Comparative glycolysis and Krebs cycle metabolism of the bovine and murine mammary gland determined with [13C6]glucose and mass spectrometry

Abstract Text:

The compositions of bovine and murine milk differ significantly with respect to the proportions of lactose, protein, and fat.  To better understand the metabolic origins of this difference, we interrogated the crossroads of glycolysis and the Krebs cycle in the mammary gland of cows and mice using a glucose stable isotope (¹³C) tracer approach in vitro.  Mammary tissue was collected from mid-lactation dairy cows (n=4) and day 15 of lactation mice (n=6) then sliced to form explants (0.5 mm thick, 100-150 mg).  Explants were incubated for 3 h (5% CO₂) at 37⁰C in DMEM containing a 50:50 mix of unlabeled and [¹³C₆] labeled glucose at 2.5, 5, 7.5 or 10 mM concentrations.  Following incubation, explants were rinsed in PBS and stored at -80⁰C.  Intracellular metabolites were extracted and derivatized for determination of ¹³C-isotopomer enrichments employing gas chromatography-mass spectrometry.  Alanine, glutamate, and aspartate ¹³C-isotopomer enrichments were monitored as representative surrogates of their Krebs cycle counterparts pyruvate, α-ketoglutarate, and oxaloacetate, respectively.  These data provided the inputs to calculate glycolytic and Krebs cycle fluxes.  In bovine mammary tissue, increasing media glucose concentration increased glycolytic flux as represented by an increasing contribution of glucose to pyruvate flux.  However, the proportion of pyruvate derived from glucose catabolism reached a plateau (44−46%) at 7.5 mM glucose.  Similarly, in murine mammary tissue, glycolytic rate increased with increasing media glucose concentration, though no plateau was attained and glucose contributed to 43% of pyruvate flux at the highest glucose concentration.  Krebs cycle flux was assessed by the relative activities of pyruvate dehydrogenase (PDH) vs. pyruvate carboxylase (PC) based on [¹³C] tracer kinetics.  For bovine mammary explants, PDH flux activity increased to a maximum at 5.0 mM glucose whereas PDH vs. PC activities of murine mammary tissue was not responsive to glucose concentration.  The current study suggests that the dairy cow mammary gland shifts from high anapleurotic flux rates into the Krebs cycle to energy-producing (oxidative) fluxes with increasing glucose concentration whereas the murine mammary gland maintains a more rigid metabolic balance, and thus is less adaptive to glucose availability.

Keywords: bovine, murine, mammary metabolism