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Combined metabolomic and fluxomic assays suggest the existence of multiple pools of glucose‐6‐phosphate in perfused muscle
Author(s) -
Previs Stephen,
Herath Kithsiri,
Petrov Aleksandr,
CastroPerez Jose
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.42.2
Subject(s) - glucose uptake , glucose 6 phosphate , glycogen , citric acid cycle , skeletal muscle , chemistry , biochemistry , glucose transporter , carbohydrate metabolism , glycerol , medicine , carbohydrate , citrate synthase , metabolism , endocrinology , glucose homeostasis , soleus muscle , phosphate , homogenization (climate) , biology , insulin , insulin resistance , enzyme , biodiversity , ecology
Glucose uptake and utilization (e.g. deposition in glycogen vs oxidation to CO2) by skeletal muscle represents a critical step to ensuring normal metabolic homeostasis. The ability to modulate glucose flux in muscle is of interest since it may affect better glucose control in subjects with impaired insulin action and/or overt diabetes. We initiated studies that used isolated perfused rat hindlimb as a model system in order to identify molecules that could alter glucose uptake and utilization, metabolomic and fluxomic analyses were used to track the fate of glucose molecules. Typical studies relied on 30–60 min open circuit perfusions, soleus and gastrocnemius were then dissected and freeze clamped. Samples were processed using standard homogenization steps and subjected to LC‐MS analyses. The data suggest greater uptake of 2‐deoxyglucose in soleus vs gastrocnemius under baseline conditions. The incorporation of [13C]glucose was then followed into glucose‐6‐ and glycerol‐3‐phosphate, lactate and several TCA cycle intermediates. The observed [13C]labeling patterns clearly demonstrate the generation of [13C]lactate but suggest minimal conversion of [13C]glucose to [13C]TCA cycle intermediates. Parallel labeling studies with [13C]acetate demonstrate [13C]labeling in the TCA intermediates, implying that mitochondria are active in the resting perfused muscle but that the muscle does not completely oxidize glucose. A striking observation however centers on a discrepancy between the enrichments of glucose‐6‐ vs glycerol‐3‐phosphate, the latter being much higher than the former (consistent in both muscles). The discrepancy in the [13C]labeling patterns can be explained by the existence of multiple pools of glucose‐6‐phosphate. These observations agree with previously reported data in hepatocytes and have important implications if/when one aims to use concentration profiles to infer aspects of metabolic control. For example, concentrations of glucose‐6‐phosphate are believed to play a central role in effecting glucose uptake (e.g. feedback inhibition). However, the existence of multiple pools complicates interpretations when one aims to link a change in metabolite concentration to a change in metabolic flux. We believe that one can use tracers to estimate overall flux rates but one should exercise caution when using concentration profiles to explain metabolic control points and therein identify reactions that are targeted by pharmacological manipulations. Support or Funding Information All authors were employed by Merck during the completion of these studies.