycogenolysis, but later increase net glycogen synthesis. Long-term glucagon infusions also were suggested to increase gluconeogenesis. We concluded that PEPCK gene expression in normal cows is down-regulated potently by insulin to offset increased glycogenolysis and gluconeogenesis during glucagon infusions. PEPCK gene expression in susceptible cows may be different from that in normal cows, but probably is not involved in the pathogenesis of bovine ketosis. Also, there was no evidence for increased lipolysis from adipose tissue caused by glucagon infusions.</p

Leucine Supplementation of Drinking Water Does Not Alter Susceptibility to Diet-Induced Obesity in Mice1–3

Branched-chain amino acids (BCAA), Leu, and the signaling pathways they regulate have been reported to either improve or worsen adiposity and insulin sensitivity. Therefore, it is unclear whether dietary supplementation of Leu would be beneficial. To help address this question, we examined the effect of adding Leu (150 mmol/L; Expt. 1 and Expt. 2) or BCAA (109 mmol/L of each; Expt. 3) to the drinking water on diet-induced obesity (induced with a 60-kJ% fat diet) in singly housed C57BL6/J male mice for at least 14 wk. Liquid and solid food intakes were evaluated weekly along with body weight. During the last few weeks, several blood samples were taken at different times for plasma glucose, total cholesterol, or Leu measurements. Metabolic rate by indirect calorimetry, locomotor activity by light beam breaking, body composition by H1-NMR, and insulin tolerance were also determined. Compared with control, supplementation did not affect body weight, food intake, oxygen consumption, locomotor activity, body composition, insulin tolerance, or total cholesterol. In fed mice, this method of Leu supplementation only increased plasma Leu by 76% when the supplemented group was compared with control. On the other hand, after overnight food deprivation, the plasma Leu did not differ between these 2 groups, even though the mice in the supplemented group had continuous access to Leu-containing water during the solid food deprivation. Taken together, the results do not provide evidence that either Leu or BCAA supplementation of drinking water ameliorates diet-induced obesity in mice, although it may improve glycemia

Disruption of BCATm in Mice Leads to Increased Energy Expenditure Associated with the Activation of a Futile Protein Turnover Cycle

SummaryLeucine is recognized as a nutrient signal; however, the long-term in vivo consequences of leucine signaling and the role of branched-chain amino acid (BCAA) metabolism in this signaling remain unclear. To investigate these questions, we disrupted the BCATm gene, which encodes the enzyme catalyzing the first step in peripheral BCAA metabolism. BCATm−/− mice exhibited elevated plasma BCAAs and decreased adiposity and body weight, despite eating more food, along with increased energy expenditure, remarkable improvements in glucose and insulin tolerance, and protection from diet-induced obesity. The increased energy expenditure did not seem to be due to altered locomotor activity, uncoupling proteins, sympathetic activity, or thyroid hormones but was strongly associated with food consumption and an active futile cycle of increased protein degradation and synthesis. These observations suggest that elevated BCAAs and/or loss of BCAA catabolism in peripheral tissues play an important role in regulating insulin sensitivity and energy expenditure

A new large area MCP-PMT for high energy detection

Abstract 20-inch Large area photomultiplier tube based on microchannel plate (MCP-PMT) is newly developed in China. It is widely used in high energy detection experiments such as Jiangmen Underground Neutrino Observatory (JUNO), China JinPing underground Laboratory (CJPL) and Large High Altitude Air Shower Observatory (LHAASO). To overcome the poor time performance of the existing MCP-PMT, a new design of large area MCP-PMT is proposed in this paper. Three-dimensional models are developed in CST Studio Suite to validate its feasibility. Effects of the size and bias voltage of the focusing electrodes and MCP configuration on the collection efficiency (CE) and time performance are studied in detail using the finite integral technique and Monte Carlo method. Based on the simulation results, the optimized operating and geometry parameters are chosen. Results show that the mean ratio of photoelectrons landing on the MCP active area is 97.5%. The acceptance fraction of the impinging photoelectrons is close to 100% due to the emission of multiple secondary electrons when hitting the MCP top surface. The mean transit time spread (TTS) of the photoelectrons from the photocathode is 1.48 ns