Cytochrome P450 1B1: An unexpected modulator of liver fatty acid homeostasis.

Cyp1b1-ko mice exhibit decreased adiposityA. Experimental design to determine effect of Cyp1b1 deletion on diet responses.B. Average body weight (BW) gain in male and female C57BL/6j (WT) and Cyp1b1-ko (KO) mice for, respectively, 11 and 36 weeks post-weaning.C. Average BW of male and female WT and KO mice fed the HFD for 6, 11 and 36 weeks post-weaning. The bar graphs indicate the average BW ± SEM. (Male WT: n=20, 6-week; n=23, 11-week; n=11, 36-week. Male KO: n=4, 6-week; n=17, 11-week; n=6, 36-week. Female WT: n=13, 6-week; n=12, 11-week; n=3, 36-week. Female KO: n=7, 6-week; n=10, 11-week; n=3, 36-week.)D. Epididymal and inguinal fat pad mass in male and female WT and KO mice fed the LFD or HFD for 11 weeks post-weaning. Each dot represents an individual animal. The horizontal line represents the average fat pad mass of each group. (WT males: n=22 LFD, n=23 HFD. KO males: n=16 LFD, n=17 HFD. WT females: n=10 LFD, n=12 HFD. KO females: n=12 LFD, n=8 HFD.)*p<0.05, ** p<0.01.

Anomalous characteristics of adipose of Cyp1b1-ko miceA. Reformatted fat-only MRI images (coronal and sagittal reformats) of representative C57BL/6j (WT) and Cyp1b1-ko (KO) mice after 11 weeks on the HFD.B. H&E staining (HFD) and quantification of sub-dermal adipose tissue layers (HFD and LFD) from WT and KO mice after 11 weeks on the respective diets. The bar graph represents the average number of adipose layers ± SEM (WT: n=4 LFD and HFD; KO: n=2 LFD, n=4 HFD).C. H&E stained epididymal fat pad adipocytes of representative male WT and KO mice after 11 weeks on the LFD and HFD.D. Image J analysis of relative adipocyte area with correlation to fat pad mass. The bar graph represents the average area ± SEM of 50 adipocytes per image from 4 mice per strain/treatment group.*p<0.05, ** p<0.01.

Glucose levels and tolerance in Cyp1b1-ko mice: Effects of selective breeding and backcrossing with WT mice on HFD responsivenessA. Fasting blood glucose levels in C57BL/6j (WT) and Cyp1b1-ko (KO) male mice fed the LFD or HFD for 11 weeks. The bar graph represents the average fasting glucose level ± SEM of WT (n=4) and KO (n=3) mice under LFD and HFD conditions, respectively.B. Glucose tolerance testing (GTT) on WT and KO male mice fed the LFD or HFD for 11 weeks. The area under the curve (AUC) provides a measure of glucose sensitivity. The bar graph represents the average fasting glucose level ± SEM for WT mice under LFD (n=4) and HFD (n=3) conditions, respectively.C. GTT of WT and KO female mice fed the LFD or HFD for 33 weeks. The area under the curve (AUC) provides a measure of glucose sensitivity. The bar graph represents the average fasting glucose level ± SEM of WT (n=4) and KO (n=3) mice under LFD and HFD conditions, respectively.D. Breeding design used to restore the suppressed obesity phenotype in a colony of KO mice that had become suppression-resistant with prolonged inbreeding. Male and female KO mice from the colony with the lowest and highest BW responses to HFD, respectively, were bred and their progeny (S1&2, L1&2) were compared for HFD responsiveness. Resistant KO mice from the colony were also bred with WT mice to provide heterozygote progeny (Het). These mice were then bred with resistant KO mice. Progeny from het mothers (Hm) and het fathers (Hf) were compared for HFD responses.E. BW and non-fasting blood glucose levels of female WT, S1&2 and L1&2 selectively bred KO mice. The red dotted line corresponds to the mean BW for fully suppressed KO mice (Figure 1). Each dot represents the BW of an individual animal (WT, n=8; S1&2, n=8; L1&2, n=12). The bar graph represents the average non-fasting glucose level ± SEM for 9 mice in each of the three groups.F. BW and non-fasting blood glucose levels in female resistant (R) and mated het x KO mice (het mother, Hm; het father, Hf,). The red dotted line corresponds to the mean BW for fully suppressed KO mice (Figure 1). Each dot represents the BW of an individual animal (R, n=4; Hm, n=8; Hf, n=10). The bar graph represents the average non-fasting glucose level ± SEM of the mice presented in the dot plot. *Significantly different from WT, p<0.05; **Significantly different from WT, p<0.01.

Enhanced decline of serum leptin and body weight in Cyp1b1-ko mice during fasting. Obesity of leptin-deficient Cyp1b1-ko miceA. Experimental design of serum leptin and body weight (BW) evaluation.B. Ad libitum and fasting serum leptin levels in C57BL/6j (WT) and Cyp1b1-ko (KO) mice after 11 weeks on LFD or HFD. The bar graph represents the average leptin concentration ± SEM of WT (n=4) and KO (n=3) ad libitum fed and fasted (n=4 in each of the respective groups) mice.C. Correlation between ad libitum serum leptin level and epididymal fat mass (n=7 in each of the respective WT and KO groups).D. BW loss during acute fasting (9AM to 1PM). Each dot represents an individual animal. The horizontal line represents the average BW loss for WT (n=8 LFD, n=6 HFD) and KO (n=4 LFD and HFD) mice.E. Leptin-deficient (ob/ob) mice are resistant to the adiposity-suppression effects of Cyp1b1 depletion introduced by cross-breeding. Each dot represents an individual animal. The horizontal line represents the average epididymal fat pad mass for ob/ob (n=4) and ob/ob-ko (n=8) mice.*p<0.05, ** p<0.01.

Cyp1b1-ko mice exhibit decreased liver steatosis, but increased glycogen retentionA. Liver mass at 6, 11 and 36 weeks of HFD exposure in C57BL/6j (WT) and Cyp1b1-ko (KO) mice. The bar graphs indicate the average liver mass ± SEM for male WT (n=19, 6-week; n=22, 11-week; n=11, 36-week) and KO (n=4, 6-week; n=19, 11 week; n=6, 36-week) and female WT (n=13, 6-week; n=12, 11-week; n=4, 36-week) and KO (n=4, 6-week; n=10, 11-week; n=3, 36-week) mice.B. Oil Red O staining for hepatic sinusoidal lipid droplets in representative WT and KO mice fed the HFD for 11 weeks.C. Pas staining for hepatic sinusoidal glycogen granules in representative male WT and KO mice fed the HFD for 11 weeks (for expanded region see Figure S4A).D. H&E stained liver sections of representative female WT and KO mice after 11 and 36 weeks of HFD exposure. Increased lipid droplets in WT livers are shown by lipid vacuoles.*p<0.05, ** p<0.01.

Effects of constitutive PPARα suppression on liver gene expression in Cyp1b1-ko miceA. Functional association of Cyp1b1 deletion-responsive genes involved in fatty acid homeostasis, which are controlled either by leptin and AMPK or by PPARα activity.B. Expression of PPARα-regulated genes, Acot1 versus Cyp4a10, for individual mice from the 4 treatment groups can be highly correlated (n=13 mice).C. Effect of stimulation of PPARα activity by Wy-14,643 treatment (WT males, 48 hours, oral gavage) is inversely correlated with the effect of Cyp1b1 deletion on the constitutive expression of 23 genes shown in Table 3.D. PPARα-responsive gene stimulation (Vnn1 and Cyp4a14) is retained in KO mice treated with Wy-14,463. WT and KO (n=2) mice were treated with Wy-14,463 for 48 h.

Real-time PCR quantification and correlation of microarray gene expression of liver genes that control triglyceride and glycogen levelsA. Correlation of adropin and Cyp7b1 gene expression in, respectively, male and female C57BL/6j (WT) and Cyp1b1-ko (KO) mice fed the LFD and HFD for 11 and 36 weeks, respectively (n=13 male and n=8 female mice).B. Real-time PCR (qPCR) quantification of the relative mRNA expression of Scd1, PPARγ and CD36 in livers from three C57BL/6j (WT) or Cyp1b1-ko (KO) male and female mice after 11 or 36 weeks of LFD or HFD exposure, respectively. The 11 week mRNA samples were those examined in the microarray analyses. Microarray and qPCR responses are also compared in Tables S4 and S5.C. qPCR quantification of the relative mRNA expression for three mice in each of the treatment groups of the glycogen synthase regulators, Ppp1r3c and Ppp1r3g.*p<0.05, ** p<0.01.

Serum lysophosphatidylcholine (LPC) as a marker of obesity and PPARα activity. Potential role of PCTP in mediating the effects of Cyp1b1 deletion on triglyceride and LPC synthesisA. In the hepatocyte, the phosphatidyl choline (PC) transfer protein (PCTP) and its partnering protein, Acot13, mediates the transfer of PC between membranes and affects synthesis of PC, lysophosphatidyl choline (LPC) and triglyceride (TG) from acyl-CoA by decreasing mitochondrial oxidation. Direct interaction of PCTP with Acot13 on the outer mitochondrial membrane increases cleavage of acyl CoA, thus decreasing formation of acyl carnitines and transfer into the mitochondria and subsequent oxidation. Both PCTP and Acot13 are stimulated by PPARα [55] and fall within the group of genes (depicted in light green) that exhibit constitutive suppression in Cyp1b1-ko mice.B. The ratio of 18:0/16:0 LPC provides an optimal marker for suppression of DIO and PPARα activity in Cyp1b1-ko (KO) mice, relative to their C57BL/6j (WT) counterparts. Serum LPC levels were measured in the mice depicted in Figure 1. The 18:0/16:0 LPC ratio parallels the trend observed in BW gain and adiposity. The bar graph represents the ratio of the average 18:0 concentration to the average 16:0 concentration ± SEM in the blood serum of WT (n=4 LFD, n=6 HFD) and KO (n=7 LFD, n=6 HFD) mice.C. Serum 18:1 LPC levels decline in WT mice fed a HFD for 11 weeks, and are further attenuated with Cyp1b1 deletion. The bar graph represents the average 18:1 LPC serum concentration ±SEM in WT (n=4 LFD, n=5 HFD) and KO (n=7 LFD, n=6 HFD) mice.D. Hepatic Scd1 expression parallels serum 18:1 LPC levels. The bar graph represents the average hepatic Scd1 mRNA expression level ±SEM in WT (n=11 LFD, n=13 HFD) and KO (n=12 LFD, n=9 HFD) mice.E. Hepatic Pctp gene expression, measured by microarray analysis, parallels serum 18:0 LPC levels [38]. The bar graph represents the average hepatic Pctp mRNA expression level ±SEM in WT (n=3 LFD and HFD) and KO (n=4 LFD, n=3 HFD) mice.F. Hepatic Pctp gene expression is highly correlative with adiposity. Data represents the compilation of WT and KO mice across all treatment groups (n=13 mice).*p<0.05, ** p<0.01.

Representation of proposed systemic effects of Cyp1b1 deletion that increase effects of leptin and growth hormone on the liverCyp1b1 deletion effects on liver gene expression indicate increased leptin and growth hormone action, each of which are regulated in the hypothalamus. Hypothesis: Cyp1b1 deletion targets two important activities A) The endothelial-pericyte niche (EPN), which provides local regulation of adjacent cells, is disrupted in Cyp1b1-ko mice, B) Estradiol (E2) levels are increased, particularly in the hypothalamus, when local removal of E2 (produced by aromatase) via Cyp1b1 metabolism is decreased. Increased transfer of leptin across the blood brain barrier (BBB) increases activation of sympathetic neurons via the ventromedial hypothalamus (VMH) and release of GH releasing hormone (GHRH) from the arcuate nucleus (AN), which stimulates pulsatile release of GH from the pituitary [21, 101]. Similar EP niche activity functions in liver sinusoids (endothelia and stellate pericytes) [71] and adipose [32] to provide local controls over adjacent hepatocytes and adipocytes, respectively.