Biochemical Pharmacology

Supplementary data tables to paper "MiR-155 and other microRNAs downregulate drug metabolizing cytochromes P450 in inflammation" by Kugler et al., Biochemical Pharmacolgy, 2019

Gckr-P446L mice of PP, PL and LL genotypes were fed on a high-fat high-sugar diet (HFHSD) without (-, open bar) or with (+, filled bar) 3 mg/kg AZD1656 for 20 wk. A) Body weight at the start and end of the 20 wk study. B) Lack of effect of AZD1656 on Gckr mRNA and Gck mRNA (ratio to 18S). C) Liver GKRP and GK Immunostaining in 8 wk old P446L mice on regular diet (RD): showing no nuclear GKRP staining and cytoplasmic GK distribution in LL mice. D) Representative immunoblots for GKRP and GK in P446L mice on either RD or after 20 wk on HFHSD. E) GKRP immunoactivity on RD or after 20 wk HFHSD study showing lower protein in LL mice. F) GK immunoactivity on RD or after 20 wk HFHSD study showing lower protein in LL mice. G) GK/GKRP immunoactivity on RD or after 20 wk HFHSD showing higher GK/GKRP in LL mice. H) Representative GKRP and GK immunostaining after 20 wk HFHSD. I-L) Nuclear (N-H) and cytoplasmic (C-H) H-scores for GKRP and GK on RD and at the end of the 20 wk study (n=4) I) Lower GKRP N-H scores in LL mice. J) AZD1656 decreased GK N-H scores in LL mice. K) AZD1656 increased GK C-H scores in PP (wild-type) mice. L) AZD1656 decreased GK nuclear distribution (N-H/C-H) in LL mice. M) AZD1656 increased liver GK activity in PL and LL mice. *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

Experimental details were as in Figure-1. A) Blood triglycerides and cholesterol after 20 wk on HFHSD showing increased cholesterol (total and LDL) by LL genotype and by AZD1656 treatment in PP but not LL mice. B) Lack of effect of AZD1656 on liver triglyceride and cholesterol and liver enzymes (ALT, alanine aminotransferase; AST, aspartate aminotransferase) in plasma. Decrease in plasma inorganic phosphate by genotype but not by AZD1656. C) Lack of effect of AZD1656 on liver steatosis and fibrosis scores in PP and LL mice. *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

Gckr-P446L mice of PP, PL and LL genotypes were fed on a high-fat high-sugar diet (HFHSD) without (-, open bar) or with (+, filled bar) 3 mg/kg AZD1656 for 20 wk. A) Body weight at the start and end of the 20 wk study. B) Lack of effect of AZD1656 on Gckr mRNA and Gck mRNA (ratio to 18S). C) Liver GKRP and GK Immunostaining in 8 wk old P446L mice on regular diet (RD): showing no nuclear GKRP staining and cytoplasmic GK distribution in LL mice. D) Representative immunoblots for GKRP and GK in P446L mice on either RD or after 20 wk on HFHSD. E) GKRP immunoactivity on RD or after 20 wk HFHSD study showing lower protein in LL mice. F) GK immunoactivity on RD or after 20 wk HFHSD study showing lower protein in LL mice. G) GK/GKRP immunoactivity on RD or after 20 wk HFHSD showing higher GK/GKRP in LL mice. H) Representative GKRP and GK immunostaining after 20 wk HFHSD. I-L) Nuclear (N-H) and cytoplasmic (C-H) H-scores for GKRP and GK on RD and at the end of the 20 wk study (n=4) I) Lower GKRP N-H scores in LL mice. J) AZD1656 decreased GK N-H scores in LL mice. K) AZD1656 increased GK C-H scores in PP (wild-type) mice. L) AZD1656 decreased GK nuclear distribution (N-H/C-H) in LL mice. M) AZD1656 increased liver GK activity in PL and LL mice. *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

Experimental details were as in Figure-1. A) Blood triglycerides and cholesterol after 20 wk on HFHSD showing increased cholesterol (total and LDL) by LL genotype and by AZD1656 treatment in PP but not LL mice. B) Lack of effect of AZD1656 on liver triglyceride and cholesterol and liver enzymes (ALT, alanine aminotransferase; AST, aspartate aminotransferase) in plasma. Decrease in plasma inorganic phosphate by genotype but not by AZD1656. C) Lack of effect of AZD1656 on liver steatosis and fibrosis scores in PP and LL mice. *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

Gckrwt/wt and Gckrdel/wt were fed on a HFD without (open bar) or with (shaded bar) 3 mg/kg AZD1656 for 16 wk. A) Representative liver GKRP immunostaining of Gckrwt/wt, Gckrdel/wt and Gckrdel/del genotypes on regular diet. B) Body weight after 16 wk on HFD -/+ AZD1656. C) Representative liver immunostaining for GKRP and GK at the end of the study on HFD -/+ 3 AZD1656. D) GKRP N-H scores; GK N-H scores, GK C-H scores and GK N-H/C-H ratio showing lower GKRP and GK nuclear staining and greater cytoplasmic GK distribution by del/wt genotype. E) GKRP-immunoactivity by Western blot showing more modest GKRP deficiency in del/wt genotype (30%) than in LL (80%) genotype. F) GKRP and GK immunoactivity after 16 wk study in Gckrwt/wt showing an increase in GK / GKRP immunoactivity ratio by AZD1656 treatment. G) GKRP and GK immunoactivity after 16 wk study in Gckrdel/wt mice showing an increase in GK and GK / GKRP immunoactivity ratio by AZD1656 treatment. H) Lower liver GK activity in the Gckrdel/wt mice and increase by AZD1656 treatment. *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

Gckrwt/wt and Gckrdel/wt were fed on a HFD without (open bar) or with (shaded) 3 mg/kg AZD1656 for 16 wk. A) Lowering of blood glucose by AZD1656 after 4 wk and 14 wk in Gckrwt/wt and Gckrdel/wt mice. B) Higher plasma insulin by Gckrdel/wt genotype at 4 wk and 14 wk. C) Impaired glucose tolerance in AZD1656-treated Gckrdel/wt mice. D) Increased plasma FFA by AZD1656 treatment in Gckrdel/wt mice. E) Increased plasma cholesterol by AZD1656 treatment in Gckrdel/wt mice. F) Increased liver / body weight ratio by AZD1656 treatment in Gckrdel/wt mice. G) Increased liver triglyceride by AZD1656 treatment in Gckrdel/wt mice. H) Higher hepatocyte steatosis, microvesicular steatosis and fibrosis scores by AZD1656 treatment in Gckrdel/wt mice. I) Representative haematoxylin and eosin (H&E) and Sirius red fast green (SRFG) images for histopathology scores, scale bar 200µm. J) Liver mRNA levels (RT-qPCR) for the genes indicated expressed as a ratio to RplpO showing increased expression of Fgf21, Cidea and Cidec by AZD1656 treatment in the Gckrdel/wt mice: n= 10 and 11 for Gckrwt/wt (-/+) and n= 6 and 10 for Gckrdel/wt (-/+) *P < 0.05 effect of AZD1656; #P < 0.05 effect of genotype.

A) Venn diagram for differentially expressed genes (DEGs adjusted P < 0.05) by AZD1656 treatment: PP- vs PP+ (26 DEGs) and LL- vs LL+ (13 DEGs); or by genotype PP- vs LL- (150 DEGs), PP+ vs LL+ (25 DEGs). Blue, common genes similar direction; red, common genes opposite direction. B) Ingenuity Pathway Analysis: showing top enriched pathways by genotype (PP-vs LL-) or by AZD1656 treatment (PP-/PP+ or LL-/LL+) showing Z-scores for pathway flux and –log (P-value). C-E) Genes that are differentially expressed by PP- vs LL- genotype show smaller but directionally similar changes by AZD1656 treatment in PP mice. Z-scores for gene counts and median and range of genes shown. **P<0.001, *P<0.02 effect of AZD1656; ##P<0.0001 genotype effect, one-way ANOVA. C) Z-scores for DEGs by PP>LL genotype (150 DEGs adjusted P < 0.05) showing relative gene counts for the 4 groups of mice (PP-, PP+, LL-, LL+) of the upregulated DEGs (n=49, upper panel) and downregulated DEGs (n=101). D) Z-scores of gene counts of GK and GKRP-responsive genes that are significantly elevated by LL genotype. E) Z-scores of gene counts of cholesterol homeostasis linked genes that are significantly up-regulated or down-regulated by LL genotype. F) Correlation of phenotypic traits (liver weight, liver cholesterol, liver triglyceride, blood glucose and insulin and blood triglycerides and blood cholesterol Total, LDL< HDL) with corresponding gene counts of GK-GKRP responsive genes identified in [35] and cholesterol homeostasis linked genes.

A) Gckrdel/wt study: body weight and insulin tolerance test, blood glucose after insulin challenge and area under the curve (AUC) performed after 12-wk of HFD -/+ AZD1656 (3 mg/kg body wt) in mice aged 30 wk. B) Gckr:P446L study: body weight and insulin tolerance test performed after 12 wk of HFHSD -/+ AZD1656 (3 mg/kg body wt) in mice were aged 20 wk. * P < 0.05 effect of AZD1656.

Experimental details were as in Figure 1. A) Similar blood glucose by genotype at the start (0 wk) of the 20 wk study and sustained blood glucose lowering by AZD1656 (3mg/kg) after 19 wk compared to 2 wk in wild-type (PP) but not in LL mice. B) Difference (delta ∆) in blood glucose by AZD1656 treatment after 2 wk (white stripe) and 19 wk (grey stripe) relative to start of study (0 wk) showing lower efficacy at 19 wk in LL mice. C) Lack of effect of AZD1656 on plasma insulin. D) Glucose tolerance test after 2h food withdrawal determined at 18wk: glucose excursion and area under the curve (AUC). E) Representative immunostaining of pancreas for insulin, glucagon and GK after 20 wk HFHSD -/+ AZD1656 in PP and LL mice. F) Pancreatic islet % area determined from insulin staining. G) Islet GK-staining intensity showing higher intensity by AZD1656 treatment in wild-type, PP mice. *P < 0.005 effect of AZD1656; #P < 0.05 effect of genotype; $P < 0.01; $$ P< 0.005 19 wk versus 2 wk.