GROWTH HORMONE AND PPARα IN THE REGULATION OF GENES INVOLVED IN HEPATIC LIPID METABOLISM. Caroline Améen

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1 GROWTH HORMONE AND PPARα IN THE REGULATION OF GENES INVOLVED IN HEPATIC LIPID METABOLISM Caroline Améen Department of Physiology and Wallenberg Laboratory for Cardiovascular Research Sahlgrenska Academy at Göteborg University 2004

2 Caroline Améen A doctoral thesis at a university in Sweden is produced either as a monograph or as a collection of papers. In the latter case, the introductory part constitutes the formal thesis, which summarises the accompanying papers. These papers have already been published or are in manuscripts at various stages (in press, submitted or in manuscript). ISBN

3 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism The most exciting phrase to hear in science, the one that heralds new discoveries, is not Eureka! (I found it!) but rather, "hmm... that's funny..." ISAAC ASIMOV 3

4 Caroline Améen ABSTRACT GROWTH HORMONE AND PPARα IN THE REGULATION OF GENES INVOLVED IN HEPATIC LIPID METABOLISM. Caroline Améen, Department of Physiology and Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, SE Göteborg, Sweden (2004). Growth hormone (GH) plays a key role in the regulation of lipid and lipoprotein metabolism. Its sexually dimorphic secretory pattern regulates many sex-differentiated functions in the liver, such as triglyceride synthesis and VLDL secretion. GH also increases insulin secretion, and the importance of increased insulin levels for the effects of GH in vivo was therefore investigated in hypophysectomised (Hx) rats. GH increased the hepatic triglyceride secretion rate and triglyceride content, as well as fatty acid synthase (FAS), stearoyl-coa desaturase-1 (SCD-1) and sterol regulatory element binding protein-1c (SREBP-1c) mrna expression. Insulin suppressed the effect of GH on hepatic triglyceride secretion rate and content, but this was not through changed gene expression of lipogenic enzymes or microsomal triglyceride transfer protein (MTP), the rate-limiting protein in VLDL assembly. The regulation of lipogenic genes and MTP by the sex-differentiated GH secretory pattern was studied both in Hx rats administered GH in a mode that mimics either the female or male plasma pattern of GH, and in intact males feminised with respect to the plasma pattern of GH. SREBP-1c, FAS, glycerol-3- phosphate acyltransferase (GPAT) and MTP levels were higher in females compared to males and specifically upregulated by the female-like GH plasma pattern in Hx rats. The expression of SCD-1 mrna was not sex-differentiated and increased by GH irrespective of administration mode. Only FAS and GPAT mrna levels were increased in males with feminised GH plasma pattern, possibly due to decreased insulin sensitivity. Increased expression of FAS, GPAT and MTP could therefore help to explain the previously described stimulatory effects of female sex and GH secretory pattern on VLDL assembly and secretion. Hepatic MTP expression and activity were also increased by the peroxisome proliferator-activated receptor α (PPARα) agonist WY 14,643 (WY), both in vivo (mice and rats) and in vitro (primary mouse and rat hepatocyte cultures). The increase in MTP expression was paralleled by a change in apob-100 secretion, which shows that the stimulatory effect of WY on apob-100 secretion could be mediated by MTP. Key words: Growth hormone, insulin, SREBP-1, lipogenic enzymes, microsomal triglyceride transfer protein, PPARα, LXRα, triglycerides, liver. 4

5 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat. Fredrik Frick*, Daniel Lindén*, Caroline Améen, Staffan Edén and Jan Oscarsson. Am J Physiol Endocrinol Metab 283: E1023-E1031, II. III. IV. Effects of gender and growth hormone secretory pattern on sterol regulatory element binding protein-1c and its downstream genes in rat liver. Caroline Améen, Daniel Lindén, Britt-Mari Larsson, Agneta Mode, Agneta Holmäng and Jan Oscarsson. Submitted. Sex difference in hepatic microsomal triglyceride transfer protein expression is determined by the growth hormone secretory pattern in the rat. Caroline Améen and Jan Oscarsson. Endocrinology 144(9): , PPARα activation increases microsomal triglyceride transfer protein expression and activity in the liver. Caroline Améen, Ulrika Edvardsson, Anna Ljungberg, Lennart Asp, Anna Tuneld, Sven-Olof Olofsson, Daniel Lindén and Jan Oscarsson. Submitted. *Both authors contributed equally to this article. 5

6 Caroline Améen ABBREVIATIONS ACC acetyl-coa carboxylase Apo apolipoprotein APOBEC-1 apob mrna-editing enzyme catalytic peptide-1 bhlh-zip basic helix-loop-helix leucine zipper BMI body mass index CETP cholesterol ester transfer protein Cis cytokine-inducible SH2-containing protein CPT-I carnitine palmitoyl transferase-i DGAT diacylglycerol acyltransferase DR direct repeat ER endoplasmic reticulum FAS fatty acid synthase FFA free fatty acid GH growth hormone GHBP growth hormone-binding protein GHRH growth hormone-releasing hormone GPAT glycerol-3-phosphate acyltransferase HDL high-density lipoprotein HL hepatic lipase HMG-CoA 3-hydroxy-3-metylglutaryl-CoA HNF hepatocyte nuclear factor Hx hypophysectomy/hypophysectomised IDL intermediate-density lipoprotein IGF-I insulin-like growth factor-i IRS insulin receptor substrate JAK janus kinase LCAT lecithin-cholesterol acyltransferase LDL low-density lipoprotein LPL lipoprotein lipase LRP LDL receptor-related protein receptor LXR liver X receptor MAPK mitogen-activated protein kinase MTP microsomal triglyceride transfer protein PDI protein disulfide isomerase PLA 2 phospholipase A 2 PPAR peroxisome proliferator-activated receptor PPRE peroxisome proliferator response element PUFA polyunsaturated fatty acid 6

7 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism RXR retinoic X receptor S1P site-1 protease S2P site-2 protease SCAP SREBP cleavage-activating protein SCD stearoyl-coa desaturase SEM standard error of the mean SOCS suppressors of cytokine signalling SR-BI scavenger receptor class B type I SRE sterol regulatory element SREBP sterol regulatory element binding protein STAT signal transducer and activator of transcription T4 thyroxine VLDL very-low density lipoprotein WY WY 14,643 7

8 Caroline Améen TABLE OF CONTENTS ABSTRACT LIST OF PUBLICATIONS... 5 ABBREVIATIONS TABLE OF CONTENTS... 8 INTRODUCTION.. 10 GENERAL INTRODUCTION. 10 LIPOPROTEIN METABOLISM Lipoproteins Exogenous lipoprotein pathway. 11 Endogenous lipoprotein pathway.. 12 Atherogenic dyslipidemia 13 APOB AND VLDL ApoB 14 VLDL assembly. 15 MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN 16 Regulation of MTP 17 STEROL REGULATORY ELEMENT BINDING PROTEINS 17 The two-step cleavage process of SREBPs.. 18 SREBP-1c and regulation of lipogenesis. 19 LIPOGENIC ENZYMES Acetyl-CoA carboxylase.. 19 Fatty acid synthase Stearoyl-CoA desaturase. 20 Glycerol-3-phosphate acyltransferase.. 21 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS PPARα and lipid metabolism. 22 GROWTH HORMONE.. 22 The GH secretory pattern in females and males. 23 GH receptor and GH signalling. 24 Influence of the GH secretory pattern on GH signalling.. 25 Insulin-like and diabetogenic effects of GH 25 GH and lipoprotein metabolism in humans. 26 GH and lipoprotein metabolism in laboratory animals 26 8

9 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism AIMS OF THE THESIS 28 METHODOLOGICAL CONSIDERATIONS 29 ANIMALS Hypophysectomised rats.. 29 HORMONAL TREATMENT.. 29 Thyroxine (T4) Glucocorticoids. 29 Growth hormone Insulin. 31 Sex steroids 31 HEPATOCYTE CULTURES SUMMARY OF RESULTS PAPER I.. 34 PAPER II. 35 PAPER III PAPER IV GENERAL DISCUSSION REGULATION OF LIPOGENESIS AND VLDL SECRETION BY GH.. 39 REGULATION OF LIPOGENESIS AND VLDL SECRETION BY INSULIN.. 41 REGULATION OF SREBP-1c AND LIPOGENIC ENZYMES 43 REGULATION OF MTP - POSSIBLE MECHANISMS OF GH ACTION 44 IMPORTANCE OF CHANGED MTP LEVELS FOR THE EFFECTS OF PPARα. 46 MTP AND DIFFERENT REGULATION OF APOB-48 AND APOB SUMMARY AND CONCLUSIONS 49 ACKNOWLEDGEMENTS REFERENCES

10 Caroline Améen INTRODUCTION GENERAL INTRODUCTION Obesity and type 2 diabetes are major health problems in Western society. They increase in prevalence in both developed and developing countries, and are major risk factors for cardiovascular disease (CVD). One key feature of these conditions is abnormal lipid and lipoprotein metabolism. Growth hormone (GH) is known to play an important role in the regulation of lipoprotein metabolism, and both GH deficiency and GH excess are associated with a 50% increased risk for CVD [1, 2]. The secretion of GH from the pituitary is moreover altered in both obesity and type 2 diabetes [3], indicating changed GH action in these conditions. Knowledge about the importance of changed GH secretion in the regulation of lipid and lipoprotein metabolism is therefore crucial to better understand the development of dyslipidemia and ultimately CVD. LIPOPROTEIN METABOLISM Lipoproteins Lipids play an essential role in energy metabolism, and their transport between tissues is therefore important to maintain energy balance in the body. As lipids are highly hydrophobic molecules with limited water solubility, they are transported in plasma associated with other molecules. Free fatty acids are bound to albumin, while larger lipids are circulating in plasma bound to lipoproteins. Lipoproteins are spherical watersoluble particles consisting of lipids and specialised proteins called apolipoproteins. Their ability to carry lipids in aqueous surroundings is due to their amphipathic nature, characterised by a hydrophilic outside and a hydrophobic inside. The inner core mainly consists of triglycerides and cholesterol esters, and is surrounded by a monolayer of phospholipids and unesterified cholesterol in which the apolipoproteins are dispersed. There are many different types of apolipoproteins; the major ones being apolipoprotein (apo) A-I, A-II, apob-48, apob-100, apoc-i, apoc-ii, apoc-iii and apoe. The apolipoproteins are important for the structural integrity of the lipoprotein (e.g. apob), but they also function as enzyme activators (e.g. apoa-i and apoc-ii) and receptor ligands (e.g. apob-100 and apoe). There are several distinct lipoprotein types with characteristic lipid and apolipoprotein composition, and they are most often classified in terms of their density. In increasing order of density, the major classes of lipoproteins are: chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL) and high-density lipoprotein (HDL) particles (Table 1). Since lipids have a lower density than proteins, the density of the lipoprotein particle is inversely related to its lipid content. The two classes of lipoproteins with the lowest density, i.e. chylomicrons and VLDL, are therefore 10

11 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism triglyceride-rich particles, while the lipoprotein particles with higher density, i.e. IDL, LDL and HDL, contain less triglycerides and are mainly carriers of cholesterol esters. An alternative way of classifying lipoproteins is to divide them into apob-containing (chylomicrons, VLDL, IDL and LDL) and non-apob-containing (HDL) lipoproteins. Table 1. Density classes and apoprotein distribution of lipoprotein subclasses. Lipoprotein class Density (g/ml) Size (nm) Major apoproteins Sources Chylomicrons VLDL IDL LDL HDL < A, B-48, C, E B-48/B-100*, C, E B-48/B-100, E B-100 A, C-II, E Intestine Liver Catabolism of VLDL and chylomicrons Catabolism of VLDL Intestine, liver, catabolism of VLDL and chylomicrons *Both apolipoprotein B-48 and apob-100 are synthesised in rodent liver, while only apob-100 is synthesised in human liver. Exogenous lipoprotein pathway Chylomicrons transport dietary fat from the intestine to peripheral tissues, mainly adipose tissue, heart and muscle (Figure 1). In addition to apob-48, chylomicrons secreted from enterocytes contain apoa-i, A-II and A-IV, while apolipoproteins C and E are soon acquired in the circulation by exchange with HDL. In the capillaries of peripheral tissues, the triglycerides in chylomicrons are hydrolysed by the action of lipoprotein lipase (LPL). This enzymatic reaction is activated by apoc-ii and allows the delivery of free fatty acids to the cells, either to be stored as triglycerides or consumed as energy. After hydrolysis of the core triglycerides, the chylomicron is transformed into a particle of smaller size that is enriched in cholesterol esters. These particles are referred to as chylomicron remnants, and they are rapidly taken up by the liver via the LDL receptor or LDL receptor-related protein receptor (LRP) with apoe serving as ligand. In the liver, cholesterol and fatty acids can be used either for VLDL production, or stored as cholesterol esters and triglycerides, respectively. The fatty acids can also be degraded by β-oxidation, while cholesterol can be excreted in the bile as free cholesterol or as bile acids. 11

12 Caroline Améen LDL Intestine Liver LDL-R LRP/ LDL-R HL SR-BI LRP/ LDL-R HDL IDL LPL LPL Chylomicrons Remnants VLDL FFA Adipose tissue Muscle Figure 1. Exogenous and endogenous lipoprotein pathways. VLDL, very low-density lipoprotein; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; HL, hepatic lipase; LDL-R, LDL receptor; LRP, LDL receptor-related protein receptor; FFA, free fatty acids. Endogenous lipoprotein pathway VLDL has a similar role as chylomicrons, but transports triglycerides that are synthesised in the liver (Figure 1). The size of the VLDL particles that are secreted varies, mostly due to the amount of triglycerides in the particle. The main subclasses of VLDL particles are large lipid-rich VLDL 1 and the smaller and denser VLDL 2 that contains less triglycerides. Just like chylomicrons, triglycerides in VLDL particles are hydrolysed by LPL in the capillaries to provide tissues with energy or to build up a triglyceride depot. After LPL-induced lipolysis, the VLDL particle is converted into IDL. IDL can either be taken up by the liver via apoe-recognising receptors, or further metabolised into LDL via a lipolytic enzyme in the liver called hepatic lipase (HL). LDL particles are taken up by LDL receptors that are present on the cell surface and 12

13 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism bind lipoproteins containing apob-100 and apoe. The LDL receptor is expressed on all cells, but the major amount is present in the liver where at least 50% of all LDL catabolism occurs. The LDL receptor is continuously internalised from the cell surface by endocytosis, with or without bound lipoproteins, and then recycled back to the cell surface. The expression of the LDL receptor is controlled by the cholesterol level inside the cell, i.e. when the cell is in need of cholesterol the LDL receptor is upregulated and vice versa. HDL is produced in the liver and small intestine, but can also be formed from surface excess lipids and apolipoproteins during catabolism of VLDL and chylomicrons. Nascent HDL can moreover be formed in the periphery by the action of ABCA1-transporters that pump out phospholipids and cholesterol of the cells to circulating apoa-1, which are acceptors of these lipids. The combined action of apoa-1 and ABCA1-transporters is the first step in a process called reverse cholesterol transport that is responsible for the transfer of cholesterol from peripheral tissues to the liver. The enzyme lecithin-cholesterol acyltransferase (LCAT) present on the surface of HDL converts phosphatidylcholine (lecithin) and cholesterol to cholesterol esters, which enter the core of the HDL particle. Cholesterol-loaded HDL can then be transported to the liver, where it is taken up via scavenger receptor B-I (SR-BI). In humans, cholesterol esters can also be transferred from HDL to apobcontaining lipoproteins in exchange of triglycerides by the action of cholesterol ester transfer protein (CETP). CETP is not present in rodents, however, which contributes to the HDL based lipoprotein profile in rodents as compared to the more LDL based lipoprotein characteristic of humans. Atherogenic dyslipidemia Disturbances of lipoprotein metabolism can result in the development of atherosclerosis and subsequently coronary heart disease, which is a major cause of deaths in the Western society. There is a fundamental notion that an elevated serum LDL cholesterol level is a major risk factor for CVD, reflecting an unbalance in forward and reverse cholesterol transport. For example, subjects with familial hypercholesterolemia have raised LDL cholesterol levels due to a mutation in the LDL receptor, resulting in premature atherosclerosis. Although LDL indeed is an important atherogenic lipoprotein, looking only at LDL levels may not be sufficient to identify the risk for CVD. In subjects with insulin resistance or with disorders such as metabolic syndrome and type 2 diabetes mellitus [4], elevated plasma triglycerides (in particular large VLDL 1 particles) together with an increased number of small dense LDL particles and a low level of HDL cholesterol [5, 6] have emerged as a significant lipid risk profile for CVD. These three abnormalities are closely related metabolically as the increase in triglyceride-rich particles generates small dense LDL and low HDL cholesterol levels. This is due to the fact that increased VLDL 1 levels favour the exchange of core lipids between VLDL 1 and LDL and HDL. In this reaction, LDL 13

14 Caroline Améen and HDL particles are depleted in cholesterol esters and enriched in triglycerides, which makes them good substrates for HL. Core triglycerides in LDL and HDL particles are hydrolysed by HL and small dense LDL and HDL particles are thus formed. Small dense LDL particles are particularly atherogenic as they readily bind to the proteoglycans of the arterial intima and are susceptible to oxidation, a modification that enhance foam cell formation that in turn initiates the development of atherosclerotic plaques. Smaller HDL particles, on the other hand, have an enhanced catabolic rate and the number of circulating HDL particles will therefore be reduced. Thus, raised VLDL triglycerides levels, small dense LDL and low HDL levels constitute an atherogenic lipoprotein profile. Together with raised LDL cholesterol, these alterations in lipid metabolism represent a fourfold entity that is characteristic of atherogenic dyslipidemia [5]. All these parameters are potential targets of therapy to reduce the risk for CVD. Statins and fibrates are the main drugs that are in clinical use, either by themselves or in combination. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG- CoA) reductase, the rate-limiting enzyme in cholesterol synthesis. As LDL receptors are controlled by the cholesterol level in the cell, statins indirectly upregulate the LDL receptor and are therefore highly effective in lowering LDL cholesterol level. Fibrates, on the other hand, are very efficient agents in lowering the plasma concentration of triglycerides and, as a consequence, also reduce plasma levels of small dense LDL [7]. These compounds are agonists of specific nuclear receptors called peroxisome proliferator-activated receptors (PPARs) and they both inhibit the secretion of VLDL triglycerides and accelerate VLDL clearance (for reviews see [4, 8, 9]). APOB AND VLDL ApoB A single apob molecule is an integral part of each VLDL particle and is a prerequisite for VLDL production. The apob structure is unusual in comparison with other lipoproteins, having amphipathic β-strands of importance for lipid binding. ApoB is one of the largest single chain mammalian polypeptides and exists in two distinct forms termed apob-100 and apob-48. ApoB-100 represents the full-length protein containing 4536 amino acids (512 kda), whereas apob-48 constitutes the amino terminal 2152 amino acids of the full-length form (250 kda). The molecular weight of apob-48 is approximately 48% of the full length apob-100, thereby its name. ApoB- 48 is synthesised from the same primary transcript as apob-100 by a posttranscriptional modification of a single mrna nucleotide called mrna editing. In this editing process, a cytidine at nucleotide position 6666 is deaminated to form uridine, which changes the codon for glutamine (CAA) to a stop codon (UAA) [10, 14

15 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism 11]. The translation is therefore terminated at this site and apob-48 is formed. The apob mrna editing process is catalysed by a multicomponent enzyme complex consisting of the catalytic subunit APOBEC-1 (apob mrna-editing enzyme catalytic peptide 1) [12] in addition to auxiliary proteins [13]. Editing of apob mrna occurs in the small intestine of all mammals, producing apob-48 molecules that associate with chylomicrons. In the liver, however, there is no editing in humans in contrast to rodents. This results in the exclusive production of the apob-100 form in human liver, whereas rodents synthesise and secrete both apob-48 and apob-100 from the liver (reviewed in [14-16]). Rough ER membrane apob TG TG MTP Pre-VLDL Smooth ER membrane TG MTP TG Lipid droplet Mature VLDL Figure 2. The two-step assembly of apob-containing particles. MTP, microsomal triglyceride transfer protein; TG, triglycerides; VLDL, very low-density lipoprotein. VLDL assembly ApoB-containing VLDL particles are synthesised in hepatocytes in a two-step process [16-18] (Figure 2). In the first step, a partially lipidated apob molecule called pre- VLDL is formed. This occurs by the addition of small amounts of lipids to apob during its translation and coincident translocation into the lumen of the rough endoplasmic reticulum (ER) [18-20]. This reaction is catalysed by a lipid transfer protein called microsomal triglyceride transfer protein (MTP), further described below. If the lipidation is incorrect, apob will not fold properly and is directed to degradation. This occurs mainly through retrograde translocation of misfolded apob from the ER lumen back to the cytosol, where apob is ubiquitinated and subsequently degraded by the proteasome [21, 22]. Although proteasomal degradation appears to be the major 15

16 Caroline Améen route for degradation of apob, there are indications that lysosomal enzymes also may be involved [23]. In the second step in VLDL assembly, the mature VLDL particle is formed by fusion of the pre-vldl particle with a preformed apob-free lipid droplet. The lipid droplets are produced in the smooth ER, while the fusion process is thought to occur in the junction between rough and smooth ER [16, 17]. Once the mature VLDL particle is formed, it is transported to the Golgi apparatus and secreted from the cell. The assembly of apob-containing lipoproteins in the intestine, i.e. chylomicrons, is thought to occur by similar mechanisms but have not been studied in detail. MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN Microsomal triglyceride transfer protein (MTP) is essential for assembly of apobcontaining lipoproteins. This 97-kDa lipid transfer protein heterodimerises with a 58- kda multifunctional chaperone called protein disulfide isomerase (PDI). MTP is primarily found in the lumen of microsomes in major apob-secreting organs, i.e. liver and small intestine [24], while PDI is expressed ubiquitously [25, 26]. MTP is responsible for the transport of neutral lipids, preferentially triglycerides and cholesterol esters, to developing apob molecules in the lumen of the ER [27]. This occurs via physical interaction between apob and MTP [28, 29], which is likely to place the lipid-binding cavity of MTP close to the lipid-binding sites in apob [30]. The MTP-mediated transfer of lipids has a stabilising effect on nascent apob molecules [31-35]. The role for PDI in apob secretion is not clear, but it has been suggested that it is necessary to maintain MTP within the ER [36, 37] and/or to maintain the stability of MTP [38]. The level of MTP expression has been shown to determine the secretion rate of apob-containing lipoproteins [32, 39-41]. In humans, mutations in MTP cause abetalipoproteinemia, a rare disorder that results in an inability to secrete apobcontaining lipoproteins from the liver and the intestine [42]. Together these observations show that the secretion of both VLDL and chylomicrons is critically dependent on the presence of MTP. MTP also has a role in the second step in VLDL assembly, in which the major amount of lipids is added to the primordial VLDL by fusion with a preformed triglyceride-rich droplet. Electron microscopy studies have shown that VLDL-sized lipoproteins, i.e. the apob-free lipid droplets, are absent in the Golgi stacks in mice lacking hepatic MTP expression [43]. Thus, this demonstrates that the formation of these lipid droplets requires the presence of MTP. However, the fusion process between the pre-vldl particle and the lipid droplet appears to be MTP- independent [44]. 16

17 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism Regulation of MTP To date, only a few factors have been shown to regulate MTP expression. Insulin and high concentrations of glucose reduce the level of MTP mrna in HepG2 cells [45], which is in line with the finding of a negative insulin response element in the human MTP promoter [46]. In insulin resistance models, such as fructose-fed Syrian hamsters [47, 48] and obese diabetic ob/ob mice [49], the hepatic expression of MTP is conversely increased together with increased secretion of triglyceride-rich apobcontaining lipoproteins. Diets enriched in triglycerides have also been found to upregulate MTP expression in hamster [50, 51] and rat [52]. Similarly, cholesterol increases hepatic concentrations of MTP mrna in hamsters [53] and in HepG2 cells [46], which may contribute to a coordinated response to hepatic cholesterol accumulation leading to increased VLDL secretion. Conversely, cholesterol depletion of HepG2 cells lowers the level of MTP mrna and protein [54], which is due to the upregulation of sterol regulatory element binding proteins (SREBPs) [54]. However, in some models the levels of both SREBP and MTP are increased [49, 55, 56], indicating that SREBP instead upregulates MTP. STEROL REGULATORY ELEMENT BINDING PROTEINS Sterol regulatory element binding proteins (SREBPs) belong to the basic helix-loophelix leucine zipper (bhlh-zip) family of transcription factors and regulate the biosynthesis of cholesterol, fatty acids and triglycerides. Three isoforms of SREBPs have been identified, designated SREBP-1a, SREBP-1c and SREBP-2 [57]. SREBP-1a and SREBP-1c are encoded from the same gene through the use of alternate promoters and differ only in their first exon [58]. This makes the length of the amino-terminal transactivation domain shorter in SREBP-1c, giving rise to a less potent transcriptional activator than SREBP-1a [59]. SREBP-2 is transcribed from a separate gene and has about 50% identity with the SREBP-1 amino acid sequence [58]. Most organs, including liver and adipose tissue, predominantly express the SREBP-1c and SREBP-2 isoforms. In contrast, most cell lines mainly express SREBP-1a in addition to SREBP- 2 [60]. Studies with transgenic mice that overexpress SREBP-1a, SREBP-1c or SREBP-2 in the liver have demonstrated distinct roles for the different SREBP isoforms [55, 61-64]. Each type of SREBP-overexpressing animal presented a different pattern of synthesis and accumulation of fatty acids and/or cholesterol in the liver. These data suggest that SREBP-1c is more selective in activating genes involved in fatty acid and triglyceride synthesis, while SREBP-2 is more specific for genes involved in cholesterol metabolism. SREBP-1a, on the other hand, is a regulator of genes involved both in fatty acid, triglyceride and cholesterol metabolism [61]. 17

18 Caroline Améen The two-step cleavage process of SREBPs SREBPs are synthesised as inactive precursors bound to the ER membrane and nuclear envelope [57]. Each SREBP precursor is structurally composed of three domains: 1) an amino-terminal domain that contains the DNA-binding bhlh -Zip region as well as a transactivation region, 2) a central domain that contains two transmembrane segments linked by a short loop projecting into the ER lumen, and 3) a carboxyl-terminal regulatory domain. In order to activate target genes, the SREBP precursor must undergo a two-step proteolytic process to release the amino-terminal domain so it can function as a transcription factor (Figure 3) [65, 66]. This cleavage process requires the presence of SREBP cleavage-activating protein (SCAP) in addition to two proteases termed Site-1 protease (S1P) and Site-2 protease (S2P). SCAP is a sensor of cholesterol and upon cholesterol depletion it transports the SREBP precursor to the Golgi apparatus where the two proteases reside. In contrast, when the cholesterol content of cells increases, the SCAP-SREBP complex is retained in the ER. In the Golgi apparatus, SP1 cleaves SREBP in the luminal loop between its two membranespanning regions, generating two membrane-bound segments. The amino-terminal of SREBP is then liberated via a second cleavage mediated by S2P, producing the mature form of SREBP that enters the nucleus and activates transcription. Like other members of the bhlh-zip family, SREBPs recognise palindromic sequences called E-boxes in the promoter region of SREBP target genes. However, due to the presence of a unique tyrosine residue in the DNA-binding domain, SREBPs can also bind to sterol response elements (SREs) or related sites [67]. This dual binding property makes it possible for SREBPs to bind a large variety of target genes in both lipogenic and cholesterol synthesis pathways. Reg TA SCAP ER - Sterols Reg SCAP Golgi TA S2P S1P Reg Nucleus SRE Transcription TA Figure 3. Model for the sterol-mediated cleavage of membrane-bound SREBPs. Reg, regulatory domain; TA, transactivating domain; SCAP, SREBP cleavage-activating protein; S1P, Site-1 protease; S2P, Site-2 protease; SRE, sterol response element. 18

19 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism SREBP-1c has been shown to activate transcription even in the presence of cholesterol, suggesting that the sterol-regulated proteolytic cleavage system is not as specific for SREBP-1c as for SREBP-2 [68]. The induction of SREBP-1c has instead been shown to be mainly at the mrna level [68-71], which correlates with both the precursor and mature form of SREBP-1 [72]. Thus, while SREBP-2 controls cholesterol synthesis almost completely at the cleavage level, SREBP-1c largely regulates fatty acid synthesis by changing its own transcription level. SREBP-1c and regulation of lipogenesis The liver is the principal organ for lipogenesis, i.e. the production of fatty acids and triglycerides from excess dietary carbohydrate. Under lipogenic conditions, glucose in the cell is converted to pyruvate via the glycolytic pathway. Pyruvate is next converted into acetyl-coa, which is used as a building block in the synthesis of long chain fatty acids. The produced fatty acids can in turn be used for esterification of glycerol-3- phosphate to generate triglycerides. Thus, in this pathway carbohydrates are converted to fat. The enzymes catalysing the lipogenic reactions, thus designated lipogenic enzymes, are mostly regulated at the transcriptional level during different nutritional and hormonal states. One of the classic actions of insulin is to induce the entire lipogenic program and this is particularly observed during fasting-refeeding treatments to rodents. Moreover, several studies show that SREBP-1c mediates the stimulatory effect of insulin on lipogenic enzymes [55, 69, 70, 73, 74]. In contrast, polyunsaturated fatty acids (PUFAs) are negative regulators of hepatic lipogenesis through suppression of SREBP-1 [71]. LIPOGENIC ENZYMES The major enzymes in the lipogenic pathway include not only genes for fatty acid synthesis, such as acetyl-coa carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-coa desaturase-1 (SCD-1), but also a gene involved in triglyceride synthesis; glycerol-3-phosphate acyltransferase (GPAT) (Figure 4). E-boxes/SRE-like sequences have been identified in the promoters of all these genes through which the mature SREBP-1c protein can exert it transcriptional activation [67, 75-77]. Acetyl-CoA carboxylase Acetyl-CoA carboxylase (ACC) is responsible for the first committed step in fatty acid synthesis by catalysing the ATP-dependent formation of malonyl-coa from acetyl- CoA and bicarbonate [78]. Malonyl-CoA is both a substrate for the next enzyme in the lipogenic pathway (fatty acid synthase) and regulates the oxidation of fatty acids by 19

20 Caroline Améen Acetyl-CoA Malonyl-CoA Palmitic acid Fatty acyl-coa Monoacylglycerol 3-phosphate Triglycerides and phospholipids Acetyl-CoA carboxylase (ACC) Fatty acid synthase (FAS) Stearoyl-CoA desaturase (SCD) Monounsaturated fatty acids Glycerol-3-phospate acyl transferase (GPAT) Figure 4. The lipogenic pathway inhibiting carnitine palmitoyl transferase-i (CPT- I), an enzyme responsible for the transport of fatty acyl-coa into the mitochondria. Thus, ACC has important regulatory functions both in fatty acid synthesis and fatty acid oxidation. ACC exists in two isoforms, ACC-1 and ACC-2, and it has been speculated that ACC-1 is mostly involved in the regulation of fatty acid synthesis, while ACC-2 is suggested to control β-oxidation. Apart from being transcriptionally regulated, ACC is also regulated in the short-term at the posttranslational level by covalent modifications, such as phosphorylation/dephosphorylation [78]. Insulin has been shown to increase ACC activity by dephosphorylation, while glucagon and epinephrine decrease the activity by phosphorylation [78]. Fatty acid synthase Fatty acid synthase (FAS) is a multifactorial enzyme responsible for the next step in the elongation of fatty acids by catalysing the formation of palmitate (16:0) from malonyl-coa and acetyl-coa. FAS is predominantly expressed in the liver and adipose tissue, i.e. tissues with a high degree of fatty acid synthesis. A high level of glucose has been shown to be required for insulin to induce FAS transcription [79], and glucose is also important for the stabilisation of FAS mrna [80]. Stearoyl-CoA desaturase Stearoyl-CoA desaturase (SCD) is a microsomal enzyme that catalyses the production of monounsaturated fatty acids by insertion of a double bond into saturated fatty acids [81]. The preferred substrates for SCD are palmitoyl (16:0) CoA and stearoyl (18:0) CoA, which are converted to palmitoleoyl (16:1) CoA and oleoyl (18:1) CoA, respectively. There are two rat SCD isoforms termed SCD-1 and SCD-2 [63]. Most tissues express both isoforms, but the liver is exceptional in that it only expresses SCD-1. Observations in mouse strains that have a natural mutation in the SCD-1 gene (asebia mice) [82] or a targeted disruption of the SCD-1 gene [83] demonstrate that the 20

21 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism oleoyl-coa and palmitoleoyl-coa produced by SCD-1 are necessary to synthesise sufficient amounts of cholesterol esters and triglycerides in the liver. The regulation of SCD is also physiologically important because it changes the ratio between saturated and monounsaturated fatty acids in the cell. This in turn affects membrane fluidity that is known to be critical for many cellular processes [81]. Glycerol-3-phosphate acyltransferase Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed and probably rate-limiting step in triglyceride and phospholipid biosynthesis (reviewed in [84]). There are two isoforms of GPAT, one located in the mitochondrial outer membrane and the other on the ER. These two isoforms have different substrate preferences, where mitochondrial GPAT preferentially uses saturated fatty acyl-coa, while microsomal GPAT uses saturated and unsaturated fatty acyl-coas equally well. Moreover, only mitochondrial GPAT is cloned and also seems to be more extensively regulated by the nutritional and hormonal status. The mitochondrial GPAT is expressed mainly in lipogenic tissues, and mice deficient in this form of GPAT have a lower hepatic content and plasma concentration of triglycerides [85]. These findings indicate an important role of mitochondrial GPAT in the synthesis of triglycerides. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that exert a general regulatory effect on lipid homeostasis. There are three different types of PPARs, termed PPARα, γ and δ, which have distinct functions and tissue distribution. PPARα is predominantly expressed in liver, heart, and kidney, where it controls the transcription of genes that participate in fatty acid catabolism, most notably those involved in peroxisomal and mitochondrial β-oxidation [86]. PPARγ target genes are mostly implicated in lipogenic pathways in the adipose tissue, where PPARγ also is highly expressed. PPARδ is abundantly expressed in most tissues, with the exception of its very low expression in liver [87]. Several fatty acids and their derivatives are natural ligands for PPARs, although they bind with different affinity. PPARα has a clear preference for binding long chain unsaturated fatty acids. To be active as transcription factors, PPARs must heterodimerise with retinoic X receptor (RXR) α, another nuclear receptor. This complex binds PPAR-response elements (PPREs) in the promoter region of target genes and activates transcription upon ligand binding. The PPRE consists of a direct repeat of the nuclear receptor hexameric DNA recognition motif (AGGTCA) that is separated by one nucleotide, thus called DR-1. 21

22 Caroline Améen PPARα and lipid metabolism PPARα is a central regulator of hepatic fatty acid metabolism by controlling genes involved in several fatty acid pathways, including uptake by the cells, intracellular binding as well as oxidation. The importance of PPARα is demonstrated during conditions when efficient fatty acid oxidation is required. For example, PPARα expression and activation are markedly induced during fasting in order to stimulate β- oxidation and production of ketone bodies. When PPARα-deficient mice are fasted, they accumulate massive amounts of lipids in their liver, reflecting the impaired expression of fasting-induced PPARα target genes in these mice [88, 89]. Fibrates are clinically used hypolipidemic drugs that lower triglycerides and increase HDL cholesterol concentrations through PPARα activation. PPARα mediates fibrate action on HDL levels in humans by inducing transcription of the major HDL apolipoproteins, apoa-i and apoa-ii. However, in contrast to humans, fibrates do not increase HDL concentrations in rats due to sequence differences in the rat and human apoa-i gene promoters [90]. Fibrates increase the LPL expression and reduce the apoc-iii expression, which leads to increased lipolysis of triglyceride-rich lipoproteins and thus accelerated clearance of these particles from the circulation [91]. In the liver, increased β-oxidation as well as reduced triglyceride synthesis [92] may further contribute to the lipid-lowering effect of fibrates by decreasing substrates for VLDL secretion. Thus, the triglyceride-lowering effect of fibrates occurs as a consequence of both enhanced catabolism of plasma triglyceride-rich lipoproteins and reduced secretion of VLDL triglycerides from the liver. GROWTH HORMONE Growth hormone (GH) is produced by the somatotropic cells in the anterior pituitary. In humans, GH consists of a single peptide of 191 amino acids that is crosslinked by two disulfide bridges. Rat GH is one amino acid shorter and has 66% homology with its human counterpart [93]. The secretion of GH into the circulation is primarily regulated by two peptide hormones with opposite effects: GH-releasing hormone (GHRH), which stimulates the secretion of GH, and somatostatin, which inhibits the secretion of GH. These hormones are produced in the hypothalamus and reach the pituitary via the portal vascular system. The GH secretion is also regulated by a number of other factors, such as insulin-like growth factor-i (IGF-I), thyroid hormone, glucocorticoids, fasting and type 1 and type 2 diabetes mellitus (for review see [94]), indicating the complexity of the regulation of GH secretion. 22

23 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism The GH secretory pattern in females and males There is a marked sex difference in the pattern of GH secretion in most adult species, including rodents [95] (Figure 5) and man [96]. Male rats secrete GH regularly in large pulses every hours with low or undetectable levels between the peaks [95, 97]. Female rats, on the other hand, secrete GH in a near continuous fashion with lower pulse amplitudes and higher baseline levels than male rats [95]. The mean plasma level of GH, however, is similar in both sexes [98]. The sexually dimorphic secretory pattern of GH in humans is similar to that in rodents but is less pronounced [96]. The average daily GH concentration, however, is higher in women compared to men [99, 100]. GH GH Time Time Figure 5. The sex-differentiated GH secretory pattern in rats. The difference in the secretory pattern of GH does not become apparent until after the onset of puberty. This suggests that the GH secretory pattern is highly controlled by gonadal steroids. Both neonatal and prepubertal gonadectomy of male rats result in elevated baseline GH levels during adult life, which can be completely reversed by sustained testosterone treatment [98]. However, GH pulse height is only decreased after neonatal gonadectomy, with unchanged pulse heights after prepubertal gonadectomy [98]. This suggests that a neonatal testosterone surge is needed to maintain normal GH pulse amplitudes in adult male rats, while a continuous presence of testosterone is necessary for preserving low GH basal levels in adult male rats. Neonatal gonadectomy of female rats only mildly affects the female GH secretory pattern [98]. However, the importance of estradiol in maintaining the sexually dimorphic GH secretory is shown by the feminisation of the GH secretory pattern in intact males after estradiol treatment, i.e. higher baseline GH levels, lower pulse heights and more frequent GH bursts [101, 102]. The mechanism of sex steroid action is probably by modulation of the GHRH and somatostatin levels, as well as by direct effects on GH secretion in the pituitary. The 23

24 Caroline Améen greater peaks that are observed in male rats compared to female rats may be due to the androgen-induced increase of both GHRH in the hypothalamus and GH secretion from the pituitary [103]. Some studies also show that androgens might enhance the somatostatin levels between peaks, which could account for the lower GH baseline level in male rats. Moreover, GHRH inhibits its own secretion and increases the secretion of somatostatin [104]. These properties in male rats are therefore likely to account for the cyclic GH secretory pattern through feedback mechanisms. In female rats, there is no such cyclic variation in somatostatin levels due to the inhibition by estradiol [105], which explains why female rats secrete GH more continuously than male rats. GH receptor and GH signalling The GH receptor is a member of the large cytokine receptor superfamily. This family includes receptors for more than 25 ligands, such as prolactin, multiple interleukins, leptin and erythropoietin [106]. Cytokine receptors are generally composed of an extracellular region, a single transmembrane domain and an intracellular region. A soluble form of the extracellular region of the GH receptor is found in plasma. This glycoprotein is called growth hormone-binding protein (GHBP) and it binds up to 60% of circulating GH [107]. The initial step in GH signalling is the sequential binding of GH to two GH receptors that results in receptor dimerisation [108]. This moves the receptors in close proximity of each other, which increases the affinity of the GH receptor for the tyrosine kinase JAK2. As the GH receptor itself lacks tyrosine kinase activity, each JAK2 transphosphorylates the other JAK2 molecule and they are both thereby activated [109]. Activated JAK2 proteins subsequently phosphorylate the GH receptor at several tyrosine residues, converting them to docking sites for other signalling molecules [110]. One such molecule is the signal transducer and activator of transcription 5 (STAT5), which upon binding becomes phosphorylated by the action of JAK2 [111, 112]. This phosphorylation event activates STAT5 proteins, triggering their dimerisation, nuclear translocation and activation of gene transcription [112, 113]. There are two closely related isoforms of STAT5, termed STAT5a and STAT5b, of which STAT5b appears to be most important in GH signalling [114]. Other signalling pathways that are activated by GH include the mitogen-activated protein kinase (MAPK) pathway, the insulin receptor substrates (IRS)-1 and IRS-2, and protein kinase C. The activation phase is usually transient, which means that effective shut off mechanisms by which the GH signalling pathway is inhibited must occur in the cell. The most important negative regulators of GH signalling are members of the cytokine- 24

25 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism inducible gene family, termed suppressors of cytokine signalling (SOCS) [115]. These proteins are induced in response to GH or other cytokines by increased transcription via JAK-STAT activation. The SOCS proteins subsequently inhibit GH signalling by reducing the kinase activity of JAKs [115]. As the SOCS switch off the signalling pathway that initially led to its production, these proteins are involved in a classical negative feedback loop. Influence of the GH secretory pattern on GH signalling The second messenger system of intracellular signalling is activated differently by the intermittent and continuous GH stimulus. This is demonstrated by the activation of STAT5b by the intermittent GH secretory pattern in contrast to the continuous GH secretory pattern [116]. STAT5b has therefore been suggested to play a key role in the regulation of the sexual dimorphic gene expression in the liver that is induced by the male pulsatile GH secretory pattern. The hepatic expression of Cis (cytokine-inducible SH2-containing protein), a member of the SOCS family, is higher in female rats compared to males due to the continuous GH secretory pattern of females [117]. As Cis is responsible for the desensitisation of GH-induced STAT5b signalling, a more pronounced expression of Cis could result in less expression of male-characteristic genes in female rats. There is not much known about signalling that is specifically induced by the female secretory pattern of GH. However, one study shows that incubation of rat hepatocytes with GH stimulates female-specific CYP2C12 expression via upregulation of phospholipase A 2 (PLA 2 ). This effect is dependent on the subsequent P450-catalysed formation of an arachidonic acid metabolite [118] that may function as an intracellular second messenger. Insulin-like and diabetogenic effects of GH GH exerts both insulin-like and diabetogenic (antiinsulin-like) effects in adipose tissue and skeletal muscle (for review see [119]). The insulin-like effects occur soon after GH exposure and involve increased glucose utilisation and decreased lipolysis. When tissues are exposed to GH for a longer time, responsive cells are turned into unresponsive cells towards insulin-like actions, which is termed the refractory effect of GH. As GH is secreted endogenously throughout the day, causing a constant refractory state, the insulin-like GH effects probably have no physiological role. The late diabetogenic effects of GH that occur after prolonged GH exposure are therefore considered to better reflect the physiological situation. These effects include impaired glucose utilisation, hyperglycemia, stimulation of lipolysis, and induction and maintenance of the refractory state to insulin-like effects. The mechanism by which GH induces refractoriness to the insulin-like effects is thought to occur via upregulation of SOCS-3 and thereby blocking JAK2 activation [120]. 25

26 Caroline Améen GH and lipoprotein metabolism in humans GH regulates a number of important functions in cholesterol and lipoprotein metabolism (Table 2). This is particularly emphasised during conditions of GH deficiency and GH excess (acromegaly), which are both associated with an abnormal lipid profile. GH-deficient subjects have elevated levels of total cholesterol, LDL cholesterol and triglycerides, and a reduced HDL cholesterol level [2]. This serum lipid profile is atherogenic and GH-deficient patients indeed have an increased risk for CVD [121]. GH replacement therapy in these patients has been shown to be favourable with respect to the changed plasma lipoprotein profile. Most notably, GH administration lowers the LDL cholesterol and increases HDL cholesterol, while plasma triglyceride levels are principally unchanged [ ]. GH therapy stimulates the secretion of VLDL-apoB from the liver [125], but despite this the plasma concentration of apob and LDL cholesterol decreases [122, 123]. This is probably due to the increased clearance of these particles after GH treatment [125], which might at least partly be explained by upregulation of LDL receptors [126, 127]. Moreover, GH treatment reduces the activity of CETP in GH-deficient patients, which increases the HDL cholesterol level and further contributes to the decreased LDL cholesterol level [128]. However, GH therapy has also been reported to increase the level of the atherogenic lipoprotein (a) [123, 124], which may contribute to the increased risk for CVD in acromegaly [1]. These patients also have increased triglyceride levels, decreased HDL levels and are often insulin resistant [129, 130], which might further contribute to the increased prevalence of CVD in acromegaly. GH and lipoprotein metabolism in laboratory animals GH plays an important role in cholesterol and lipoprotein metabolism also in rodents (Table 2). GH is known to stimulate lipolysis in adipose tissue, which increases the flux of free fatty acids to the liver and other tissues such as skeletal muscle. A continuous GH infusion to Hx rats has also a stimulatory effect on some hepatic lipogenic enzymes in vivo [ ] and on triglyceride synthesis in hepatic cultures derived from these rats [135, 136]. Although this results in a stimulated secretion of VLDL [136, 137], no increase in serum levels of VLDL has been found in GH-treated Hx rats [138], which is in line with the findings in humans. This indicates that GH increases both production and clearance of VLDL. The increased turnover of VLDL in rats can partly be explained by the fact that GH enhances the editing of apob mrna. This results in an increased proportion of secreted apob-48-containing VLDL particles [139], known to have a considerably shorter half-life than particles containing apob- 100 [140]. GH also has a stimulatory effect on LPL activity in skeletal muscle, which may further contribute to a more rapid catabolism of secreted VLDL particles [141, 142]. In addition, the secretion of apoe is increased in hepatocytes isolated from Hx rats treated with a continuous infusion of GH [143]. As apob-48-containing 26

27 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism lipoproteins can be removed via interaction of apoe with LRP and LDL receptors, an increased content of apoe may add to the enhanced turnover of lipoproteins in response to GH. HDL cholesterol levels are increased by GH treatment in both rats [138] and mice [144], which in rats has been shown to be due to the female GH secretory pattern [138]. The LDL cholesterol level is conversely decreased by GH treatment in hypophysectomised rodents [138]. This is probably mainly due to the stimulatory effect that GH exerts on hepatic LDL receptor expression [126, 127], but increased editing of apob mrna [139] and increased HL activity [142] may also be involved. In conclusion, even though it is clear that GH increases VLDL secretion from the liver, the mechanisms behind the stimulatory effect of GH on VLDL assembly and secretion is not known. Table 2. Effects of GH on lipoprotein metabolism in humans and laboratory animals. *Hx rats. Humans Lab. animals* HDL cholesterol LDL cholesterol TG or or ApoB ApoE * * Lp(a) * Hepatic apob mrna editing Hepatic TG synthesis * VLDL-secretion * LDL receptor CETP LPL in adipose tissue LPL in skeletal muscle *Effect dependent on mode of GH administration 27

28 Caroline Améen AIMS OF THE THESIS The general aim of this thesis was to investigate the effects of GH and PPARα on key genes of importance for hepatic lipogenesis and VLDL assembly. The specific aims of Paper I-IV were: To study the role of increased insulin levels for the effects of GH on lipoprotein metabolism in vivo To study the effects of gender and the sex-differentiated GH secretory pattern on the mrna expression of SREBP-1c and lipogenic enzymes To study the effects of gender and the sex-differentiated GH secretory pattern on MTP expression To study whether PPARα activation increases MTP expression and activity 28

29 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism METHODOLOGICAL CONSIDERATIONS Detailed descriptions of the assays that have been used are given in each paper and references therein. In this section, additional considerations concerning the in vivo and in vitro models are commented upon. ANIMALS Hypophysectomised rats Sprague Dawley rats were hypophysectomised (Hx) by the temporal approach at 50 (Paper I and III) or (Paper II and III) days of age (Møllegaard Breeding Center Ltd, Ejby, Denmark). The pituitary deficiency in hypophysectomised animals is total, meaning that the endogenous secretion of all hormones normally released from the pituitary is abolished. The hormonal treatment was initiated days after hypophysectomy. The body weight gain was monitored for about one week during this period to determine the completeness of hypophysectomy. A body weight gain of more than 0.5 g/day was considered as incomplete hypophysectomy and used as an exclusion criterion. An alternative way of controlling the completeness of Hx is by examining the sella turcica for remaining GH activity using RIA. This has previously been done and the results of these measurements indicate that monitoring weight gain is a sensitive alternative of investigating completeness of Hx. Gonadectomised (Gx) rats (Paper III) and PPARα deficient mice (Paper IV) were also used as described in the respective papers. HORMONAL TREATMENT Thyroxine (T4) The plasma level of thyroxine is very low two weeks after hypophysectomy [145] due to the lost action of thyroid-stimulating hormone. This was substituted for by giving the hypophysectomised rats a daily subcutaneous injection of 10 µg L- thyroxine/kg/day (Nycomed, Oslo, Norway) diluted in saline. This dose results in somewhat higher thyroxine levels than in normal rats [139, 146], but has been shown to be within the physiological range as measured by the effect of different thyroxine doses on longitudinal bone growth [147]. However, the level of the active form of thyroxine, i.e. T3, has not been measured after this substitution. Glucocorticoids To substitute for the lack of adrenocorticotropic hormone, all Hx rats were given a daily subcutaneous injection of 400 µg cortisol phosphate/kg/day (Solo-Cortef, 29

30 Caroline Améen Upjohn, Puurs, Belgium) diluted in saline. A replacement dose of 500 µg cortisone/kg/day has been shown to be within the physiological range with respect to body growth and longitudinal bone growth [148] as well as GH binding to adipocytes [149]. The dose of cortisol was adjusted to 400 µg/kg/day due to the higher potency of cortisol than cortisone. In the in vitro studies, 1 nm dexamethasone was added to the medium since glucocorticoids exert a permissive action on some GH effects [150, 151]. The dose of 1 nm dexamethasone, or 0.39 ng/ml, corresponds to 32.5 ng/ml corticosterone after correction for the different potencies of these glucocorticoids. This dose of dexamethasone is somewhat below the physiological range of corticosterone concentration in serum ( ng/ml) [152]. Growth hormone Recombinant bovine GH was given in a dose of mg/kg/day diluted in 0.05 M phosphate buffer (ph 8.6) containing 1.6% glycerol and 0.02% sodium azide. Bovine GH was chosen rather than human GH, since bovine GH only binds to the GH receptor in contrast to human GH that also binds the prolactin receptor. Compared to rat GH, bovine GH is more stable and also much more available on the market. GH were administered to the hypophysectomised rats either as a continuous infusion via an osmotic mini-pump (Alza Corp., Palo Alto, CA, USA) implanted subcutaneously between the scapulae, or as two daily subcutaneous injections at 12-h intervals (0800 and 2000 h). These modes of GH administration have been shown to experimentally imitate the female and the male GH secretory pattern, respectively, with respect to feminisation and masculinisation of P450 enzyme levels in rat liver [101, 153]. GH injections, however, will result in fluctuations of GH plasma levels during the day with the possible outcome that GH effects on mrna species with high turnover are not detected. The hepatic expression was therefore analysed both at 2 and 6 hours after the last GH injection. The normal secretory rate of GH in days old female rats is 1.3 mg/kg/day as calculated from the GH clearance rate (1.19 ml/min) [154] and the normal mean plasma level of GH (135 ng/ml) [155]. Thus, both the in vitro dose of GH (100 ng/ml, Paper II) and the continuous infusion of 1.5 mg GH/kg/day to Hx rats (Paper I) are within the physiological range. When the hypophysectomy model was used to investigate the regulatory role of the feminine and masculine GH secretory pattern, a lower dose of GH (0.7 mg/kg/day) was administered (Paper II and III). This dose was chosen to assure very low or undetectable GH levels between the GH injections analogous to the GH secretory pattern of the male. Although lower than the normal 30

31 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism GH secretion rate, this GH restitution is sufficient as indicated by the increased final body weight and body weight gain in hypophysectomised rats ([156] and Paper II). To assess the role of the different GH secretory patterns without giving GH injections that result in slow diurnal variations in GH concentrations, a low dose of GH (0.5 mg/kg/day) was administered as a continuous infusion to intact rats. This mode of GH administration will feminise male rats with respect to the GH secretory pattern without any major changes in the total mean plasma level of GH [157, 158]. The final body weight, body weight gain and IGF-I mrna expression did not change by this GH treatment in Paper II and III, indicating the total GH exposure was not particularly affected in our studies. Insulin A slow-release form of insulin (Insulatard, 100 IU/ml, Novo Nordisk A/S, Denmark) diluted in saline was given as a daily subcutaneous injection at 1600 h to the Hx animals in Paper I. To avoid insulin-induced fatal hypoglycemia, the dose of insulin was gradually increased from 1 IU to 2 IU/day. This insulin treatment has been shown to result in serum insulin levels similar to those in sham-operated animals [159]. In the in vitro studies, the cells were plated in a medium containing 16 nm insulin and then cultured in the presence of 3 nm insulin. The insulin concentration in the portal blood of fasting rats is 0.34 nm [160] and is several-fold higher in fed rats. The in vitro doses of insulin are therefore near or above the physiological range. Sex steroids Testosterone was diluted in propylene glycol and given at a dose of 0.5 mg/kg/day as a daily subcutaneous injection to Gx male rats (Paper III). A similar dose of testosterone has been shown to increase the level of plasma testosterone in Gx rats to that of intact male rats [161], and also to masculinise the secretory pattern of GH [155]. 17βestradiol was diluted in propylene glycol and administered at a dose of 0.1 mg/kg/day as a daily subcutaneous injection to female Gx rats (Paper III). The serum level of estradiol in intact female rats varies between 2 and 50 pg/ml throughout the cycle [162]. As treatment of intact rats with 0.01 mg estradiol/kg/day increases the plasma estradiol level to approximately 51 pg/ml [163], our higher dose of estradiol (0.1 mg/kg/day) would therefore probably result in supraphysiological serum levels of estradiol in Gx rats. 31

32 Caroline Améen HEPATOCYTE CULTURES Hepatocytes were isolated by nonrecirculating collagenase perfusion through the vena porta of anaesthetised female Sprague Dawley rats or PPARα null and wild type mice [151, 164, 165]. During the first 5-6 minutes, the liver was perfused with a medium containing EGTA at a flow rate of ml/min for rats and ml/min for mice. EGTA has a Ca 2+ -chelating function and therefore facilitates the disruption of Ca 2+ - dependent cell-to-cell interactions. For the next 7-9 minutes, the liver was perfused with a second medium supplemented with collagenase IV. Collagenase is a metalloprotease that degrades collagen and this further helps separating the cells inside the liver capsule. After the perfusion, the liver capsule containing the liver cells was excised. The cells were filtered trough a 250 µm pore size mesh nylon filter followed by a 100 µm pore size mesh nylon filter to remove undigested part of the liver. Thereafter, the cell suspension was repeatedly washed in plating medium in order to remove collagenase and cells with lower density, such as Kuppfer cells and fat-storing Ito cells. The cells were seeded at ~ cells/cm 2 in petri dishes coated with a layer of Matrigel (Figure 6). The Matrigel contains large amounts of laminin, proteoglycan and collagenase type IV, which support the hepatocytes in the subendothelial space of normal liver. In comparison with primary hepatocytes that are cultured on plastic or collagen, hepatocytes cultured on Matrigel have been shown to better maintain their characteristic properties [166, 167], such as expression of functional GH receptors [168]. In experiments where protein concentrations were measured, the matrigel was first removed by adding PBS supplemented with 5 mm EDTA in order to eliminate contaminating proteins from the Matrigel. During the first night of culturing, the medium contained a high dose of insulin (16 nm) as this has been shown to enhance plating efficiency and formation of dispersed monolayers [169]. This medium also contains glucose (28 nm), which has been shown to maintain the expression of GH receptors [170]. After plating, the cells were cultured in a medium without insulin or with a lower insulin concentration (3 nm), and with the hormone/substance of interest supplemented. Dexamethasone (1 nm) was also added to the culture medium due to its permissive actions on GH effects [151]. The hepatocytes were incubated during the next 3 days unless otherwise stated. The constant Figure 6. Primary rat hepatocytes in culture 32

33 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism presence of GH in the culture medium is similar to the continuous GH infusion in vivo that mimics the feminine GH secretory pattern. The in vitro results of GH incubation can therefore be used as an indication of the direct or indirect nature of the continuous GH secretory pattern on hepatic functions in vivo. The effects in the hepatocytes cultures were related to the DNA content in each culture dish to correct for differences in cell number (Paper III and IV). 33

34 Caroline Améen SUMMARY OF RESULTS PAPER I Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat. GH affects many parameters involved in lipoprotein metabolism, and it also increases insulin secretion from β-cells [171]. To investigate the importance of increased serum insulin levels for in vivo effects of GH on lipoprotein metabolism, Hx rats were treated with GH or insulin alone or with GH and insulin in combination. GH treatment of Hx rats decreased LDL cholesterol and increased HDL cholesterol levels independently of concomitant insulin treatment (Fig. 1A, Paper I). Similarly, GH decreased the level of LDL triglycerides and somewhat increased the VLDL triglyceride level, effects that were not affected by the presence of insulin (Fig. 1B, Paper I). The triglyceride secretion rate from the liver was increased by GH treatment, but this effect was suppressed by combined insulin treatment (Fig. 2B, Paper I). The hepatic triglyceride content changed in parallel with the secretion of triglycerides by the hormonal treatments (Fig. 2D, Paper I), suggesting that the rate of triglyceride secretion is dependent on triglyceride availability. The mrna expression of two lipogenic enzymes was therefore measured. GH increased the mrna levels of FAS and SCD-1 (Fig. 4, Paper I), which is likely to contribute to the increased hepatic triglyceride content and secretion after GH treatment. The effect of GH on FAS and SCD mrna expression was similar in the presence of insulin, indicating that the GHantagonistic action of insulin on triglyceride secretion rate and content is not via decreased mrna expression of these genes. The mrna level of SREBP-1c, a known regulator of lipogenic enzymes, was also increased by GH treatment (Fig. 4, Paper I). In contrast to FAS and SCD, however, the effect of GH was counteracted by insulin, showing that upregulation of SREBP-1c mrna is not needed for increased expression of FAS and SCD mrna. Insulin treatment alone increased SCD and tended to increase FAS mrna levels, suggesting that the stimulatory effect of GH on these genes is partly mediated by insulin. The serum level of apoe and editing of apob mrna were increased in GH-treated Hx rats (Fig. 3 and Table 2, Paper I). These findings indicate an increased turnover of VLDL particles after GH treatment, which agrees well with the calculated triglyceride clearance rate that is increased by GH treatment (Fig 7). ApoB mrna editing was also elevated by insulin alone and the effect of GH was less marked in Hx rats given insulin compared to Hx rats not given insulin. This indicates that part of the effect of GH on apob mrna editing could be mediated by insulin. 34

35 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism Triglyceride clearance rate (ml/min) 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0 * N Hx * Hx GH Hx Insulin * Hx GH Insulin Figure 7. Effect of GH and insulin treatment on triglyceride clearance rate in Hx rats. Values are based on 3-6 observations ±SEM (* p < 0.05 vs. Hx, one-way ANOVA followed by Bonferroni s test). In conclusion, GH treatment increased the hepatic triglyceride content and secretion, probably by enhancing lipogenesis (FAS and SCD mrna). Upregulation of SREBP-1c mrna was not required for increased FAS and SCD mrna levels. Insulin antagonised the effects of GH on hepatic TG content and secretion, but this was not through decreased FAS and SCD mrna expression. GH treatment also increased clearance of the secreted VLDL particles. PAPER II Effects of gender and growth hormone secretory pattern on sterol regulatory element binding protein-1c and its downstream genes in rat liver. It is known that the hepatic triglyceride synthesis and VLDL secretion is higher in female rats compared to males due to the more continuous GH secretory pattern characteristic of the female rat [136, 137], but the mechanisms behind this are not fully known. As the availability of fatty acids limits triglyceride synthesis and hence formation and secretion of VLDL, the effects of gender and GH secretory pattern on the expression of genes involved in lipogenesis were investigated. The mrna levels of SREBP-1c, FAS and GPAT were higher in female rats than in male rats (Fig. 1, Paper II). Moreover, these genes were increased by GH administered as a continuous infusion to Hx female rats, thus mimicking the female GH secretory pattern, while GH given as two daily injections, thus mimicking the male GH secretory pattern, had no effect compared to Hx control rats (Fig. 2, Paper II). In contrast to the in vivo results, however, GH treatment in vitro decreased the mrna levels of FAS and 35

36 Caroline Améen GPAT, and did not affect SREBP-1c mrna expression (Fig. 3, Paper II). This suggests that the effect of GH on these genes in vivo is indirect. The GH plasma pattern of intact males was feminised by giving a low dose of GH as a continuous infusion. These rats were shown to be less insulin sensitive (Table 2, Paper II) and had higher levels of FAS and GPAT mrna, indicating that continuous GH infusion could exert its stimulatory effect on FAS and GPAT mrna expression through decreased insulin sensitivity. SREBP-1c mrna expression, however, did not change in these animals, again showing that upregulation of SREBP-1c is not required for the stimulatory effect of the continuous GH infusion on FAS and GPAT mrna (compare Paper I). The ACC-1 mrna expression was not sex-differentiated but was specifically upregulated in Hx rats administered GH as a continuous infusion (Fig. 1 and 2, Paper II). However, ACC-1 mrna levels were not affected in intact males given a low dose of GH as a continuous infusion, which suggests yet another regulation of this gene. In Paper I it was shown that the hepatic expression of SCD-1 increased after continuous infusion of GH to Hx rats. In this study, we extend those findings by showing that SCD-1 mrna expression is upregulated by both female- and male-like GH administration to Hx rats (Fig. 2, Paper II) and that the mrna level of SCD-1 was increased by GH in vitro (Fig. 3C, Paper II). Together these results show that SCD-1 is upregulated by a direct effect of GH on hepatocytes that is not dependent on the mode of GH exposure. The mrna expression of liver X receptor (LXR) α, known to mediate the effects of insulin on SREBP-1c and lipogenesis, was decreased by GH both in vivo and in vitro (Fig. 2 and 3, Paper II) and could thus not explain the effects of GH on SREBP-1c and its downstream genes in vivo. In conclusion, FAS and GPAT mrna levels are specifically upregulated by the female GH secretory pattern, while SCD-1 mrna expression is increased by GH irrespective of administration mode. Increased FAS and GPAT, but not SCD-1 mrna levels, could thus explain the stimulatory effect of the female GH secretory pattern on hepatic triglyceride synthesis. Decreased insulin sensitivity, but not changed LXRα mrna expression, could be responsible for these effects of GH in vivo. PAPER III Sex difference in hepatic microsomal triglyceride transfer protein expression is determined by the growth hormone secretory pattern in the rat. In this paper, the mechanism behind the upregulation of VLDL secretion by the GH secretory pattern in female rats was further studied. As MTP is known to regulate the 36

37 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism assembly and secretion of VLDL particles, we investigated whether the expression of MTP also is influenced by gender and the sexually dimorphic GH secretory pattern. The expression of MTP mrna and protein was found to be higher in female rats than male rats (Fig. 1A and B, Paper III). This sex difference was abolished by gonadectomy, but restored by 17β-estradiol and testosterone treatment to Gx female and male rats, respectively (Fig. 1C, Paper III). The mrna and protein expression of MTP was increased in male rats that were feminised with respect to their GH secretory pattern (Fig. 2, Paper III), while a continuous infusion of GH to female rats did not affect the expression of MTP. This suggests that the feminine, more continuous GH secretory pattern is responsible for the higher MTP expression in female rats. This is supported by the finding that Hx of female rats markedly decreased the level of MTP mrna and protein, while Hx of male rats had no effect on MTP mrna expression (Fig. 3, Paper III). Similarly, the mrna and protein expression of MTP was specifically upregulated by the female-characteristic GH administration (continuous GH infusion) in Hx female rats (Fig. 4, Paper III). In Paper I it was shown that the hepatic triglyceride secretion was increased by a continuous infusion of GH and that this effect was diminished by conbined insulin treatment (Fig. 2B, Paper I). In this study, we investigated whether the inhibitory effect of insulin on triglyceride secretion could be due to a decrease in MTP levels. However, insulin did not affect the expression of MTP mrna, neither alone nor in the presence of GH (Fig. 5, Paper III). Thus, this indicates that insulin is not likely to mediate its inhibitory effect on GHinduced triglyceride secretion through decreased MTP mrna expression. In conclusion, the MTP expression is sexually differentiated with a higher expression in female rats due to the stimulatory effect of the feminine GH secretory pattern. These results might help to explain the effects of gender and GH on VLDL assembly and secretion. PAPER IV PPARα activation increases microsomal triglyceride transfer protein expression and activity in the liver. PPARα agonists have previously been shown to enhance the secretion of apob-100 in primary rat hepatocytes despite decreased triglyceride synthesis [92]. As the level of MTP is known to determine the secretion of apob, we investigated whether the effect of PPARα activation on apob-100 secretion could be explained by increased MTP expression. 37

38 Caroline Améen Treatment of mice with the PPARα agonist WY 14,643 (WY) increased both expression and activity of MTP in the liver, but had no effect in the intestine (Fig. 1, Paper IV). WY treatment also increased MTP expression and activity in rat liver (Fig. 2, Paper IV), showing that the effect of WY was not specific to mice. Incubation of cultured mouse hepatocytes with WY also increased the mrna expression of MTP, while a PPARγ agonist (rosiglitazone) did not have an effect on MTP mrna levels (Fig. 3, Paper IV). This shows that MTP responds to PPARα, but not to PPARγ activation. Moreover, MTP mrna expression in primary hepatocytes isolated from PPARα null mice was not changed by WY incubation, indicating that WY increases MTP expression specifically through PPARα activation (Fig. 4, Paper IV). Addition of the RXR ligand 9-cis-retinoic acid (cra) to the medium, however, increased the expression of MTP both in primary hepatocytes from wildtype mice as well as PPARα null mice (Fig. 4, Paper IV), demonstrating that the effect of cra is not dependent on PPARα. In line with the previous findings [92], WY increased the secretion of apob- 100 from primary rat hepatocytes but had no effect on apob-48 secretion (Fig. 5C and D, Paper IV). In the present study, we extend these findings by showing the induction time for the increase in apob secretion after WY treatment. MTP mrna levels increased between 6 and 24 h, and MTP protein expression and apob-100 secretion increased between 24 and 72 h (Fig. 5A and B, Paper IV). The similar time courses of these effects thus indicate that the stimulatory effect of the PPARα agonist on apob- 100 secretion could be mediated by an increased expression of MTP. In conclusion, WY-induced PPARα activation stimulates MTP expression through a direct effect on hepatocytes. The increase in MTP levels is paralleled by a change in apob-100 secretion, which indicates that MTP expression could mediate the stimulatory effect of WY on apob-100 secretion. 38

39 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism GENERAL DISCUSSION REGULATION OF LIPOGENESIS AND VLDL SECRETION BY GH Fatty acids taken up by the liver are known to increase VLDL secretion, but before being incorporated into VLDL particles most fatty acids seem to enter a cytosolic storage pool of triglycerides (for review see [16, 172]). This indicates that the size of the cytosolic triglyceride storage pool may be of importance for the rate of VLDL secretion. This concept agrees with the effects of GH seen in Hx rats, where the changes in hepatic triglyceride content were paralleled by changes in triglyceride secretion rate (Paper I). It thus seems as if GH controls VLDL secretion by changing the availability of triglycerides for VLDL assembly. However, several aspects of the turnover of this cytosolic triglyceride storage pool are far from elucidated. For instance, there is still little known about the enzymes responsible for hydrolysis of the cytosolic triglyceride pool and the subsequent transport of the products (acyl-coa and diacylglycerol) to ER for reesterification by diacylglycerol acyltransferase-2 (DGAT- 2) [172]. As these processes may be major determinants of the size of the secretioncoupled triglyceride pool, it would be of interest to investigate the regulation of these steps, especially with respect to GH. Substrates for the triglyceride storage pool are mainly derived from three sources: 1) free fatty acids bound to albumin in the plasma, 2) uptake of lipoproteins and 3) de novo synthesis of fatty acids and triglycerides. Under normal conditions, de novo lipogenesis quantitatively accounts for only a minor part of the triglycerides secreted; about 9% in rats [173] and < 5% in humans [174]. In spite of this, de novo lipogenesis has been intimately connected to changes in VLDL secretion in some nutritional and metabolic conditions [175]. For example, the contribution of hepatic de novo fatty acid synthesis to triglyceride secretion in obese Zucker rats is increased to 44% (as compared to 9% in lean littermates) in association with enhanced triglyceride secretion [173]. In the present study it was shown that continuous GH administration increased the hepatic triglyceride secretion rate and content (Paper I) along with increased mrna expression of the lipogenic enzymes ACC-1, FAS, SCD, GPAT and their regulator SREBP-1c (Paper I and II). These findings suggest that one mechanism by which GH controls triglyceride synthesis and secretion is through stimulation of the gene expression of enzymes involved in both de novo fatty acid and triglyceride synthesis. Thus, the GH-induced stimulation of triglyceride secretion also seems to be linked to the degree of lipogenesis in the liver. However, the GH effect on lipolysis in adipose tissue may also contribute to the hepatic triglyceride pool. These fatty acids may be incorporated into triglycerides via enhanced activity of GPAT. Moreover, 39

40 Caroline Améen DGAT mrna levels have been shown to be increased in GH transgenic mice [176], suggesting that GH also may influence triglyceride synthesis and storage pool via activation of this enzyme. Female rats have higher triglyceride synthesis and secretion than males [161, 177], which could be explained by the sexually differentiated GH secretory pattern [136]. It is therefore interesting to note that SREBP-1c, FAS and GPAT mrna levels were higher in female rats than in males, and that these genes in addition to ACC-1 were specifically upregulated by the continuous female-like GH administration in Hx (Paper II). Moreover, the mrna expression of FAS and GPAT was increased in intact males with feminised plasma pattern of GH, again suggesting that the female-like GH secretory pattern upregulates these genes. Thus, increased mrna expression of at least FAS and GPAT could help to explain the specific stimulatory effect of the female pattern of GH secretion on hepatic triglyceride synthesis and secretion. The effect of the continuous female-like GH plasma pattern on lipogenic enzymes might also be important in humans. It has been shown that the hepatic de novo fatty acid synthesis is higher in women than in men [178]. It can therefore be speculated that the higher fatty acid synthesis in women compared to men is due to a sex difference in gene expression of FAS as shown in rat liver (Paper II). Moreover, the GH secretory pattern is sexually dimorphic in humans just like in rodents, with higher basal GH levels between peaks in women compared to men [94, 96]. The sex difference in GH secretion has also been implicated in the regulation of some human hepatic functions, like CYP3A4 activity [179], plasma levels of lipoprotein (a) and expression of apo(a) [180, 181]. Thus, available data indicate that there is a sex difference in hepatic lipogenesis and that the sex-differentiated GH secretory pattern also influences hepatic functions in man. Interestingly, the secretion of GH is more frequent in obese males with type 2 diabetes mellitus than in BMI-matched healthy controls. The GH secretory pattern in these patients is therefore more or less continuous with few or no periods of undetectable GH levels [3], thus resembling the female GH secretory pattern. In contrast, obese males without type 2 diabetes have reduced GH secretion compared to non-obese males, probably due to the lower testosterone [182] or the higher serum FFA levels [183] in these subjects. The obese non-diabetic subjects therefore have a low GH secretion and male-characteristic diurnal periods of very low or undetectable GH levels between GH peaks. This indicates that the diabetic state per se has a stimulatory effect on GH secretion frequency that outweighs the inhibitory effect of obesity on GH secretion in patients with type 2 diabetes. It could therefore be speculated that obese men that develop type 2 diabetes have an increased hepatic lipogenesis and a 40

41 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism subsequent increased hepatic VLDL secretion [9] due to their continuous GH secretion. The reason for the changed GH secretion as well as the importance of the changed GH secretion in type 2 diabetes would be an important area for future research. In Paper II it was shown that the increased mrna expression of FAS and GPAT could be explained by decreased insulin sensitivity, as indicated by a lower glucose infusion rate after continuous GH administration compared to controls. The hepatic glucose output only tended to increase in these animals and therefore it cannot be concluded that continuous GH had an effect on insulin sensitivity in the liver. However, as the absolute changes in glucose infusion rate and hepatic glucose output were similar it can be speculated that the increase in hepatic glucose output nevertheless was responsible for the decrease in glucose infusion rate. Moreover, stimulation of hepatic lipogenesis results in increased conversion of glucose to fatty acids and this may lead to underestimation of the increase in hepatic insulin resistance measured as hepatic glucose output. The lipogenic effect of continuous GH infusion might therefore reduce the hepatic glucose output with the outcome that the change in hepatic insulin sensitivity is underestimated. Thus, although the mechanisms are not clear the femalecharacteristic secretion of GH may increase lipogenesis via decreased insulin sensitivity in the liver. It is interesting to note that women with type I diabetes are less insulin sensitive than men with the same condition and that differences in GH secretion between men and women were presumed to be responsible for this difference [184]. Thus, the different GH secretion in males and females might change the response to insulin also in humans. REGULATION OF LIPOGENESIS AND VLDL SECRETION BY INSULIN The stimulatory effect of GH on hepatic triglyceride content and secretion was suppressed by insulin, but this did not occur via decreased FAS and SCD mrna levels (Paper I). The inhibitory effect of insulin on GH stimulation of these parameters in the liver was neither mediated by decreased MTP mrna expression (Paper III). It should be remembered that it is not known how combined GH and insulin treatment affects the protein expression or activity of these gene products, neither what happens with other lipogenic genes such as GPAT or ACC-1 after combined insulin and GH treatment. Due to the antilipolytic effect of insulin on adipose tissue, the GHantagonistic effect of insulin on hepatic triglyceride secretion and content might also be explained by a decreased flux of fatty acids to the liver. Moreover, insulin has been shown to inhibit the stimulatory effect of GH on phosphatidate phosphohydrolase in vitro [185], a gene that generates diacylglycerol by cleaving a phosphate group from 41

42 Caroline Améen phosphatidic acid. As diacylglycerol can form triacylglycerol (triglyceride) that in turn takes part in VLDL assembly, the inhibitory effect of insulin on phosphatidate phosphohydrolase might reduce triglyceride secretion. However, the inhibitory effect of insulin on GH-stimulated phosphatidate phosphohydrolase activity has not been investigated in vivo. The hepatic VLDL production is increased in type 2 diabetes [9, 186]. There is also a corresponding increase in hepatic secretion of triglyceride-rich lipoprotein particles in animal models with insulin resistance and hyperinsulinemia [187]. However, the hepatic production of VLDL particles has been shown to be inhibited by short-term hyperinsulinemia both in vitro and in vivo (for review see [188]), but this inhibitory action of insulin is lost in insulin resistant states [189]. The acute inhibitory effect of insulin on VLDL production in vivo may be explained by the antilipolytic effect of insulin on adipose tissue, leading to a decreased flux of fatty acids to the liver. However, lowering of FFAs by using the antilipolytic agent (acipimox) in humans did not fully imitate the acute effect of insulin on VLDL secretion [190]. Moreover, elevation of plasma FFAs during hyperinsulinemia did not completely abolish the inhibitory effect of insulin on VLDL triglyceride production. These results therefore suggest that changes in plasma FFAs only partly explain the acute inhibition of VLDL triglyceride production by insulin and also that insulin might have a direct inhibitory effect on VLDL production in the liver. A direct effect of insulin on VLDL production is supported by the finding that insulin inhibits apob secretion in primary rat hepatocytes, an effect that is thought to occur through activation of phosphoinositide 3-kinase (PI 3-K) [191]. In contrast, insulin treatment for 2 weeks increases hepatic triglyceride production [192]. This finding was repeated in another long-term study (7 days of treatment) [193], which also showed that the stimulated triglyceride secretion in hepatocytes derived from hyperinsulinemic rats could be decreased by insulin added in vitro. These results therefore suggest that other metabolic alterations than hyperinsulinemia are responsible for the lack of inhibition of VLDL secretion by insulin in insulin resistant states. Our observations that long-term insulin treatment (7 days) tended to decrease the hepatic triglyceride secretion rate and significantly suppressed the effect of GH on hepatic triglyceride secretion rate (Paper I) are thus different from the effect of exogenous insulin on hepatic triglyceride secretion seen in other 1-2 week studies [192, 193]. These findings are instead more similar to shortterm effects of insulin in terms of triglyceride secretion, showing a unique feature of our Hx model. 42

43 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism REGULATION OF SREBP-1c AND LIPOGENIC ENZYMES Whereas cholesterol synthesis almost completely is regulated by SREBPs, the synthesis of fatty acids also depends on other transcription factors. LXR is activated by oxidised cholesterol and mainly regulates the transcription of genes controlling cholesterol homeostasis [194]. However, this nuclear receptor is also involved in the regulation of fatty acid synthesis, suggesting that LXR is an important factor in the regulatory interplay between cholesterol and fatty acid metabolism. The FAS promoter contains a binding element for the nuclear receptor LXR (a direct repeat of AGGTCA separated by 4 nucleotides, DR4), which allows a low level of transcription in response to LXR ligands even when SREBPs are suppressed [195]. As GH decreased both LXR and FAS mrna (Paper II), it is possible that LXR mediates the inhibitory effect of GH on FAS mrna expression in vitro. The reduced LXRα mrna expression might be responsible also for the lower GPAT mrna level in GH-treated primary rat hepatocyte cultures (Paper II), but a functional LXR response element has not been identified in the GPAT promoter as yet. PPARα agonists have been shown to increase hepatic LXRα expression [196]. As GH decreases PPARα expression in liver both in vivo and in vitro [197, 198], reduced PPARα signalling might mediate the lower LXRα mrna expression seen after GH treatment in vitro and in vivo. Thus, GH may inhibit the hepatic expression of lipogenic enzymes through a direct effect on the hepatocyte, possibly involving changed LXRα mrna levels. In contrast to the in vitro situation, however, continuous GH treatment of Hx rats increased FAS and GPAT mrna levels in spite of decreased LXRα mrna expression (Paper II). Moreover, the mrna expression of LXR was decreased irrespective of GH administration mode. This shows that decreased LXRα expression cannot explain the specific stimulatory effect of the female secretory pattern of GH on mrna expression of FAS and GPAT in vivo. It has recently been discovered that SREBP-1c also is a target for LXR [199] and an oxysterol-inducible LXR-binding site has accordingly been identified in the mouse SREBP-1c promoter [200]. The increase in SREBP-1c transcription by oxysterols allows for continued synthesis of fatty acids even in cholesterol-loaded cells, which makes it possible to produce inert cholesterol esters. This mechanism has therefore been suggested to be important for the protection of cells from excess free cholesterol. Moreover, the stimulatory effect of insulin on expression of SREBP-1c and its downstream genes is dependent on the presence of LXR [201], and insulin treatment also increases the hepatic LXRα expression [201]. It is therefore conceivable that insulin mediates its stimulatory effects on SREBP-1c and lipogenesis by increasing LXRα expression. However, GH administration decreased the LXRα mrna level, but the same treatment still increased the expression levels of SREBP-1c in vivo (Paper 43

44 Caroline Améen II). This shows that GH does not mediate its effects through the same mechanisms as insulin. In contrast to FAS and GPAT mrna expression, decreased insulin sensitivity was not likely to contribute to upregulation of SREBP-1c mrna (Paper II), suggesting that another factor than changed insulin sensitivity is involved in the regulation of this gene. GH has been reported to stimulate the enzymatic activity of rat hepatic cholesterol 7α-hydroxylase [202], the rate-limiting enzyme in bile acid synthesis. Moreover, overexpression of cholesterol 7α-hydroxylase in McArdle rat hepatoma cells increased the cellular content of mature SREBP-1 [56], probably due to inactivation of oxysterol repressors. Thus, GH might have induced SREBP-1c expression indirectly by stimulation of cholesterol 7α-hydroxylase activity (Paper I and II). It is interesting to note that a functional LXRE has been identified in the promoter region of the rat cholesterol 7α-hydroxylase gene through which LXR exerts a stimulatory effect [203]. Moreover, a previous study has demonstrated increased cholesterol 7α-hydroxylase activity in spite of decreased mrna expression after GH treatment in Hx rats [202]. Thus, even though GH may reduce the amount of cholesterol 7α-hydroxylase mrna via decreased LXR mrna expression, the same treatment might increase the activity of cholesterol 7α-hydroxylase via another mechanism. This could lead to reduced oxysterol levels and a concomitant increase in mature SREBP-1 protein as well as lipogenic enzyme levels. In future studies it would therefore be interesting to investigate the regulation of cholesterol 7α-hydroxylase activity by the sexually dimorphic secretory pattern of GH. Moreover, it would be interesting to investigate whether cholesterol 7α-hydroxylase activity is unaffected by GH in vitro just like SREBP-1c, which then could explain the lack of effect by GH on SREBP-1c. An alternative explanation for the effect of GH on SREBP-1c mrna levels could be that GH regulates the expression of IRS-2, a protein important for insulin signalling and insulin sensitivity. Insulin resistant ob/ob mice have a decreased expression of IRS-2 and increased expression of SREBP-1 [204]. Moreover, IRS-2 knockout mice have increased expression of SREBP-1c, indicating that decreased expression of IRS-2 increase SREBP-1c expression [205]. Thus, continuous GH infusion may alternatively mediate its stimulatory effects on SREBP-1c through decreased IRS-2 expression. REGULATION OF MTP - POSSIBLE MECHANISMS OF GH ACTION The expression of MTP was specifically induced by the female-like GH secretory pattern (Paper III), but it is not known how this effect is mediated. The MTP mrna 44

45 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism level has been shown to be increased in mice that overexpress SREBP-2 [55], and both MTP and SREBP-1c levels are upregulated in insulin-resistant models [49]. Likewise, stimulation of MTP mrna expression was linked to increased cellular content of mature SREBP-1 in rat hepatoma cells transfected with cholesterol 7α-hydroxylase [56]. As cholesterol 7α-hydroxylase decreases the level of oxysterols in these cells, the suppression of the sterol-sensitive SREBP cleavage is likely to be alleviated. This could explain the increased levels of mature SREBP-1, which in turn may stimulate MTP expression. Since GH increases cholesterol 7α-hydroxylase activity [202], it could be hypothesized that the continuous GH administration stimulated the expression of MTP in the liver by induction of SREBP-1 due to increased cholesterol 7α-hydroxylase activity. However, this hypothesis is not likely since the regulation of SREBP-1c and MTP by GH was not similar (compare Paper II and Paper III). Paradoxically, a reporter gene study in HepG2 cells suggests that SREBP inhibits MTP transcription [54] and a negative modified SRE site has also been found in the human MTP promoter [46]. The reason for these discrepant results is difficult to explain, but may be due to species or model differences. Triglyceride-rich diets exert a stimulatory effect on MTP expression [50-52] and it has therefore been suggested that the fatty acid availability in the liver is an important factor determining MTP expression. It might hence be speculated that GH also exerts its stimulatory effect via similar mechanisms. This is consistent with the known lipolytic effect of GH, and also with the increased hepatic lipid content (Paper I) and expression of lipogenic enzymes (Paper I and II) after continuous GH administration in vivo. Studies of GH and fatty acid regulation of MTP in vitro using cultured hepatocytes would resolve this issue. In this context it is interesting to note that PPARα is activated by long-chain unsaturated fatty acids and that we in Paper IV show increased MTP expression by PPARα activation. It could therefore be speculated that the stimulatory effect of triglyceride-rich diets on MTP is mediated via PPARα. To decisively establish the relationship between unsaturated fatty acids, PPARα and MTP, it would be valuable to use PPARα knockout mice given a diet enriched in triglycerides containing unsaturated fatty acids. The increase in MTP expression by triglyceride-rich diets might be also mediated by hepatocyte nuclear factor-4 (HNF-4), since this transcription factor is a major activator of genes involved in lipoprotein metabolism and is responsive to fatty acids. Indeed, consensus recognition sequences for HNF-4 have been identified in the human MTP promoter [46] and HNF-4α null mice show reduced levels of MTP expression [206]. Whether HNFs also mediate the stimulatory effect of the female GH secretory pattern on MTP is not known, but several observations indicate this possibility. One strong candidate transcription factor for mediating the specific effect of the female-like GH 45

46 Caroline Améen secretory pattern on MTP is HNF-6. This transcription factor is expressed at about 2- fold higher levels in female than male liver [207], and the female pattern of GH secretion induces liver HNF-6 mrna about 2-fold more efficiently than the male pattern [207]. Moreover, continuous GH administration stimulates transcription of the female-specific CYP2C12 gene via HNF-6 binding to the CYP2C12 promoter [207], linking HNF-6 to the regulation of a sex-differentiated gene. Binding sites for HNF-6 have been identified by homology search in the promoter regions of several other genes that are known to be regulated by the sexually dimorphic GH secretory pattern [207]. This indicates that HNF-6 may be involved in the sex-dependent control also of other GH-regulated genes. The HNF-6 gene is known to be stimulated by HNF-4 [208] and the decreased MTP expression in HNF-4α KO mice hence denotes a connection between HNF-6 and MTP. Thus, HNF-6 may contribute to the stimulatory effect of the female-characteristic GH secretory pattern on MTP expression. IMPORTANCE OF CHANGED MTP LEVELS FOR THE EFFECTS OF PPARα The general effect of fibrates in dyslipidemic patients is characterised by a marked decrease in plasma triglycerides and an increase in HDL cholesterol levels. In particular, treatment with fibrates results in decreased plasma levels of VLDL 1 [4]. Overproduction of large triglyceride-rich VLDL 1 particles in type 2 diabetes is especially detrimental with respect to atherosclerosis, mainly because they are the precursors of atherogenic small dense LDL [9]. It is therefore interesting to note that treatment of primary rat hepatocytes with the PPARα agonist WY 14,643 increases apob-100 secretion despite decreased triglyceride synthesis, which results in a secretion shift from large triglyceride-rich VLDL particles to smaller triglyceride-poor apob-containing lipoproteins [92]. These triglyceride-poor particles do not generate small dense LDL to the same extent as large triglyceride-rich VLDL particles and would therefore improve the atherogenic lipid profile. In Paper IV, we found that the mechanism for the stimulatory effect of WY on apob-100 secretion could be via increased hepatic expression and activity of MTP. Thus, PPARα agonists may promote a beneficial lipid profile by increasing MTP expression in addition to reducing triglyceride synthesis, supporting an increase in hepatic secretion of apob on smaller triglyceride-poor lipoprotein particles. Increased hepatic MTP activity might also be responsible for the small and variable effects of fibrates on LDL cholesterol levels [209] that in some studies even result in an increase in LDL cholesterol levels [210]. In Paper IV, the serum level of apob was decreased in mice treated with WY in spite of increased MTP expression and probably also increased apob-100 secretion. This observation could reflect the formation of smaller less triglyceride-rich apobcontaining lipoproteins as these particles promote the production of LDL particles with 46

47 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism shorter half-lives than those generated from large triglyceride-rich VLDLs [4]. Fibrate treatment is moreover known to exert its lipid-lowering effect through a reduction in apoc-iii and increased LPL expression [86], which also contributes to enhanced clearance of apob-containing particles from the circulation. Thus, fibrate treatment might induce a favourable lipid profile in terms of atherosclerosis by promoting both the secretion of less atherogenic lipoproteins particles and by increasing their turnover. MTP AND DIFFERENT REGULATION OF APOB-48 AND APOB-100 In contrast to the stimulatory effect of WY incubation on apob-100 secretion, WY did not affect the secretion of apob-48 in primary rat hepatocytes (Paper IV). This is consistent with previous findings, showing increased secretion of apob-100 only [92]. In that study, it was also shown that WY inhibited the cotranslational degradation of apob-100 but not of apob-48. Together these observations highlight the fact that there are differences between the assemblies of apob-48- and apob-100-containing particles, at least in primary rat hepatocytes. There are studies indicating that hepatic MTP deficiency might affect the secretion of apob-100 more than the secretion of apob-48 [41, 43]. For example, inactivation of the MTP gene in mouse liver reduced the plasma level of apob-100 with more than 95%, while apob-48 in the plasma only was reduced by 20% [43]. It could be speculated that all apob-48 in the plasma instead originated from the intestine, since the intestine produces only apob-48 and MTP was specifically knocked out in the liver. However, the plasma apob-48 level in mice lacking apob production in the intestine is not significantly different from that in control mice [211]. Thus, these findings indicate that most of the apob-48 in mouse plasma originates from the liver and that apob-48 can be secreted in the absence of MTP. That apob-100 secretion is more dependent on MTP than secretion of apob-48 has also been indicated in experiments using MTP inhibitors. This is exemplified by the finding that MTP inhibition only blocked the secretion of apob polypeptides that had reached 65% or more of full length apob-100 in HepG2 cells [31]. Thus, the requirement of MTP activity for apob secretion seems to be dependent on the length of the apob polypeptide - the longer the apob polypeptide, the more dependent on MTP. This might be explained by the existence of clusters of lipid-associating domains in the carboxy-terminal part of apob-100 [212]. As the apob polypeptide elongates more lipophilic β-sheets are formed, and the requirement of MTP activity thus increases [212]. It could therefore be speculated that the reason for the discrepant effects of PPARα activation on apob-48 and apob-100 secretion is that apob-100 secretion is more sensitive to changed expression of MTP than apob-48 and that this might be due to the different lengths of the apob proteins. 47

48 Caroline Améen In mice overexpressing MTP in the liver, however, apob-48 and apob-100 was shown to equally contribute to an increased secretion of VLDL-triglycerides [39]. This indicates that the difference in sensitivity between apob-48 and apob-100 might be specific for MTP-deficient conditions. The MTP expression was increased in our study, but there was still a differentiated regulation of apob-48 and apob-100 secretion (Paper IV). As the triglyceride synthesis also was decreased in the primary rat hepatocytes, it could be speculated that a distinction between apob-48 and apob-100 secretion is prominent either when the availability of triglycerides is scarce or when MTP levels are insufficient, i.e. during conditions that limit VLDL assembly. To test this possibility it would be interesting to study whether the changes in apob-48 and apob-100 secretion would be similar to those after PPARα activation if the triglyceride synthesis specifically is blocked and MTP is over-expressed. These studies could determine whether the specific effect of PPARα on apob-100 secretion could be solely explained by changed triglyceride synthesis and increased MTP activity or if other effects of PPARα are involved. It would also be interesting to investigate whether the increase in MTP expression was due to a transcriptional effect of the PPARα agonist on the MTP gene. This is likely to be the case, since a functional DR1 element, characteristic of PPREs, has been identified in the MTP promoter [213]. 48

49 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism SUMMARY AND CONCLUSIONS In this thesis, the effects of GH and PPARα on lipid metabolism have been investigated, especially with respect to hepatic genes involved in lipogenesis and VLDL assembly. The importance of increased insulin levels for the effects of GH on lipoprotein metabolism was investigated in Hx rats. GH increased the triglyceride secretion rate and content in the liver (Figure 8), which could be explained by increased gene expression of enzymes in lipogenesis. Insulin suppressed the effect of GH on the hepatic triglyceride secretion rate and content, but this was not due to decreased expression of SCD-1, FAS or MTP. Insulin also suppressed the effect of GH on SREBP-1c mrna. The GH-antagonistic effect of insulin may therefore be explained by changed expression of other SREBP-1c regulated genes or decreased availability of FFA. In conclusion, insulin does not mediate the effects of GH but inhibits the stimulatory effect of GH on hepatic SREBP-1c expression and hepatic triglyceride secretion rate and content. TG FAS, GPAT mrna TG synthesis* + + Cytosol apob TG Pre-VLDL ER lumen TG MTP TG TG synthesis* + + GHc TG Figure 8. The effects of continuous GH (GHc) administration in this thesis. *TG (triglyceride) synthesis was measured as total TG biosynthesis in the whole cell (results from Sjöberg et al, 1996 [136]). 49

50 Caroline Améen The regulation of hepatic lipogenic genes and MTP by the sex-differentiated GH secretory pattern was studied in Hx rats administered GH in a mode that mimics either the female or male plasma pattern of GH. FAS and GPAT mrna levels were increased in females compared to males and specifically upregulated by female-like GH administration, probably due to decreased insulin sensitivity. SCD- 1 was not sex-differentiated and increased by GH irrespective of administration mode. The MTP expression was higher in female rats due to the female GH secretory pattern. In conclusion, increased expression of FAS, GPAT and MTP could help to explain the previously described stimulatory effects of female sex and the female GH secretory pattern on VLDL assembly and secretion (Figure 8). Treatment of mice and rats with a PPARα agonist (WY) increased MTP expression and activity in mouse and rat liver. Incubation of primary cultures of mouse and rat hepatocytes with WY also increased MTP expression. By using primary hepatocyte cultures from PPARα KO mice, this effect of WY was shown to be PPARα specific. In rat hepatocytes incubated with WY, MTP protein expression and apob- 100 secretion increased between 24 and 72 h of incubation. In conclusion, PPARα activation increases MTP expression and activity that could explain the increased apob-100 secretion from hepatocytes following PPARα activation (Figure 9). - Co-translational degradation* TG TG synthesis* - Cytosol apob TG Pre-VLDL ER lumen TG MTP TG synthesis* + PPARα TG + + Small TG-poor VLDL* ApoB-100 Large TG-rich VLDL* - Figure 9. The effects of PPARα treatment in this thesis. *Results from Lindén et al, 2002 [92]. TG (triglyceride) synthesis was measured as total TG biosynthesis in the whole cell. 50

51 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism ACKNOWLEDGEMENTS I am grateful to all the persons who in one way or another have contributed to this thesis or who have made my life as a PhD student much more enjoyable. I would especially like to thank: Jan Oscarsson, my supervisor, for all your support and excellent guidance throughout the years. You have shared both your great knowledge and enthusiasm for science with me, which I have very much appreciated. Past and present members of the Endocrine division, for all the good times and for creating such a nice atmosphere to work in. It was sad leaving you but I am very happy for the warm welcome at Wallenberg laboratory! My time here has been a pleasure thanks to all nice people and the cheerful atmosphere! All co-authors, for help with lab work and valuable comments on the manuscripts. My present fellow PhD students, Anna Ljungberg, for being a good friend and for sharing my passion for apples and aversion for cold, and Ulrika Edvardsson, for nice company in the lab and for being my personal Maniatis. My former fellow PhD students, Linda Carlsson, for good company in the booth and for the way you kept the Endocrine division in order, Daniel Lindén, for nice collaboration that is still ongoing, and Fredrik Frick, for your sense of humour that makes everyone feel good at work. Mikael Alsterholm, for being such a nice person and for always drinking tea with me. I miss you at the lab (are you coming back soon?)! Heimir Snorrason, the enchanting wizard of computers, for invaluable help with everything concerning computers. I have really appreciated all your kind help! All my friends outside the lab - no one mentioned, no one forgotten. I m sorry that I have been asocial the last couple of months Now I will definitively try to spend more time with you, my excellent friends! Jonas Boström, for all the fun we had throughout the years and for being my best friend. Especially many thanks for your excellent Thai food and the warm bath that saved me from the radioactivity disaster. 51

52 Caroline Améen My parents, Ulla and Torkel, for all your love and for always always being there. Kajsa, the best sister you can think of! Thanks for being such a cool and kind sister (except when you don t get food in time) and, of course, for regularly getting rid of the ball. Sandra, for endless love and support, thank you for everything. And remember, Acknowledgements is NOT the important part of a thesis! Now go on read from the beginning 52

53 Growth hormone and PPARα in the regulation of genes involved in hepatic lipid metabolism 53