rich lipoprotein metabolism in the metabolic syndrome

DOI: 10.1111/j.1365-2362.2008.02019.x Blackwell LOSS ORIGINAL WEIGHT Publishing ARTICLE ON THE Ltd METABOLIC SYNDROME

ORIGINAL ARTICLE

Effect of weight loss on markers of triglyceride-rich lipoprotein metabolism in the metabolic syndrome D. C. Chan*, G. F. Watts*, T. W. K. Ng*, S. Yamashita† and P. H. R. Barrett* *

School of Medicine and Pharmacology, University of Western Australia, Perth, Australia, †Osaka University Graduate School of Medicine, Osaka, Japan

ABSTRACT Backgroud Hypertriglyceridaemia, a consistent feature of dyslipidaemia in the metabolic syndrome (MetS), is related to the extent of abdominal fat mass and altered adipocytokine secretion. We determined the effect of weight loss by dietary restriction on markers of triglyceride-rich lipoprotein (TRL) metabolism and plasma adipocytokines. Design Thirty-five men with MetS participated in a 16 week randomized controlled dietary intervention study. Apolipoprotein (apo) C-III, apoB-48, remnant-like particle (RLP)-cholesterol, total adiponectin, high-molecular weight (HMW) adiponectin, and retinol-binding protein-4 (RBP-4) concentrations were measured using immunoassays. Results Compared with weight maintenance (n = 15), weight loss (n = 20) significantly decreased body weight, plasma insulin, triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol and lathosterol (P < 0·05). Weight loss also decreased plasma concentrations of apoC-III (–33%), apoB-48 (–37%), very low-density lipoprotein (VLDL)-apoB (–43%), RLP-cholesterol (–48%), and RBP-4 (–20%), and significantly increased plasma total (+20%) and HMW-adiponectin (+19%) concentrations. In the weight loss group, reduction in plasma apoC-III was associated (P < 0·05) with reduction in plasma apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides. Increase in total adiponectin was associated (P < 0·05) with the reduction in plasma VLDL-apoB and triglycerides. The changes in HMW-adiponectin and RBP-4 were not associated with changes in plasma apoB-48, apoC-III, VLDL-apoB, RLP-cholesterol or triglycerides. In multiple regression analysis including changes in visceral fat, insulin and total adiponectin concentrations, the fall in plasma apoC-III concentration was an independent predictor of the reductions in plasma apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides concentrations. Conclusions In men with MetS, weight loss decreases the plasma concentrations of apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides. This effect could partly relate to concomitant changes in plasma apoC-III and adiponectin concentrations that accelerate the catabolism of TRLs. Keywords Adipocytokines, cardiovascular disease, lifestyle intervention, obesity, triglyceride-rich lipoproteins. Eur J Clin Invest 2008; 38 (10): 743–751

Introduction Hypertriglyceridaemia is a consistent feature of the metabolic syndrome (MetS) [1,2], and a risk factor for cardiovascular disease (CVD) and diabetes mellitus [3,4]. The underlying mechanism for dyslipidaemia in MetS may relate to over secretion, reduced hydrolysis, and/or impaired clearance of triglyceride-rich lipoproteins (TRLs) and their remnants [2,5,6]. These abnormalities may be consequent on central obesity and insulin resistance. Apolipoprotein (apo) B-48, remnant-like particle (RLP)-cholesterol and VLDL-apoB-100 are static markers for the metabolism of TRLs [2]. Fasting plasma apoB-48 levels predict

postprandial lipaemia [7], a risk factor for atherosclerosis [8]. Increased plasma RLP-cholesterol concentrations have been associated with coronary heart disease, diabetes mellitus and other lipid disorders [9,10]. VLDL-apoB levels are the major determinant of plasma triglyceride concentrations in both normolipidaemic and MetS subjects. ApoC-III is also involved in TRL metabolism, via its effect in inhibiting lipoprotein lipase (LPL) activity and TRL remnant uptake by hepatic lipoprotein receptors [11]. Elevated apoC-III may accordingly result in accumulation in plasma of TRLs and increased risk of CVD European Journal of Clinical Investigation Vol 38

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[12]. We previously demonstrated that centrally obese individuals have insulin resistance and increased concentrations of apoB48, VLDL-apoB, apoC-III and remnant-like particle (RLP)-cholesterol [2]. Regulation of these abnormalities via lifestyle or pharmacotherapy is important to reduce the associated risk of CVD. Adiponectin and retinol-binding protein 4 (RBP-4) are two adipocytokines that potentially impact on lipid metabolism, and link obesity with insulin resistance and/or metabolic and cardiovascular risk factors [13–18]. Adiponectin may stimulate fatty acid oxidation in skeletal muscle [16], decreasing intramyocellular accumulation of triglycerides and potentially accelerating the catabolism of TRLs [19,20]. Low plasma adiponectin in obese and insulin resistance subjects may therefore contribute to impaired clearance of VLDL. Recently, adiponectin was shown to circulate in plasma in several multimeric forms, of which the high–molecular weight (HMW) form is most biologically active [21]. We previously demonstrated that adiponectin is inversely associated with plasma apoB-48, apoC-III, RLP-cholesterol and triglycerides [19]. Recent animal and human studies suggest that RBP-4 induces insulin resistance and is associated with both insulin resistance and obesity [17,18]. RBP-4 stimulates hepatic gluconeogenesis and inhibits insulin signalling in skeletal muscle, thereby inducing insulin resistance in the liver and peripheral tissue [17], but the relationship between RPB-4 and abnormalities in TRLs in obesity remains unclear. Weight loss by dietary restriction is the first line approach to correct dyslipidaemia in MetS [22]. We previously demonstrated that in obese men 5 to 10 kg weight loss with a low caloric diet effectively decreases plasma concentrations of triglycerides and LDL-cholesterol, as well as improves insulin sensitivity [23,24]. However, the corresponding effects of weight loss on markers of TRL metabolism and plasma adipocytokines have not yet been formally investigated. Whether the changes in plasma concentrations of triglycerides, apoB-48 and RLP-cholesterol with weight loss could be explained by changes in plasma apoC-IIII and adipocytokines also needs clarification. We previously reported the kinetic effects of weight loss on apoB and apoA-I metabolism in subjects with MetS [24]. We extend that study by investigating the effects of weight loss on markers of TRL metabolism (apoB-48, apoC-III and RLP-cholesterol) and plasma adipocytokines (total, HMW adiponectin and RBP-4) and the potential associations among these changes.

Materials and methods Subjects We studied 35 non-smoking, centrally obese Caucasian men with the metabolic syndrome [25]. None had diabetes mellitus, CVD, apoE2/E2 or E4/E4 genotype, macro proteinuria, creatinaemia

(> 120 μmol L−1), hypothyroidism, abnormal liver enzymes, nor consumed > 40 g alcohol/day or were taking agents affecting lipid metabolism. The study was approved by the Royal Perth Hospital Ethics Committee.

Study design and clinical protocols Subjects entered a randomized, controlled dietary intervention study. After weight stabilization for 4 weeks, they were randomized to either a weight reducing hypocaloric diet for 14 weeks, immediately followed by a 2 week weight stabilization period or to weight maintenance on an isocaloric diet for 16 weeks. All tests were carried out at stable body weight. Body weight, height, waist circumference and blood pressures were recorded. Subcutaneous abdominal adipose tissue and visceral adipose tissue volumes and masses were estimated following magnetic resonance imaging, as described previously [19,20]. All subjects were studied after a 14 h fast. Venous blood was collected for biochemical measurements at baseline and week 16. Subjects were requested to maintain their usual level of physical activity and alcohol intake. These were assessed by 7 day recall questionnaires and alcohol diaries. Three day dietary diaries were completed every three weeks by both groups and analysed using DIET 4 Nutrient Calculation Software (Xyris Software, Brisbane, Australia).

Biochemical analyses Fasting plasma cholesterol, triglycerides and HDL-cholesterol were determined by standard enzymatic methods (interassay CVs were < 3%). LDL-cholesterol was calculated using the Friedewald’s equation or by direct measurement with triglycerides > 4·5 mmol L–1. Non-HDL-cholesterol was derived as total cholesterol minus HDL-cholesterol. Plasma non-esterified fatty acids (NEFAs) and insulin were measured by enzymatic methods (Boehringer Mannheim, Mannheim, Germany; CV < 3%). Glucose was measured by a hexokinase method. Insulin resistance was estimated by HOMA score [26]. Plasma lathosterol concentration (a surrogate marker of cholesterol synthesis) was measured by GCMS (Hewlett Packard, Maryland, USA). The VLDL fraction was isolated from 3 mL plasma by ultracentrifugation (Kontron Instruments, Milan, Italy) at 147 000 g for 16 h at 4° C. VLDL-apoB fraction was precipitated with 50% isopropanol. Extraction of the precipitate with 100% isopropanol removed neutral lipids (triglycerides, cholesterol and cholesterol esters). The delipidated apoB was then made soluble in alkaline deoxycholate solution and protein content estimated by the Lowry method, with bovine serum albumin as standard (interassay CV was < 5%). Plasma apoB-48 levels were measured by a sandwich ELISA (Fujirebio, Tokyo, Japan) as reported previously (interassay CV was < 5%) [27]. Plasma apoC-III was measured by immunoturbidimetric assay (Daiichi, Toyko, Japan). Plasma RLP-cholesterol was determined from plasma with a

744 © 2008 The Authors. Journal Compilation © 2008 Blackwell Publishing Ltd

WEIGHT LOSS ON THE METABOLIC SYNDROME

JIMRO-II (Japan Immunoresearch Laboratories, Takasaki, Japan) assay kit using an immunoseparation technique (interassay CV was < 5%) [28]. Total and HMW adiponectin concentrations were determined using an immunoassay kit (Alpco Diagnostics, New Hampshire, USA). Plasma RBP-4 concentrations were determined using enzyme immunoassay kit (Immunodiagnostik, Bensheim, Germany, the interassay CV was < 10%).

Statistical analyses All analyses were carried out using SPSS 15 (SPSS Inc, Chicago, USA). Baseline characteristics of the subjects by groups were compared by independent t-tests. Changes in the weight loss group were compared with the weight maintenance group using general linear models, with adjustments made for baseline covariates. Student’s paired t-test was used to evaluate significant changes within groups. Associations were examined using simple, partial correlations and both simple and multivariate linear regression methods. Statistical significance was defined as P < 0·05.

Table 1 Clinical and biochemical characteristics of the subject studied Characteristics

Mean ± SD

Age (years)

46 ± 8

Weight (kg)

107 ± 11

−2

BMI (kg m )

34 ± 3

Waist circumference (cm)

113 ± 7

Systolic blood pressure (mmHg)

132 ± 18

Diastolic blood pressure (mmHg)

76 ± 9

Glucose (mmol L−1)

5·6 ± 0·6

Insulin (mU L−1)

16 ± 9

HOMA score

4·0 ± 2·4

Cholesterol (mmol L−1)

5·9 ± 1·1

−1

Triglyceride (mmol L )

3·1 ± 2·3 −1

HDL-cholesterol (mmol L )

1·0 ± 0·2

LDL-cholesterol (mmol L−1)

3·5 ± 0·8

Results

Non-HDL-cholesterol (mmol L )

Baseline characteristics

BMI, body mass index; HOMA, homeostasis model assessment; HDL, high density lipoprotein; LDL, low density lipoprotein.

−1

Table 1 shows the clinical and biochemical characteristics of the subjects at baseline. On average, they were middle-aged, centrally obese, normotensive, insulin resistant and dyslipidaemic (elevated plasma triglycerides and total apoB, and low HDL-cholesterol). There were no significant group differences in any of the variables in Table 1. Average daily energy and nutrient intake of the 35 obese subjects studied (mean ± SD) was: 10 045 ± 2406 kJ, 36 ± 6% energy from fat, 38 ± 8% energy from carbohydrates, 20 ± 3% energy from protein and 6 ± 6% energy from alcohol. Nutrient intake did not differ between patients randomized to weight loss or weight maintenance.

Dietary intervention Table 2 summarizes the dietary composition and nutrient intake of subjects during the study. Subjects in the weight reduction

4·9 ± 1·1

group significantly reduced their total energy and fat intake (percentage of energy) and significantly increased their carbohydrate intake (percentage of energy) during the active weight loss period. Nutrient intake did not change in the subjects in the weight maintenance group during the 16 week intervention. That the subjects on the weight loss diet consumed an isocaloric diet from week 14 to week 16 was supported by the fact that body weight did not vary by more than 1% during this period. There was also no change in reported physical activity levels during the study in either the weight loss or weight maintenance groups (data not shown). Table 3 shows the anthropometric characteristics, plasma lipid, lipoprotein, apolipoprotein and measures of insulin resistance

Table 2 Daily nutrient intakes in the weight loss and weight maintenance groups Weight loss (n = 20)

Total energy (kJ)

Weight maintenance (n = 15)

0

6 weeks

14–16 weeks

0

6 weeks

14–16 weeks

9782 ± 438

6143 ± 363*

6979 ± 312*

10 246 ± 634

9914 ± 705

9738 ± 397

Fat (% energy)

37 ± 1

26 ± 2*

35 ± 3

35 ± 2

37 ± 1

39 ± 2

Carbohydrate (% energy)

37 ± 2

48 ± 2*

39 ± 4

40 ± 2

39 ± 2

38 ± 2

Protein (% energy)

19 ± 1

21 ± 1

20 ± 1

20 ± 1

19 ± 1

20 ± 2

Alcohol (% energy)

10 ± 5

5±1

6±1

5±1

4±2

4±1

All values are mean ± SEM. Statistical significance for changes in the weight loss group (wk0 vs. wk6 or wk0 vs. week 14–16) were compared to the weight maintenance group using general linear models with adjustment for baseline covariates. Student’s paired t-test was used to evaluate significant changes within the weight maintenance group. *P < 0·001.

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Figure 1 Associations between the changes in plasma apolipoprotein (apo) C-III concentrations with (a) triglycerides, (b) apoB-48, (c) remnant-like particle (RLP)-cholesterol and (d) very low-density lipoprotein (VLDL)-apoB following weight loss.

before and after 16 weeks intervention in the weight loss and weight maintenance groups. As anticipated, the weight loss diet resulted in a significant reduction in body weight (–12%, P < 0·001), BMI (–13%, P < 0·001), waist circumference (–9%, P < 0·001), visceral (–23·5%, P < 0·001) and subcutaneous (–22·5%, P < 0·001) abdominal adipose tissue masses (ATM), and mean arterial pressure (–9%, P < 0·01) Compared with the weight maintenance group, weight loss also resulted in significant decreases (P < 0·05) in plasma cholesterol (–12%), triglycerides (–43%), non-HDL cholesterol (–42%), LDL cholesterol (–8%) and total apo B (–16%), lathosterol (–23%), as well as insulin (–41%) and HOMA score (–46%). However, there were no significant effects of weight loss on plasma concentrations of HDL-cholesterol, apoA-I, NEFAs and glucose.

Markers of TRL metabolism and plasma adipocytokines The pre- and post-intervention plasma apoC-III, apoB-48, VLDL-apoB, RLP-cholesterol and adipocytokine concentrations

are shown in Table 4. Compared with weight maintenance, weight loss had a significant effect of lowering plasma apoC-III (–33%, P = 0·004), apoB-48 (–37%, P = 0·02), VLDL-apoB (–43%, P = 0·003) and RLP-cholesterol (–48%, P < 0·001). These were accompanied by significant increases in both total adiponectin (+20%, P = 0·038) and HMW-adiponectin (+19%, P = 0·027), and a significant reduction in RBP-4 (–20%, P = 0·021) concentrations.

Correlational analysis in weight loss group As shown in Table 5, reduction in plasma apoC-III with weight loss was significantly and directly (P < 0·05 for all) associated with a reduction in apoB-48, VLDL-apoB, RLP-cholesterol and triglycerides (Fig. 1). The fall in apoC-III was also significantly associated with a reduction in insulin (r = 0·456, P < 0·05), with a borderline significant association with reduction in HOMA score (r = 0·423, P = 0·07). The reduction in plasma apoB-48 was significantly associated with the reduction in plasma VLDL-apoB, RLP-cholesterol and triglycerides (P < 0·05 for all). There was



Table 3 Anthropometric characteristics, plasma lipids and lipoproteins and measures of insulin resistance before and after weight loss and weight maintenance Weight loss (n = 20) 0 Weight (kg)

16 week

109 ± 2

BMI (kg m−2)

96 ± 3*

35 ± 1·0

Waist circumference (cm)


31 ± 0·7*

112 ± 2

103 ± 2*

0 105 ± 3 33 ± 0·7 113 ± 2

16 week 109 ± 2 35 ± 0·9 113 ± 2

Visceral ATM (kg)

7·1 ± 0·5

5·4 ± 0·4*

6·9 ± 0·4

6·7 ± 0·4

Subcutaneous ATM (kg)

8·4 ± 0·7

6·5 ± 0·4*

9·6 ± 0·7

9·9 ± 0·7

95·4 ± 2·8

86·4 ± 2·8†

96·6 ± 3·0

94·7 ± 3·1

Mean blood pressure (mmHg) −1

Cholesterol (mmol L )

6·0 ± 0·3

5·2 ± 0·2†

6·0 ± 0·2

6·0 ± 0·2

Triglyceride (mmol L−1)

3·5 ± 0·6

2·0 ± 0·2*

2·9 ± 0·6

2·7 ± 0·4

1·0 ± 0·04

1·1 ± 0·05

1·0 ± 0·04

1·0 ± 0·04

LDL-cholesterol (mmol L )

3·3 ± 0·2

3·0 ± 0·2‡

3·9 ± 0·2

3·9 ± 0·29

Non-HDL-cholesterol (mmol L−1)

4·9 ± 0·3

4·2 ± 0·2†

4·8 ± 0·2

4·9 ± 0·2

Total apoB-100 (g L−1)

1·2 ± 0·06

1·0 ± 0·06†

1·2 ± 0·06

1·2 ± 0·05

1·3 ± 0·05

1·3 ± 0·04

1·2 ± 0·04

1·2 ± 0·02

−1

HDL-cholesterol (mmol L ) −1

−1

Total apoA-I (g L ) −1

Lathosterol (μmol L ) Glucose (mmol L−1) −1

Insulin (mU L ) HOMA score Non-esterified fatty acids (mmol L−1)

17·4 ± 3·4

11·9 ± 2·4‡

14·5 ± 2·1

14·4 ± 2·0

5·7 ± 0·2

5·3 ± 0·1

5·4 ± 0·2

5·5 ± 0·3

14 ± 2

8 ± 1*

18 ± 3

16 ± 2

3·7 ± 0·5

2·0 ± 0·2†

4·6 ± 0·8

4·0 ± 0·6

0·96 ± 0·07

0·86 ± 0·08

0·81 ± 0·05

0·82 ± 0·05

All values are mean ± SEM. Effect of weight loss was tested using general linear modelling after adjusting for the weight maintenance group. *P < 0·001, †P < 0·01 and ‡P < 0·05. BMI, body mass index; ATM, adipose tissue mass; HDL, high-density lipoprotein; LDL, low-density lipoprotein; apo, apolipoprotein; HOMA, homeostasis model assessment.

Table 4 Plasma apoC-III, apoB-48, VLDL-apoB, RLP-cholesterol and adipocytokines before and after weight loss and weight maintenance Weight loss (n = 20) 0

0

16 week

171 ± 16

114 ± 9†

131 ± 16

139 ± 15

9·9 ± 1·4

6·3 ± 1·0‡

8·1 ± 1·5

7·0 ± 1·4

103 ± 18

59 ± 6†

86 ± 18

96 ± 14

36 ± 8

19 ± 3*

37 ± 9

35 ± 7

2·9 ± 0·3

3·4 ± 0·3‡

2·3 ± 0·2

2·3 ± 0·3

HMW adiponectin (mg L )

1·0 ± 0·2

1·2 ± 0·2‡

0·5 ± 0·1

0·5 ± 0·1

RBP-4 (mg L−1)

38 ± 2

30 ± 2c

36 ± 3

35 ± 3

ApoC-III (mg L−1) ApoB-48 (mg L−1) −1

VLDL-apoB (mg dL ) RLP-cholesterol (mg L−1) −1

Total adiponectin (mg L ) −1

16 week


All values are mean ± SEM. Statistical significance for changes in the weight loss group (wk0 vs. wk16) were compared with the weight maintenance group using general linear models with adjustment for baseline covariates. *P < 0·001, †P < 0·01 and ‡P < 0·05. Apo, apolipoprotein; VLDL, very low-density lipoprotein; RLP, remnant-like particle; HMW, high-molecular weight; RBP-4, retinol-binding protein-4.


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Table 5 Associations (Pearson correlation coefficients) between changes in markers of TRL metabolism and plasma adipocytokines in the weight loss group ApoC-III

ApoB-48

VLDL-apoB

ApoB-48

0·569*

VLDL-apoB

0·533†

0·733*

RLP-cholesterol

0·633*

0·745*

0·950*

Triglyceride

0·551†

0·558†

0·751*

RLP-cholesterol

Triglyceride

Total adiponectin

HMW adiponectin

0·508†

Total adiponectin

–0·127

–0·228

–0·493†

–0·369

–0·522†

HMW adiponectin

0·146

0·203

–0·002

0·047

–0·209

0·812*

RBP-4

0·380

0·022

0·046

0·207

0·004

0·362

0·250

P-value. * < 0·01, † < 0·05; TRL, triglyceride-rich lipoprotein; apo, apolipoprotein; VLDL, very low-density lipoprotein; RLP, remnant-like particle; HMW, high-molecular weight; RBP-4, retinol-binding protein-4.

also a significant association between the reduction in VLDL-apoB and plasma RLP-cholesterol and plasma triglycerides (P < 0·01 in both). Moreover, the increase in total adiponectin was significantly and directly associated with an increase in HMWadiponectin (P < 0·001) and inversely associated with the reduction in plasma VLDL-apoB and triglycerides (P < 0·05 in both). The rise in total adiponectin was also significantly and inversely associated with a reduction in visceral adipose tissue mass (r = –0·467, P < 0·05) By contrast, neither HMWadiponectin nor RBP-4 changes were associated with changes in visceral ATM, plasma insulin, apoC-III, apoB-48, VLDL-apoB, RLP-cholesterol or triglycerides. In partial correlational analysis adjusting for changes in visceral ATM, insulin and total adiponectin concentrations, the fall in plasma apoC-III concentration still remained significantly correlated with the reductions in plasma apoB-48 (r = 0·576, P = 0·025), VLDL-apoB (r = 0·579, P = 0·024), RLP-cholesterol (r = 0·691, P = 0·004) and triglycerides (r = 0·775, P = 0·001) concentrations. Inclusion of changes in RBP-4 or fat intake in the analyses did not alter the findings (data not shown). Likewise, the increase in adiponectin remained significantly associated with the reduction in VLDL-apoB concentration (r = –0·587, P = 0·02) after adjusting for changes in visceral ATM, insulin and apoC-III concentrations. However, the significant association between changes in adiponectin and triglycerides was lost after adjusting for changes in visceral ATM, insulin and apoC-III concentrations (P > 0·05). In multiple regression analysis including changes in visceral ATM, insulin and total adiponectin concentrations, the fall in plasma apoC-III concentration was an independent predictor of the reductions in plasma apoB-48 (β-coefficient = 0·509, P = 0·008), VLDL-apoB (β-coefficient = 0·568, P = 0·004), RLP-cholesterol (β-coefficient = 0·577, P = 0·001) and triglycerides (βcoefficient = 0·743, P = 0·001) concentrations.

Discussion We extend our previous studies and represent new data on the effects of weight loss on plasma apoC-III, apoB48 and RLP-cholesterol with measurement of HMW adiponectin in subjects with the metabolic syndrome. We demonstrate that weight loss with a low fat diet significantly lowers plasma triglycerides, apoB-48, apoC-III, VLDL-apoB and RLP-cholesterol. These effects were accompanied by increases in plasma total and HMW adiponectin, as well as a reduction in RBP-4 concentrations. The reduction in apoC-III concentration was a significant predictor of the fall in plasma triglycerides, apoB-48, VLDL-apoB and RLP-cholesterol independent of changes in insulin, adiponectin or visceral adipose tissue mass. Few studies have specifically examined the effect of weight loss on markers of TRL metabolism. Fernamdez et al. reported that in postmenopausal women, a 6 month weight loss with a low fat diet reduced plasma apoC-III [29]. However, this study was uncontrolled and no information provided on changes in other markers of TRL metabolism. We previously reported that in 12 overweight men, a weight loss with a low fat diet for 16 weeks resulted in a non-significant reduction in apoB-48 concentrations, but the study was underpowered to show a significant treatment effect on apoB-48 [30]. Other studies reporting that weight loss with dieting alters plasma adiponectin concentrations have not employed a weight maintenance comparator group [31,32], limiting the strength of inference. We have therefore extended the previous reports by examining all these markers of TRL metabolism in a controlled intervention trial. Disturbances in TRL metabolism relate to insulin resistance and accumulation of abdominal fat [33,34]. Therefore, weight loss with a low fat diet could correct the abnormalities in TRL metabolism, potentially by an improvement in insulin sensitivity and reduction in visceral fat. Improved insulin sensitivity increases blood flow



to skeletal muscle and stimulates LPL, with the compound effect of enhancing hydrolysis of endogenously and exogenously derived TRLs. Improved insulin sensitivity also decreases secretion of VLDL-apoB [23,24,35], and up-regulates expression and activity of LDL receptors that catabolize TRL remnants. Pioglitazone, an insulin sensitizing agent, has interestingly been reported to reduce plasma triglyceride levels by increasing VLDL-triglyceride clearance rate with no effect on hepatic secretion of VLDL apoB [36]. The lack of reduction in hepatic VLDL secretion in that study was unexpected, however. This could relate to the duration of intervention and to insufficient improvement in insulin sensitivity. It is noteworthy that in that study pioglitazone also resulted in decreases in the hepatic secretion and plasma concentration of apoC-III. In vitro studies also show that insulin resistance up-regulates the expression of the apoC-III gene [37]. Improvement in insulin sensitivity with weight loss may therefore restore the sensitivity to the normal insulin-mediated suppression of apoC-III gene expression. Consistent with this, we found that the fall in apoC-III was associated with a reduction in insulin. However, the significant association between these changes was not strong suggesting that other factors (e.g. changes in dietary intake and composition), in addition to improved insulin sensitivity, could also account for the fall in plasma apoC-III [38]. Reduction in adipose mass also decreases the flux of free fatty acids in the portal vein, thereby suppressing gluconeogenesis and triglyceride synthesis and the hepatic secretion of VLDL secretion [34,39]. Although plasma free fatty acid levels did not alter in our study, these may not reflect the corresponding portal or hepatic concentrations that regulate apoB metabolism. Taken together, these effects potentially decrease the plasma concentration of remnant lipoproteins containing apoB-100 and decrease competition for hepatic uptake between chylomicron and VLDL remnants. This concept is consistent with our observation that the concentrations of plasma triglycerides, VLDL-apoB, apoB-48, apoC-III and RLP-cholesterol decreased with weight loss. Obesity and expansion in adipose tissue mass is associated with low plasma adiponectin and elevated RBP-4 concentrations in obesity [40,41]. One might expect that changes in these adipocytokines would be reversed with fat loss. Consistent with this, we found that weight loss resulted in an increase in plasma adiponectin (total and HMW) and a reduction in RBP-4 concentrations, in agreement with other observations [32,42]. Furthermore, recent data also suggest that the production of adiponectin and RBP-4 is stimulated and suppressed, respectively, by activation of peroxisome proliferator-activated receptor (PPAR)-γ [17,21]. Since this PPAR-γ pathway is found to be activated by negative energy balance and weight loss [43], it is conceivable that this mechanism could partly account for the effects of weight loss on plasma adiponectin and RBP-4 concentrations. It is also noteworthy that the activation of

PPAR-gamma by a thiazolidinedione usually results in weight gain but the effect is probably owing to increased fluid intention with a redistribution of fat from visceral to subcutaneous regions. Observational data show a positive association between plasma apoC-III and triglycerides, apoB-48, RLP-cholesterol and non-HDL-cholesterol. In this study, we demonstrate that after weight loss changes in plasma apoC-III were strongly associated with changes in these markers of TRL metabolism in subjects with the metabolic syndrome. More importantly, we found that these associations were independent of changes in visceral adipose tissue mass, insulin and adipocytokines. The findings reinforce the direct role of apoC-III in modulating TRL metabolism that a decrease in apoC-III concentration would enhance the lipolysis of VLDL-triglyceride by LPL and the hepatic uptake of TRL remnants by LDL receptors [11]. Using VLDL-apoB kinetic data from the same subjects [24], we also found that the fall in plasma apoC-III concentration was significantly and inversely correlated with the increase in VLDL-apoB fractional catabolic rate (r = –0·468, P < 0·05), but not the reduction in VLDL-apoB secretion rate (r = 0·173, P > 0·05). That the increase in total plasma adiponectin was strongly associated with a reduction in plasma triglycerides is also consistent with the notion that adiponectin may decrease the accumulation of triglycerides in skeletal muscle by enhancing fatty acid oxidation through activation of AMP kinase [16]. However, such associations between adiponectin and triglycerides was not independent of the reduction in visceral ATM, insulin and apoC-III, suggesting that adiponectin may not play a direct role in regulating triglyceride metabolism following weight loss. Nevertheless, we found that in the weight loss group the significant and inverse association between the changes in plasma adiponectin and VLDL-apoB concentrations were independent of changes in visceral ATM, insulin and apoC-III. This agrees with our previous cross-sectional analyses showing that adiponectin was an independent predictor of plasma VLDL-apoB concentration [20]. We did not find a significant association between changes in HMW-adiponectin and triglycerides. The precise reasons for this remain unclear. Bluher et al. has also reported that HMW-adiponectin is not better than total adiponectin in assessing measures of insulin sensitivity and lipid profile or their response to exercise [44]. Despite the significant reduction in plasma RBP-4 concentration with weight loss, there were no significant associations with changes in markers of TRL metabolism. This is consistent with a recent uncontrolled study of morbidly obese individuals following bariatric surgery [45]. The role of RPB-4 in regulating lipoprotein metabolism remains unclear and requires further investigation. There are limitations to our study. We did not specifically measure plasma apoC-III in the VLDL and other TRLs. However, we anticipate that the majority of plasma circulating apoC-III is


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bound in the TRL fraction and therefore this limitation would not confound our results. Our study was carried out in the postabsorptive state. However, both fasting apoB-48 and RLP-C have been shown to predict postprandial response [7,46]. Our data do not support the superiority of HMW over total adiponectin in assessing metabolic variables in response to weight loss. Further studies, using additional methods to measure HMW adiponectin, are needed to confirm these findings [44]. Only obese Caucasian men were studied, and it is possible that the lipoprotein effects of weight loss might have been different in women. Plasma apoC-III concentrations are typically elevated in MetS [2,6] and are strongly associated with hypertriglyceridaemia and progression of CVD [12,47]. Our study provides new insight as to how changes in apoC-III metabolism with weight loss could impact directly on TRL metabolism. From a clinical perspective, further studies should examine the additive effect of weight loss, together with other therapeutic agents such as PPAR-α agonists that are known to reduce plasma apoC-III concentrations. Also, the kinetic bases for the reduction in apoC-III with weight loss also warrants investigation.

Acknowledgements This work was supported by the National Heart Foundation of Australia and National Health and Medical Research Foundation (NHMRC). D.C.C. is an NHMRC Career Development Fellow. P.H.R.B. is an NHMRC Senior Research Fellow and is supported in part by National Institutes of Health Grant NIBIB P41-EB-001975. We thank the study participants for their time and commitment. We also thank the nursing and laboratory staff of the Metabolic Research Centre, School of Medicine and Pharmacology (Royal Perth Hospital, University of Western Australia) for providing expert assistance. Address Metabolic Research Centre, School of Medicine and Pharmacology, University of Western Australia, Perth, WA, Australia (D. C. Chan, G. F. Watts, T. W. K. Ng, P. H. R. Barrett), Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan (S. Yamashita). Correspondence to: Gerald F. Watts, School of Medicine and Pharmacology, University of Western Australia, Royal Perth Hospital, Perth, WA, Australia. Tel.: 61 89224 0245; fax: 61 89224 0246; e-mail: [email protected] Received 5 May 2008; Accepted 31 July 2008

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