Extended Data Fig. 2: Protein kinases A and Cs are not activated by fasting in liver, and lipid-driven mTORC1/C2 activation is leptin, IGF-1 or insulin-independent.
From: mTORC2–NDRG1–CDC42 axis couples fasting to mitochondrial fission
(a) Experimental plan for generation of insulin-deficient diabetic mice using streptozotocin (STZ). (b) Serum insulin levels in 3 mo-old STZ-treated C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. N values for number of mice in each group are in parenthesis. Fed (n = 7); fed + STZ (n = 7); fed + corn oil (n = 8); fed + STZ + corn oil (n = 8); fasted (n = 8); fasted + STZ (n = 8); fasted + corn oil (n = 8); fasted + STZ + corn oil (n = 8). (c) Blood glucose levels 6 weeks after the first injection with STZ (n = 16 mice). (d) Representative IB, and (e-g) quantification for indicated proteins normalized to corresponding total protein in livers of 3 mo-old STZ-treated C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. For e, fed (n = 6); fed + STZ (n = 6); fed + corn oil (n = 6); fed + STZ + corn oil (n = 6); fasted (n = 5); fasted + STZ (n = 6); fasted + corn oil (n = 5); fasted + STZ + corn oil (n = 5). For f and g, all indicated groups consist of n = 5 mice each. (h, i) Serum (h) leptin and (i) IGF-1 levels in 3 mo-old C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. For h, fed (n = 8); fed + corn oil (n = 8); fasted (n = 6); fasted + corn oil (n = 9). For i, all indicated groups consist of n = 9 mice each. (j) IB and (k) quantification for the indicated proteins in livers of 2–10 mo-old mice fed or fasted for the indicated durations. N values for number of mice analyzed at each time-point for individual phosphoproteins are indicated in parentheses. P-PKAThr197/PKA: 0 h (n = 3), 3 h-20 h (n = 5); P-PKCα/βIIThr638/641/PKCα (all time-points are n = 5); P-PKCδThr505/PKCδ (all time-points are n = 5); P-PKCδ/θSer643/676/PKCδ (all time-points are n = 5); and P-PKCζ/λThr410/403/PKCζ: 0 h (n = 4), 3 h-20 h (n = 5). Ponceau is the loading control. Individual replicates and mean values are shown. *P < 0.05, **P < 0.01 and ****P < 0.0001, 2-way ANOVA followed by Tukey’s multiple comparisons test (b, e, f, h and i); two-tailed unpaired Student’s t-test (c). ns=not significant. Please refer to Supplementary Table 10_statistical summary. Source numerical data are available in SourceData_Table 1, and unprocessed blots are available in Source Data Extended Data Fig. 2.
