Increased Lipid Deposition in the Livers of STAT6 Knock-out Mice

Hepatocytes fulfill a complex metabolic role both in lipid and glucose homeostasis.

The proteome of the livers of the STAT6 knock-out mice revealed differences in the expression levels of several enzymes involved in both processes. The functions of the different lipid and glucose metabolic enzymes identified by the proteomic comparison and discussed in the following two chapters are summarized in Figure 8.

Liver fatty acid binding protein (FABP1) is involved in the uptake, intracellular established by direct quantification of liver lipid content which was significantly increased in the knock-out mice (Fig. 6b). Previous studies conducted in the STAT6-deficient mice failed to describe morphological signs of hepatic lipid accumulation when examined by hematoxylin-eosin staining (46). In accordance with these studies we found no gross structural alterations in similarly stained liver sections (Fig. 6c, upper panels). However, when applying the neutral lipid dye Oil-red-O, the generalized lipid deposition in the livers of the knock-out mice became evident (Fig. 6c, lower panels).

Increased lipid accumulation can reflect increased fatty acid uptake, an increase in the rate of fatty acid synthesis or a decrease in mitochondrial fatty acid β-oxidation. In contrast to the increased lipid storage we actually observed a decrease in the amount of ATP-citrate synthase (ACLY), an enzyme involved in the first step of fatty acid and cholesterol biosynthesis (Table I, #18). To better characterize the changes in fatty acid synthesis we assessed the mRNA expressions of two key regulatory lipogenic enzymes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), which are regulated at the transcriptional level. None of the two enzymes showed significant alterations in their mRNA levels confirming that the observed lipid accumulation is not due to enhanced fatty acid synthesis (Fig. 6d). Exogenous fatty acids taken up by the liver can be oxidized during starvation or be used for triglyceride synthesis in fed conditions. FABP1 has been demonstrated to regulate fatty acid uptake and esterification but also mitochondrial β-oxidation under starving

conditions when circulating fatty acid levels are high. Increased FABP1 levels in the STAT6 knock-out mice may therefore indicate increased triglyceride synthesis but also enhanced fatty acid β-oxidation. Indeed, we identified two β-oxidation-related enzymes whose expression was up-regulated: the medium-chain acyl-CoA synthetase (ACSM1) and the 3-ketoacyl-CoA thiolase (ACAA2) (Table II, # 17 and 24). Moreover, an increase in fatty acid β-oxidation is also indirectly suggested by the upregulation of the mitochondrial ATP synthase F1 complex (ATP5B) (Table II, # 22) involved in the oxidation of the NADH and FADH2 produced during this process and in the Kreb’s cycle. The rate limiting step of

β-oxidation is the acyl-CoA transport across the mitochondrial membrane by the carnitin-palmytoil CoA transferase 1 (CPT1). CPT1 mRNA levels were not different between wild type and knock-out mice suggesting that the increase in fatty acid oxidation was within the limits of physiological capacity of this transporter (Fig. 6d). Fatty acid uptake in the liver is elevated during starvation. This elevation leads to increased triglyceride synthesis but also to enhanced acetyl-CoA production which might saturate the Kreb’s cycle’s eliminating capacity. Accumulation of acetyl-CoA results in the formation of beta-hydroxybutyrate (BHB) and acetoacetate, commonly referred to as “ketone bodies”. In line with the similar mRNA expression levels of CPT1 we found identical BHB and acetoacetate levels in wild type and knock-out mice regardless if mice were fed or were challenged by overnight starving (Fig. 6e). This finding again suggests that the increased liver lipid content found in the STAT6 knock-out mice is the result of enhanced triglyceride synthesis and that the alterations in mitochondrial β-oxidation enzymes reflect an increase in fatty acid availability most likely due to the up-regulation of FABP1 expression.

iTRAQ analysis also revealed decreased expression of several enzymes involved in the cholesterol / bile acid synthesis pathway e.g. acetyl CoA acetyltransferase (ACAT2), farnesyl pyrophosphate synthetase (FPP Synthase) and cysteine sulfinic acid decarboxylase (CSAD) (Table I, #17, 14 and 20). In accordance with the decreased expression of these proteins we found diminished mRNA expression of CYP7A1, the rate-limiting enzyme of bile acid synthesis in rodents (Fig. 6d).

Liver plays a crucial role in the metabolism of bile acids and heme, the major functional component of hemoglobin. Bile acids are conjugated with taurin or glycin, while the degradation product of heme, bilirubin, is conjugated with glucorinic acid. The livers of STAT6 knock-out mice showed decreased expression of two key enzymes of these two

conjugating pathways: the cysteine sulfinic acid decarboxylase (CSAD) and the UDP-glucose 6-dehydrogenase (UGDH) (Table I, #20 and 13). The decreased expression of CSAD is in line with the decreased expression of several enzymes of the cholesterol/bile acid synthesis pathway. The physiological importance of the observed decrease in UGDH expression was reflected by a significant decrease in the ratio of conjugated bilirubin in the STAT6 knock-out mice (Fig. 6f).

FIG. 6. Changes in liver lipid homeostasis in STAT6 knock-out mice (see next page). A. mRNA expression of the Fatty Acid Binding Protein 1 (FABP1). Each bar represents the average mRNA level expressed as normalized to the mean of the controls ±S.E.M.; n=5, *=p≤0.05. B. Total liver chloroform/methanol-extractable lipid content. n=9, *= p≤0.05. C. Hematoxylin-eosin (H.E.) (magnification 20x) and Oil red O (magnification 10x) staining of liver sections. D. mRNA expression of enzymes involved in lipid metabolism. Each bar represents the average mRNA level expressed as arbitrary units normalized to the mean of the wild type controls ± S.E.M. ACC: Acetyl CoA Carboxylase, FAS: Fatty Acid Synthase, CPT1:

Carnitin Palmoyltransferase 1, CYP7A1: Cytochrome 7A1. n=5, *=p≤0.01. E. Ketone body concentrations.

Each bar represents the mean values ±S.E.M. n= 9, ###=p≤0.001 fed vs. starved state in mice of the same genotype. HO-butyrate: hydroxybutyrate. F. Ratio of conjugated plasma bilirubin. Data are expressed as the mean ± S.E.M. n= 9, *= p≤ 0.05.