Fig 1: Loss of ALT2 reduces hepatocyte alanine metabolism and alanine-mediated gluconeogenesis(A) Representative western blots for ALT2, ALT1, or GAPDH using liver lysates from WT and LS-Gpt2-/- mice demonstrating loss of ALT2 protein.(B) Glucose concentrations in the media of hepatocytes isolated from WT or LS-Gpt2-/- mice stimulated with glucagon in the presence of no substrate, alanine, pyruvate, or glutamine. Some cells were also treated with the transaminase inhibitor ß-chloroalanine (ß-Cl) (n = 7). *Indicates significant differences (p < 0.05).(C) Schematic depicting incorporation of 13C-alanine into pyruvate and TCA cycle intermediates. Black circles indicate 13C. White circles indicate 12C. Created with BioRender.com.(D) Hepatocyte citrate enrichment from 13C-alanine is shown. *Significant differences (p < 0.05) between hepatocytes from different genotypes of mice.(E) Hepatocyte M3 citrate enrichment from 13C-alanine is shown. *Significantly different (p < 0.05) from WT hepatocytes treated with vehicle. **Significantly different (p < 0.05) from all other groups.(F) Media glucose enrichment from 13C-alanine is shown. *Significant differences (p < 0.05) between hepatocytes from different genotypes of mice. For (D)–(F), a representative experiment (of 3) performed in triplicate is shown. Data are presented as mean ± SEM.(G) Blood glucose concentrations during ATT, QTT, and pyruvate tolerance test (PTT) analyses using lean WT or LS-Gpt2-/- mice.
Fig 2: Hepatic ALT2 knockdown results in decreased blood glucose concentrationMice (db/+ and db/db) were infected with an adenovirus expressing shRNA targeting LacZ or Gpt2.(A) Gpt2 gene expression was assessed in adenovirus-treated db/+ (n = 11) and db/db (n = 9–13) mice 6 days post infection. *p < 0.05 versus indicated groups.(B) Representative western blot image for ALT2 or mitochondrial complex III as a loading control using mouse liver lysates from adenovirus shRNA-treated db/+ or db/db mice 6 days after adenovirus administration.(C) Total liver ALT activity in db/+ and db/db liver (n = 4–7 per group) after treatment with adenovirus expression shRNA against LacZ or Gpt2. *p < 0.05 versus indicated groups.(D) Random fed blood glucose concentrations in adenovirus shRNA-treated db/+ or db/db mice 5 days after adenovirus injection (n = 13–18 per group). *p < 0.05 versus indicated groups.(E) Plasma insulin concentrations in adenovirus shRNA-treated db/+ or db/db mice 7 days after adenovirus injection. Plasma was collected at sacrifice after a 4 h fast (n = 8–10 per group). *p < 0.05 versus indicated groups.(F) Plasma amino acid concentrations in db/+ or db/db mice 7 days after administration of adenovirus expressing shRNA against LacZ or Gpt2. Plasma was collected at sacrifice after a 4 h fast (n = 4–5 per group). *p < 0.05 versus shLacZ db/db mice. **p < 0.05 versus db/+ mice.
Fig 3: Diet-induced obese and db/db mice exhibit increased hepatic ALT abundance and exacerbated hyperglycemia from gluconeogenic amino acids(A and B) (A) Representative western blot images for ALT2 and GAPDH proteins and (B) expression of Gpt2 mRNA in livers of mice fed either a control low-fat diet (LFD) (n = 5) or a 60% high-fat diet (HFD) (n = 5).(C and D) Blood glucose concentrations after an i.p. injection of L-alanine (C) or L-glutamine (D) in diet-induced obese (HFD; n = 5) and lean (LFD; n = 5) mice.(E) Representative western blot image for ALT2 and mitochondrial complex III from either db/+ or db/db mouse liver homogenates.(F) Expression of Gpt2 in liver RNA from db/+ (n = 8) and db/db (n = 5, 7) mice (bottom panel).(G and H) Blood glucose concentrations after an i.p. injection of L-alanine (G) or L-glutamine (H) in db/+ (n = 7, 8) and db/db mice (n = 6, 7). For (A) and (E) densitometric quantification of ALT2/loading control band intensity is provided numerically between the blots. Data are presented as mean ± SEM. *p = 0.05 for LFD versus HFD or db/+ versus db/db.
Fig 4: ALT2 is expressed in human liver and is reduced after significant weight loss(A) Schematic of hepatic alanine metabolism. ALA, alanine; GLU, glutamine; MPC, mitochondrial pyruvate carrier; ALT, alanine transaminase; OAA, oxaloacetate; aKG, a ketoglutarate. Created with BioRender.com.(B) Western blotting analysis for ALT2, ALT1, and HSP60 conducted using lysates from obese human liver (n = 2),Huh7 cell lysates (treated with scramble [scr] or GPT2 siRNA), and mouse liver lysates (WT or LS-Gpt2-/-).(C) Western blot image for ALT2, ALT1, and tubulin from liver biopsies collected from cadaveric donor livers (n = 3) or during RYGBS (n = 2) from patients with obesity.(D) Expression of mRNA for GPT and GPT2 in obese subjects before (pre) (n = 8) and 6 months after (post) (n = 8) bariatric surgery.(E) Expression of the indicated genes encoding mitochondrial amino acid transporters in obese subjects during (pre) (n = 7) and 6 months after (post) (n = 7) bariatric surgery.(D and E) Data are presented as mean ± SD. *p = 0.05 for pre versus post.
Supplier Page from Abcam for Anti-Alanine Transaminase antibody [EPR19616]