Noninvasive Measurement of Murine Hepatic Acetyl-CoA¹³C-Enrichment Following Overnight Feeding with¹³C-Enriched Fructose and Glucose

Abstract

The 13C-isotopomer enrichment of hepatic cytosolic acetyl-CoA of overnight-fed mice whose drinking water was supplemented with [U-13C]fructose, and [1-13C]glucose and p-amino benzoic acid (PABA) was quantified by 13C NMR analysis of urinary N-acetyl-PABA. Four mice were given normal chow plus drinking water supplemented with 5% [1-13C]glucose, 2.5% [U-13C]fructose, and 2.5% fructose (Solution 1) overnight. Four were given chow and water containing 17.5% [1-13C]glucose, 8.75% [U-13C]fructose and 8.75% fructose (Solution 2). PABA (0.25%) was present in both studies. Urinary N-acetyl-PABA was analyzed by 13C NMR. In addition to [2-13C]- and [1,2-13C]acetyl isotopomers from catabolism of [U-13C]fructose and [1-13C]glucose to acetyl-CoA, [1-13C]acetyl was also found indicating pyruvate recycling activity. This precluded precise estimates of [1-13C]glucose contribution to acetyl-CoA while that of [U-13C]fructose was unaffected. The fructose contribution to acetyl-CoA from Solutions 1 and 2 was 4.0 ± 0.4% and 10.6 ± 0.6%, respectively, indicating that it contributed to a minor fraction of lipogenic acetyl-CoA under these conditions.

A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice

“…Overnight urine was collected and stored at 4°C until further processing. N -acetyl-PABA was purifi ed and processed and analyzed via 1 H and 2 H NMR as previously described ( 38 ). The total 2 H enrichment of carbon 2 from the acetyl moiety of N -acetyl PABA was calculated by comparing the area of N -Ac-PABA CH 3 (2.1 ppm) signal with the mean areas of the pair of aromatic PABA-d 4 (2.0% 2 H) signals at 7-8 ppm, which serve as the internal standard.…”

Identifying Sources of Hepatic Lipogenic Acetyl-CoA Using Stable Isotope Tracers and NMR

“…In tissues such as the heart, where pyruvate cycling fluxes are typically low and randomization of 13 C at the level of pyruvate is therefore insignificant, contributions of up to four different types of substrates to the acetyl-CoA pool may be determined by selecting a mixture of 13 Cenriched substrates that generate each of the four possible acetyl-CoA isotopomers [4][5][6]. In the liver, the randomization of pyruvate limits the resolution of acetyl-CoA 13 Cisotopomers from glycolytic 13 C-enriched substrates but can nevertheless provide novel and important information on the contributions of important glycolytic sources such as fructose and glucose to the lipogenic acetyl-CoA pool [7].…”

“…Metabolites that retain the acetyl-CoA 13 C-isotopomer signature include the fatty acid products of DNL, as well as glutamate, ketone bodies, acetyl carnitine, and acetylated xenobiotics such as sulfomethoxazole [18] and PABA [7]. A comparison of acetyl-CoA isotopomer readouts by different metabolites has revealed a substantial heterogeneity in hepatic acetyl-CoA enrichment from 13 C-enriched substrates [19][20][21][22].…”

“…Therefore, the 13 C terminal and penultimate fatty acid signals that are observed in these experiments invariably consist of a singlet and doublet component reflecting the presence of discrete acetyl 13 C-isotopomers. This is illustrated in Figure 2(a), which shows 13 C NMR signals of hepatic lipids that were isolated postmortem from a mouse that had previously ingested a mixture of [1-13 C]glucose and [U-13 C]fructose in its drinking water during overnight feeding [7]. As described by Carvalho et al, this substrate mixture generated the predicted [2-13 C]acetyl-CoA and [1,2-13 C 2 ]acetyl-CoA isotopomers from conversion of [1-13 C]glucose and [U-13 C]fructose to acetyl-CoA via glycolysis and pyruvate oxidation.…”

“…As described by Carvalho et al, this substrate mixture generated the predicted [2-13 C]acetyl-CoA and [1,2-13 C 2 ]acetyl-CoA isotopomers from conversion of [1-13 C]glucose and [U-13 C]fructose to acetyl-CoA via glycolysis and pyruvate oxidation. However, a significant fraction of [1-13 C]acetyl-CoA was also generated indicating the randomization of the [1-13 C]glucose label into carbons 2 and 3 of pyruvate via pyruvate recycling [7].…”