Effets d'une modeste perte de poids sur les sous-fractions des lipoprotéines à jeun et en post-prandial chez les patients diabétiques de type II

Thesis

Reference

Effets d'une modeste perte de poids sur les sous-fractions des lipoprotéines à jeun et en post-prandial chez les patients diabétiques

de type II

MAKOUNDOU, Vincent

Abstract

Nous avons évalué l'efficacité d'une perte pondérale modeste (1.5 ± 0.3 Kg) combinée à une amélioration simultanée du contrôle glycémique sur les sous-fractions des lipoprotéines plasmatiques à jeun et post-prandiales chez 9 patients présentant un diabète de type 2 bien contrôlé (HbA1c=7.3 ± 0.1%) et un excès pondéral (BMI=28 ± 1.7 Kg/m2). Les patients ont suivi une diète hypocalorique équilibrée (1561 ± 9 Kcal/j) pendant les 10 jours d'observation, avec un contenu en graisse réduite de 96 ± 12 g/j à 62 ± 4 g/j (P

MAKOUNDOU, Vincent. Effets d'une modeste perte de poids sur les sous-fractions des lipoprotéines à jeun et en post-prandial chez les patients diabétiques de type II. Thèse de doctorat : Univ. Genève, 2005, no. Méd. 10459

URN : urn:nbn:ch:unige-3848

DOI : 10.13097/archive-ouverte/unige:384

Available at:

http://archive-ouverte.unige.ch/unige:384

Disclaimer: layout of this document may differ from the published version.

1 / 1

Service d'enseignement thérapeutique pour maladies chroniques

Thèse préparée sous la direction du Professeur Alain GOLAY

« Effets d'une modeste perte de poids

Sur les sous-fractions des lipoprotéines à jeun Et en post-prandial chez les patients diabétiques

Type II »

Thèse

présentée à la Faculté de Médécine de l'Université de Genève

pour obtenir le grade de Docteur en médecine

par

Vincent MAKOUNDOU

de Genève Thèse N. Méd

Genève, 2005

Table of contents :

I-Introduction (version française)………1-10 II-Introduction...11-18 III-Materials and methods...19-24 IIIa-Subjects...19-20 IIIb- Protocol...20-22 IIIc-Results...22-24 IV-Effects of weight loss on lipoprotein sub-fractions...25-28 V-Discussion...29-32 VI-Appendix( tables and graphics)...33-34 VII-References...35-45

Nous avons évalué l’efficacité d’une perte pondérale modeste (1.5 ± 0.3 Kg) combinée à une amélioration simultanée du contrôle glycémique sur les sous-fractions des lipoprotéines plasmatiques à jeun et post-prandiales chez 9 patients présentant un diabète de type 2 bien contrôlé (HbA1c=7.3 ± 0.1 %) et un excès pondéral (BMI=28 ± 1.7 Kg/m2). Les patients ont suivi une diète hypocalorique équilibrée (1561 ± 9 Kcal/j) pendant les 10 jours d’observation, avec un contenu en graisse réduite de 96 ± 12 g/j à 62 ± 4 g/j (P<0.05). Les taux plasmatiques des lipides ainsi que des sous-fractions des lipoprotéines ont été mesurés à jeun (F) ainsi que 4 heures après deux repas standards (pp1 et pp2, respectivement) au début et à la fin de l’étude. Cette perte pondérale modeste a permis la diminution significative de deux facteurs de risque cardiovasculaires reconnus : l’hyperglycémie et l’hyperlipémie post- prandiales (P<0.05, chacune). La clearance des triglycérides post-prandiaux a été augmentée de 23% à la fin de l’étude (P<0.02) parallèlement à la diminution de l’enrichissement des VLDL-2 en triglycérides (P<0.05). Finalement, les taux de LDL-cholestérol à jeun et post- prandiaux ont diminué (P<0.05) avec amélioration des rapports du LDL-2/LDL-3 post- prandiaux (P<0.05).

Conclusions : Même une perte pondérale modeste chez des patients diabétiques de type 2 bien contrôlés permet d’observer une amélioration significative de 2 facteurs de risque cardiovasculaire, les triglycérides post-prandiaux et le rapport LDL-2/LDL-3 indépendamment de l’amélioration des profiles glycémiques.

I-Introduction

L’incidence de la maladie athérosclérotique est très marquée chez les patients diabétiques de type II^1-4. L’excès de maladies cardiovasculaires qui en découle, est plus important encore en présence d’une obésité⁵. Le concept fondamental du profil d’athérogénéité, responsable de la dyslipidémie et de la maladie athérosclérotique établi à ce jour, met en évidence : des VLDL et des LDL élevées associées à une baisse des HDL⁶. Si ce profil reste considéré comme prédictive des maladies coronariennes, il ne se corrèle pas avec toutes pathologies vasculaires athéromateuses chez différents individus présentant un profil dyslipidémique similaire⁷. Des études montrent l’existence des altérations spécifiquement individuels liées aux variations des sous-fractions des lipoprotéines^8-11.

Les sous-fractions des VLDL révèlent leur importance par exemple du fait que la variation des triglycérides plasmatiques se traduit principalement par des changements adaptés de la fonction des VLDL1¹².

Dans tous les cas, un niveau élevé des VLDL1 reste un point central qui accompagne le syndrome métabolique¹³.

Concernant les LDL, on sait que des valeurs de triglycérides supérieures à 1.5mmol/l entraînent une augmentation des LDL2 qui, finalement, se transforment en LDL3. Il est également établi que la délipidation des VLDL très riches en

triglycérides se transforment préférentiellement en LDL3¹³. Ces sous-fractions LDL3 étant plus denses que les LDL2 et LDL1, se caractérisent évidemment par une athérogénéité plus importante.

Schéma hypothétique des délipidations VLDL et LDL

^LPL

^TG

LPL LPL LPL HL

TG

^{LPL LPL/HL}

^TG

LPL LPL/HL

TG : triglycérides;

LPL : lipoprotéine lipase;

HL : lipase hépatique;

CETP : Protéine de transfert du cholestérol ester.

FOIE TG

TG

1 2

IDL2 LDL1

VLDL2 IDL1

LDLII ^Smaller

LDL

VLDL1

Remnants LDLIII Smaller

LDL

Larger

VLDL 1 Remnants ^{LDL IV Smaller} _LDL

Nous nous sommes intéressés alors à d’autres facteurs de risques dont l’un d’entre eux est la modification des lipoprotéines post-prandiales^14,15.

Les maladies cardiovasculaires ne peuvent donc pas être expliquées uniquement et complètement par les facteurs de risques cardiovasculaires conventionnels comme le tabac, l’hypertension artérielle, la sédentarité et l’hyperlipémie.

Krent et al¹⁶ affirment dans leur étude que les patients diabétiques type II ont un risque élevé d’athérosclérose et devraient bénéficier, par conséquent, de mesures préventives.

Dans le cadre de l’obésité, selon le degré de développement des adipocytes, la détérioration de l’action de l’insuline induit une accélération de l’hydrolyse des triglycérides intracellulaires avec une libération des acides gras libres. Ceux-ci constituent un substrat important pour la synthèse et l’altération à partir du foie des lipoprotéines plasmatiques

Smaller LDL

Physiologie des lipoprotéines

HDL3

LCAT

CEPT

ApoA1

Nascent « preBéta-HDL »

Schéma du métabolisme des HDL

FC

IDL

L’augmentation des VLDL plasmatiques sont associées à l’hyperlipémie post-prandiale aggravée par la baisse de l’activité des lipoprotéines lipases. Cette montée constitue un facteur indépendant lié aux maladies coronariennes. Ces particules résiduelles de VLDL contiennent plus de cholestérol pour les macrophages que les particules de LDL-C.

Plusieurs études sont rapportées sur ce sujet, en dépit de la longue étude de Zilversmit sur l’hypothèse concernant l’athérogénéité potentielle des lipoprotéines post-prandiales¹⁴.

CE

CE CE

FC/PL ABCA-1

FC FC

LCAT

Maggi et al¹⁷ ont démontré récemment que les lipoprotéines remnants (RLP) en post-prandiale contribuent d’une manière significative au dysfonctionnement endothéliale.

Koba et al¹⁸ en étudiant la tolérance aux graisses en post- prandiale chez 32 patients avec infarctus aigus du myocarde ont conclu que l’augmentation post-prandiale des fractions larges de VLDL et les particules remnants contribuent à la formation des LDL de petite densité chez les patients avec maladies coronariennes.

Egalement, Fukushima et al¹⁹, dans leur étude sur la valeur pronostique des particules RLP sur les maladies coronariennes chez des patients diabétiques, affirment que le niveau élevé des particules RLP cholestérol constitue un facteur de risque cardiovasculaire indépendant et prédictif chez des patients diabétiques type II souffrant de maladies coronariennes.

Parallèlement, d’autres études ont montré récemment que les maladies cardiovasculaires se corrèlent étroitement, d’une manière significative, au degré de l’hyperlipémie post- prandiale^20,21.

Rivellese et al²² se sont intéressés aux anomalies de la lipémie post-prandiale exogène ou endogène chez des diabétiques de type II ayant un bon contrôle glycémique et des taux de triglycérides normaux. Ils ont mis en évidence, même dans ce contexte métabolique, une augmentation significative des

VLDL post-prandiales quelle que soit l’origine des lipides par apport extérieur ou par synthèse endogène.

L’augmentation du niveau des triglycérides à partir d’un état de jeun et 4 heures après ingestion de lipides peut permettre de distinguer les sujets normo-insulinémiques à ceux avec hyperinsulinémie.

Par ailleurs, le rôle des particules riches en triglycérides qui sont sécrétées par le foie et l’intestin reste une question ouverte. Néanmoins, il y a des études qui soutiennent que ces particules (d’origine endogène ou exogène) riches en triglycérides contribuent en partie à la résistance à l’insuline et à l’hyperinsulinémie aussi bien chez des patients ayant des degrés différents de intolérance au glucose^23-24, que chez les sujets saints^25-28.

Le phénomène d’élévation des triglycérides associé à une élévation réelle du taux d’insuline totale, à une sensibilité réduite de l’insuline avec une assimilation réduite du glucose, se traduisant par une augmentation du rapport plasmatique insuline/glucose, et à une masse élevée du tissu adipeux intra- abdominal en dépit d’un index de masse corporelle normal, est rapporté dans le cadre du syndrome métabolique²⁹.

McGarry²⁷ a affirmé que quelle que soit son origine, l’hyperinsulinémie peut influencer négativement le tissu adipeux, en augmentant son accumulation intra-abdominale, avec un ralentissement de la ré-estérification des acides gras

libres. Ces perturbations dues au niveau du métabolisme des acides gras libres^14,20 et des lipoprotéines peuvent induire la résistance à l’insuline et ainsi contribuer au développement du syndrome métabolique³⁰.

Couillard et al³¹, dans leur étude sur les apolipoprotéines en post-prandiale chez des hommes avec obésité abdominale associée à un ralentissement de la lipolyse, rapportent que l’augmentation des triglycérides en post-prandiale est le résultat de l’élévation des lipoprotéines contenant les apoB-48 et B-100 (soit les VLDL, LDL et les IDL). Ils concluent, dans cette même étude, que la baisse de la lipolyse due à l’obésité abdominale peut contribuer à la péjoration de l’hyperlipémie post-prandiale.

Reinehr et al³² en étudiant les changements de facteurs de risque d’athérogénéité selon la perte de poids, affirment que la perte de poids est associée à une amélioration de la sensibilité à l’insuline et à celle du profil de l’athérogénéité.

Plusieurs études mettent en évidence une amélioration de la lipémie^28,29,33 : après une perte de poids chez des sujets non- diabétiques^34-37, chez ceux avec une intolérance au glucose³⁸ et aussi chez des diabétiques^39,40.

Dans des études précédentes⁴¹, le profil des lipoprotéines chez des diabétiques de type II a été déterminé chez des sujets avec un mauvais contrôle glycémique. Le bénéfice d’un régime équilibré aussi bien à jeun qu’en post-prandial sur le contrôle

glycémique, le cholestérol total, les triglycérides et le HDL- cholestérol constitue un fait bien établi. Néanmoins, l’augmentation des triglycérides et des sous-fractions VLDL en post-prandiale n’était pas influencée par le contrôle métabolique alors que le poids des patients restait stable.

Maksvytis et al⁴² ont trouvé que les femmes obèses d’âge moyen présentent un profil lipidique caractérisé par une élévation des apoliprotéines B et du rapport apoB sur apoA-I avec ou sans altérations du cholestérol total et des triglycérides.

Dans le cadre des mesures diététiques appropriées, la perte de poids qui survient permet la normalisation de la résistance à l’insuline chez des sujets diabétiques obèses de type II^43-45. Jeff et al⁴⁶, en comparant les effets de deux régimes hypocaloriques sur la lipémie post-prandiale (un faible en glucide et un autre faible en graisse), ont mis en évidence une baisse significative de la lipémie post-prandiale dans les deux groupes sujets.

Ainsi, nous avons donc décidé d’étudier l’efficacité d’une perte de poids modeste associée à un bon contrôle glycémique, sur les sous-fractions lipoprotéines (VLDL1, VLDL2, VLDL3, LDL1, LDL2, LDL3) aussi bien à jeun qu’en post-prandial chez des sujets diabétiques de type II bien contrôlés ayant observé un régime équilibré légèrement hypocalorique pendant 2 semaines.

II-Introduction

Non-insulin dependent diabetes mellitus (NIDDM) patients are subject to a markedly increased incidence of atherosclerotic diseases^1-4. The excess of cardiovascular diseases is more important when diabetes is coupled with obesity.

The atherogenicity profile of fundamental concept responsible for dyslipidemia and leading to atherosclerotic diseases is characterized by elevated very low-density lipoprotein (VLDL) and low-density lipoproteins levels (LDL), reduced high-density lipoprotein (HDL) levels⁵. However, although this view is predictive of coronary artery disease (CAD)⁶ the extent of various atherosclerotic diseases varies greatly between individuals with similar lipid profiles⁷. More recent findings indicate that specific alterations in individual lipoprotein subclasses may account for these variations^8-11. The significance of VLDL subclasses was revealed by the observation that variation in plasma triglyceride levels is principally a function of changing VLDL1 levels¹². In any case, elevated VLDL1 remain one of the key point in metabolic syndrome¹³.

Regarding LDL subclasses, it is known that a plasma triglyceride levels of 1.5mmol/l is the threshold value, above which either LDL2 is increasingly converted to LDL3 in the circulation or LDL3 rather than LDL2 is the preferred product of

VLDL delipidation¹³.That LDL3 subclasses being more dense than LDL2 and LDL1 has obviously more atherogenic impact.

VLDL and LDL production hypothetical metabolic scheme

^LPL

^TG

LPL LPL LPL HL

TG

^{LPL LPL/HL}

^TG

LPL LPL/HL

TG : triglycerides, LPL : lipoprotein lipase, HL : hepatic lipase,

CETP : cholesterol ester transfer protein, VLDL : very low density lipoprotein,

IDL : intermediate density lipoprotein, LDL : low density lipoprotein.

LIVER TG

TG

1 2

IDL2 LDL1

VLDL2 IDL1

LDLII ^Smaller

LDL

VLDL1

Remnants LDLIII Smaller

LDL

Larger

VLDL 1 ^{Remnants LDL IV Smaller} _LDL

This has led to consideration to other factors, one of which is lipoprotein modifications in the post-prandial phase^14,15.The excess of cardiovascular diseases cannot be fully explained by conventional risk factors.

According to Krentz¹⁶, patients with type 2 diabetes are at high risk from complications associated with atherosclerosis and should therefore receive preventive intervention. At the level of adipocyte, impaired insulin action leads to increased rates of intracellular hydrolysis of triglycerides with the release of NEFA. The rise in NEFA provides substrate for the liver that, in the presence of impaired insulin action and relative insulin deficiency, is associated with complex alterations in plasma lipids. Plasma VLDL levels are raised and increased VLDL levels are associated with post-prandial hyperlipidaemia that is compounded by impaired LPL activity. The latter may be independently associated with CAD. Remnant particles can deliver more cholesterol to macrophages than LDL-C particles.

The latter have been largely overlooked despite the Zilversmit’s long-standing hypothesis concerning the atherogenic potential of post-prandial lipoproteins¹⁴.

Physiology of lipoproteins

HDL3 CEPT

ApoA1

Nascent « preBéta-HDL »

Scheme of HDL metabolism

FC

IDL

It is known that Maggi et al¹⁷ C-RLPS contribute significantly to the endothelial dysfunction occurring during the post-prandial lipemia.

Koba et al¹⁸ in this study, performed an oral fat tolerance test in 32 patients with acute myocardial infarction and the results suggest that post-prandial increase of large VLDL fractions and RLPs contribute to the formation of small dense LDL in CHD.

Besides, increased levels of RLP cholesterol are a significant and independent risk factor of CAD and predict future coronary events in patients with CAD and type 2 diabetes¹⁹. Several

CE

CE CE

FC/PL ABCA-1

FC FC

LCAT

reports have recently concluded that coronary heart disease is significantly related to the degree of post-prandial lipemia^20,21. Rivellese et al²² studied post-prandial lipemia anomaly after exogenous load or endogenous lipid synthesis for balanced type II diabetic subject with normal triglyceride level. They demonstrated that, even in this metabolic case, a significant post-prandial VLDL increase indiscriminately for both exogenous or endogenous synthesis.

But the respective role of triglyceride-rich particles secreted by the liver or the intestine is still an open question. Post-prandial accumulation of triglyceride (TG)-rich lipoproteins of endogenous and exogenous origins is significantly related to the degree of insulin resistance and/or hyperinsulinemia in patients with various degrees of glucose tolerance^23-24 as well as in healthy volunteers^25-28.

The increase in triglyceride level from the fasting level at baseline, 4 hours after the lipid load, could be used to discriminate between people with normo-insulinaemia and those with hyperinsulinemia.

A phenomenon of triglyceride high response, higher total insulin and true insulin levels, a reduced sensitivity of insulin in terms of reduced glucose utilization, an elevated plasma insulin/glucose ratio, and a higher intra-abdominal adipose tissue mass despite a normal body mass index have been described with the metabolic syndrome²⁹.

McGarry²⁷ has proposed that whatever the underlying cause, hyperinsulinaemia can lead to abnormalities in adipose tissue, leading to intra-abdominal fat deposition, with an eventual decrease in re-esterification of FFA. These abnormalities in FFA and lipoprotein metabolism^14,20 could act as promoters for insulin resistance, thus contributing to the metabolic syndrome³⁰.

In their study, Couillard et al³¹ regarding post-prandial apolipoproteins to abdominal obese men with decreased lipolysis, they reported that triglyceride increase is due to increase of apoB-48 and B-100 (or VLDL, LDL and IDL) containing lipoproteins. They also concluded that the worsening post-prandial lipemia is a result of abdominal obesity related to decreased lipolysis.

Reinehr et al³² demonstrated that weight loss is followed by improvement of the insulin sensitivity and the atherogeneicity profile.

Numerous studies have demonstrated improvements in lipid levels and insulin resistance^28,29,33 with weight loss in non- diabetic^34-37, glucose-intolerant³⁸ and diabetic subjects^39-40. In previous studies, we have characterized the lipoprotein profile of poorly controlled NIDDM patients and then shown the fasting and post-prandial benefits of short-term dietary improvement in glycemic control on total cholesterol, triglycerides, HDL-cholesterol; nevertheless, the incremental

post-prandial rises in triglycerides and VLDL sub-fractions were not influenced by improved metabolic control whilst patients weight remained stable⁴¹.

It is known that middle-aged obese women lipid profile is characterised by increase of Apo-B containing lipoproteins and increase of ApoB/ApoA1 ratio with or without total cholesterol and triglycerides alterations⁴².

Weight loss by means of dietary treatment allows normalization of insulin sensitivity in obese type 2 diabetic patients^43-45.

Two hypoenergetic diets (very-low carbohydrate and low-fat diets) versus placebo showed a significant reduction of post- prandial lipemia in both regims⁴⁶.

Thus, we consequently aimed at assessing the efficacy of a modest weight loss and simultaneous rapid improvement in glycemic control on fasting and post-prandial lipoprotein sub- fractions (VLDL1, VLDL2, VLDL3, LDL1, LDL2, LDL3) in a well controlled type 2 diabetic patient group who followed a non drastic hypocaloric balanced diet for two weeks.

III-Materials and Methods IIIa-Subjects

Nine Type II Diabetes Mellitus (DM) patients (3 females, 6 males), aged 51 ± 1.9 years old^43,63, according to the WHO criteria were recruited among those hospitalised for two weeks at the Service of Therapeutic Education for Chronic Diseases of Geneva University Hospital (CH) over a 6-month period.

Inclusion criteria were the following : moderately well controlled type II DM (HbA1c < 8%), slight overweight (defined as Body Mass Index between 25 and 30 kg/m²) and normal fasting plasma total cholesterol and triglycerides concentrations (< 6.5 mmol/L and < 2.5 mmol/L, respectively).

Exclusion criteria included any of the causes for secondary dyslipidemia other than uncontrolled DM and being on a lipid lowering drug for the last 6 months.

Patients were in general good health and underwent an elective hospital admission aiming at performing a thorough medical check-up including a screening for diabetic micro and macro-angiopathic complications and improving their daily metabolic control through dietary counselling and guidelines.

Extensive review of their medical records disclosed diabetes duration of 9.9 ± 4.9 years and the absence of micro/macro- angiopathic complications. Two out of nine patients presented

with high blood pressure which was well controlled with angiotensin-converting enzyme inhibitors.

Patients signed a written informed consent after thorough explanation of the aims and procedures of the study. The study was submitted and approved by the local ethics committee.

IIIb-Protocol

On day 1 of the first week of hospital admission, all patients had a one-hour interview with a registered dietician who carefully recorded their dietary habits prior to the present hospitalisation and prescribed a test diet. Its implementation was to be introduced on day 2.

Three blood samples (30 ml each) were drawn (fasting -F-, 4 hours after standard breakfast -pp1-and 4 hours after standard lunch -pp2-) for each patient on day 2 of the first week of hospitalisation and repeated ten days later, on day 5 of the second week of hospitalisation. Simultaneously, serial capillary triglyceride and blood sugar levels were measured for every patient each two hours during day 2 and then ten days later.

On blood-sampling days all patients ingested identical test meals which consisted of a moderately hypo-caloric equilibrated multi-fractioned (3 meals and 3 snacks) diet averaging 1561 ± 39 kcal/day. Their assigned caloric intake was calculated as being 25 kcal/kg of ideal body weight. The quantitative caloric restriction averaging 606 ± 168 kcal/day

was imposed on our patient for the entire study period (table 1).

The percent distribution of nutrients was : 50.3 % carbohydrates, 35.5 % fat and 16.9 % protein in accordance to the dietary guidelines proned by the American Diabetes Association. The distribution of the daily caloric intake was breakfast 20 %, lunch 35 %, dinner 30 % and snacks 15 % (-5% each-). The prescribed diet contained less than 10 % as saccharose.

Quantitative determination of triglyceride concentrations in capillary blood was performed using a Reflotron device from Boehringer Manheim, based on a previously described test principle⁴⁷, whose range of measurement is (0.80-6.86 mmol/l).

Quality control determinations were performed using the control serum Precinorm ^R U for Reflotron ^R (4 x 2 ml, Cat No.

745154). Capillary and venous triglyceride samples disclosed an excellent correlation (r=0.896; P<0.0001).

Subcutaneous fat deposition and abdominal adiposity were determined by skinfold thickness measurements and the waist- to-hip ratio (WHR) respectively on day 2 of the first week of hospitalisation and repeated ten days later, on day 5 of the second week of hospitalisation.

Plasma lipid and apolipoprotein levels were measured as described previously⁴⁸. Sub-fractions of very low density (VLDL) and low density (LDL) lipoproteins were obtained by cumulative flotation ultra-centrifugation^39,40.

Results are expressed as mean ± SD. Statistical comparisons before and after weight loss were made using the ANOVA of repeated measures. Pearson´s correlation test was employed to analyse correlation between variables. Multiple linear regression analyses were performed using the observed lipid changes as dependent variables and weight loss and blood glucose changes as independent variables. P<0.05 was considered statistically significant.

IIIc-Results

Physical characteristics of the study population at entry are depicted in Table 2. Biochemical lipid parameters prior to and after weight loss are reflected in Table 3.

Under the above-mentioned changes, our nine patients disclosed a significant weight loss averaging 1.5 ± 0.3 kg (P<0.001). Skinfold thickness measurement prior to and after weight loss allowed an estimation of the subcutaneous fat loss which averaged 1.4 ± 0.3 kg. Therefore, according to our estimates, 97 % of the total weight loss accomplished during the study period corresponded to subcutaneous adipose tissue.

Fasting (08h00) glycaemias upon admission averaged 7.3 ± 2.1 mmol/l and by the end of the study they had descended to 6.7 ± 2.6 mmol/l (P=NS). Post-prandial glycaemias averaged 10.2 ± 3.4 mmol/l (10h00), 9.6 ± 4.5 mmol/l (12h00), 8.2 ± 4.5 mmol/l (14h00), 7.0 ± 3.0 mmol/l (16h00) and 5.9 ± 1.5 mmol/l

(18h00) mmol/l at entry and were 7.3 ± 2.1 (10h00), 5.5 ± 1.2 mmol/l (12h00), 5.5 ± 1.8 mmol/l (14h00), 6.8 ± 1.7 mmol/l (16h00) and 6.5 ± 1.9 mmol/l (18h00) mmol/l by the end of the study. Significant improvements were noted at the 10h00, 12h00 and 14h00 time-points (Fig. 1a).

As depicted in Table 3, fasting total cholesterol was significantly lowered from 6.1 ± 0.7 to 5.3 ± 0.6 mmol/l (P<0.005) while the pp2 total cholesterol was significantly reduced from 6.2 ± 0.6 to 5.3 ± 0.5 mmol/l (P<0.01) throughout the study period. Neither Apo A-1 nor Apo B disclosed significant changes before and after weight loss.

Fasting plasma triglyceride concentrations were significantly lowered from 2.1 ± 0.7 to 1.5 ± 0.4 mmol/l (P<0.005) while pp2 triglyceride values were significantly lowered from 2.6 ± 1.0 to 2.1 ± 0.6 mmol/l (P<0.01). Neither fasting HDL-cholesterol concentrations nor the total cholesterol/HDL-cholesterol or the LDL-cholesterol/HDL-cholesterol ratios showed significant changes during the study period. Upon admission, post- prandial capillary triglyceride values showed a significant (P<0.001) rise from baseline (fasting value) at four over five post-prandial sampling periods (12, 14, 16 and 18 hours). After weight loss, post-prandial capillary triglycerides showed a significant (P<0.05) rise from baseline only at one post-prandial sampling period (14 hours) (Figure 1b). Moreover, the area under the curve for capillary triglyceride concentrations (AUC-

TG) was significantly reduced by 23 ± 0.2 % (P<0.02) by the end of the study period. Interestingly, a significant correlation (r=0.893; P<0.002) was found between weight loss and delta AUC-TG (AUC-TG upon admission – AUC-TG at the end of the study).

Multiple linear regression analyses disclosed weight loss as an independent variable accounting for the ability to predict delta AUC-TG changes (dependent variable) (P<0.05). Blood glucose improvement (BG upon admission – BG at the end of the study at each time-point) which was also entered as a parameter did not appear a variable being able to predict AUC- TG changes (P=0.420).

IV-Effects of weight loss on lipoprotein sub-fractions

Figure 2 depicts both fasting and post-prandial modifications of the different lipoprotein sub-fractions prior to and after weight loss. As indicated in the graphics, the main involved sub- fractions were VLDL-1, VLDL-2, LDL-3 and the LDL-2/LDL-3 mass ratios.

After weight loss, VLDL-1 concentrations were significantly decreased both fasting (62.9 ± 37.6 mg/dl vs 23.7 ± 16.1 mg/dl; P<0.002) and at pp1 (76.2 ± 54.5 mg/dl vs 36.2 ± 17.2 mg/dl; P<0.05). At pp2 VLDL-1 concentrations decreased without reaching statistical significance (95.9 ± 63.4 mg/dl vs 68.2 ± 22.7 mg/dl; P=NS). Post-prandial (pp1 and pp2) VLDL-1

concentrations were significantly higher than fasting levels (23.7 ± 16.05 mg/dl - F- vs 36.2 ± 17.2 mg/dl–pp1- vs 68.2 ± 22.7 mg/dl–pp2-; P<0.01) after weight loss while the post- prandial rise (pp1 and pp2) in VLDL-1 concentrations upon admission did not reach statistical significance.

A significant correlation was found between delta ([pp2] – [F]) VLDL1-cholesterol and delta ([pp2] – [F]) triglycerides both prior to (r= 0.897 ; P<0.02) and after weight loss (r= -0.928;

P<0.01).

Moreover, multiple linear regression analyses disclosed delta fasting TG (before-after weight loss) as an independent variable accounting for the ability to predict delta fasting VLDL- 1 (before-after weight loss) changes (dependent variable) (P<0.05).

After weight loss, VLDL-2 concentrations were significantly decreased both at F (33.3 ± 23.2 mg/dl vs 54.7 ± 30.5 mg/dl;

P<0.05), at pp1 (36.6 ± 11.6 mg/dl vs 63.7 ± 36.8 mg/dl;

P<0.05) and at pp2 (44.3 ± 21.1 mg/dl vs 72.4 ± 44.3 mg/dl;

P<0.05). Interestingly, a significant correlation between delta ([pp2] – [F]) VLDL-2 cholesterol and delta ([pp2] – [F]) triglyceride concentrations found at entry (r=0.933: P<0.01) was no longer apparent after weight loss (r=-0.173: P=NS).

No significant alterations were found for the smaller VLDL-3 sub-fractions or between post-prandial triglyceride and VLDL-3 concentrations.

Upon admission, LDL-1 concentrations significantly increased post-principally from 47.8 ± 15.8 mg/dl at F to 56.7 ± 19.3 mg/dl at pp1; (P<0.05). After weight loss, LDL-1 concentrations remained unchanged post-prandially while their pp1 and pp2 concentrations significantly decreased when compared to admission pp1 and pp2 values (56.7 ± 19.3 mg/dl vs 45.4 ± 14.1 mg/dl; (P<0.02) –pp1- and 54.4 ± 13.6 vs 44.4 ± 16.5 mg/dl; (P<0.001)–pp2-), respectively.

No significant fasting or post-prandial modifications were noted for the LDL-2 sub-fractions at entry and after weight loss.

LDL-3 concentrations were significantly decreased post- prandially after weight loss (117.5 ± 67.2 mg/dl vs 53.8 ± 20.9 mg/dl; P<0.01 at F) and (105 ± 48 mg/dl vs 58 ± 10 mg/dl;

P<0.05 at pp2).

The LDL-2/LDL-3 mass ratio showed a non-significant post- prandial rise upon admission while an opposite post-prandial behaviour was observed after weight loss when the fasting LDL-2/LDL-3 mass ratio significantly decreased from 4.5 ± 1.5 to 3.5 ± 1.9 at pp1 (P<0.02). Additionally, close-to-significance correlations were initially found between the LDL-2/LDL-3 mass ratio and TG (r=-0.625; P=0.07) at fasting was no longer apparent after weight loss (r=-0.234; P=NS). Moreover, a close-to-significance correlation between LDL-2/LDL-3 mass ratio and TG found initially at pp2 (r=-0.695; P=0.055) disappeared after weight loss (r=-0.348; P=NS). Interestingly,

multiple linear regression analyses disclosed delta fasting TG (before-after weight loss) as an independent variable accounting for the ability to predict delta fasting LDL-2/LDL-3 mass ratio (before-after weight loss) changes (dependent variable) (P<0.05).

After weight loss, HDL-2 concentrations at F, pp1 and pp2 were increased from their F, pp1 and pp2 time-points counterparts upon admission even though differences did not reach statistical significance.

HDL-3 concentrations disclosed significant decreases both at F (212.4 ± 55.5 vs 168 ± 29.2 mg/dl; P<0.005) and at pp1 (244 ± 56.2 vs 186 ± 18.4 mg/dl; P<0.02) after weight loss. The HDL- 3 decrements at pp2 did not reach statistical significance.

Additionally, HDL-3 concentrations disclosed a significant increment post-prandially after weight loss (167.6 ± 29.2 at F vs 193.7 ± 36.4 mg/dl at pp2; P<0.05).

V-Discussion

A small group of type II diabetic patients who were well controlled and slightly overweight underwent a rapid improvement (-10 days-) in post-prandial lipemia and post- prandial glycemic excursions through a modest weight loss.

The main lipoproteins sub-fractions involved were VLDL-1, VLDL-2, LDL-3 and the LDL-2/LDL3 mass ratio.

The design of this study was such that in order to minimize the effects of glycemic control amelioration on lipoprotein sub- fractions inclusion criteria accurately targeted a population of well controlled NIDDM patients. Thus, we were able to assess minimal changes in daily glycemic control throughout the study period.

Weight loss was found to account for the ability to significantly predict changes in the post-prandial triglyceride excursions (delta Triglyceride Area Under the Curve (TG-AUC) while Blood Glucose improvement did not show the ability to predict these changes.

As depicted in Figure 1, the post-prandial triglyceride excursions were significantly diminished after weight loss even though their kinetics very much paralleled every other one (prior to- vs -after weight loss). Moreover, fasting triglyceride values were, as expected, significantly lowered and the Triglyceride Area Under the Curve (TG-AUC) was significantly diminished by 23% at the end of the study. Thus, modest

weight loss in otherwise well controlled NIDDM patients rapidly and significantly improves triglyceride clearance and/or intolerance independently of improvements in glycemic control.

The latter is one of several key features in the constellation of findings the define the metabolic syndrome^49-50. Additionally, triglyceride intolerance/impaired clearance is one key feature of insulin resistance observed through various degrees of glucose tolerance^23;24;51 and whose phenotypic characterization is inherited as a dominant trait^52,53.

Post-prandial triglyceride enrichment of VLDL sub-fractions was indirectly assessed by correlating their respective increments in [VLDL] vs the [TG] increments at identical time- points (pp2 [VLDL] – F [VLDL]) vs (pp2 [TG] – F [TG]). Weight loss did not have a significant effect on neither VLDL-1 nor VLDL-3 post-prandial triglyceride enrichment. Interestingly enough, the VLDL-2 sub-fractions disclosed the disappearance of triglyceride enrichment post-prandially at the end of the study.

The net effects of weight loss on LDL-2 sub-fractions disclosed a blunted post-prandial rise (both pp1 and pp2) even though the fasting LDL-2 concentrations were non-significantly raised after weight loss.

LDL-3 cholesterol concentrations decreased after ten days both at fasting and at pp2 sampling periods. Again this finding supports the concept of rapid improvement in the clearance of

LDL-2 sub-fractions by modest weight loss and is in accordance with the available literature⁵⁴.

LDL-3 cholesterol post-prandial kinetics prior to and after weight loss were symmetrically opposed. Whilst fasting LDL-3 cholesterol concentrations decreased by 19 % at pp1 and by 11 % at pp2 prior to weight loss they increased by 36 % at pp1 and by 8 % at pp2.

LDL-2/LDL-3 mass ratios disclosed a significant rise at fasting at the end of the study while the post-prandial increments did not reach statistical significance. Again, these ratios displayed symmetrically opposed kinetics between prior to and after weight loss. Thus, the ratio increased by 28 % at pp1 and by 24 % at pp2 at entry and decreased by 22 % at pp1 and by 14

% at pp2 after weight loss.

LDL-2/LDL-3 mass ratios did not show any significant correlation with either weight loss or blood glucose improvement.

LDL-2/LDL-3 mass ratios correlation with serum triglycerides is a finding which has already been reported in a previous study by our group in which stepwise multiple regression analysis identified a three-parameter model comprising triglycerides, HbA1c, and high density lipoprotein cholesterol as best defining the variations in the LDL-2/LDL-3 mass ratio⁵⁵. We could not find these correlations in our patient group probably due to reduced sample size and overall better controlled

diabetes. These observations are consistent with an independent impact of diabetes on the LDL distribution profile and the possibility that the latter may be subjected to multiple pathological influences in diabetic patients.

In conclusion, a modest weight loss in overweight well controlled type II diabetic patients is associated with a significant improvement in post-prandial triglyceride excursions and the LDL2/LDL3 mass ratio kinetics independently from glycaemic control improvements.

VI-Annexes

Tables and figures

Table 1. Dietary habits and prescribed diet. Results are expressed as mean ± SD; *: P<0.05 between dietary habits recorded and prescribed data. Dietary habits: data collected from the dietary habits recorded at entry. Prescribed diet: diet followed during the study period.

Table 2. Physical characteristics before and after weight loss.

Results are expressed as mean ± SD; *: P<0.05 between before and after weight loss.

Table 3. Mean values ± SD are given for fasting and post- prandial blood samples obtained 4h after breakfast (pp1) and lunch (pp2). *: P<0.01 before vs after weight loss, **: P<0.005 before vs after weight loss.

Figure 1. Fasting and post-prandial capillary blood glucose values before and after weight loss. *: P<0.05 (before vs after weight loss).

Figure 2. Basal and post-prandial capillary triglyceride values before and after weight loss. *: P<0.05 (before vs after weight loss). #: P<0.05 (08:00 AM vs any other daytime)

Figure 3. VLDL-1, VLDL-2 and VLDL-3 cholesterol kinetics before and after weight loss. *: P<0.05 (before vs after weight loss). #: P<0.05 (F vs pp1/pp2).

Figure 4. LDL-2, LDL-3 cholesterol and LDL-2/LDL-3 mass ratio kinetics before and after weight loss. *: P<0.05 (before vs after weight loss). #: P<0.05 (F vs pp1/pp2).

Dietary habits Prescribed diet P

Kcal/day 2167 ± 178 (1600-3146) 1561 ± 39 (1400-1700) 0.01

Carbohydrates (gr.) 197 ± 13 196 ± 4 NS

Carbohydrates (%) 37.8 ± 3.3 50.3 ± 1.4

Fat (gr.) 96 ± 12 62 ± 4 0.03

Fat (%) 39.7 ± 3.2 35.5 ± 1.0

Protein (gr.) 92 ± 8 66 ± 3 0.02

Protein (%) 17.2 ± 1.2 16.9 ± 7.0

NaCl (gr./day) 5 5 NS

(Results are expressed as: mean values SD)± .

Before weight loss After weight loss

Weight (Kg) 79.2 ± 4.2 77.6 ± 4.0 *

Body Mass Index (Kg/m²) 28.0 ± 1.7 27.4 ± 1.6

Body Fat Mass (Kg) 27.8 ± 1.3 26.3 ± 1.9*

Waist circumference (cm) 95.0 ± 1.4 93.0 ± 3.0

Hip circumference (cm) 100.0 ± 3.0 98.0 ± 2.0 *

Waist/Hip ratio 0.95 ± 0.1 0.93 ± 0.1

Fasting blood sugar (mM/l) 7.3 ± 2.1 6.7 ± 2.6

Post-prandial blood sugar (mM/l) 8.2 ±2.6 6.5 ± 1.9 *

HbA1C (%) 7.3 ± 0.1 ---

Systolic Blood Pressure (mm Hg) 147 ± 6.0 142 ± 4.0

Diastolic Blood Pressure (mm Hg) 85 ± 3.0 82 ± 3.0

Before weight loss After weight loss

Fasting pp1 pp2 Fasting pp1 pp2 Cholesterol (mmol/L) 6.1 ± 0.7 6.1 ± 0.9 6.2 ± 0.6 5.3 ± 0.6** 5.2 ± 0.7 5.3 ± 0.5*

HDL-cholesterol (mmol/L) 1.1 ± 0.3 1.2 ± 0.3 1.2 ± 0.3 1.0 ± 0.3 1.0 ± 0.3 1.0 ± 0.3 Apo A-I (g/L) 111 ± 26 115 ± 22 118 ± 27 101 ± 25 103 ± 25 106 ± 26 Apo B (g/L) 93 ± 38 98 ± 21 99 ± 17 86 ± 14 83 ± 14 86 ± 13 Triglycerides (mmol/L) 2.1 ± 0.7 2.3 ± 0.9 2.6 ± 1.0 1.5 ± 0.4** 1.6 ± 0.5 2.1 ± 0.6*

0.00 1.00 2.00 3.00 4.00 5.00

08:00 10:00 12:00 14:00 16:00 18:00

Triglycerides (mmol/L)

BEFORE AFTER

* *

*# #

#

*

#

0 200

BEFORE AFTER

VLDL-1 (mg/dl)

*

*

#

0 200

BEFORE AFTER

VLDL-2 (mg/dl)

* * *

0 200

BEFORE AFTER

VLDL-3 (mg/dl)

F pp1 pp2

0 500

LDL-2 (mg/dl)

0 250

LDL-3 (mg/dl)

* *

0 8

BEFORE AFTER

LDL-2/LDL-3

F pp1 pp2

* * ^#

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