Application Note 45
Metabolic Isotopic Analysis Reveals Mitochondrial Loss of Function in pRb-Deficient Cells in vivo
Brandon N. Nicolay
Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology
Harvard Medical School, Charlestown, MA USA
Metabolic isotopic analysis (MIA) utilizes stable isotope-enriched substrates and mass spectrometry to study metabolism in living cells and tissue. Because isotope-labeled substrates make the determination of metabolic pathways and flux in living cells or tissue possible, MIA has become an integrated platform in oncology research.
This application note presents an example of MIA for studying metabolism in mice and cultured human cells used in oncology research by summarizing research conducted by Nicolay, et al. Previously, these researchers used MIA to study the inactivation of the retinoblastoma protein (pRb) tumor suppressor in the fly model. In the presented work, Nicolay and coworkers found that acute loss of pRB in adult mouse tissues led to rapid changes in glucose oxidation (Nicolay, et al., 2015). This subsequent study shows that MIA of glucose-derived intermediates in the TCA cycle for mitochondria in pRb-/- tissue are less functional than the pRb+/+ tissue. These effects are mimicked in human cell culture models of pRb loss.
Methods and Results
An in vivo isotopic analysis of 13C6-glucose (CLM-1396) in both the colon and the lung using protocols similar to previously published work was performed (Fan, et al., 2011; Lane, et al., 2011; Yuneva, et al., 2012; Sellers, et al., 2015). Briefly, mice were given a single bolus of 13C6-glucose and were sacrificed after 20 minutes for tissue isolation. An initial timecourse analysis found that 20 minutes after injection the levels of 13C6-glucose had peaked within the blood, and 13C enrichments were sustained in downstream intermediates of glycolysis and the TCA cycle within both the lung and colon (Figure 1A-D). To determine that the glucose clearance would be similar in both Rb+/+ and RbKO mice, glucose tolerance tests (GTT) (of the same concentrated bolus of 13C6-glucose) showed no differences between genotypes 96 hrs after Cre induction (Figure 1E). These control experiments demonstrated that qualitative differences in glucose-derived metabolites could be determined using this methodology.
In support of the GTT results, analysis of 13C6-glucose uptake from the serum revealed no differences between the Rb+/+ and RbKO colon or lung tissues (Figure 2A). In contrast, loss of Rb1 induced a significant enrichment (two-fold) of M+2 citrate in both tissues (Figure 1F). No difference was seen in the amount of M+3 pyruvate or M+3 lactate produced in either tissue (Figure 1G, H). Interestingly, no increase in glycolysis was detected following Rb1 loss (as indicated by pyruvate and lactate production (Figure 2B,C)). This curious result suggests that RbKO tissues had increased entry of glucose into the TCA. In agreement with this, a ratiometric that qualitatively measures pyruvate dehydrogenase (PDH) activity (M+2 citrate/M+3 pyruvate) was significantly elevated upon loss of Rb1 (Figure 1I). Furthermore, a significant enrichment of M+2 acetyl-CoA in both RbKO tissues was observed, as well as a significant increase in total acetyl-CoA in the RbKO lung (Figure 1J-K). Using this technique we could not qualitatively measure the TCA cycle activity directly. Nevertheless, these results show that the loss of pRb in vivo did not produce a glycolytic phenotype, and, despite increased entry of glucose into the TCA cycle, ATP output decreased under comparable energetic conditions.
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