Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the [U-C(6)]-d-glucose tracer in mice

Abstract

Previous genetic and proteomic studies identified altered activity of various enzymes such as those of fatty acid metabolism and glycogen synthesis after a single toxic dose of valproic acid (VPA) in rats. In this study, we demonstrate the effect of VPA on metabolite synthesis flux rates and the possible use of abnormal (13)C labeled glucose-derived metabolites in plasma or urine as early markers of toxicity. Female CD-1 mice were injected subcutaneously with saline or 600 mg/kg) VPA. Twelve hours later, the mice were injected with an intraperitoneal load of 1 g/kg [U-(13)C]-d-glucose. (13)C isotopomers of glycogen glucose and RNA ribose in liver, kidney and brain tissue, as well as glucose disposal via cholesterol and glucose in the plasma and urine were determined. The levels of all of the positional (13)C isotopomers of glucose were similar in plasma, suggesting that a single VPA dose does not disturb glucose absorption, uptake or hepatic glucose metabolism. Three-hour urine samples showed an increase in the injected tracer indicating a decreased glucose re-absorption via kidney tubules. (13)C labeled glucose deposited as liver glycogen or as ribose of RNA were decreased by VPA treatment; incorporation of (13)C via acetyl-CoA into plasma cholesterol was significantly lower at 60 min. The severe decreases in glucose-derived carbon flux into plasma and kidney-bound cholesterol, liver glycogen and RNA ribose synthesis, as well as decreased glucose re-absorption and an increased disposal via urine all serve as early flux markers of VPA-induced adverse metabolic effects in the host.

Fig. 1

Principles of mass isotopomer analysis (MIDA). The intraperitoneally injected [U-¹³C₆]-d-glucose tracer [shown with all ¹³C carbons with red filled circles] breaks down via enzymatic steps of glycolysis while via gluconeogenesis it loses, exchanges and dilutes its carbon skeleton with ¹²C [shown with empty circles] before reappearing in plasma via hepatic glucose production or gets deposited into cellular glycogen in tissues. Various positional ¹³C glucose isotopomers observed in plasma or glycogen depict specific reactions that contribute to gluconeogenesis, hepatic glucose production and glucose dependent futile cycles in the liver (plasma glucose) or tissues (glycogen glucose obtained from organs) (OA = oxaloacetate). (Color figure online)

Fig. 2

Intraperitoneal ¹³C tracer glucose tolerance test (IPGTT). Glucose ¹³C labeled fractions as per cent of total glucose in plasma (Y axis, Σm_n=C1–C6) which includes all ¹³C-labeled positional isotopomers of glucose at 0, 60, 120 and 180 min (X axis) after tracer load. Control and VPA-treated animals show virtually identical ¹³C labeled glucose fractions in plasma indicating similar absorption and clearance of tracer glucose at all time points. Control (lower continuous line) or VPA (upper broken line) glucose ¹³C-labeled plasma fractions as independent variables are compared using regression analysis to corresponding plasma cholesterol ¹³C-labeled fractions as the dependent variable to determine glucose to cholesterol flux by liver metabolism in Tables 1 and 2 (mean + standard deviation (SD); n = 8)

Fig. 3

All ¹³C labeled glucose isotopomers (including the [U-¹³C]-d-glucose tracer) in plasma. Positional glucose ¹³C isotopomer fractions as per cent of the total ¹³C enriched fraction in plasma (Y axis, Σm_{n=C1,2,3,4,5,6}) at 0, 60, 120 and 180 min (X axis) after tracer load. The various isotopomers indicate: M1, pyruvate carboxylase activity; M2, pyruvate dehydrogenase activity and complete TCA cycle turnaround; M3, glucose generated from [U-¹³C₃]-dl-lactate and the Cori cycle; M4, non-oxidative pentose cycle; and M5, oxidative pentose cycle (mean; n = 8) (Lee et al. 1991)

Fig. 4

Acetyl-CoA saturation curves of plasma cholesterol from the [U-¹³C]-d-glucose tracer. Please also see Table 1 (control vehicle treatment) and Table 2 (VPA treatment) regression statistics (mean ± standard deviation (SD); n = 8)

Fig. 5

Liver glucose (glycogen) and ribose (total RNA) ¹³C-labeled fractions 180 min after tracer load. a Glucose ¹³C labeled fractions as per cent of total in liver glycogen (Y axis), b The ¹³C labeled fraction in RNA ribose (mean + standard error of the mean (SEM); n = 8)

Fig. 6

All ¹³C labeled glucose isotopomers in aspirated urine from bladder 3 h after ¹³C tracer glucose load. Glucose ¹³C isotopomer fractions are shown as per cent of the total ¹³C enriched fraction in urine (Y axis, Σm_{n=C1,2,3,4,5,6}) in control (C) and valproic acid treated (VPA) animals (X axis). Control and VPA treated animals showed distorted ¹³C labeled glucose isotopomer fractions in urine indicating increased tracer disposal via urine (M6, [U-¹³C₆]-d-glucose tracer injected at time 0; average + SEM, n = 8; *P = 0.016) in VPA treated animals. The various isotopomers indicate: M1, pyruvate carboxylase activity (*P = 0.029); M2, pyruvate dehydrogenase activity and complete TCA cycle turnaround; M3, glucose generated from [U-¹³C₃]-dl-lactate and the Cori cycle; M4, non-oxidative pentose cycle; and M5, oxidative pentose cycle