Chylomicron and apoB48 metabolism in the JCR:LA corpulent rat, a model for the metabolic syndrome

JCR:LA-cp rat as a model of PP lipaemia

Animal models of obesity and the metabolic syndrome usually have defects in the metabolism of leptin and its receptor. Some of these models include the ob/ob and db/db mice, the Zucker (fa/fa) and the JCR:LA-cp rat [15,16]. Phenotypically, these rodent models develop symptoms associated with the metabolic syndrome including obesity, IR, hypertriglyceridaemia and/or hyperleptinaemia [15,16]. Of these animal models, only the JCR:LA-cp rat model has been reported to spontaneously develop pathological complications, such as atherogenesis and myocardial ischaemia that are consistent with CVD complications seen in men [15].

The plasma lipid profile of the JCR:LA-cp (cp/cp) rat has been characterized extensively over recent years [15]. The cp/cp phenotype has mildly higher total plasma cholesterol levels compared with lean (+/?) counterparts [15]. The observed increase in total cholesterol has been associated with the VLDL (very-low­-density lipoprotein), and not the LDL fraction, similar to that observed in the pre-diabetic state in humans. As a result, the JCR:LA-cp model provides a unique opportunity to study dyslipidaemia in the pre-diabetic state.

Recently, we assessed the metabolism of PP CMs in the JCR:LA-cp rat. Our approach for these studies has been to develop a novel, oral fat challenge test for our rodents analogous to the existing approach used in clinical studies [17,18]. After an overnight fast (16 h), animals are offered a 5 g food pellet supplemented with dairy fat (approx. 30% carbohydrate, 60% fat, 10% protein w/w). Our methodology uses a well-established standardized conscious, non-restraint protocol [19]. Given that the PP phase (0–4 h) predominantly represents plasma apoB48 derived from the intestine, we utilized this as a guide to assess CM metabolism in the JCR:LA-cp rat [19]. In addition, our group has developed techniques to accurately detect the presence of apoB48 in plasma using a highly sensitive Western blotting/enhanced chemiluminescent procedure [19–21].

Using our approach in the JCRL:LA-cp rat, we have observed significant lipaemia associated with apoB48-containing particles, particularly in the early PP phase. Importantly, we have determined that fasting concentrations of apoB48 in this model, despite having contributions from both intestinal and hepatic sources [19,22,23], can predict the corresponding change in PP response, as measured by the incremental area under the curve [19]. We have also observed that the kinetic profile of PP apoB48 demonstrates a significant and exacerbated delay following an oral fat challenge. These results are consistent with reports in human subjects that also show elevated levels of fasting apoB48 in individuals at risk of CVD [11,14]. Thus an oral fat load (and/or challenge) in these animals is likely to contribute to pro-atherogenic processes by either facilitating the saturation of lipolytic pathways, reducing the clearance capacity of cholesterol-rich lipoproteins, or exacerbating the permeability and retention of cholesterol in arterial vessels [11,14]. Consequently, evidence from our studies supports the hypothesis that the JCR:LA-cp model provides a unique means to clarify further the pathological role of PP lipaemia during the pre-diabetic state.

Hypertriglyceridaemia and VLDL overproduction: a major feature of IR

An important characteristic of patients with IR is the overproduction of VLDL, with increased secretion of both apoB100 and TAG [24]. Physiologically, it is also essential to consider the PP phase and the often hyperphagic-like behaviour observed during clinical obesity. Humans (and animals) are potentially in a PP state continuously for up to 16–20 h per day. This in turn contributes to a sustained secretion of CM-associated TAG from the intestine, which contributes to circulating TAG levels. Increases in the dietary carbohydrate substrate can also facilitate increased synthesis of lipid at the site of the liver. Thus hyperphagia and IR collectively generate significant biochemical modulations in the liver including the up-regulation and hypersecretion of VLDL [15]. Consistent with observation in humans, the JCR:LA-cp model also presents with classic hypertriglyceridaemia and VLDL oversecretion which appears to develop in response to several lipidogenic factors, including SREBP-1c (sterol regulatory element-binding protein-1c) regulation [15,19].

SREBPs are a family of transcription factors that regulate cholesterogenesis and lipogenesis. In vivo studies have shown that the SREBP-1c isoform is primarily regulated by insulin [25]. SREBP-1c levels are increased in the liver of obese, insulin-resistant and hyperinsulinaemic ob/ob mice [26,27], and the oversecretion of VLDL in JCR:LA-cp animals has been attributed to the dysregulation of endogenous fatty acid synthesis via increased expression of SREBP-1c [15]. Further contributions to increased plasma TAG concentrations may be due to down-regulation in muscle LPL (lipoprotein lipase) activity and increased adipose LPL activity observed in the JCR:LA-cp rat [15]. Very recent evidence also implicates the overexpression of PCSK-9 (proprotein convertase subtilisin kexin type 9) in VLDL oversecretion [28]. However, the regulatory aspect of PSCK-9 during IR in influencing LDL (apoB100/E) receptor expression and impact on apoE-containing lipoproteins remains largely unknown.

The role of insulin in intestinal CM production and metabolism

CMs are synthesized in the enterocyte and deliver endogenous and dietary lipids to the circulation via the lymphatic system. Insulin has been shown to have an acute inhibitory effect on apoB48 production in enterocytes isolated from chow-fed animals, but this effect is not seen in animals that have hyperinsulinaemia [29,30]. Studies in humans have shown that modest delays in PP apoB48 peak response may be associated with insulin levels in both individuals with fasting hyperinsulinaemia and normal controls [31]. Collectively, it appears that the enterocyte is sensitive to circulating insulin levels, but in conditions of chronic hyperinsulinaemia, the enterocyte may become resistant to these effects [30,31]. However, very little is known regarding the effect of the amount and type of dietary lipid on CM synthesis and clearance in the circulation, particularly in IR. Today, few laboratories have the expertise to undertake in vivo lymphatic isolation of CMs in order to measure CM intestinal synthesis and secretion directly. Preliminary data from our laboratory have shown that in the hyperinsulinaemic JCR:LA-cp phenotype, the lymphatic apoB48 concentration is doubled following a lipid meal (1188 μg/ml) compared with normo-insulinaemic animals (555 μg/ml, P<0.05, n=5). This is associated further with a reduced TAG/apoB48 ratio (indicative of the size of particles) in the hyperinsulinaemic phenotype, suggesting that dietary lipids may play a key role in the regulation of PP CM synthesis. Indeed high levels of insulin may exacerbate an overproduction of apoB48 in the PP phase following a lipid-containing meal.

In addition, evidence indicates that intestinal overproduction of apoB48 may be associated with alterations in insulin signalling pathways in the enterocyte [30]. Specifically, acute studies in primary cultured enterocytes isolated from fructose-fed hyperinsulinaemic hamsters have shown alterations in phosphorylated ERK (extracellular-signal-regulated kinase) and the associated MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2] [30]. This represents a possible mechanism linking the dysregulation of insulin-signalling in the hyperinsulinaemic state and elevated intestinal apoB48 production. Thus these recent findings provide the impetus to extend the research focus onto studying the chronic regulation of intestinal apoB48 production in IR using the JCR:LA-cp model of pre-diabetes.

Effect of n−3 PUFA (polyunsaturated fatty acid) supplementation on PP lipaemia in the JCR:LA-cp rat

The literature demonstrates that consumption of n−3 PUFAs decreases total plasma TAG and lipogenesis, potentially by inhibiting the activation of SREBP-1c [32]. We have assessed the effect of n−3 PUFA supplementation on PP lipid, adipokine and inflammatory markers in the JCR:LA-cp rodent model. We have discovered that relative to obese control animals, treatment of JCL:LA-cp rats with n−3 PUFA significantly reduces concentrations of fasting plasma TAG, total cholesterol, leptin and apoB48. Furthermore, n−3 PUFAs can significantly improve the PP response (area under the curve) for both TAG and apoB48 in this animal model. In this meeting, we report evidence supporting the view that modest dietary n−3 PUFA supplementation can potentially reduce both PP dyslipidaemia and the pro-inflammatory status associated with IR [33].

CMRs: increased atherogenicity for the metabolic syndrome

Evidence now suggests that remnant lipoproteins (including intestinal apoB48-containing CMRs) can contribute to atherogenesis and are a significant risk factor for CVD [4–7,34–36]. We have compared the delivery and efflux of both CMR lipoproteins and LDL in the vessel wall in order to further understand the factors that regulate cholesterol accumulation in early atherogenesis [37]. Our results indicate that, whereas LDL particles have a higher rate of delivery, they efflux more readily from arterial tissue compared with the larger CMRs. Collectively, our findings have highlighted that lipoproteins permeate through arterial tissue differently, and the flux of particles may be dependent on the phenotype and potential interactions with extracellular matrix components such as arterial proteoglycans. In disease states, such as diabetes, the arterial wall demonstrates an exacerbated uptake and retention of lipoprotein-derived cholesterol, including apoB48-containing CMR particles [34–36]. Thus we have hypothesized that the response-to-retention aetiology of atherosclerosis should extend beyond circulating concentrations of cholesterol to include the rate of lipoprotein particle accumulation and retention in the sub-endothelial space within the arterial wall (see the model proposed in Figure 1).

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Figure 1 Proposed mechanism of increased atherogenicity due to accumulation of CMRs during metabolic syndrome

There is evidence from several studies (using both animal models and humans) that an equivalent fat load (challenge) will result in very divergent response of the intestine when comparing normal and insulin-resistant conditions. The exaggerated secretion of CMs from an ‘insulin-resistant’ intestine is now thought to contribute to the accumulating presence of TAG-rich particles in plasma. Furthermore, delayed clearance of apoE-containing CMR particles via the LDL-R (LDL receptor) and LRP-R (LDL receptor-related protein receptor) pathways ensures a continued exposure of atherogenic particles to the arterial wall. This in turn provides a platform for modified vascular function and exacerbated atherogenesis both during pre-diabetes and the metabolic syndrome.

Among the series of pathogenic mechanisms that contribute to atherosclerosis during IR is the remodelling of extracellular proteoglycans [37,38]. The proliferation of vascular smooth muscle cells can stimulate the secretion of arterial proteoglycans, which in turn can increase the capacity of lipoprotein binding (in vitro) [39,40]. More specifically, transforming growth factor (TGF) β-1 has been identified in atherosclerotic vessels and has been shown to stimulate the synthesis of chondroitin sulfate and dermatan sulfate-containing proteoglycans by arterial smooth muscle cells [40]. JCR:LA-cp rats also have increased circulating concentrations of TGFβ-1 [41]. Recent data from our group suggest that relative to lean controls, there is a significant increase in biglycan protein core content in the obese JCR:LA-cp rats, which increases with both age and progressive stages of IR (Figure 2). Moreover, there is also a significant positive correlation between aortic biglycan protein core content and fasting insulin levels at 6-, 12- and 32-week time points in obese rats (Figure 2). Chait and colleagues have demonstrated that TGFβ-1 can increase proteoglycan–lipoprotein binding due to the increased length of the glycosaminoglycan chain [40]. Furthermore, Scott et al. [42] have shown (using a comparable cell culture model of fibroblasts) that TGF β-1 can have profound effects on the regulation of both biglycan and decorin.

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Figure 2 Content of biglycan (protein core) from aorta of lean and obese (JCR:LA-cp) rats with increasing age

(A) Aorta from at least five animals for both phenotype and each age 6, 12 and >32 weeks (representative of pre-, during and developed IR in this model) was subjected to proteoglycan extraction and purification, as well as GAG digestion, separation by SDS/PAGE and Western blotting and incubation with an antibody cocktail for biglycan. Bars represent pooled animal samples (n≥5) that were repeatedly blotted (n=3) and measured by densitometry (means±S.E.M.). *P<0.01 between phenotype at each age; †P<0.01 for increasing content of biglycan with increasing age in obese animals. (B) Correlation of aortic biglycan protein (μg/μl) with increasing fasting plasma insulin concentrations.