Glutamate dehydrogenase activator BCH stimulating reductive amination prevents high fat/high fructose diet-induced steatohepatitis and hyperglycemia in C57BL/6J mice

Individuals with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) induced by high calorie western diet are characterized by enhanced lipogenesis and gluconeogenesis in the liver. Stimulation of reductive amination may shift tricarboxylic acid cycle metabolism for lipogenesis and gluconeogenesis toward glutamate synthesis with increase of NAD+/NADH ratio and thus, ameliorate high calorie diet-induced fatty liver and hyperglycemia. Stimulation of reductive amination through glutamate dehydrogenase (GDH) activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced both de novo lipogenesis and gluconeogenesis but increased the activities of sirtuins and AMP-activated kinase in primary hepatocytes. Long-term BCH treatment improved most metabolic alterations induced by high fat/high fructose (HF/HFr) diet in C57BL/6J mice. BCH prevented HF/HFr-induced fat accumulation and activation of stress/inflammation signals such as phospho-JNK, phospho-PERK, phospho-p38, and phospho-NFκB in liver tissues. Furthermore, BCH treatment reduced the expression levels of inflammatory cytokines such as TNF-α and IL-1β in HF/HFr-fed mouse liver. BCH also reduced liver collagen and plasma levels of alanine transaminase and aspartate transaminase. On the other hand, BCH significantly improved fasting hyperglycemia and glucose tolerance in HF/HFr-fed mice. In conclusion, stimulation of reductive amination through GDH activation can be used as a strategy to prevent high calorie western diet-induced NAFLD and T2D.

Measurement of SIRT activity. Deacetylase activities of SIRT1, SIRT3 and SIRT5 were determined by measuring fluorescence released from different peptides supplied from kits.

Measurement of glucose incorporation into lipid.
De novo lipogenesis was determined by measuring radio-labeled glucose ( 14 C-glucose) accumulated in triacylglycerol (TG) in hepatocytes. Briefly, cells (1x10 6 ) incubated with 14 C-glucose (0.1 μCi/ml, PerkinElmer) for 3 h were collected by differential centrifugation (500g, 5 min). Cell pellets were suspended in 1 ml of chloroform: methanol (2:1) mixture. Following an addition of 0.3 ml of water, lipids in the organic phase were separated by differential centrifugation (4,000g, 10 min) and saved in a new tube. The radioactivity in the lipid fraction was determined using a scintillation counter (Tri-Carb 2100TR, Packard, USA).

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Glucose production assay in primary hepatocytes. Hepatocytes were cultured in glucosefree DMEM supplemented with 20 mM lactate and 2 mM pyruvate for 2 h. Glucose concentration in the media was measured by using an Accu-Check glucometer (Roche, Mannheim, Germany).
Ex Vivo fat oxidation in liver tissues. Livers were quickly removed from mice and placed in 25-ml flasks fitted with center wells containing 1 N NaOH and filter paper strip to trap 14 CO 2 .
Flasks were capped with bottle stoppers. The incubation media contained 3 ml of Krebs-Ringer phosphate buffer, 2 μCi of [1-14 C] oleic acid and cold oleic acid (0.6 mM final concentration) in complex with BSA. Tissues were incubated in a 37 °C shaking water bath for 30 min. One ml of 0.5 N sulfuric acid was injected into the media to release 14 CO 2 . Flasks were maintained at 50 °C for 3 h to transfer 14 CO 2 to NaOH in the center well. After acid treatment, contents in the center well were transferred to scintillation fluid and [ 14 C] radioactivity was measured.
Measurement of energy expenditure. Energy expenditure was assessed with a metabolic monitoring system (comprehensive animal metabolic monitoring system, CLAMS: Columbus Instruments) for 4 days for each mouse after 2 days of acclimation followed by 2 days of measurement as described previously (Proc Natl Acad Sci USA 104, 16480-16485. 2007 ).
Respiratory quotient (RQ) was calculated from gas exchange data. RQ was the ratio of VCO 2 to VO 2 . Energy expenditure (EE) was calculated with the following formula: EE = (3.815 + 1.232 x RQ) x VO 2 . Hourly energy expenditure and energy intake values were averaged for the 24 h period.

Culture of INS-1 beta cells. INS-1 rat insulinoma cells kindly supplied by Dr. Wollheim
(University of Geneva Medical Center, Geneva, Switzerland) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 g/mL streptomycin, and 10 mM HEPES at 37 °C in a humidified atmosphere containing 95% air and 5% CO 2 .
Oil Red O staining. Liver tissue frozen in OCT compound were cut at 5 μm thickness, mounted onto slides, fixed with 10% formalin, and stained in 0.5% Oil Red O solution. Red oil drops were observed under light microscope.
Sirius Red staining. Paraffin sections were de-waxed and hydrated with phosphate-buffered saline. Collagens were stained in Sirius Red solution (Abcam) for one hour. After washing with acidified water, tissues were dehydrated with 100% ethanol. Nuclei were stained with hematoxylin.
Histological assessment. Liver tissues fixed with phosphate buffered saline containing 4% paraformaldehyde were embedded in paraffin, sliced (4 μm sections), mounted onto slides,   condition, glutamate is presumed to be synthesized from alpha-ketoglutarate and ammonia through reductive amination using the same enzyme, concomitantly releasing NAD+.
However, large amounts of GTP synthesized in highly respiratory conditions may block the reductive amination reaction due to its inhibitory action for GDH. Thus, TCA intermediates preferably siphon into DNL. BCH, an analogue of leucine, can stimulate the reductive amination reaction for the production of glutamate and NAD+ in a feeding condition since it has been reported that leucine could displace GTP from the allosteric inhibitor binding sites in GDH. Stimulation of reductive amination through GDH activation reduces DNL and gluconeogenesis. It also augments ketogenesis because of a relative deficiency of TCA cycle intermediates. Stimulation of reductive amination also provides NAD+.