Fig. 2: period mutants exhibit high metabolic rate due to mitochondrial uncoupling.

Relative to controls (gray), per⁰¹ mutants (green) exhibited: a increased feeding rate (n = 6 vials of ten flies/condition, p < 0.01); b decreased survival upon starvation (n ≥ 99 flies per condition, p < 0.001); c lower baseline levels of lipids (left) and increased rate of lipid utilization after 24 h of starvation (right), as shown by quantification of triacylglyceride (TAG) levels (n ≥ 4 samples/condition, 5 flies/sample, both p < 0.0001); d increased respiration, which was reverted by ubiquitous overexpression of Per during adulthood (n = 6 groups of 10 flies per condition); e higher CO₂ production over the circadian day (n = 6 groups of 10 flies/condition and timepoint); f higher respiration rates after 24 h of feeding with rotenone and oligomycin, but not with 2,4-DNP, or stearic acid (n ≥ 5 groups of 10 flies/condition); g increased oxygen consumption rate when stimulated through complexes I, II, and IV relative to controls (n = 4–6 oxygraph runs per condition); h increased leak respiration, using high-resolution respirometry on purified mitochondria (n = 5 oxygraph runs per condition, p < 0.001); i lower membrane potential, measured by JC-1 staining of purified mitochondria (n = 10 mitochondrial preps per condition, p < 0.001); and j faster recovery from cold shock (n = 26–30 flies per condition, p < 0.001). See Supplementary Information for n if not listed here; ^n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; p values were obtained by unpaired two-tailed t-test (a, c, e, g–i), ANOVA followed by Tukey’s post-hoc test (d, f), and log-rank analysis (b, j); error bars represent SEM.