PFKFB2 is Pivotal for Metabolic Flexibility and Differential Glucose Utilization

Figures

• 

  Figure 1.. PFKFB2 loss moderately impacts cardiac mitochondrial metabolic flexibility. A, PDH enzyme activity was measured across groups in isolated cardiac mitochondria from fed and fasted, control (CON) and PFKFB2 knockout (cKO) hearts (n=7–9). B, Pyruvate (Pyr)- and malate-dependent mitochondrial respiration was measured as oxygen consumption in the ADP-dependent state (state 3; n=7–9). C-D, Relative abundance of total pyruvate dehydrogenase (PDH; C) and phosphorylated pyruvate dehydrogenase (p-PDH; D) were measured by western blot in the mitochondrial fraction of hearts (n=7–9). E, Pyruvate dehydrogenase kinase 4 (PDK4) abundance was measured by western blot in whole heart homogenate (n=7–9). F, Palmitoyl carnitine (PC) and malate-dependent respiration was measured in state 3 (n=7–9). G, The ratio of PC/Pyr- dependent State 3 respiration was calculated using measures from B and F (n=7–9). A statistical outlier in PC and malate-dependent respiration was detected in the fed CON group via a ROUT test with Q=1% and excluded in panels F and G. H, Carnitine palmitoyl transferase 1 (CPT1) activity was measured in isolated mitochondria(n=5). Statistics were performed as two-way ANOVA.…

• 

  Figure 2.. Key Metabolite Changes and Correlations Associated with PFKFB2 Loss and Fasting. Metabolites were measured using a combination of liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) on PFKFB2 knockout (cKO) or control (CON) hearts (n=4–7). A, Metabolites which were significantly affected by fasting include Aspartate (LC-MS), Serine (GC-MS), Succinic Acid (GC-MS), and 3-Hydroxybutyrate (GC-MS). Data are shown as mean ± SD. B, Metabolites which are significantly correlated with cKO genotype (left, n=9–13) or fasted status (right, n=11) are shown, with those metabolites whose correlation achieves statistical significance of p<0.10 listed in tables below. C, Pathways correlated with cKO genotype or fasted status with statistical significance of p<0.05 are listed. D, Principal component analysis on metabolites with correlation significance of p<0.10 (n=4–7). Circles indicate fed status, squares indicate fasted status, grey indicates control, and red indicates cKO. A, Metabolites were analyzed by two-way ANOVA with Sidak’s post hoc test for multiple comparisons between groups which varied by a single factor. *p≤0.05, **p≤0.01, ***p≤0.001,…

• 

  Figure 3.. O-GlcNAcylation is increased in fed, but not fasted, cKO hearts, suggesting substrate-level regulation. Glutamine-fructose 6-phosphate amidotransferase (GFAT1), O-GlcNAc transferase (OGT), O-GlcNAcase (OGA (MGEA5)), and overall protein O-GlcNAcylation were measured by western blot, using heart homogenate from fed and fasted, control (CON) and PFKFB2 knockout (cKO) hearts (n=7–9). O-GlcNAc levels on western blot were measured using the average of signals from two different pan-specific antibodies targeting the modification (mouse monoclonal (RL2 clone) and rabbit monoclonal (R mAb)), and quantified either as the full lane of proteins (left) or only proteins which are 50 kDa or larger (right). O-GlcNAcylation of specificity protein 1 (Sp1) was measured by immunoprecipitation of Sp1, subsequently blotting for both Sp1 and O-GlcNAc (RL2 antibody), and taking the quotient. Sp1 was quantified directly, and the ratio of RL2 signal/Sp1 signal was used to determine the relative O-GlcNAcylation of Sp1 (n=5). Data were analyzed by two-way ANOVA with Sidak’s post-hoc test for multiple comparisons between groups which differed by one factor. *p≤0.05, ***p≤0.001, ****p≤0.0001. Data are shown…

• 

  Figure 4.. Loss of cardiac PFKFB2 enhances insulin signaling and glucose utilization in the fasted state. A-D, Relative abundance of total Akt (A), phosphorylated Akt (p-Akt, B), and the ratio of p-Akt/Akt (C) were measured by western blot in fed and fasted PFKFB2 knockout (cKO) and control (CON) hearts (n=13–16). A sample blot is shown with p- Akt in green and total Akt in red (D). E, Blood glucose was measured immediately prior to sacrifice (n=5–7). F, Plasma insulin was measured from blood collected by cardiac puncture. G, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated from blood glucose and plasma insulin measures (n=5–7). H-I, Total Akt was measured by western blot in mitochondrial fraction (n=7–9). Data are shown as mean ± SD. All data were analyzed by two-way ANOVA with Sidak’s post-hoc test for multiple comparisons between groups varying by one factor. *p≤0.05, **p≤0.01, ***p≤0.001.

• 

  Figure 5.. Acute stimulant stress promotes differential systemic glucose homeostasis in cKO mice. A, In the first group of mice, blood glucose was measured in the fed state. Mice were then fasted for 12 hours, after which blood glucose was measured a second time (fasted, pre-stress). Following the second blood glucose measurement, a stimulant stress of caffeine and epinephrine was injected intraperitoneally. 20 minutes after injection, a third blood glucose measurement was acquired (fasted, post-stress). B-C, Average blood glucose levels across fed, fasted pre-stress, and fasted post-stress states (B) are plotted alongside individual trends (C) in fed and fasted CON and cKO (n=5–9). D-E, Change in blood glucose was calculated, first with fasting alone (D) and then with both fasting and stimulant stress (E, n=5–9). F, In another group of mice, blood glucose was measured in the fed state. The stimulant stress of caffeine and epinephrine was then administered to fed mice and blood glucose was measured again 20 minutes post-injection. G, Change in blood glucose with stimulant stress in fed or fasted, CON or cKO mice is shown (n=5–10). H-I, Relative liver glycogen (H) and liver triglycerides…

• 

  Figure 6.. Stimulant stress drives increased cardiac O-GlcNAcylation in CON and cKO mice. Relative levels of protein O-GlcNAcylation of proteins 50 kDa or larger were measured using a pan-specific antibody in stressed (caffeine and epinephrine) and baseline (unstressed), fed and fasted PFKFB2 knockout (cKO) and control (CON) hearts (n=5). Data are shown as mean ± SD and were analyzed by three-way ANOVA with Sidak’s post-hoc test for multiple comparisons. *p≤0.05, **p≤0.01, ****p≤0.0001.