Diabetic dyslipidaemia: from basic research to clinical practice*
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The recognition that the increase of plasma triglyceride rich lipoproteins (TRLs) is associated with multiple alterations of other lipoproteins species that are potentially atherogenic has expanded the picture of diabetic dyslipidaemia. The discovery of heterogeneity within major lipoprotein classes VLDL, LDL and HDL opened new avenues to reveal the specific pertubations of diabetic dyslipidaemia. The increase of large VLDL 1 particles in Type 2 diabetes initiates a sequence of events that generates atherogenic remnants, small dense LDL and small dense HDL particles. Together these components comprise the atherogenic lipid triad. Notably the malignant nature of diabetic dyslipidaemia is not completely shown by the lipid measures used in clinical practice. The key question is what are the mechanisms behind the increase of VLDL 1 particles in diabetic dyslipidaemia? Despite the advances of recent years, our understanding of VLDL assembly and secretion is still surprisingly incomplete. To date it is still unclear how the liver is able to regulate the amount of triglycerides incorporated into VLDL particles to produce either VLDL 1 or VLDL 2 particles. The current evidence suggests that the machinery driving VLDL assembly in the liver includes (i) low insulin signalling via PI-3 kinase pathway that enhances lipid accumulation into "nascent " VLDL particles (ii) up-regulation of SREBP-1C that stimulates de novo lipogenesis and (iii) excess availability of "polar molecules" in hepatocytes that stabilizes apo B 100. Recent data suggest that all these steps could be fundamentally altered in Type 2 diabetes explaining the overproduction of VLDL apo B as well as the ability of insulin to suppress VLDL 1 apo B production in Type 2 diabetes.
Recent discoveries have established the transcription factors including PPARs, SREBP-1 and LXRs as the key regulators of lipid assembly in the liver. These observations suggest these factors as a new target to tailor more efficient drugs to treat diabetic dyslipidaemia.
KeywordsLipids apoliproteins insulin Type 2 diabetes liver fat coronary heart disease stable isotopes transcription factors
ATP cassette binding protein-1
ADP ribosylation factor 1
- ATP III
Adult Treatment Panel III
cholesterylester transfer protein
Diabetes Atherosclerosis Intervention Study
de novo lipogenesis
gradient gel electrophoresis
Heart Protection Study
intermediate density lipoprotein
the LDL receptor-related protein
microsomal transfer protein
- PI-3 kinase
phospohotidylinositol 3 kinase
phospholipid transfer protein
peroxisome proliferator-activator receptor
Svedberg flotation rate
sterol regulatory element-binding protein
scavenger receptor BI
triglyceride rich lipoproteins
Veterans Affairs HDL Intervention Study
In Type 2 diabetes dyslipidaemia is an important and common risk factor for coronary heart disease (CHD) that is the leading cause of morbidity and mortality worldwide [1, 2]. The International Diabetes Federation (IDF) foresees a rise of epidemic proportions in Type 2 diabetes that portends a daunting increase in CHD . Solid evidence has confirmed that people with Type 2 diabetes have a similar risk of heart attack as people without diabetes who have already had a heart attack [4, 5]. Adult Treatment panel III (ATP III) designated diabetes as a CHD risk equivalent and LDL was identified the first priority of lipid lowering .
Recent developments have recognized the complex nature of diabetic dyslipidaemia that is a cluster of potentially atherogenic lipid and lipoprotein abnormalities . Two core components of diabetic dyslipidaemia are increased plasma triglycerides and low concentrations of HDL cholesterol. More recently recognized features are small dense LDL and excessive postprandial lipemia. These components are not isolated abnormalities but metabolically closely linked to each other. The aim of this review is to build up a concept that recognizes the heterogeneity of lipoprotein classes and the co-ordinated regulation of different lipoprotein species and specifies the perturbations associated with Type 2 diabetes. A better understanding of the pathophysiology behind diabetic dyslipidaemia will allow us to tailor targeted goals for the lipid management.
Heterogeneity of triglyceride rich lipoproteins (TRLs)
Distribution of TRL species
The size of endogenous VLDL particles varies markedly from about 350 Å to 700 Å in diameter . Most of this difference is due to variation in the amount of triglycerides in the core. The common way to isolate the VLDL subfractions is by density gradient ultracentrifugation. Endogenous VLDL particles can be separated into two main subclasses: large buoyant VLDL 1 particles (Sf 60 –400) and smaller and more dense VLDL 2 particles (Sf 20–60). VLDL subclasses show distinct differences in the size, composition and metabolic function. Both endogenous and exogenous TRL particles are processed via the same delipidation cascade. The lipolytic rate of TRLs is regulated by two enzymes: lipoprotein lipase (LPL) and hepatic lipase (HL), as well as by the composition of TRL particles. The first step in the clearance of TRLs is the hydrolysis of lipoprotein triglycerides by LPL. A number of studies have reported that LPL activity is frequently low or subnormal in subjects with Type 2 diabetes . In the second step of the hydrolytic process hepatic lipase is responsible for the conversion of IDL particles into LDL particles. Substantial evidence indicates that hepatic lipase activity is commonly increased in Type 2 diabetes and this can enhance the conversion of IDL into LDL [12, 13, 14].
An important observation is that in a population survey of normolipidaemic subjects 75 % of the variation in plasma triglycerides seems to be determined by the VLDL 1 concentration . So far very little attention has been paid to VLDL subclass distribution in diabetes although the increase of plasma triglycerides is a consistent feature of diabetic dyslipidaemia. Recently it was reported  that Type 2 diabetic patients had more larger lipid rich VLDL particles than non-diabetic control subjects. We observed a proportionally higher increase of VLDL 1 particles compared with VLDL 2 particles in a small group of Type 2 diabetic patients . Likewise another study  reported a relatively higher increase of VLDL 1 than that of VLDL 2 particles in a small cohort of Type 2 diabetic subjects. Therefore we asked the question whether the increase of VLDL 1 is the key determinant of plasma triglycerides in diabetic dyslipidaemia and what would be the consequences with respect to the metabolism of other lipoproteins? We separated VLDL 1 and VLDL 2 fractions using the density gradient ultracentrifugation from a cohort of 229 Type 2 diabetic patients recruited for the FIELD study in Helsinki  as well as from a cohort of non-diabetic subjects matched for age and sex (n=74). The concentration of VLDL 1 triglycerides was higher in Type 2 diabetic subjects than in non-diabetic subjects (mean ± SD; 0.80±0.55 vs 0.34±0.24 mg/dl, p<0.001). The respective numbers for VLDL 2 triglycerides were 0.33±0.16 and 0.17±0.08 mg/dl (p<0.001). Notably the relative increase of triglycerides was 2.3-fold in VLDL 1 but 1.8-fold in VLDL 2. Altogether these observations suggest that in Type 2 diabetic subjects the increase of VLDL 1 particles contributes more than that of VLDL 2 particles to the increase of TRLs in the fasting state.
Alteration of apoproteins in TRLs
In addition to apo B100, C and E apoproteins are important constituents of VLDL particles. Apo C III, which is synthesized mainly in the liver, is the most abundant apolipoprotein in VLDL particles. Apo C III modulates the metabolism of VLDL particles at different steps [9, 20]. Firstly, apo C III is a direct inhibitor of LPL activity . In the subsequent step apo C III interferes with the apo E-mediated receptor binding, impairing the hepatic uptake of remnants [22, 23]. Plasma apo C-III concentrations are increased in hypertriglyceridaemic subjects and there seems to be a correlation between plasma apo C III and plasma Tg concentrations [20, 24]. Of interest, apo CIII containing TRLs represent most triglyceride-rich species that are catabolized slowly [25, 26]. Emerging evidence suggest that apo CIII containing apo B particles are a better discriminator for CHD risk than particles without apo C III [27, 28]. Plasma triglycerides, apo CIII and apo CIII /B particles were independent markers of cardiovascular disease in a cross-sectional cohort including 188 Type 2 diabetic patients . Unfortunately this study did not include a non-diabetic cohort to allow a comparison. Surprisingly the data on plasma apo CIII in Type 2 diabetes is scarce and inconsistent [24, 30, 31, 32].
The surface of VLDL particles carries both apo E and C III . Apo E has a critical role in the removal process of TRL remnants as the ligand for the LDL receptor-related protein (LRP) and the LDL receptor and by interacting with proteoglycans . Although the importance of apo E in the remnant removal is well established, the data on apo E in diabetic dyslipidaemia is incomplete. Our preliminary data suggested that in Type 2 diabetic subjects, postprandial TRLs were rich in apo E . The observation that apo E enrichment of VLDL reduces the catabolic rate of particles represents a paradox, and in contrast suggests that apo E enrichment could aggravate postprandial lipemia. In a recent study, apo E content of fasting TRL, expressed as a molar ratio of apo E to apo B, was similar in Type 2 diabetic subjects and in control subjects matched for age . Likewise there was no difference between the two groups in the binding of isolated TRLs to both purified biglycan and to the LDL receptor in vitro. Regardless of the lack of information on the apoprotein distribution in TRL subclasses, it is obvious that the apo CIII to apo E ratio can be critical for the metabolic fate of these lipoproteins in diabetic dyslipidaemia as well as for determining their atherogeneity.
Pathophysiology of VLDL metabolism in Type 2 diabetes
Regulatory steps in VLDL metabolism
The metabolism of TRLs has several distinct steps that include (i) the assembly and secretion of VLDL particles, (ii) the hydrolysis of triglycerides in TRLs by LPL, (iii) the direct uptake of TRL remnants in the liver, and (iv) the flux of apo B 100 through VLDL-IDL-LDL delipidation cascade in circulation. Most of the data on VLDL assembly and secretion has been derived from in vitro cell cultures or from animal experiments [36, 37]. Insulin has been shown acutely to inhibit the assembly and secretion of VLDL particles by mechanisms involving an increase of apo B degradation and a decrease of the expression of microsomal transfer protein (MTP) in hepatocytes [38, 39]. In contrast, hepatic expression of MTP is increased in the obese and hypertriglyceridaemic rat  and in obese diabetic mice . Recent studies using the fructose-fed Golden Hamster, an animal model of insulin resistance and dyslipidaemia, have shown that increased expression of MTP and increased lipid availability in the liver is associated with reduced degradation of apo B and are the forces that are driving the overproduction of apo B in the insulin-resistant state [42, 43].
In subsequent studies we showed that in Type 2 diabetic men insulin failed to suppress VLDL 1 apo B production during acute hyperinsulinaemia . Thus this new action of insulin was defective in Type 2 diabetic subjects in contrast to normal subjects. This failure of insulin to suppress VLDL 1 particle release in Type 2 diabetes results in inappropriate production of VLDL 1 particles in the postprandial phase when these particles are not needed in the presence of chylomicron flux. The overproduction of VLDL 1 particles can saturate the metabolic capacity and contribute to postprandial lipemia. We speculate that this perturbation is the major cause for the increase of plasma triglyceride concentrations in Type 2 diabetes.
Pertubations in the assembly and secretion of VLDL particles
To date it is still unclear how liver is able to regulate the amount of triglycerides incorporated into VLDL particles to produce predominantly either large VLDL 1 particles versus VLDL 2 particles. The current evidence suggests that factors modulating VLDL assembly and secretion in the liver are (i) expression of MTP, (ii) activation of phosphatidylinositol 3 kinase (PI 3-kinase), (iii) ADP-ribosylation factor (ARF-1) by activating phospholipase D (PLD), and (iv) availability of lipids in hepatocytes. The latter component is closely linked to the influx of NEFA into the liver as well as to de novo lipogenesis (DNL).
The current concept is that the assembly of VLDL particles in the liver involves two distinct steps [51, 52, 53]. The first step of the secretory pathway produces "pre-VLDL" particles that involves the co-translational lipidation of apo B catalysed by the microsomal triglyceride transfer protein (MTP). This step takes place in the rough endoplasmic reticulum (ER). In the second step the major amount of lipids is incorporated into the core of "pre-VLDL" particles that are transformed into larger VLDL particles. The second step and the transport of VLDL particles into the smooth membrane compartment is driven by ADP ribosylation factor-1 (ARF-1) that regulates VLDL assembly by activating phospholipase D (PLD) . The activation of PLD by ARF-1 is linked to intracellular insulin signalling. In this scenario the activated PI 3 kinase converts PI-4,5 biphosphate (PIP2) into PI-3,4,5 triphosphate. Lack of PIP2 prevents the activation of PLD that is necessary for the formation of lipid droplets and the actual assembly of lipids into "pre-VLDL " . The normal inhibitory effect of insulin on VLDL assembly is located at the second step and linked to the lack of PLD activation during normal intracellular insulin signalling. In contrast a defective activation of PI-3 kinase in insulin resistant state results in an excess of PI-4,5-biphosphate that in turn signals the activation of PLD. Thus one action site for the inhibitory effect of insulin on VLDL assembly could to be located at the second step that regulates the addition of the lipids into "pre VLDL" particles. This cascade explains why insulin in vivo acutely inhibits specifically the production rate of triglyceride-rich VLDL 1 particles and possibly explains why this action is impaired in Type 2 diabetes [47, 50, 54, 55].
An interesting turn on the potential sites of insulin in regulating VLDL metabolism in the liver arose form the observations that insulin regulates the expression of the sterol regulatory element-binding protein-1c (SREBP-1c) in the liver . The novelty of this finding is that hyperinsulinaemia per se up-regulates the expression of SREBP-1c which is followed by the activation of enzymes for lipogenesis, including fatty acid synthase (FAS) and acetyl Co A carboxylase (ACC). These enzymes are the key factors stimulating de novo lipogenesis (DNL) in the liver. Importantly, the up-regulation of SREBP-1c mRNA is seen in the setting of down-regulation of IRS-2-mediated insulin signalling pathway in the insulin-resistant state. Decisive evidence exists for the insulin induced expression of SREBP-1c . This action of insulin is explained by IRS-1-PI 3 kinase pathway . Overall SREBP-1c seems to have a crucial role in the regulation of triglyceride accumulation in the liver . High rates of DNL are invariably associated with a shift of cellular metabolism from lipid oxidation to lipid storage in the liver increasing the availability of triglycerides for VLDL assembly .
Variation of plasma NEFA and VLDL apo B production rate
The dysregulation of fat metabolism in the liver could be a key factor leading to the increase of large VLDL 1 particles in insulin resistance. Taken together the machinery driving VLDL assembly in the liver includes (i) impaired insulin signalling via PI3 kinase pathway that enhances lipid accumulation to "nascent" VLDL particles, (ii) up-regulation of SREBP-1C that stimulates de novo lipogenesis, (iii) excess availability of "fat" in hepatocytes that stabilizes apo B 100. There has been accumulating evidence showing that hepatic steatosis is a feature of dyslipidaemia and insulin resistance [69, 70, 71]. Nonalcoholic fatty liver disease (NAFLD) is closely associated with hypertriglyceridaemia, low HDL cholesterol, hyperinsulinaemia and insulin resistance and glucose intolerance . Notably there is a close positive correlation between fasting plasma triglycerides and liver fat quantitated by using proton spectroscopy in humans . Thus the factors modulating the amount of fat in the liver have become a topic of urgent interest. Whether the amount of fat in the liver is the cause or the consequence of insulin resistance remains an issue to be resolved but obviously it is highly relevant to insulin resistance and dyslipidaemia as well as Type 2 diabetes .
Is diabetic dyslipidaemia a postprandial phenomenon?
Fat intolerance in Type 2 diabetes
The mechanisms behind the excessive postprandial lipemia in Type 2 diabetes
The initial step in TRL metabolism is the hydrolysis of lipids by LPL bound to heparin sulfate proteoglycans (HSPG) at endothelial cells . Triglycerides in large lipoproteins are hydrolyzed faster than in smaller particles. Thus, the flux of chylomicrons after a fat-rich meal will impede the lipolysis of VLDL particles due to the competion for the contact with LPL [8, 78, 84, 85, 86]. Consequently the delipidation process of VLDL particles are slowed down and the residence time of these particles in circulation is prolonged. These processes are exaggerated in the situations where VLDL 1 production is excessive as exemplified by the failure of insulin to suppress VLDL 1 production in Type 2 diabetes [8, 50]. A second factor impeding the lipolytic rate is the reduction of LPL activity commonly found in Type 2 diabetes [9, 11, 85, 87]. LPL activity is regulated by insulin and its actions are impaired in insulin resistance and Type 2 diabetes. Recent data confirmed that LPL activity in adipose tissue is indeed a determinant of postprandial triglyceride excursions in non-diabetic subjects . In this study adipose tissue LPL activity also correlated negatively with the insulin resistance index. The carrier status of LPL gene mutations that are relatively common like N291S and D9 N, can aggravate the postprandial lipemia [88, 89]. A third potential mechanism is the impaired clearance of remnant particles by liver receptors (LDL receptor and LRP) [85, 86]. Lipoprotein uptake is preceeded by trapping of remnants in the vicinity of receptors. HSPG, LPL, hepatic lipase as well as apo E can act as bridging proteins and thus modulate the removal of remnants. Recently reduced synthesis of HSPG core protein perlican was reported in streptosotozin diabetic mice together with concomitant delayed clearance of apo B 48 containing lipoproteins .
Whether there is also overproduction of apo B48 containing particles in the insulin resistant state and in Type 2 diabetes is a debated issue. The induction of the insulin resistant state in fructose-fed hamsters is associated with overproduction of apo B 48 containing particles . There is also evidence that intestinal MTP is increased in diabetic animals and this could enhance the formation of apo B 48 containing particles [86, 91]. Further studies are needed to delineate the exact contribution of intestinal overproduction of apo B 48 lipoproteins in diabetes to the postprandial lipemia in humans. Taken together the regulation of postprandial TRL metabolism is a complex process where several subsequent steps could be fundamentally altered in Type 2 diabetes.
Is postprandial lipidemia a hazard?
The important implication is that postprandial lipemia has severe adverse consequences at the level of the arterial wall. A number of studies have shown that postprandial lipemia is linked with the endothelial dysfunction and generation of oxidative stress in Type 2 diabetic patients [92, 93, 94, 95]. Remnant particles particularly cholesterol ester rich remnants are considered to be highly atherogenic [8, 9, 78, 85, 86]. Recent data suggest that apo B 48 and apo B 100 containing lipoproteins are probably equally atherogenic . Both apoproteins can bind effectively to proteoglycans, the former via a newly identified binding segment of apo B 48 masked in apo B 100  and the latter via a different domain . In concert postmeal metabolic excursions comprise a cluster of potentially highly atherogenic perturbations that could be more important in terms of damaging the arterial wall than those due to hyperglycaemia . In addition lipoproteins and remnants can also interact with coagulation factors [98, 99]. In this context a persuasive hypothesis is that a fatty meal is a trigger for acute coronary syndrome . Therefore it is not suprising that the postprandial state is in the spotlight of current research.
Metabolic alterations of LDL subclasses in Type 2 diabetes
Distribution and properties of LDL species
LDL comprises a heterogeneous spectrum of particles, which differ in size, density, chemical composition and atherogeneity. The most common way to separate LDL subclasses has been the use of gradient gel electrophoresis (GGE). Small dense LDL is defined as a particle with a mean diameter of the major LDL-peak less than 25.5 nm and it is the major component in pattern B, whereas pattern A consists of LDL particles with a greater diameter . Small dense LDL differs from large LDL with respect to chemical composition and binding affinity to proteoglycans. Small dense LDL contains less polar lipids than large LDL particles (35.6 vs 63.3%) . The key variant is the cholesterol to triglyceride ratio of LDL particles that is decreased in small dense LDL particles . The conformation of apo B 100 is distinct between the LDL subspecies and this is reflected in the different exposure of apo B 100 on the surface of LDL particles. The key difference is that specific segments which mediate the binding of apo B 100 to proteoglycans are exposed on the surface of small dense LDL particles [96, 97, 101, 103].
Why is small dense LDL highly atherogenic?
A several set of observations witnesses that the reduced LDL size is linked to the increased risk of CHD [116, 117, 118, 119]. Both LDL size and the number of particles seems to increase synergistically the risk of CHD [120, 121]. Recently convincing tenets seem to explain the atherogeneity of small dense LDL particles. Solid data shows that LDL size is a strong determinant of endothelial function in Type 2 diabetic subjects as well as in normal healthy men [122, 123, 124, 125]. In our study the subjects with small dense LDL, but comparable LDL cholesterol concentration, had impaired response to endothelium-dependent vasodilator acetylcholine [124, 125]. Whether or not this association is causative is not clear but the data suggest that small dense LDL can at least partly explain the adverse effects on vascular function in Type 2 diabetes. It is generally considered that small particle size favours the penetration of LDL particles into the arterial intima . In addition the prolonged residence time of small dense LDL due to its poor binding to LDL receptor gives more time for particles to infiltrate into the arterial intima. Recently it was reported that transvascular LDL transport rate is indeed increased in patients with Type 2 diabetes . Surprisingly there was no correlation between LDL size and transvascular LDL transport. The authors suggest that the increased transvascular LDL transport reflects a general increase of transvascular permeability in diabetes. Irrespective of the mechanism, increased flux of lipoproteins results in subsequent deposition of LDL particles in the arterial wall. Small dense LDL particles have higher binding affinity to intimal proteoglycans than large LDL particles [128, 129, 130]. Additionally indirect binding via bridging molecules such as lipoprotein lipase and decorin further increase the retention of LDL in the arterial wall . The retained LDL particles in the intima are exposed to modification under conditions of oxidative stress. Small dense LDL shows an increased susceptibility to oxidation and glycation of LDL further aggravating this susceptibility to oxidation [132, 133, 134, 135]. A debated question is whether the size of LDL or its integral physical properties determine LDL oxidisability. Growing evidence suggest that free fatty compositions of LDL particles is the most important determinant of LDL oxidisability [136, 137]. Moreover the increased lability of cholesterol ester hydroperoxides in small dense LDL is a key feature of oxidative susceptibility . Ultimately oxidized LDL in the intima is the trigger that initiates a cascade of processes leading to the formation of macrophage foam cells and the plaque formation.
Metabolic alterations of HDL subclasses in Type 2 diabetes
Distribution and properties of HDL species
HDL particles can also be separated according to their concentration of the two structural apoproteins A-I and A-II. Some HDL particles contain only apo A-I but not apo A-II (LpA-I), or other particles containing both apo A-I and A-II (LpA-I:A-II). We found in a cross sectional study that both apo A-I and A-II were reduced in patients with Type 2 diabetes . As expected there was a consistent decrease in LpA-I:A-II particles in Type 2 diabetic patients. We also found that the ability of plasma from Type 2 diabetic subjects to promote cholesterol efflux in vitro from hepatoma cells was related to LpA-I:A-II concentrations rather than to LpA-I concentrations . Thus Type 2 diabetic subjects showed a deficiency of Lp:A-I mediated reverse-cholesterol transport.
Mechanisms behind lowering of HDL
There are several cogent reasons for the lowering of HDL in diabetic dyslipidaemia. The increase of plasma triglycerides drives the exchange of core lipids between TRLs and HDL particles as between TRLs and LDL particles (Fig. 6) [8, 139, 144]. This process results in the triglyceride enrichment of HDL particles like LDL particles. Triglycerides in HDL, like in LDL, are a good substrate for hepatic lipase and the hydrolysis produces smaller particles and free apo A-I that is shed from the particle and cleared by the kidneys. Triglyceride enrichment of HDL has been observed to enhance in vivo clearance of HDL apo A-I in healthy men . It has been shown that lipolysis of Tg enriched HDL particles by HL was required for the enhanced clearance rate of HDL . These results highlight the concerted action of both HDL Tg enrichment and hepatic lipase action in the pathogenesis of HDL lowering. A number of studies have confirmed that the clearance rate of HDL is indeed enhanced in Type 2 diabetic patients [147, 148, 149]. In Type 2 diabetic subjects LPL activity was inversely related to HDL Tg concentrations and thus also contributed to the compositional changes of HDL and consequently the clearance rate of the particles . Taken together there seems to be a symmetry of mechanism leading to the generation of small HDL and small LDL particles (Fig. 6).
Recent studies have shown new factors that are integral regulators of HDL metabolism and reverse cholesterol transport including the ATP cassette binding protein-1 (ABCA1) and the scavenger receptor BI (SR-BI) . There is evidence that glucose regulates the expression of mRNA levels of ABCA-1, SR-BI and phospholipid transfer protein (PLTP) . Additionally, PLTP an intravascular regulator of HDL metabolism is regulated by insulin or insulin resistance . Whether insulin or insulin resistance also influences the expression of these gate-keeper proteins of cholesterol trafficking (ABCA-1, SR-BI) as well as the precise mechanism of action are the exciting targets of future research.
Consequences of the increase of VLDL 1 particles
Solid evidence suggests that the increase of TRLs, in particular, of large VLDL 1 particles in Type 2 diabetes is hazardous with respect to atherosclerosis. These metabolic consequences include (i) the increase of plasma triglycerides, (ii) accumulation of remnant particles in circulation, (iii) the generation of small dense LDL particles, and (iv) the lowering of HDL concentration as well as the reduction of HDL size. All these alterations comprise a cluster of highly atherogenic potential and thus the increase of VLDL 1 particles seems to be the major culprit in diabetic dyslipidaemia.
The principal objective of lipid management in Type 2 diabetes
As described above diabetic dyslipidaemia is a cluster of lipid aberrations that are metabolically closely linked. A much debated question has been which individual lipoprotein aberration in diabetic dyslipidaemia is most atherogenic and should be the primary target of lipid management. This debate cannot be considered fully relevant but is at least partly sanctimonious and has given the excuse for therapeutic negligence. The most powerful atherogenic components are (i) small dense LDL, (ii) remnant particles, and (iii) the low concentration of HDL with its unfavorable compositional alterations. The coexistence of these three factors strongly aggravates the lipid accumulation in arterial wall and the formation of plaques [7, 9, 86, 126, 139]. Consequently all three factors should be the targets of therapy.
Recently Adult Treatment Panel III (ATP III) designated diabetes as a CHD equivalent . The reason for this fundamental step was the undisputed evidence that Type 2 diabetic subjects without a prior history of myocardial infarction (MI) have as a high risk of CHD as non-diabetic patients who have had a previous MI [4, 5] as well as the extremely poor short-term prognosis of Type 2 diabetic subjects after their first MI [154, 155]. ATP III and ADA guidelines identified LDL cholesterol as the first priority of lipid lowering and the optimal level was set <2.6 mmol/l [6, 156].
Landmark secondary prevention studies (4S, CARE, LIPID) have provided strong evidence of the clinical benefits of LDL-lowering in people with diabetes [157, 158, 159]. The Heart Protection study (HPS) included 5348 people with diabetes and 2279 with no previous CHD . The HPS showed that simvastatin reduced vascular events by 24% in people with diabetes independently of the initial LDL cholesterol concentration. Notably at the entry to the study the mean concentration of LDL averaged 3.23 mmol/l and 40% of the subjects had LDL cholesterol <3.0 mmol/l . Thus the Type 2 diabetic subjects included in the HPS had a very similar lipid profile to those seen in the UKPDs study  and DAIS cohort .
Thus the benefits of statins therapy in people with Type 2 diabetes can no longer be questioned recognizing also the fact that statins have additional properties that can increase their effectiveness beyond the lipid lowering that is mainly related to lowering of LDL and decrease of LDL particle number [164, 165, 166]. The secondary goal of lipid lowering is to achieve the optimal concentrations of plasma triglycerides at less than 1.7 mmol/l and HDL cholesterol greater than 1.15 mmol/l [6, 156]. Recent data from two intervention trials, Veterans Affairs HDL Intervention Study (VA-HIT) and Diabetes Atherosclerosis Intervention Study (DAIS), using fibrate (VA-HIT, gemfibrozil and DAIS; fenofibrate) compared with placebo have given encouraging results [163, 167]. In both trials the fibrate treatment raised HDL cholesterol and lowered plasma triglycerides. These changes were associated with statistically significant clinical benefits (VA-HIT) and beneficial changes in angiographic parameters of focal stenosis (DAIS). The question whether patients with LDL cholesterol lower than 3.4 mmol/l but with the atherogenic lipid triad will benefit more from correction of the lipid triad by fibrates than from lowering of LDL cholesterol by statins remains to be established in ongoing clinical trials (FIELD, Fenofibrate Intervention and Event Lowering in Diabetes and ACCORD, Action to control Cardiovascular Risk in Diabetes; http://www.accordtrial.org).
New perspectives of lipid management in Type 2 diabetes
The peroxisome proliferator-activator receptors (PPARs) play a central role in the regulation of lipid and lipoprotein metabolism. In the liver the target genes of PPAR-α action include several genes that regulate lipoprotein metabolism including the expression of apo A-I, A-II and apo C-III, LPL gene and genes of enzymes involved in beta-oxidation . A substantial wealth of data showing that the well known lipid or lipoprotein effects of fibrates can be explained by the activation of PPAR-α resulting in either stimulation or suppressing of the target genes . The discovery of PPAR-α as the target of fibrate action has opened a renaissance for fibrates to correct the key lipoprotein abnormalities of diabetic dyslipidaemia . The activation of both PPARs α and γ seems to promote the expression of ABCA1 gene via LXR activation and consequently enhance the reverse cholesterol transport [170, 171]. This action explains the consistent increase of HDL cholesterol induced by thiazolidinediones (TZDs) . Why the responses of plasma triglycerides and LDL-cholesterol are variable between different TZDs is an open issue. Most recent evidence suggests that PPARs also modulate inflammation response and lipid homeostasis of macrophages in entholial cells . Conceptually, the development of PPAR α/γ agonists represents a promising line to produce tailored compounds that can correct both pertubations of lipid metabolism and insulin resistance. Recently a dual PPAR α/γ agonist (LY 465608) has been reported to improve glycaemia as well as lipid parameters in the db/db mouse .
The recognition that overproduction of large VLDL particles is the culprit of diabetic dyslipidaemia pinpoints the regulatory steps in VLDL assembly as the critical target of drug action. Interestingly in primary hepatocytes PPAR α agonist (WY 14643) decreased the synthesis and secretion of triglycerides in the face of increased synthesis of apo B 100 . The end result was the formation of more apo B 100 containing particles with fewer triglycerides, i.e. a shift from large VLDL to smaller VLDL particles. Another interesting observation is that in the fructose-fed hamster with insulin resistance and mild hypertriglyceridaemia rosiglitazone treatment improved hepatic insulin resistance that was followed with a reduction of MTP expression and VLDL assembly and secretion linked to reduced intracellular apo B stability . Of interest, PPAR-α deficiency is associated with lipid accumulation in the liver . The complement to these studies is the observation that PPAR-γ agonist in humans reduced fat in the liver . These observations indicate that the activation of PPAR α and PPAR-γ can influence the VLDL assembly at several sites and provide an option to tailor more efficient drugs that target the initial sites behind diabetic dyslipidaemia in the liver.
The rapid increase of our understanding of the complex pathophysiology of diabetic dyslipidaemia will hopefully be translated to better a treatment of diabetic dyslipidaemia. Although the clinical benefits of statin therapy are clear, it is widely accepted that there is a need for therapy beyond statins based on the specific features of diabetic dyslipidaemia. It is quite clear that the focus will shift to the liver and the complex but exciting world of transcription factors including PPARs, SREBP-1C and LXRs. Future research will hopefully provide new more specific drugs for vascular protection to relieve the heavy burden of CHD in diabetes. If Claude Bernard would live today he would probably state "The liver is a factory that produces lipids and glucose in excess, both products being hallmarks in diabetes and hazards for vascular wall".
This 2002 Claude Bernard Lecture of European Association for the Study of Diabetes is based on the studies of lipids and lipoprotein pathophysiology and metabolism in people with Type 2 diabetes and the relationship to CHD by the author, her Finnish and international collaborators and colleagues over the last two decades. Additionally the review aims to cover the most important literature published in the field over the last 5 years and seminal prior contributions.
The authors's work cited in this review was supported by grants from the Sigrid Juselius Foundation, the Finnish Heart Foundation, Helsinki University Central Hospital Research Foundation, The Academy of Finland and the Finnish Diabetes Association, Helsinki, Finland. The author gratefully acknowledges the fundamental contributions of her collaborators and postdoctoral fellows to the work discussed in this review. The skillful assistance of the research nursing and laboratory staff of the Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland is greatly appreciated. Ms. H. Laakkonen and A.-M. Syrjänen provided excellent secretarial assistance in the preparation of this manuscript. The artistic work by Ms. S. Aarnio for helping with the figures is also appreciated.
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