Egg white hydrolysate enhances insulin sensitivity in high-fat diet-induced insulin-resistant rats via Akt activation.

Agents that block the renin-angiotensin system (RAS) improve glucoregulation in the metabolic syndrome disorder. We evaluated the effects of egg white hydrolysate (EWH), previously shown to modulate the protein abundance of RAS component in vivo, on glucose homeostasis in diet-induced insulin-resistant rats. Sprague-Dawley rats were fed a high-fat diet (HFD) for 6 weeks to induce insulin resistance. They were then randomly divided into four groups receiving HFD or HFD supplemented with different concentrations of EWH (1, 2 and 4 %) for another 6 weeks in the first trial. In the second trial, insulin-resistant rats were divided into two groups receiving only HFD or HFD+4 % EWH for 6 weeks. Glucose homeostasis was assessed by oral glucose tolerance and insulin tolerance tests. Insulin signalling and protein abundance of RAS components, gluconeogenesis enzymes and PPARγ were evaluated in muscle, fat and liver. Adipocyte morphology and inflammatory markers were evaluated. In vivo administration of EWH increased insulin sensitivity, improved oral glucose tolerance (P < 0·0001) and reduced systemic inflammation (P < 0·05). EWH potentiated insulin-induced Akt phosphorylation in muscle (P = 0·0341) and adipose tissue (P = 0·0276), but minimal differences in the protein abundance of tissue RAS components between the EWH and control groups were observed. EWH treatment also reduced adipocyte size (P = 0·0383) and increased PPARγ2 protein abundance (P = 0·0237). EWH treatment yielded positive effects on the inflammatory profile, glucose tolerance, insulin sensitivity and adipocyte differentiation in HFD-induced insulin resistance rats. The involvement of local RAS activity requires further investigation.

Increased risk of cardiovascular disease in Type 1 diabetes: arterial exposure to remnant lipoproteins leads to enhanced deposition of cholesterol and binding to glycated extracellular matrix proteoglycans.

AIMS: To determine fasting and postprandial metabolism of apolipoprotein B48 (apoB48) remnant lipoproteins in subjects with Type 1 diabetes and the relationship to progressive cardiovascular disease, and to investigate the impact of remnant lipoprotein cholesterol accumulation associated with arterial wall biglycan using a rodent model of Type 1 diabetes. METHODS: Normolipidaemic subjects (n = 9) with long-standing Type 1 diabetes (and advanced cardiovascular disease) and seven healthy control subjects were studied. Fasting and postprandial apoB48 concentration was determined following a sequential meal challenge. A rodent model of streptozotocin-induced diabetes was used to investigate the ex vivo retention of fluorescent-conjugated remnants. Binding of remnant lipoproteins to human recombinant biglycan was assessed in vitro. RESULTS: A significantly higher concentration of fasting plasma apoB48 remnants was observed in patients with Type 1 diabetes compared with control subjects. Patients with Type 1 diabetes exhibited a greater total plasma apoB48 area under the curve (AUC) and an increased incremental AUC following a second sequential meal compared with control subjects. The arterial retention of remnants ex vivo and associated cholesterol was increased sevenfold in Type 1 diabetes rats relative to controls. Remnants were shown to bind with significant affinity to human biglycan in vitro and a further 2.3-fold increased binding capacity was observed with glycated biglycan. Remnants were shown to colocalize with both arterial biglycan and glycated matrix proteins in the Type 1 diabetes rodent model. CONCLUSION: Impaired metabolism of remnant lipoproteins associated with enhanced binding to proteoglycans appears to contribute to the arterial cholesterol deposition in Type 1 diabetes. Our findings support the hypothesis that impaired remnant metabolism may contribute to accelerated progression of atherosclerosis in the hyperglycaemic and insulin-deficient state.

Evolution and obesity: resistance of obese-prone rats to a challenge of food restriction and wheel running.

The adaptive hypothesis that an obese-prone genotype confers a fitness advantage when challenged with food restriction and food-related locomotion was tested using a rat model. Juvenile (35-40 days) and adolescent (45-50 days) JCR:LA-cp rats, obese prone (cp/cp) and lean prone (+/?), were exposed to 1.5 h daily meals and 22.5 h of voluntary wheel running, a procedure that normally leads to self-starvation. Genotype had a dramatic effect on survival of rats when exposed to the challenge of food restriction and wheel running. Although similar in initial body weight, obese-prone juveniles survived twice as long, and ran three times as far, as their lean-prone counterparts. Biochemical measures indicated that young obese-prone animals maintained blood glucose and fat mass, whereas lean-prone rats depleted these energy reserves. Corticosterone concentration indicated that obese-prone juveniles exhibited a lower stress response to the survival challenge than lean-prone rats, possibly due to lower energy demands and greater energy reserves. Collectively, the findings support the hypothesis that an obese-prone genotype provides a fitness advantage when food supply is inadequate, but is deleterious during periods of food surfeit, such as the energy-rich food environment of prosperous and developing societies worldwide.

Dietary supplementation of n-3 PUFA reduces weight gain and improves postprandial lipaemia and the associated inflammatory response in the obese JCR:LA-cp rat.

BACKGROUND: Postprandial dyslipidaemia occurs in obesity and insulin resistance (IR), and is associated with an increased risk of developing cardiovascular disease. We have recently established that the JCR:LA-cp rodent model develops postprandial dyslipidaemia concomitant with complications of the metabolic syndrome. Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) are proposed to modulate plasma lipids, serum hormone levels, lipoprotein metabolism and the inflammatory state; however, results remain inconsistent during conditions of IR. AIM: To assess the acute metabolic and inflammatory effects of dietary fish oil supplementation on existing postprandial dyslipidaemia in the JCR:LA-cp model. METHODS: JCR:LA-cp rats (14 weeks of age) were fed either a control, isocaloric, lipid balanced diet (15% w/w total fat, 1.0% cholesterol, P:S ratio 0.4), a lipid balanced diet with 5% n-3 PUFA [fish oil derived eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA)] or a lipid balanced diet with 10% n-3 PUFA for 3 weeks. Fasting plasma lipid, cytokine levels, postprandial chylomicron (apoB48) metabolism and the postprandial inflammatory response [haptoglobin and lipopolysaccharide binding protein (LBP)] were assessed following a standardized 'oral fat challenge'. RESULTS: n-3 PUFA treatment resulted in a significant improvement (i.e. decrease) in the postprandial response for triglyceride (45%) (p < 0.05), apoB48 (45%) (p < 0.03) and LBP (33%) (p < 0.05) compared to controls (measured as area under the clearance curve). In contrast, we observed a significant elevation in postprandial haptoglobin (165%) (p < 0.001) in obese rats supplemented with 10% n-3 PUFA. Treatment with 5% n-3 PUFA in the JCR:LA-cp obese animals resulted in a complementary decrease in total body weight gain (6%) (p < 0.001) and an increase (i.e. improvement) in adiponectin (33%) (p < 0.05) compared to controls, without a concomitant reduction in food intake. CONCLUSION: Acute dietary n-3 PUFA dietary supplementation can improve fasting as well as postprandial lipid metabolism and components of the associated inflammatory response in the JCR:LA-cp rat. Further, moderate dose n-3 PUFA supplementation may reduce corresponding body weight during conditions of hypercholesterolaemia and/or modulate inflammation associated with obesity and the metabolic syndrome.

Effects of feeding fish oil on mesenteric lymph node cytokine responses in obese leptin receptor-deficient JCR:LA-cp rats.

OBJECTIVE: berrant immune responses have been identified in obesity; however, immune cells of lymph nodes residing in the inflammatory environment of visceral adipose tissue have been largely overlooked. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can reduce inflammation and modify T-cell function and therefore may improve immune function in obesity. Thus, we determined the effects of feeding fish oil (FO) containing EPA and DHA on mesenteric lymph node (MLN) immune cell function. METHODS: In this study, 14-week-old obese, leptin receptor-deficient JCR:LA-cp rats (cp/cp) (n=10 per group) were randomized to one of three nutritionally adequate diets for 3 weeks: control (ctl, 0% EPA+DHA), low FO (LFO, 0.8% w/w EPA+DHA) or high FO (HFO, 1.4% w/w EPA+DHA). Lean JCR:LA-cp (Cp/cp or Cp/Cp) rats (n=5) were fed ctl diet. MLN cell phospholipid (PL) fatty acid composition, phenotypes and cytokine production were measured. RESULTS: Obese ctl rats produced more IL-1beta, IL-4 and IL-10, despite a higher proportion of (n-3) polyunsaturated fatty acids (PUFAs) and a lower (n-6):(n-3) PUFA ratio in MLN PL compared with lean ctl rats (P<0.05). Concanavalin A-stimulated IL-2 production did not differ from lean rats even though obese ctl rats had a lower proportion of CD4(+)CD25(+) cells (P<0.05). Feeding FO to obese rats increased the incorporation of (n-3) PUFA into MLN PL and normalized production of IL-1beta (HFO only), IL-4 and IL-10 to the levels similar to lean ctl rats (P<0.05). CONCLUSION: We demonstrate for the first time that obese JCR:LA-cp rats have impaired responses of MLN immune cells to mitogen stimulation and altered PL fatty acid composition. Feeding FO lowered the ex vivo inflammatory response (HFO only) and production of Th2 cytokines, without changing IL-2 production from ConA-stimulated splenocytes, which may occur independent of leptin signalling.

Methods to assess impaired post-prandial metabolism and the impact for early detection of cardiovascular disease risk.

Post-prandial lipaemia has emerged as a key contributor to cardiovascular disease (CVD) risk and progression. Specifically, delayed clearance of chylomicrons (CM) and their remnants increase the delivery of triglyceride and cholesteryl ester to the vessel wall and can accelerate the progression of atherosclerosis, which may be particularly pertinent to individuals with insulin resistance and/or obesity. As the number of studies linking post-prandial metabolism and chronic disease increases, interest has grown in the use of parameters reflecting CM metabolism as a possible indicator of early CVD risk. This, in turn has raised the question of what method might be most appropriate to detect CM and their remnants in plasma accurately. However, the handful of techniques able to measure CM metabolism (triglyceride-rich lipoprotein fractions; remnant-lipoprotein cholesterol; retinyl esters, CM-like emulsion; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; immunoblotting, enzyme-linked immunoabsorbent assays; C(13) breath test; capillary finger prick) differ in their specificity, cost and applicability in research and in the clinical setting. In this review, we explore the scientific and clinical implications of CM methodology to better understand early risk assessment of CVD. We raise ongoing issues of the need to appreciate differential separation of very low-density lipoprotein and CM fractions, as well as to identify the technical basis for imprecision between assays for apolipoprotein B48. We also highlight emerging issues with respect to the practicality of measuring post-prandial metabolism in large clinical studies and offer opinions on the appropriateness of existing techniques in this field.

Impaired postprandial apolipoprotein-B48 metabolism in the obese, insulin-resistant JCR:LA-cp rat: increased atherogenicity for the metabolic syndrome.

AIM: Postprandial lipaemia is a significant contributor to the development of dyslipidaemia and cardiovascular disease, which has more recently been shown as a potential risk factor for obesity and pre-diabetes. Clinically however, the diagnosis of early insulin-resistance remains confounded due to the fact that aberrations in lipid metabolism are not often readily identified using classic indicators of hypercholesterolemia (i.e. LDL). METHODS: In this study, we assessed the metabolism of apolipoprotein-B48 (apoB48)-containing lipoproteins in an animal model of obesity and insulin-resistance, the JCR:LA-cp rat. The contribution of lipoproteins from the intestine was assessed by measuring plasma apoB48 concentration in the postprandial period following an oral fat load. Plasma apoB48 was measured by improved enhanced chemiluminescent detection and other biochemical parameters measured by established analysis. RESULTS: Fasting concentrations of plasma apoB48, postprandial apoB48 area under the curve (AUC), as well as incremental-AUC (iAUC), were all significantly greater in the obese phenotype compared to lean controls. Fasting apoB48 correlated significantly with apoB48-iAUC, triglyceride (TG)-iAUC and insulin-iAUC. In addition, there was a highly significant association with fasting insulin and the postprandial ratio of TG:apoB48, a relationship not often detected in humans during insulin-resistance. CONCLUSIONS/INTERPRETATION: We conclude that the JCR:LA-cp rat can be used as a model of postprandial lipemia to explore chylomicron metabolism during the onset and development of insulin-resistance, including the increased cardiovascular complications of the metabolic syndrome.

Increased insulin sensitivity and reduced micro and macro vascular disease induced by 2-deoxy-D-glucose during metabolic syndrome in obese JCR: LA-cp rats.

BACKGROUND AND PURPOSE: The metabolic syndrome, characterized by obesity, insulin resistance and dyslipidemia, is a major cause of cardiovascular disease. The origins of the syndrome have been hypothesized to lie in continuous availability of energy dense foods in modern societies. In contrast, human physiology has evolved in an environment of sporadic food supply and frequent food deprivation. Intermittent food restriction in rats has previously been shown to lead to reduction of cardiovascular risk and a greater life span. The non-metabolizable glucose analogue, 2-deoxy-D-glucose (2-DG) is taken up by cells and induces pharmacological inhibition of metabolism of glucose. We hypothesized that intermittent inhibition of glucose metabolism, a metabolic deprivation, may mimic intermittent food deprivation and ameliorate metabolic and pathophysiological aspects of the metabolic syndrome. EXPERIMENTAL APPROACH: Insulin resistant, atherosclerosis-prone JCR:LA-cp rats were treated with 2-DG (0.3% w/w in chow) on an intermittent schedule (2 days treated, one day non-treated, two days treated and two days non-treated) or continuously at a dose to give an equivalent averaged intake. KEY RESULTS: Intermittent 2-DG-treatment improved insulin sensitivity, which correlated with increased adiponectin concentrations. Further, intermittent treatment (but not continuous treatment) reduced plasma levels of leptin and the inflammatory cytokine IL-1 beta. Both 2-DG treatments reduced micro-vascular glomerular sclerosis, but only the intermittent schedule improved macro-vascular dysfunction. CONCLUSIONS AND IMPLICATIONS: Our findings are consistent with reduction in severity of the metabolic syndrome and protection against end stage micro- and macro-vascular disease through intermittent metabolic deprivation at a cellular level by inhibition of glucose oxidation with 2-DG.

Sequential Feeding of Lipid Supplement Enriches Beef Adipose Tissues with 18:3n-3 Biohydrogenation Intermediates.

The present study was designed to determine if feeding steers extruded flaxseed and hay (25 and 75%; DM basis) together as a total mixed ration (TMR), or sequentially (non-TMR) would result in different enrichments of polyunsaturated fatty acids (PUFA) and their biohydrogenation intermediates (BHI) in beef adipose tissues [subcutaneous (SC) vs perirenal (PR) fat]. Forty-eight Angus cross steers (325 ± 16 kg) were stratified by weight to six pens, and pens were randomized to either TMR or non-TMR and fed ad libitum for an average of 242 days. The concentrations of α-linolenic acid increased by 18 mol% in both SC and PR in non-TMR steers compared to TMR steers (P < 0.01). trans 18:1 isomers were more concentrated in PR than SC (14.4 vs 9.5 mol%; P < 0.01) and increased by 10 mol% in both fat depots for non-TMR (P < 0.01). Other BHI including non-methylene-interrupted 18:2 (atypical dienes), conjugated linoleic acids and conjugated linolenic acids (CLnA) were affected by diet × tissue interactions (P < 0.01). The CLnA and CLA contents were higher in both fat depots when feeding the non-TMR, but the effect of diet was more pronounced in PR than in SC (P < 0.01). Atypical dienes were highest in PR from non-TMR and lowest in TMR fed steers (4.3 and 3.6 mol%) with SC contents being intermediate. The sequential feeding of lipid supplement can thus profoundly affect the enrichment of PUFA and their BHI in beef fat and their differentially enrichment is also fat depot dependant.

Effects of feeding steers extruded flaxseed on its own before hay or mixed with hay on animal performance, carcass quality, and meat and hamburger fatty acid composition.

The objective of the present experiment was to determine if carcass quality and fatty acid profiles of longissimus thoracis (LT) and hamburger would be affected by feeding steers extruded flaxseed on its own followed by hay (non-TMR) compared to when hay and extruded flaxseed were fed together (TMR). Forty-eight steers in six pens were assigned to TMR or non-TMR for an average of 242days. Dry matter intake was lower for non-TMR versus TMR steers (10.56 vs. 11.42kg/d; P=0.02), but final live weight (610±0.50kg) and average daily gain (1.18±0.02kg/d) did not differ. Compared to TMR, feeding non-TMR enriched LT and hamburger with α-linolenic acid (ALA; 18:3n-3) by 14%, vaccenic acid (VA; t11-18:1) by 44%, rumenic acid (RA; c9,t11-18:2) by 40%, and conjugated linolenic acid (CLnA) by 58%. Overall, feeding extruded flaxseed separately from hay in a non-TMR was more effective at enhancing deposition of ALA, VA, RA and CLnA in beef.

Koch's postulates, microbial dysbiosis and inflammatory bowel disease.

Over the past 20 years, a growing amount of evidence supports the role of microbes and an imbalanced microbiota in inflammatory bowel disease (IBD). While many reviews have been written on the microbiota in IBD, few have considered how they fulfil the Koch's postulates. In this review, we consider how the Koch's postulates might be modified so that they can be fulfilled for polymicrobial diseases, and we discuss the progress made to date in fulfilling them.

Differential expression of hypothalamic, metabolic and inflammatory genes in response to short-term calorie restriction in juvenile obese- and lean-prone JCR rats.

BACKGROUND: Childhood obesity is an important early predictor of adult obesity and associated comorbidities. Common forms of obesity are underpinned by both environmental and genetic factors. However, the rising prevalence of obesity in genetically stable populations strongly suggests that contemporary lifestyle is a premier factor to the disease. In pediatric population, the current treatment/prevention options for obesity are lifestyle interventions such as caloric restriction (CR) and increase physical activity. In obese individuals, CR improves many metabolic parameters in peripheral tissues. Little is known about the effect of CR on the hypothalamus. This study aimed to assess the effect of CR on hypothalamic metabolic gene expression of young obese- and lean-prone animals. METHODS: Male juvenile JCR:LA-cp obese-prone rats were freely fed (Obese-FF) or pair fed (Obese-FR) to lean-prone, free-feeding animals (Lean-FF). A group of lean-prone rats (Lean-FR) were matched for relative average degree of CR to Obese-FR rats. RESULTS: In free-feeding conditions, obese-prone rats consumed more energy than lean-prone rats (P<0.001) and showed greater increases in body weight, fat mass, plasma glucose, insulin and lipids (P<0.01). These metabolic differences were associated with alterations of feeding-related neuropeptides expression in the hypothalamus, as well as pro-inflammatory cytokines and oxidative stress markers. When submitted to the same degree of CR, the two genotypes responded differently; hypothalamic inflammatory and oxidative stress gene expression was improved in Obese-FR, while it was worsened in Lean-FR rats. CONCLUSIONS: We demonstrate in JCR rats that the metabolic and inflammatory response of the brain to CR is genotype dependent.

Retention of chylomicron remnants by arterial tissue; importance of an efficient clearance mechanism from plasma.

Atherosclerosis is thought to begin with the trapping of cholesterol rich lipoproteins within the intima of arterial vessels. Thereafter a complex inflammatory cascade involving recruitment and transformation of leukocytes, accumulation of sterols in macrophages and cellular proliferation, can lead to a progressive occlusion in blood flow, or an unstable arterial lesion prone to prothrombotic events. Primary intervention strategies aimed at reducing atherogenesis are designed to achieve reductions in sterol rich lipoproteins, primarily low density lipoproteins, given the hypothesis that decreased exposure will attenuate the rate of arterial cholesterol accumulation. Epidemiological evidence has clearly identified a positive relationship between poor dietary (fat) habits and the onset and progression of atherosclerosis. However lipoproteins which mediate the transport of dietary lipid, that is chylomicrons, are not normally considered to be directly involved in atherogenesis, because of their larger size and inability to efficiently penetrate arterial tissue. In contrast, this article reviews recent evidence which suggests that once chylomicrons are hydrolysed to their remnant form, the triglyceride depleted chylomicron remnants penetrate arterial tissue and moreover, become preferentially trapped within the subendothelial space as concentrated focii. Ongoing studies demonstrate that significant chylomicron remnant accumulation can occur in a number of primary and secondary lipid disorders and in normolipidemic subjects with coronary artery disease. Chylomicron remnant dyslipidemia in conditions prone to premature atherosclerosis is consistent with the putative atherogenicity of these particles and can be explained by increased arterial exposure to cholesterol rich chylomicron remnants.

Arterial intimal retention of pro-atherogenic lipoproteins in insulin deficient rabbits and rats.

Recent observations that remnants of triglyceride rich lipoproteins become trapped within the subendothelial of arterial vessels gives rise to the possibility that these particles could initiate the atherogenic cascade. Increased frequency and progression of atherosclerosis in diabetes might in part be a consequence of raised concentrations in plasma of remnant lipoproteins. In addition, diabetes may lead to changes in the arterial vasculature which exacerbate arterial retention of pro-atherogenic lipoproteins. To explore these possibilities, in this study we determined aortic retention of chylomicron remnants, which are of intestinal origin, and of hepatically derived low-density lipoproteins (LDL) in insulin deficient rabbits and rats. The two species were selected because of their disparate susceptibility to develop atherosclerosis in the presence of diabetes induced hyperlipidemia. Chylomicron remnants and LDL were differentially radiolabelled with a residual marker and injected simultaneously into conscious rabbits or rats. Arterial retention was determined 2 h after injection, and relative retention was expressed as a percentage of mean arterial exposure. We found in insulin deficient rabbits and rats that intimal and medial retention of chylomicron remnants was positively related to the degree of hyperglycemia and was significantly greater than in non-diabetic control groups. In contrast, insulin deficiency did not influence arterial retention of LDL. Rabbits which are susceptible to diabetes induced atherogenesis had significantly greater intimal retention of chylomicron remnants compared to rats. Results from this study support the hypothesis that chronic hyperglycemia promotes arterial retention of triglyceride rich remnant lipoproteins and that atherosclerotic susceptibility might be related to degree of remnant entrapment within the subendothelial space. Greater retention of remnant lipoproteins could in part explain the increased prevalence of atherogenesis in diabetes.

Arterial fatty lesions have increased uptake of chylomicron remnants but not low-density lipoproteins.

BACKGROUND: This study compares the accumulation of pre-formed chylomicron-remnants, chylomicrons and low-density lipoproteins (LDL) in rabbit thoracic aorta. To determine whether lipoproteins are delivered via the vasa vasorum, the aortic uptake of lipoproteins was compared to that of the common carotid artery. The uptake of chylomicron remnants and LDL were compared in lesioned and non-lesioned aortic tissue from Watanabe heritable hyperlipidaemic (WHHL) rabbits. METHODS: Chylomicrons, chylomicron-remnants and LDL were radio-labelled with tyramine-cellobiose and injected into rabbits. Arterial uptake was determined after 2 h as the percentage of injected lipoproteins associated with arterial tissue and also expressed as a fraction of mean arterial exposure. RESULTS: Aortic accumulation of radio-labelled chylomicron-remnants was substantially greater than for chylomicrons, and both were significantly greater than LDL. The data suggests that chylomicrons must first be hydrolysed to smaller particles before uptake. In normal rabbits, there was no difference in uptake of the lipoproteins between the aorta and carotid vessels, suggesting that the vasa vasorum is not significantly involved in lipoprotein delivery. However, in WHHL rabbits there was significantly greater aortic uptake of chylomicrons and LDL compared to the carotid vessel and, in cholesterol-fed rabbits, significantly greater aortic uptake of chylomicrons, suggesting that in hypercholesterolaemia the lipoprotein retention properties of some arterial beds change. In arterial fatty lesions from WHHL and cholesterol-fed rabbits there was an exclusive increase in chylomicron remnant uptake, whereas LDL uptake was similar to non-lesioned tissue. CONCLUSIONS: Chylomicron remnants and not their precursors might be primary atherogenic lipoprotein because they penetrate arterial tissue efficiently and are selectively retained in sites of lesion formation.

A highly sensitive assay for quantitation of apolipoprotein B48 using an antibody to human apolipoprotein B and enhanced chemiluminescence.

We describe a method for the rapid quantification of serum apolipoprotein B48 using a commercially available anti-apolipoprotein (apo)B antiserum and compare it to analytical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with coomassie blue R250 staining. The method described here eliminates the need for de-lipidation of samples and only requires a one-step overnight ultracentrifugation. Western-blotting and enhanced chemiluminescence (ECL) visualization of proteins was approximately 10 times more sensitive than coomassie staining and generally took no longer to complete than staining/destaining of SDS-PAGE gels. The sensitivity of the antiserum/ECL technique enabled quantitation of fasting apolipoprotein B48 which could not be resolved by SDS-PAGE and Coomassie staining.

Detection of LDL receptor by ligand blotting with chylomicron remnants labelled with colloidal gold.

The LDL receptor plays a pivotal role in the clearance of pro-atherogenic lipoproteins, and LDL receptor deficiency may be the underlying cause of several primary and secondary dyslipidaemic conditions. Intervention strategies are often targeted to increase hepatic LDL receptor expression. It is difficult to quantitate hepatic LDL receptor activity and to monitor changes post-therapy. In order to avoid liver biopsy, human skin fibroblasts or circulating mononuclear cells have often been used as surrogate markers for the hepatic receptor. Fibroblasts, and particularly mononuclear cells, are relatively easy to isolate and can be stored for extensive lengths of time without significant loss of LDL receptor expression. Leucocytes or fibroblasts are normally probed with isotopically or gold-labelled LDL. However, the specific activity of the LDL conjugate is usually too low to enable accurate quantitation of differences, or changes, in LDL receptor expression. In this study, we describe an enhanced colloidal gold-labelling procedure for the detection of LDL receptor binding activity. The binding of colloidal gold-labelled chylomicron remnants to human hepatocytes (HepG2 cells) was compared with that of gold-conjugated LDL. Labelled remnants bound specifically to a cell surface protein with a molecular weight of approximately 130 kDa. Binding was blocked in the presence of unlabelled remnants, LDL, or antiserum specific to the LDL receptor. The binding of gold-labelled remnants was substantially greater than that of gold-labelled LDL. Compared with gold-labelled LDL, we found a much clearer demarcation of remnant binding with hepatocytes incubated in the presence or absence of sterols. Our observations suggest that, because of the greater affinity of the LDL receptor for lipoproteins containing apolipoprotein E, changes in LDL receptor expression might be more readily identified using gold-labelled remnants. We conclude that gold-conjugated chylomicron remnants might provide a useful means of detecting subtle changes in LDL receptor expression.

Types of oilseed and adipose tissue influence the composition and relationships of polyunsaturated fatty acid biohydrogenation products in steers fed a grass hay diet.

The current study evaluated the composition and relationships of polyunsaturated fatty acid biohydrogenation products (PUFA-BHP) from the perirenal (PRF) and subcutaneous fat (SCF) of yearling steers fed a 70 % grass hay diet with concentrates containing either sunflower-seed (SS) or flaxseed (FS). Analysis of variance indicated several groups or families of structurally related FA, and individual FA within these were affected by a number of novel oilseed by fat depot interactions (P < 0.05). Feeding diets containing SS increased the proportions of non-conjugated 18:2 BHP (i.e., atypical dienes, AD) and conjugated linoleic acids (CLA) with the first double bond from carbon 7 to 9, trans-18:1 isomers with double bonds from carbon 6 to 12, and these PUFA-BHP had greater proportions in SCF compared to PRF (P < 0.05). Enrichment of conjugated linolenic acids, AD and CLA isomers with the first double bond in position 11 or 12, and t-18:1 isomers with double bonds from carbon 13 to 16 were achieved by feeding diets containing FS, with PRF having greater proportions than SCF (P < 0.05). Principal component analysis visually confirmed interaction effects on these groups/families of FA, and further confirmed or suggested a number of relationships between PUFA-BHP. Feeding SS or FS in a grass hay diet and exploiting adipose tissue differences, therefore, present unique opportunities to differentially enrich a number of PUFA-BHP which seem to have positive health potential in humans (i.e., t11-18:1, c9,t11-18:2 and c9,t11,c15-18:3).

Elevated remnant lipoproteins may increase subclinical CVD risk in pre-pubertal children with obesity: a case-control study.

OBJECTIVES: Current clinical guidelines to assess paediatric cardiovascular disease (CVD) risk heavily rely on cholesterol parameters that are generally normal for obese children. Remnant lipoproteins have emerged as a critical CVD risk factor particularly in adults with normolipidemia. We assessed remnant lipoprotein concentration (measured by apolipoprotein [apo] B48) and its relationship with other traditional CVD risk biomarkers in pre-pubertal children with obesity. METHODS: Pre-pubertal children (n = 78) with obesity (n = 39, 9.9 ± 0.3 years old) as well as sex-matched normal-weight controls (n = 39, 9.8 ± 0.3 years) were assessed for anthropometry, blood pressure and fasting plasma biochemical parameters for remnant lipoprotein, lipid and glucose/insulin metabolism, and inflammatory status. RESULTS: Children with obesity had striking 2-fold higher apoB48-containing remnant lipoproteins concentrations relative to normal-weight peers; the magnitude of elevation in the remnant lipoproteins is comparable to the levels previously reported for adults with established CVD and type-2 diabetes. Fasting apoB48 was positively correlated with fasting triglyceride concentration in children with obesity (r = 0.51, P < 0.001) and their normal-weight peers (r = 0.45, P < 0.01). Traditional CVD biomarkers including low-density lipoprotein cholesterol showed no difference between groups and remained within the normal range for a paediatric population. CONCLUSION: Elevated apoB48-containing remnant lipoprotein is a stronger biomarker for paediatric CVD risk compared to traditional cholesterol parameters and may be associated with early adaptation of the intestine during obesity. Further investigation of abnormalities associated with the secretion and/or clearance of atherogenic remnant lipoproteins during the postprandial state may yield insight into our understanding of and therapeutic targets for managing risk for CVD in children with obesity.

Effects of feeding flaxseed or sunflower-seed in high-forage diets on beef production, quality and fatty acid composition.

Yearling steers were fed 70:30 forage:concentrate diets for 205 d, with either grass hay (GH) or red clover silage (RC) as the forage source, and concentrates containing either sunflower-seed (SS) or flaxseed (FS), each providing 5.4% oil to diets. Feeding diets containing SS versus FS significantly improved growth and carcass attributes (P<0.05), significantly reduced meat off-flavor intensity (P<0.05), and significantly increased intramuscular proportions of vaccenic (t11-18:1), rumenic (c9,t11-CLA) and n-6 fatty acids (FA, P<0.05). Feeding diets containing FS versus SS produced significantly darker and redder meat with greater proportions of atypical dienes (P<0.05). A significant forage × oilseed type interaction (P<0.05) was found for n-3 FA, α-linolenic acid, and conjugated linolenic acid, with their greatest intramuscular proportions found when feeding the RC-FS diet. Feeding GH versus RC also significantly improved growth and carcass attributes, sensory tenderness (P<0.05) and significantly influenced intramuscular FA composition (P<0.05), but overall, forage effects on FA profiles were limited compared to effects of oilseed.