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

Abstract

The role of hepaticde novolipogenesis (DNL) in promoting fatty liver disease and hypertriglyceridemia during excessive nutrient intake is becoming firmly established. Certain nutrients such as fructose promote hepatic DNL activity and this has been at least partly attributed to their efficient conversion to the acetyl-CoA precursors of DNL. However, tracer studies indicate a paradoxically low level of fructose incorporation into lipids, which begs the question of what the actual lipogenic acetyl-CoA sources are under these and other conditions. Here, we describe novel approaches for measuring substrate contributions to lipogenic hepatic acetyl-CoA using13C-tracers and13C-NMR analysis of lipids and acetyl-CoA probes. We review and address aspects of hepatic intermediary fluxes and acetyl-CoA compartmentation that can confound the relationship between13C-precursor substrate and lipogenic13C-acetyl-CoA enrichments and demonstrate novel methodologies that can provide realistic estimates of13C-enriched substrate contributions to DNL. The most striking realization is that the principal substrate contributors to lipogenic acetyl-CoA have yet to be identified, but they are probably not the so-called “lipogenic substrates” such as fructose. The proposed methods may improve our insight into the nutrient sources of DNL under various feeding and disease states.

De novo lipogenesis in non‐alcoholic fatty liver disease: Quantification with stable isotope tracers

“…With isotopic tracers, the fraction of hepatic or VLDL lipid that was synthesized via DNL can be measured. The principal tracer approaches for quantifying fractional DNL rates have involved carbon‐based tracers such as 13 C or 14 C acetate as well as labelled water ( 2 H 2 O or 3 H 2 O) 41 . Carbon‐based tracers result in obligatory enrichment of the acetyl‐CoA precursor pool followed by passage of the carbon label through subsequent intermediates of the DNL pathway.…”

“…In the case of the labelled hydrogens of water (which from this point on will focus on the stable 2 H isotope), these are partially incorporated at the level of acetyl‐CoA or precursor substrates as well as in the subsequent hydration and reduction steps of fatty acyl synthase (FAS). The terminal methyl hydrogens of synthesized fatty acyls are directly descended from the initial acetyl‐CoA molecule that became bound to FAS and therefore reflects the isotopic enrichment of acetyl‐CoA at the moment of recruitment by DNL 41 …”

“…Second, hepatic intermediary metabolism features a number of substrate and metabolite recycling pathways that, among other things, randomize the positional 13 C enrichment during conversion of a 13 C‐enriched substrate to 13 C‐enriched acetyl‐CoA. Thirdly, there is substantial evidence that hepatocytes have distinctive intracellular acetyl‐CoA pools that are enriched to different levels by 13 C‐enriched acetyl‐CoA precursors 41 . Thus, for determining 13 C‐enriched substrate contributions to DNL, either the true cytosolic acetyl‐CoA precursor pool must be selectively sampled, 57,63 or alternatively, enrichment of acetyl‐CoA units within newly synthesized fatty acids needs to be measured 61 .…”

Dose-dependent quantitative effects of acute fructose administration on hepatic de novo lipogenesis in healthy humans

“…The decrease in hepatic acetyl-CoA enrichment over time with fructose administration represents dilution of the intrahe-patic acetyl-CoA pool; however, the identity of the acetyl-CoA sources that supply the majority of DNL carbons is still debated (21). This parameter is itself metabolically informative and provides novel information about substrate load and the liver's biosynthetic response to it.…”

Metabolic Complementation between Glucose and Amino Acid Drives HepaticDe NovoLipogenesis and Steatosis

“…The obese state drives DNL through multiple mechanisms, but the principle substrate source of DNL and its regulation in the obese state remains unsettled 16 . Work by Clark and colleagues showed that glucose and fructose were not suitable for DNL in primary hepatocytes when compared to lactate, pyruvate, acetate, and alanine 17 .…”