Pentose phosphate pathway activity parallels lipogenesis but not antioxidant processes in rat liver

Abstract

The pentose phosphate pathway (PPP) is widely assumed to play a key role in both reductive biosynthesis and protection from oxidative stress because it is the major source of NADPH. However, little is known about the activity of the PPP in fatty liver, which is characterized by both oxidative stress and lipogenesis. This study was designed to test whether the PPP is active in parallel with lipogenesis and antioxidant processes in the fatty liver of whole animals. Eight- and 16-wk-old obese Zucker diabetic fatty rats and their lean littermates received [U-¹³C₃]glycerol, and ¹³C labeling patterns of glucose and triglycerides were analyzed for the assessment of hepatic PPP activity and the potentially related processes simultaneously. Oxidative stress, antioxidant activity, and NADPH-producing enzymes in the liver were further examined. Both PPP activity and lipogenesis increased in the fatty liver of young obese Zucker rats but decreased together in older obese Zucker rats. As expected, lipid peroxidation measured by malondialdehyde increased in the fatty liver of obese Zucker rats at both ages. However, evidence for antioxidant processes such as [glutathione] or activities of glutathione reductase, glutathione peroxidase, and catalase was not altered. Hepatic PPP activity paralleled lipogenesis but was dissociated from biomarkers of oxidative stress or antioxidant processes. In summary, NADPH from the PPP was presumably consumed for reductive biosynthesis rather than antioxidant defense in the fatty liver.

Keywords: NADPH; gluconeogenesis; glutathione; glycerol; lipid peroxidation.

Fig. 1.

[U-¹³C₃]glycerol incorporation into triglycerides (TG) and glucose. Direct [U-¹³C₃]glycerol incorporation into fatty acid esterification and gluconeogenesis produces triple-labeled glycerol backbones of TG and glucose, respectively (“direct” contribution). A fraction of [U-¹³C₃]glycerol is metabolized to [U-¹³C₃]pyruvate, entering the TCA cycle prior to incorporation into TG or glucose, producing double-labeled glycerol backbones or glucose (“indirect” contribution via the TCA cycle). [1,2,3-¹³C₃]glusose 6-phosphate (G6P), produced through [U-¹³C₃]glycerol-gluconeogenesis, may enter the full cycle of the PPP, producing [1,2-¹³C₂]fructose 6-phosphate (F6P) and consequently [1,2-¹³C₂]glucose. These processes can be detected by ¹³C NMR analysis of the metabolic products such as TG-glycerol and glucose. G3P, glycerol 3-phosphate; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; TCA, tricarboxylic acid. Open circle, ¹²C; black circle, ¹³C; blue circle, =¹³C after metabolism through the TCA cycle; red circle, ¹³C after metabolism through the PPP.

Fig. 2.

TG synthesis in liver after [U-¹³C₃]glycerol administration. Overnight-fasted rats received [U-¹³C₃]glycerol, and liver was harvested after 60 min. A: ¹³C NMR of lipid extracts show signals of the glycerol backbones of TG from an 8-wk fa/fa rat and a lean littermate (+/?). Doublet (D) reflects double-labeled ([1,2-¹³C₂]- and [2,3-¹³C₂]-) glycerol, and triplet (T) arises exclusively from [U-¹³C₃]glycerol moiety of TG. B: hepatic [TG] was higher in 8-wk fa/fa rats than in lean littermates but was not altered in 16-wk fa/fa rats. Total amounts of TG in liver increased in both 8- and 16-wk fa/fa rats than in lean littermates. C: fractions of TG-[¹³C]glycerol were lower in fa/fa rats at both ages than in lean littermates. Total amounts of TG-[¹³C]glycerol in liver increased in 8-wk fa/fa rats but decreased in 16-wk fa/fa rats. D: fraction of indirect contribution of [U-¹³C₃]glycerol to TG was higher in fa/fa rats at both ages than in lean littermates. Amounts of TG-[¹³C₂]glycerol (“indirect” contribution) increased in 8-wk fa/fa rats but not in 16-wk fa/fa rats. Amounts of TG-[¹³C₃]glycerol (“direct” contribution) increased in 8-wk fa/fa rats but decreased in 16-wk fa/fa rats. D, doublet from coupling of C1 with C2 or from coupling of C2 with C3; T, triplet arising from coupling of C2 with both C1 and C3; S, singlet; open circle, ¹²C; black circle, ¹³C; *P < 0.05, **P < 0.01, ***P < 0.001; n = 5–7 in each group.

Fig. 3.

Hepatic PPP activity and gluconeogenesis from [U-¹³C₃]glycerol. Plasma glucose was derivatized for NMR analysis to estimate gluconeogenesis and hepatic PPP activity. A: spectra show signals of glucose carbons 2 and 5 from an 8-wk fa/fa rat and a lean littermate (+/?). Triple-labeled ([1,2,3-¹³C₃] and [4,5,6-¹³C₃]) glucose reflects gluconeogenesis directly from [U-¹³C₃]glycerol. Double-labeled (especially [5,6-¹³C₂]) glucose reflects [U-¹³C₃]glycerol metabolism via the TCA cycle. Hepatic PPP interruption in gluconeogenesis from [U-¹³C₃]glycerol produces additional [1,2-¹³C₂]glucose, causing the ratio difference between [1,2-¹³C₂]/[2,3-¹³C₂] and [5,6-¹³C₂]/[4,5-¹³C₂] in glucose. B: fractions of ¹³C-labeled glucose in both 8-wk and 16-wk fa/fa rats were lower vs. lean littermates. Levels of ¹³C-labeled glucose were similar between +/? and fa/fa rats at both ages. C: the fraction of [5,6-¹³C₂]glucose was not altered in 8-wk fa/fa rats but decreased in 16-wk fa/fa rats. The level of [5,6-¹³C₂]glucose increased in 8-wk fa/fa rats but was not changed in 16-wk fa/fa rats vs. lean littermates. D: hepatic PPP activity increased in 8-wk fa/fa rats but decreased in 16-wk fa/fa rats vs. lean littermates based on 1) the ratio difference between [1,2-¹³C₂]/[2,3-¹³C₂] and [5,6-¹³C₂]/[4,5-¹³C₂] in glucose, 2) [1,2-¹³C₂]glucose produced through the PPP, and 3) PPP flux relative to gluconeogenesis. D12, doublet from coupling of C1 with C2; D23, doublet from coupling of C2 with C3; Q, doublet of doublets, or quartet, arising from coupling of C2 with both C1 and C3 or from coupling of C5 with both C4 and C6; D45, doublet from coupling of C4 with C5; D56, doublet from coupling of C5 with C6; S, singlet; open circle, ¹²C; black circle, ¹³C. *P < 0.05; **P < 0.01; ***P < 0.001; n = 6–7 in each group.

Fig. 4.

Liver injury/oxidative stress/antioxidant. A: plasma alanine aminotransferase (ALT) increased in fa/fa rats at both ages than lean littermates, whereas aspartine aminotransferase (AST) tended to increase in 16-wk fa/fa rats only without statistical significance. Malondialdehyde, an indicator of lipid peroxidation, was higher in fa/fa rats at both ages than in lean littermates. B: glutathione measured by NMR analysis of liver tissue extracts was not altered in fa/fa rats at both ages. C and D: in immunoblot assays, protein levels of antioxidant enzymes (i.e., glutathione reductase, glutathione peroxidase, and catalase) are similar between +/? rats and fa/fa rats at both ages. E: enzyme activities of glutathione reductase, glutathione peroxidase, and catalase are also similar between +/? and fa/fa rats at both ages. *P < 0.05; **P < 0.01; ***P < 0.001; n = 6–7 in each group.

Fig. 5.

NADPH-producing enzymes in the livers. A: Western blot analysis was performed to measure protein levels of NADPH-producing enzymes in the liver. B: G6PDH, the rate-limiting step of the PPP, substantially increased in 8-wk fa/fa rats but was not altered in 16-wk fa/fa rats vs. lean littermates. C: ME1 slightly increased in 8-wk fa/fa rats but was not altered in 16-wk fa/fa rats. D: IDH1 was not altered in fa/fa rats at both ages, but IDH2 decreased in 8-week fa/fa rats compared to lean littermates. G6PDH, glucose-6-phosphate dehydrogenase; IDH1, cytosolic NADP⁺-dependent isocitrate dehydrogenase; IDH2, mitochondrial NADP⁺-dependent isocitrate dehydrogenase; ME1, cytosolic NADP⁺-dependent malic enzyme. *P < 0.05; **P < 0.01; ***P < 0.001; n = 6–7 in each group.