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. 2020 Aug 4;10:13110. doi: 10.1038/s41598-020-69901-3

Figure 2.

Figure 2

Microvilli of canaliculae in livers are unchanged upon induction of PI3K/Akt signalling. (a,b) Immunoblotting analyses reveal a sustained activation of pAkt signalling in perfused livers treated with 100 nM insulin (a). Quantitative analyses show the pAkt/Akt ratios over time normalised to control (0 min) (b). Note that also perfusions with buffer already led to a minor and transient activation of Akt signalling (presumably due to mechanostress) but that the insulin-induced effects were about 2–4 times as large and led to readily detectable, sustained pAkt immunosignals. Inset shows a comparison of pAkt/Akt ratios at 60 min using an adapted axis to visualise the prolonged pAkt signalling. n = 4 assays. (c,d) Scanning EM micrographs of WT liver tissue broken by shock-freezing in liquid nitrogen at low (c) and medium magnification (d; enlargement of boxed area in c). Note that low magnifications (c) provide an excellent overview over the liver tissue. Arrows mark examples of canaliculae broken open, asterisks mark examples of sinusoids (*) broken open and the cross marks a cross-break through a large blood vessel (+). At medium magnification (d), more delicate cellular structures, such as fenestrations in the sinusoids and microvilli, become visible and microvilli protruding into the Disse space (examples marked with “D”) can clearly be distinguished from canalicular microvilli (arrows), which were quantitatively evaluated. Bars, 20 µm (c); 2 µm (d). (e–g) Blinded, quantitative evaluations of canalicular microvilli diameter (e), length (f) and density (g) in WT mouse livers perfused with 100 nM insulin in KHB (grey columns) and KHB (Krebs Henseleit buffer) (control; white columns), respectively. n = 4 livers/condition à 12 pictures each; n = 48 canalicular regions of interest (ROIs) per condition (g) and n = 60 microvilli (e,f), respectively. Data, mean ± SEM. Unpaired t test and 2way ANOVA + Bonferroni’s test (b,e–g). *P < 0.05; ****P < 0.0001. For P < 0.0001, exact P values are not available. Other P values always are presented in the figures.