Hepatic Lipase (HL; EC 3.1.1.3) is an extracellular enzyme which has phospholipase A1 and triacylglycerol hydrolase activity. It has also some esterase activity but no cholesterylesterase activity. Both rat and human HL are glycoproteins bearing 2 and 4 N-coupled oligosaccharides. Most of the HL protein is synthesized in the parenchymal cells of the liver and secreted into the space of Disse (1 - 3) were it binds to heparin sulfate proteoglycans. The HL protein is also present in the steroidogenic organs as adrenal glands, ovaries and testes (4 - 7). By using heparin HL protein is displaced from its binding site similar to lipoprotein lipase (LPL). The rat and human HL protein are both homodimers, and the monomer of the rat HL has a molecular weight of 58 kDa and that of human HL a molecular weight of 65 kDa. In the metabolism of plasma lipoproteins HL plays an important role; it mediates the conversion of cholesterol-enriched HDL2 to cholesterol-poor HDL3, the conversion of IDL to LDL, and the formation of small dense LDL from large buoyant LDL. HL has a role in postprandial lipid transport where it facilitates the clearance of remnant lipoproteins by the liver (8). Besides this, in adrenals and ovaries the HL enzyme may play a role in delivery of HDL cholesterol for steroidogenesis, at least in the rat (9, 10). The amount of HL in human post-heparin plasma is determined by genetic (11, 12), hormonal and nutritional factors (13), and by body composition (14 - 16). The HL is associated with the risk on Coronary Artery Diseases (CAD). Several human association studies and transgenic animal models have shown that a high expression of HL protects against the development of atherosclerosis. However, other studies have shown that HL is pro-atherogenic (reviewed in 8). The finding that high HL expression is anti-atherogenic or sometimes pro-atherogenic depends on other genetic or metabolic factors, like concomitant hypertriglyceridemia (8, 17). A correlation has been found between high HL expression and low levels of HDL cholesterol and reduced LDL particle size (18). It was also found that humans with central obesity and insulin resistance (14) or type 2 diabetes (19, 20) show increased levels of HL expression. How HL gene expression is altered in insulin-resistant conditions is unknown. Hetero- and homozygosity for the common HL promoter -514C->T polymorphism is correlated with reduced post-heparin HL activity, and is associated with dyslipidemia and insulin resistance in healthy controls and in FCH (21). The HL gene contributes to the atherogenic lipoprotein profile (high plasma TGs, low HDL cholesterol, reduced LDL particle size) in familial combined hyperlipidemia (FCH) (22). In cell culture experiments using human HepG2 hepatoma cells, HL expression was found to be increased by elevated levels of fatty acids (23, 24) and glucose (25, 26), conditions that prevail in insulin resistance. How these metabolic factors affect HL expression is largely unknown.
2 Hepatic Lipase and the metabolism of lipoproteins
HL is besides lipoprotein lipase (LPL) one of the two important lipolytic enzymes that takes part in plasma lipoprotein metabolism (2). Unlike LPL, HL does not have an absolute requirement for an apolipoprotein cofactor in order to be enzymatically active, although different types of apolipoproteins can affect the HL activity. HL plays a role in the exogeneous and endogeneous pathway, as well in the reverse cholesterol pathway. This will be discussed next in more detail:
2.1 The exogenous pathway
In the exogenous pathway the digestion products of dietary fat are absorbed by the cells of the intestinal mucosa. In the enterocytes, triglycerides (also named as triacylglycerol or triacylglyceride, abbreviated as TG) are synthesized from absorbed monoglycerides and fatty acids at the smooth endoplasmatic reticulum, together with the synthesis of phospholipids and cholesterol. At the rough endoplasmatic reticulum the synthesis takes place of the Apolipoprotein (apo) B-48, apo A-I, apo A-II and apo A-IV. In the Golgi-apparatus these components are assembled into chylomicrons. The chylomicrons are formed discontinuously in the intestinal mucosa this depending on the amount of the neutral fat to be available and able to be absorbed.
When the supply of TGs increases this has as result that not only the number of the particles increases but also the size of the chylomicrons. The chylomicrons are secreted by the intestine into the systemic circulation via the thoracic duct after which they then re-circulate into the liver. Observations suggest that chylomicrons are synthesized continuously by human absorptive cells, both in the fasting state and after eating (27). After entering the systemic circulation, the chylomicrons have a half-life of only a few minutes and as consequence they are only found in the serum after a high-fat meal and not in fasting serum (28 -31). In the postprandial state chylomicron secretion and synthesis is induced after fat ingestion (32, 33). In first instance the chylomicrons do not have the appropriate apolipoproteins that would allow them to be taken up by the hepatic receptors in the liver. When they enter the blood apo A-IV and apo A-I are lost and apo C-I, apo C-II, apo C-III and apo E are acquired from the HDL. The apo C-II is necessary as co-factor for LPL. LPL is present on the outside of cells of vessel endothelia in fat tissue and muscle; it hydrolyses most of the TG-rich core of the chylomicrons to fatty acids and glycerol. The fatty acids are taken up by the adipose tissue and by muscle cells and the glycerol is going to the liver. Since the TGs are lost from the chylomicrons, smaller particles develop which are named the chylomicron-remnants. These particles have a too small surface to contain all the apolipoproteins. The surface components (apo C, apo A-I, also some free cholesterol, phospholipids) are released and taken up by the HDL. The apo E stays attached to the chylomicron remnant and enhances the uptake of the particle into the liver. This uptake is mediated by the LDL receptor and the LDL receptor-related protein (LRP) (34). LRP has the identity of a remnant receptor and functions as such (35 - 37); nowadays it is also named LRP1 or occasionally CD91 (38). In the hepatocytes the chylomicron remnant are degraded. In the liver, HL is present extracellular in the space of Disse. This enzyme plays an important role in the uptake of the chylomicron-remnants into the liver. Studies in which mice and rats were injected with antibodies to inhibit HL activity showed impaired chylomicron remnant-removal in rats and mice (39, 40). In transgenic mice over-expressing HL, TG rich plasma lipoproteins like the chylomicron remnants were reduced lowered compared with those in wild-type (WT) mice (41). It has been proposed, either alone or in combination with other factors such as apo E and proteoglycans, that HL may contribute to the sequestration of chylomicron remnants. Studies by confocal microscopy showed that HL co-localized with chylomicron remnant clusters in the Space of Disse in the liver of transgenic mice, suggesting that HL is a component of the LRP-proteoglycan clusters (34). HL therefore helps to direct chylomicron remnants containing dietary cholesterol and residual TGs to the liver by a nonenzymatic mechanism. Furthermore, it has been suggested that HL which is liberated from cell surface heparin sulphate proteoglycans (HSPG) binds to the chylomicron remnants and subsequently brings this remnant in close proximity with involved receptors for uptake into the liver (42) similar to the role of LPL in sequestering chylomicrons and VLDLs to the adipose and muscle tissue. Thus, HL is involved in clearance of atherogenic remnant particles by the liver, which may add to its anti-atherogenic potential.
To be included: Figure 1. Pictorial scheme of the exogenous pathway
2.2 The endogenous pathway
Synthesis of VLDL does take place in the liver similar to the synthesis of chylomicrons. The fatty acids from which the TG is synthesized are derived from "de novo" synthesized fatty acid synthesis, from HSL mediated lipolysis of TG stores in adipose tissue, or from intracellular digestion of internalized remnant particles. During the assembly of VLDL from TG, cholesterol (esters) and phospholipids, apo B 100 is used. The process of assemblage is almost identical to that of the chylomicron. The nascent VLDL is secreted directly into the circulation where it receives apo C-I, apo C-II, apo C-III and Apo E from HDL. The mature VLDL does have apo C-II that is necessary for the activity of LPL. The TG from the VLDL is converted to glycerol and fatty acids. The resulting particle, named the intermediate density lipoprotein (IDL), contains less TG and is smaller than VLDL. The fatty acids does go into the fat cells where it is stored as TG and the glycerol return to the liver. During the break down of the VLDL there are also surface fragments formed, which are taken up by the HDL. The HL present in the liver hydrolyses the TG from the IDL. When apo E and apo C-II is released from the IDL, TG-poor and cholesterol-rich LDL is formed. This LDL can be taken up via the LDL-receptor as well by the liver as the extra hepatic tissue. The LDL-receptor is also able to bind the IDL. In humans less than 20% of the VLDL is converted to LDL. 80% of the LDL precursors are taken up directly by the liver. IDL bind via apo E to the B,E receptor in the liver while IDL and LDL bind to the receptor via apo B 100 ligand. The only apolipoprotein which remains in the particle throughout the entire process is apo B which is later virtually the only apolipoprotein component of LDL. The LDL delivers cholesterol and other LDL components (incl. Fat-soluble vitamins) to the peripheral cells, which express apo B,E receptors depending on their need for cholesterol. A large part of the LDL is taken up by the liver again. 60 - 90% of the LDL catabolism is receptor mediated; 10 - 40% is removed from the plasma via the so-called scavenger pathway (43). The extent of LDL catabolism depends not only on the availability of apo B,E receptors but also on the affinity of the ligand apo B 100. With higher HL activity, more TG is hydrolysed from LDL particles resulting in TG-depleted small dense LDL. However these particles have less affinity for the LDL-receptor. Recent studies suggest that apo C-III, present on light LDL, is an accelerator of the lipolysis of LDL by HL under physiological in vivo conditions. However, apo C-III inhibits postheparin HL activity in vitro. The results suggest that apo C-III strongly inhibits hepatic uptake of VLDL and IDL overriding the opposite influence of apo E when both are present. The presence of apo C-III on dense VLDL is not associated with slow conversion to IDL, a lipoprotein lipase-dependent process. Thus when apo C-III is present on the lipoprotein particles less VLDL remnants are taken up into the liver and more LDL and sdLDL is produced. Thus, a high HL activity promotes the formation of sdLDL, which may attribute to the pro-atherogenic potential of HL.
To be included: Figure 2. Pictorial scheme of the endogenous pathway
2.3 The reverse cholesterol transport
Nascent HDL originating from de novo synthesis in the liver, and the surface remnants resulting from chylomicron degradation are HDL precursors. They are composed of phospholipids, free cholesterol and apo A-I arranged as bilayers and are referred to as discoidal HDL. The HDL takes up free cholesterol from the peripheral cells, which is the beginning of the process of cholesterol transport from the periphery to the liver, named "reverse cholesterol transport". The efflux of cholesterol and phospholipids to apo A-I is mediated by the ATP-binding cassette, subfamily A, member 1 (ABCA1) receptor on peripheral cells (44). In this process, cholesterol and phospholipids are initially integrated in the surface of the HDL. HDL also plays a role in the esterification of cholesterol in the blood plasma. HDL contains apo A-I and apo A-IV that stimulate the enzyme lecithin:cholesterol acyltransferase (LCAT), which is bound to the HDL in the plasma. This enzyme catalysis the conversion of free cholesterol into cholesterol esters with a fatty acid derived from the beta-place of lecithin.
The discoidal HDL consists of a phospholipid coat and become a spherical HDL particle due to the incorporation of the cholesterol esters by LCAT. Also, the result of the uptake of free cholesterol and the forming of cholesterol esters is the increase of the diameter of HDL. The HDL now contains on average more cholesterol esters molecules per particle. The liver then can take up the cholesterol esters from the HDL via a direct way or indirect way of the reverse cholesterol transport. In the direct pathway, HDL directly delivers cholesterol via endocytosis after binding to HDL receptors at the liver cell surface (45). The whole HDL particle ends up in the lysosome and is completely degraded. Alternatively, by docking to SR-B1 both free and esterified cholesterol can be selectively removed from the HDL particles as well (46). Here, the cholesterol ester rich HDL particle is also endocytized but then recycled back to the surface of the liver cell (44). The HDL in the endosome gets in contact with HL, and via the phospholipase A1 activity of HL internalized simultaneously the selective unloading of free and esterified cholesterol is promoted. Pre-treatment of HDL with HL as well or other phospholipases enhances its cholesterol delivery capacity to liver cells (47, 48). Selective cholesterol uptake is also evident in steroidogenic organs, in which HL and SR-B1 co-localize. In rat adrenal, HL cooperates together with SR-BI in the selective uptake of HDL (10). Via the indirect pathway, the cholesterol esters from the HDL are delivered to the liver after Cholesteryl ester transfer protein (CETP) has transferred part of the LCAT produced cholesterol esters from HDL to the apo B containing lipoproteins like IDL in exchange for TGs. The TGs in HDL are hydrolysed by HL. Subsequently HL facilitates the conversion of IDL into LDL. The LDL is then taken up into the liver via the LDL receptor. CETP may also transfers cholesterol esters from HDL to chylomicrons. LPL makes chylomicrons remnants from chylomicrons, which are subsequently taken up into the liver via the remnant receptor. Once taken up by the liver, the cholesterol esters are hydrolysed in the lysosome; part of the resulting free cholesterol is esterified again by the enzyme ACAT and another part is secreted into the bile. In humans, HDL cholesterol is transferred to the liver mainly through the indirect pathway.
Postheparin HL activity correlates inversely with low levels of HDL cholesterol in humans (49). Individuals with a functional mutation of the HL gene leading to a less active enzyme have moderately raised HDL cholesterol with enlarged TG-rich HDL particles (50). Like human patients, HL-deficient mice have increased plasma concentrations of HDL cholesterol and phospholipids compared with wild-type mice. This is even more pronounced when they are fed a high-fat and cholesterol-rich diet (51). The amount of HDL cholesterol is reduced and the size of HDL particles is smaller and denser in transgenic mice and rabbits that overexpress HL (52).
Finally, HL mediates the remodelling of cholesterol-rich HDL (HDL2) thereby producing particles which are smaller, denser and heavier (HDL3). As HDL3 is a better acceptor of peripheral cholesterol and is a better substrate for LCAT than HDL2, this action of HL may further enhance reverse cholesterol transport
The effects of HDL apolipoproteins on HL action are well described in several studies.
It has been suggested that apo A-I, apo A-II, apo C-I, apo C-II, apo C-II, and apo E in HDL inhibit the hydrolysis of TG by HL in cases where lipid emulsions have been used as model substrates. (53 - 55)
After apo A-I, apo A-II is the second most common apolipoprotein in HDL-density class (43) and plays an important role in the reverse cholesterol transport. Although Apo A-II inhibits LCAT and CETP activities and also inhibits the hepatic cholesteryl uptake from HDL (probably through the SR-BI depending pathway), it shows to activate the TG hydrolysis activity of HL on plasma lipoproteins. (56, 57)
Apo A-IV also plays an important role in the reverse cholesterol transport process. It promotes cholesterol efflux from several extrahepatic cells (58, 59) and activates LCAT as explained earlier. Upon LCAT-induced cholesterol esterification in whole plasma, lipoprotein-free apo A-IV redistributes to an HDL subpopulation with a high degree of LCAT modification (60, 61). According to Sindelar and co-worker this apo A-IV-rich HDL is the preferred substrate for HL and apo A-IV alters the substratespecificity of HL from triglyceridase towards phospholipase activity. Apo A-IV strongly stimulated the hydrolysis of phosphatidylcholine and phosphatidylethanolamine in human HDL2 and VLDL, while the hydrolysis of TGs was completely inhibited. (62)
Cholesterol accumulation stimulates macrophages to synthesis large amounts of apo E which is secreted in the form of apo E/phospholipids discs as cholesterol acceptor independently of HDL. During cholesterol esterification in the LCAT reaction, the HDL incorporates the apo E from apo E/phospholipids discs. These cholesterol ester- and apo E-rich HDLE are recognized preferentially by the apo E receptor in the liver and internalised (43). According to Thuren, Apo E mainly stimulates phospholipid hydrolysis, and this stimulation was dependent on low surface pressure (63). In a later publication, also it was also demonstrated that HL has higher activity towards apo E-rich HDL than towards apo E-poor HDL and that phosphatidylcholine (PC) is the preferred substrate (64).
Apo E also activates TG hydrolysis by hepatic lipase in emulsion substrates (65, 66). But to hydrolyze lipoprotein TG the HL enzyme must be liberated from proteoglycans at the surface of hepatocytes (67). HL activity is only measurable after releasing HL from the liver with an injection of heparin. Higher postheparin activity appears to be a marker for a larger liver depot of inactive HSPG-bound HL. HDL has been shown to displace HL from the cell surface much like heparin (67 - 69); however, the composition of HDL can affect its ability to displace HL. Apoliporproteins like apo A-I, apo A-II and Apo C-I appear to stimulate HL displacement, while other apolipoproteins have the opposite effect and block HDL from displacing cell surface HL (67 - 69). Interestingly, according to Young and co-workers higher HDL-apo E levels seems to be associated with decreased HL displacement from liver cell surface proteoglycans and may therefore affect lower vascular TG hydrolysis by HL in humans (70).
By promoting reverse cholesterol transport, HL may be anti-atherogenic. However, HL also lowers HDL. Besides its role in reverse cholesterol transport, HDL has also beneficial anti-inflammatory, anti-oxidative and anti-thrombotic properties, which will attribute to its atheroprotective potential. By lowering HDL, therefore, HL may also be pro-atherogenic. The potential pro- or anti-atherogenic functions of HL will be described in the next chapter.
To be included: Figure 3. Pictorial scheme of the reverse cholesterol transport
3 Hepatic Lipase and Coronary Artery Disease
Coronary Artery Disease (CAD) is a disease with high mortality in the Western societies (71-73). Atherosclerosis is one of the multiple factors that contribute to CAD. Multiple published human and animal studies supports the theory that HL has an impact on atherogenesis (8, 12, 74-77). Although HL has shown to modulate atherogenic risk, whether its role is either a protective or proatherogenic agent remains debateable. By meta-review and based on different experimental approaches Jansen and co-workers has described the proatherogenic and antiatherogenic functions for HL (8); this is summarized in table 1.
Table 1. Overview of arguments of the potential pro- or anti-atherogenic functions of HL (8).
Impact on Human Lipoprotein Metabolism
Pro-atherogenic
Decreases LDL size
Anti-atherogenic
Stimulates HDL cholesterol (ester) uptake (reverse cholesterol transport)
Stimulates post-prandial lipid clearing
Stimulates IDL clearing
Stimulates preβ HDL, HDL3 formation
Animal Studies
Pro-atherogenic
Deficiency attenuates atherosclerosis in apoE k.o. mice
Overexpression augments plaque size in rabbits
Anti-atherogenic
Over-expression in mice decreases aortic cholesterol deposition
Inhibition of activity in apoA-II overexpressing mice increases aortic cholesterol deposition
Animals low in HL show diet-induced hyperlipidemia
Associations with Human Lipoprotein Profile
Pro-atherogenic
Activity inversely correlated with HDL (2)
Activity inversely correlated with LDL size
Anti-atherogenic
Activity inversely correlated with post-prandial lipids
Activity inversely correlated with LpCIII:B
Association with Human Atherosclerosis Promoting Diseases and Other Conditions
Pro-atherogenic
Activity high in males compared to females
Activity positively associated with insulin-resistance and high in type 2 diabetes
Activity positively correlated with omental fat mass, fasting insulin
Activity high in FH
Anti-atherogenic
Activity low in hypothyroidism
Association with Human CAD
Pro-atherogenic
Decrease in activity during hypolipidemic drug treatment associated with increased
LDL size and decreased CAD
Anti-atherogenic
Activity low in CAD patients (if accompanied by low CETP)
Activity inversely associated with calcification in homozygote FH
Activity predictor of CAD regression after dietary intervention
Deficiency associated with increased CAD risk
Several transgenic animal models have been used to elucidate the role of HL in atherosclerosis. By genetically overexpressing HL in cholesterol fed mice the aortic cholesterol content but also plasma HDL cholesterol became reduced (78). Also, overexpression of HL in transgenic rabbits (79) as well as overexpression of apo B in transgenic mice and apo E deficient mice (80) led to reduction of circulating pro-atherogenic lipoproteins and HDL cholesterol. In contrast, HL knockout mice did not show an occurrence of increased atherosclerosis (80?). Furthermore, HL knockout mice with also apo E deficiency have increased plasma cholesterol but decreased susceptibility to develop atherosclerosis (81). These studies give indicates that HL has an important role in the development, albeit which is independent of plasma cholesterol levels and they show that other pathways are involved.
Human and animal studies have shown that variability in HL is correlated with atherosclerosis and CAD risk. Low HL activity is correlated in humans with an increased risk of CAD. However, although premature CAD has been reported in patients with complete HL deficiency, there is suspicion of ascertainment bias. Other studies did not found that decreased HL activity influences susceptibility to CAD. Contrary, it has been found in some studies that patients with CAD show an increase of HL activity. But there is a strong opposite association between HL activity and CAD in homozygous FH and hypertriglyceridemia in humans. According to two studies of Dugi et al. HL activity and CAD were stronger correlated in the homozygous FH patients than in the other patients (82, 83). In the FH patients, HL activity and lipoprotein lipase (LPL) dimmer mass together significantly accounted for 85% of the variability in coronary calcification.
In humans the promoter of the HL gene (LIPC) contains a common functional variant that is of interest due to its correlation with CAD. A -514 C>T base substitution is present which is linked with three other base variants in the proximal LIPC promoter as well (84). The collective presence of the linked variants is represented by two alleles the LIPC C- and T-allele. The T-allele is present in 17-24% of Caucasians, 40-50% of Japanese and Afro-Americans, and to 55% of Native-Americans (11). In vitro, promoter activity of the LIPC T-allele was less than the LIPC C-allele (85). Also, this T-allele is associated with a 15-30% lower post-heparin HL activity (86). But despite that human carriers of this allele have an elevated HDL cholesterol level there is a higher incidence for coronary artery disease as well among this group.
Connelly and Hegele suggested that HL deficiency, in the presence of a second genetic or environmental factor affecting lipoprotein levels, increases atherogenic risk (50). In a study of Hirano and co-workers this idea is supported where subjects with low HL activity exhibited increased CAD only in the presence of genetically determined low cholesteryl ester transfer protein (CETP) activity (87). Further support for this view was found in the REGRESS study by van Acker and co-workers (88) where genotype frequencies of CETP Taq1B (rs708272) and LIPC-514C/T (rs1800588) polymorphisms in male coronary artery disease patients were analyzed with non-symptomatic controls. The CAD patients were 7.16 more often carrier of the combined LIPC T/T (low HL) and CETP B2/B2 (low CETP) genotype than the controls. Moreover, the combined homozygosity for the LIPC T-allele and the CETP B2 allele associated within the group with a larger progression of CAD compared with other allelic combination. However, high HDL cholesterol does not protect against coronary artery disease when associated with combined CETP- and HL-lowering gene variants. The HDL cholesterol was 1.39 times higher in B2B2/TT carriers than in the B1B1/CC carriers.
3.2 Role in HDL metabolism
It has been proven in vitro as well in vivo in animal studies that HL activity is inversely correlated with HDL cholesterol levels. HL maybe lower in this instance HDL cholesterol levels by facilitates the uptake of HDL cholesterol (ester) in the reverse cholesterol transport (89, 90), this either directly and/or indirectly via lipoprotein receptors such as the putative HDL receptor (HDLR), Scavenger Receptor Class B type I (SR-BI) (91, 92). When overexpressing the HL expression in transgenic mice a decrease of HDL was observed (22), which was also observed with the hepatic overexpression of SR-BI in mice liver (93). This can be explained by the hypothesis that an increased reverse cholesterol transport causes a low HDL cholesterol level. Cholesterol efflux from peripheral tissues represents the first step in the reverse cholesterol transport. Smaller HDL subclasses (e.g. HDL3 and/or preβ HDL), produced by HL of delipidating HDL, stimulate this cholesterol efflux from cells (94). HL thus stimulates the reverse cholesterol transport by promoting HDL cholesterol (ester) uptake in the liver as well by affecting HDL functionality. Involvement of HL in the reverse cholesterol transport may influence CAD risk (95 - 97). However, as explained earlier by the study of Hirano and co-workers, this increased CAD risk by HL influence is only when low HL activity is present together with genetically determined low cholesteryl ester transfer protein (CETP) activity (87). This can be explained by the role what HL and CETP has in the reverse cholesterol transports. Once taken up by HDL and esterified by LCAT, in humans peripheral cholesterol may be transported to the liver via two different pathways (96, 97). In both pathways lipid transfer proteins are involved. In the direct route the HL is involved and the liver takes up HDL cholesterol directly. In the indirect route the CETP is involved. Here HDL cholesterol ester is first transferred to the apo B 100 or apo B 48 containing lipoprotein, and finally taken up by receptor-mediated endocytosis (eg LDLR). When one of the pathways fails, it can be compensated by the other route (i.e. lipoprotein receptors). Only in case both pathways fail there would be therefore an increase of risk on CAD.
Role in IDL/LDL metabolism
In humans, HL has a role in of regulating the LDL subclass distribution, which in turn determines the atherogenic risk (18, 98 - 100). Human as well animal studies have shown that HL also affects the metabolism of apo B 100 containing lipoproteins. Demant and coworkers (101) showed that HL deficiency causes significant distortion in the plasma lipoprotein profile in humans. The subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50% and the conversion of IDL to LDL was almost totally inhibited with 10% of normal. This is consistent with the development of a type III-like lipoproteins profile described in HL deficient subjects (50). Jansen and co-workers found with the EARS-II study that healthy young carriers of the T allele of the HL promoter variant -514C/T, which decrease the HL activity, have increased fasting lipids and HDL, and accumulation of atherogenic LpC-III:B particles (102). In children with congenital hypothyroidism, who also have conditions with decreased HL activity levels, the IDL-like lipoproteins have been accumulated (103). Patients with HL deficiency present with hypercholesterolemia or hypertriglyceridemia and accumulate β-very low-density lipoproteins (VLDLs), chylomicrons remnants, IDLs, TG-rich LDLs, and HDLs (12, 104 - 109). IDL is a major determinant of CAD risk (110). Therefore, low HL activity might lead to increased atherosclerosis due to the accumulation of IDL. Also, small-dense LDL is considered to be atherogenic, however in contrast here high HL activity does might lead to the accumulation of sdLDL. Thus lowering the HL activity as a treatment might lead to regression of CAD (75).
4 Hepatic Lipase and the Glucose metabolism / Diabetes Mellitus
4.1 Glucose metabolism and Insulin regulation
Glucose is required in the body as fuel for the generation of energy for all its functions. The glucose metabolism is regulated tightly to have a sufficient glucose supply all times to glucose-dependent organs in humans and animals. Via two ways the glucose is getting into the blood: dietary glucose derived from the intestine and glucose produced by the liver and the kidney (111). During fasting, the organs will depend primarily on the production of glucose, mainly by the liver. Glucose can be produced directly through gluconeogenesis from various substrates, such as certain amino acids, lactate and glycerol. Glucose can also be produced indirectly through a process is named glycogenolysis in which the liver is able to produce glucose through phosphorylation of glycogen, the storage form of glucose. Glucose can also be taken up first by the blood, phosphorylated by glucokinase to form glucose-6-phosphate (G6P) and then be secreted again after dephosphorylation by glucose-6-phosphatase (G6Pase). This process is named glucose cycling. The process of hepatic glucose production is tightly regulated by a variety of mechanisms. The two pathways gluconeogenesis and glycogenolysis are interconnected in such a way that when a decrease of gluconeogenesis is generally accompanied by an increase in glycogenolysis and the other way as well (112). This process of auto-regulation is not controlled by hormones. But in most of the processes of the glucose metabolism hormones do play important roles in the regulation of hepatic glucose. (111)
One of the most important hormones is insulin, which is synthesized and secreted by the β-cells of the Islets of Langerhans (113). In addition to the β-cells, the Islets of Langerhans contain α-cells that, in case of low levels of glucose, secrete Glucagon responsible for inducing hepatic glycogenolysis as well as gluconeogenesis (114). Insulin is secreted by a tightly regulated process and is triggered by a variety of stimuli including high levels of glucose. The glucose is transported into the β-cells by glucose transporter 2 (GLUT2) and subsequently metabolised, mainly through glycolysis and subsequently the tricarboxylic acid cycle. Besides glucose, other nutrients, hormones, neurotransmitters, ions, and drugs can also stimulate insulin secretion, sometimes in a glucose-dependent way. For example, fatty acids stimulate insulin secretion through an incompletely understood process that requires their oxidation. After secretion by exocytosis from the β-cells, the insulin reaches the liver via the portal vein. In the hepatocytes a high insulin:glucagon ratio does stimulate glycolysis, glycogenesis, and lipogenesis, and in contrast does suppress gluconeogenesis and glycogenolysis. Through these mechanisms, insulin indirectly stimulate peripheral glucose uptake by the hepatocytes (115). Insulin does not directly stimulate glucose uptake by the hepatocytes: the hepatocyte glucose transporter (GLUT2) is not regulated by insulin. Insulin has a multitude of effects in peripheral tissues. With respect to energy metabolism, the main target tissues for insulin are cardiac and skeletal muscle and adipose tissue. In these tissues insulin stimulates cellular uptake of glucose form the blood. This is accomplished by stimulating translocation (and activity) of glucose transporter 4 (GLUT4) from within the cell to cell membrane. Consequently, the blood glucose concentration decreases and the intracellular glucose concentration increase (113). Other hormones like Epinephrine, which is secreted by the adrenal medulla, stimulate the glycogenolysis and gluconeogenesis in the liver (116, 117). The steroid hormone Cortisol influences carbohydrate metabolism by increasing glycogen synthesis, although its role in gluconeogenesis and hepatic glucose production is debatable (118 - 121). Furthermore, fatty acids also seem to play important regulatory functions in hepatic carbohydrate metabolism (111).
4.2 Pathophysiology of Type 2 Diabetes Mellitus
Diabetes mellitus is a chronic disease, which is characterized by hyperglycaemia, and it results from defects in insulin secretion, insulin action or both (122). The WHO has diagnosed Diabetes mellitus by the classic symptoms of polyuria, polydipsia and unexplained weight loss, and/or a hyperglycaemia (123).
Type 2 diabetes mellitus (T2DM), in the past named Adult Onset Diabetes, Non-insulin dependent Diabetes Mellitus or Maturity Onset Diabetes, denotes all forms of diabetes with relative insulin deficiency, which can be caused by insulin resistance or secretory defects (122). It is the most common diabetes and according to the European Health Report 2002 of the WHO, between 85 and 95% of diabetics suffer from T2DM (122, 124). The disease normally affects usually adults over 40 years of age, but nowadays the disease is increasingly occurring among teenagers as well. Due to increasing obesity and failure to exercise T2DM is also more prevalent in society. Heredity and Genetic factors also do have some influence in the development of T2DM (125).
The main causes leading to this T2DM are the development of Insulin Resistance in the muscle, in adipose tissue or in the liver, where the cells do not accept the insulin and insufficient insulin secretion by the β-cells due to their subsequent destruction. In the beginning of the development of the disease the patients are still able to produce insulin, but the target tissue is less responsive to the insulin. To composite for this lack of response the pancreatic β-cells are increasingly secrete insulin, leading to hyperinsulinemia in the early stages of the disease. But this subsequently will further decrease the insulin responsiveness in finally all organs. At the end the Islets of Langerhans are exhausted and fail to produce some insulin. Due to the lack of insulin the blood sugar level raises strongly giving rise to full-blown T2DM phenotype. The liver of patients with this disease also produces glucose through the process gluconeogenesis, which further worsens the controlling of glucose level.
T2DM is associated with hyperglycemia and hyperlipidemia (111). The mechanisms causing hyperglycemia during T2DM are well known. During the T2DM the basal hepatic glucose production seems to be increased. A correlation exists between the rate of glucose production and degree of fasting hyperglycemia in T2DM. This increased hepatic glucose production could be explained by increased gluconeogenesis and/or increased glycogenolysis, but this cannot compensate the similar increases in peripheral glucose uptake (126, 127). Several studies suggest an interaction between the hepatic glucose production and hepatic lipid content in T2DM patients (128, 129). Increased supply of FFA to the liver, i.e. during increased lipolysis or lipid infusions, are generally associated with increased hepatic glucose production. Increased lipolysis is also associated with increased gluconeogenesis and lowering of FFA levels improve insulin resistance in T2DM (130). Observations also suggest that FFA cause mainly a decrease in the insulin-mediated suppression of glycogenolysis, leading to increased hepatic glucose productions in healthy human subjects (131).
T2DM is also characterized by hyperlipidemia, which includes also hypercholesterolemia and hypertriglyceridemia (132). Higher levels of TGs (mainly VLDL particles) and small, dense LDL particles and lower levels of HDL cholesterol are commonly found (133), giving rise to an atherogenic lipid profile. An increased hepatic VLDL secretion into the blood, increased FFA release from adipose tissue or decreased TG clearance from the blood could give rise to the hyperlipidemia. Studies with animals made diabetic as well in diabetic humans showed that increased VLDL secretion might be caused by the decreased sensitivity to the inhibitory effects on this process of insulin directly. Further, it is commonly agreed that by increased flux of FFA to the liver, the insulin also indirectly increase VLDL secretion in T2DM. A number of studies have shown that insulin has a diminished ability to suppress FFA rate of appearance in T2DM patients (reviewed in 134). However, some ex vivo studies found no effects of fatty acids on apo B secretion under basal conditions (135, 136). And contradicting, in one study a group of Pima Indians with T2DM showed unaffected VLDL production (137), which might have been related to the absence of increased levels of FFA in these patients.
A decrease of clearance of TGs from the blood in T2DM patients is linked to the impaired lipolysis of VLDL-TGs. Since the insulin-sensitive enzyme lipoprotein lipase (LPL) mediates this kind of process, insulin resistance would lead to lower levels of the LPL. Several studies show lower TG clearance (137, 138), albeit this not always confirmed by other studies (139, 140).
Finally, studies have shown that the skeletal muscle in T2DM patients have a reduced ability to oxidise the fatty acids (141, 142). The combination of increased lipolysis from adipose with a lower uptake of FFA by the skeletal muscle could result that FFA is shifted from adipose tissue and skeletal muscle to the liver (111).
4.3 Involvement of Hepatic Lipase
HL expression is increased in T2DM (20, 143). Studies with humans indicate that HL activity is positively correlated with insulin levels (13). HL activity increases with fasting insulin levels in non-diabetic, normocholesterolemic coronary artery disease patients (12), and plasma HL activity positively correlates with increased plasma insulin levels in response to an oral glucose load (144, 145). HL activity increases during conditions with high plasma insulin levels, such as in T2DM (146) and obesity-related hyperinsulinaemia (147). HL activity is also associated with parameters of insulin resistance in non-diabetic males (12, 21) and in familial combined hyperlipidemia (FCHL) (21). In an animal model, HL expression is increased upon induction of insulin resistance; this is partially reversed by treatment with an insulin sensitizer (148). However, a direct stimulating effect of insulin on HL expression has not been unequivocally identified (149). Instead, acute hyperinsulinemia actually reduces HL expression (150). Taken all together, although a direct stimulating effect has not been established, there are some aspects of insulin resistance that induces the increase in HL expression.
Besides LPL, also HL is a lipolytic enzyme that plays a key role in lipoprotein metabolism and in the remodeling of HDL and LDL (8). Elevated HL expression is associated with the dyslipidemia in the metabolic syndrome, and in type 2 diabetes (18, 149).
HL hydrolyses the TGs in IDL, and maybe also in the chylomicron remnants. An increased HL activity will increase TG lipolysis in IDL leading to the formation of small dense LDL and the reduction of HDL cholesterol levels, typical of the diabetic dyslipidemia (8, 18). Further, the HL gene is associated with the lipoprotein abnormalities in familial combined hyperlipidemia (22).
Omental fat mass, a parameter of visceral obesity with increased risk for development of T2DM, causes also a strong increase of HL activity (14). This indicates that fatty acid supply to the liver also increases HL expression may. Indeed, in vitro HL expression is increased by fatty acids (23, 24). The transcription of the HL gene in HepG2 cells is shown to be increased by glucose (25), which suggests that HL expression may be elevated in insulin resistant states as a consequence of the hyperglycemia and increased fatty acid delivery to the liver.
