MolMet Figure1: P446>L substitution in human or mouse GKRP compromises protein expressivity and GK nuclear sequestration

Hepatocytes isolated from GKRP-deficient mice were treated with adenoviral vectors (at 5x10⁶ or 10x10⁶ pfu/ml) to express human or mouse GKRP:446P/L (hP, hL, mP, mL) and where indicated with a vector to overexpress GK (-/+ GKOE: D,F,I,J,L-N) and cultured for 24-30h. Inhibitors (CX, AGK2: C-E) were added for the last 6h culture.

A) GKRP-deficient hepatocytes transfected with human GKRP:446P at 5x10⁶ or 10x10⁶ have comparable GKRP immunoactivity, lower endogenous GK and higher GKRP/GK immunoactivity ratios than wild-type hepatocytes. Means ± SEM, n=5, *P<0.05.

B) Lower expressivity of GKRP:446L variant (hL, mL vs hP, mP) in transfections at 5x10⁶ or 10x10⁶ pfu/ml is more prominent at the higher titre, n=4, #P<0.05 446L vs 446P.

C) Lower protein stability of human compared with mouse GKRP after incubation with cycloheximide (CX). Pooled data of transfections with 5x10⁶ and 10x10⁶ pfu/ml and cultured for 24h followed by 6h culture -/+ 10 μM CX, n=5, *P< 0.05 effect of CX; #P<0.05, 446L vs 446P.

D) GK overexpression by ~2-fold relative to endogenous GK promotes stabilization of human GKRP:446P/L and mouse GKRP:446L. Means ±SEM, n=5 hepatocyte preparations; pooled data of 5x10⁶ and 10x10⁶ pfu/ml transfections. *P<0.05 effect of GKOE; #P<0.05, 446L vs 446P.

E) Stabilization of GKRP:446L by a Sirt2 inhibitor (10 μM AGK2) in transfections at 5 x10⁶ pfu/ml. Means ± SEM, n=3, *P< 0.05 effect of AGK2; #P<0.05, 446L vs 446P.

F) GKRP and GK immunostaining in transfections with human (hP, hL) or mouse (mP, mL) GKRP at 10x10⁶ pfu/ml at endogenous GK (upper panels) or with GK overexpression (+GKOE).

G) Lower nuclear sequestration (N/C, nuclear/cytoplasmic ratio) of GKRP:446L in hepatocytes expressing endogenous GK, n=15-16 hepatocyte experiments.

H) Greater nuclear sequestration of endogenous GK with human compared with mouse 446P. n=7; #P<0.05, 446L vs 446P; $P<0.05 human vs mouse (G,H).

I) Nuclear sequestration of GKRP:446P or 446L is increased by GKOE, n=7.

J) Nuclear sequestration of GK during GKOE is lower with GKRP:446L, n=6; #P<0.05, 446L vs 446P; *P<0.05 effect of GKOE (I,J).

K-M) Comparison of GKRP expressivity (N+C intensity) and nuclear GKRP sequestration (N/C ratio), in a representative experiment (-/+ GKOE), with data points representing individual hepatocytes (K,L) or mean value of fields (M,N). $ P<0.05 human vs mouse; #P<0.05, 446L vs 446P; *P<0.05, 10x10⁶ vs 5x10⁶ pfu/ml; ~P<0.05 effect of GKOE (M,N).

K) At endogenous GK, with GKRP expressed at 5x10⁶ or 10x10⁶ pfu/ml, expressivity of GKRP:446L (N + C), was compromised at 10x10⁶ pfu/ml, but nuclear sequestration (N/C) of 446L was lower at both 5x10⁶ or 10x10⁶ pfu/ml.

L) With GKOE, GKRP:446L expressivity was not compromised at 10x10⁶ pfu/ml and GKRP:446L N/C was lower at both titres.

M) GKRP N/C summary for (K)-(L): showing greater N/C for human vs mouse GKRP ($) and lower N/C for 446L (#).

N) GK N/C summary showing greater N/C for human vs mouse and lower N/C for 446L vs 446P (#) with GKOE.