Fructose Consumption Aggravates Dysregulation of Postprandial Lipid Metabolism in Obese Hypertriglyceridemic Men With High Cardiometabolic Risk Profile and Associates With Liver Fat Deposition
Phase: NA
Conditions: Central Obesity, Hypertriglyceridemia
Sponsor: Marja-Riitta Taskinen

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Fructose consumption aggravates dysregulation of postprandial lipid metabolism 
in obese hypertriglyceridemic men with high cardiometabolic risk profile and 
associates with liver fat deposition 
Short title: Visceral/liver fat, postprandial lipemia, de novo lipogenesis and fructose-containing 
beverages 
Principal Investigator Marja-Riitta Taskinen1 MD, PhD; Niina Matikainen1 MD, PhD; Kirsi Pietiläinen1 MD, 
PhD; Leonie Bogl1PhD; Nina Lundbom1 MD, PhD; Jan Borén2 MD, PhD; Björn Eliasson2 MD, PhD; Martin 
Adiels2 PhD; Marcus Ståhlman2 PhD; Marju Orho-Melander3 PhD; Angela Rivellese4 MD, PhD; Gabriele 
Riccardi4 MD, PhD; Jean-Pierre Despres5 PhD; Eric Larose5 MD, Caroline Rhéaume5 MD, PhD; and Natalie 
Alméras5 PhD  
1Helsinki University, Faculty of Medicine, Helsinki, Finland; 2Sahlgrenska Academy at University of 
Gothenburg, Gothenburg, Sweden; 3Lund University Diabetes Center, Malmö, Sweden; 4University of Naples, 
Faculty of Medicine, Naples, Italy; 5 Quebec Heart and Lung Institute, Université Laval, Québec, Canada 
Objectives 
This project focuses on the deleterious effects of fructose-containing beverages on lipid metabolism. Our 
hypothesis is that consumption of fructose-containing beverages increases de novo lipogenesis (DNL) with 
numerous consequences on lipid and glucose metabolism, insulin resistance, inflammation and blood 
pressure. We further propose that fructose also decreases the removal of triglyceride-rich lipoproteins 
(TRLs) from the circulation by inhibiting the activity of lipoprotein lipase (LPL), a key enzyme in 
lipoprotein metabolism. We therefore focus our study on the effects of fructose on postprandial lipemia 
and features of ectopic fat deposition including visceral adipose tissue and liver fat content which are key 
features of individuals with metabolic syndrome and type 2 diabetes. So far, little data exist on the 
patophysiological responses to fructose-containing beverages in obese subjects with multiple 
cardiometabolic risk factors. We will test the hypothesis that hypertriglyceridemic (HTG) obese subjects are 
particularly prone to the deleterious effects of fructose-containing beverages compared to normolipidemic 
(NTG) obese subjects. We have therefore designed our study to differentiate the weight-dependent vs. 
weight-independent effects of fructose-rich diet in HTG and NTG obese subjects. 
Background 
Obesity has become a contemporary global health hazard affecting not only adults but also adolescents 
and children. The prevalence of obesity in children as well as in adults is rising rapidly both in USA and 
Europe. In parallel with obesity, the prevalence of type 2 diabetes and cardiovascular disease (CVD) is 
increasing dramatically and this portends a new wave of CVD. In the face of this major health problem a 
better understanding of factors contributing to a positive energy balance and to body fat gain is necessary, 
particularly regarding factors related to the development of visceral obesity associated with excess ectopic 
and liver fat, which is the most dangerous form of obesity.  
Notably, consumption of sugar-sweetened beverages (SSB) has increased steadily over the past decades 
and is identified as the major source of added sugar intake accounting for a major part of excess energy 
intake. A major dietary change over the past decades has in fact been the dramatic increase of fructose 
intake due to increased consumption of soft drinks and juice beverages. The fructose intake has increased 
by about 20 % per capita in USA between 1970 and 2005 and the present daily intake (approximately 55 g) 
accounts for about 10 % of the total daily caloric intake.1 Recently, large epidemiological studies have 
documented the association between SSB consumption and increased risk for obesity, type 2 diabetes and 
CVD.2-4 Furthermore, recent meta-analyses reported that the risk to develop type 2 diabetes was increased 
by 26 % in the highest quintile of SSB intake which corresponds to the consumption of 1-2 soft drinks per 
day.4  
Strong evidence indicates that added fructose, a component of sucrose and high fructose corn syrup, has 
specific deleterious cardiometabolic effects as compared to glucose.5 A high fructose diet is linked with 
dyslipidemia, insulin resistance/glucose intolerance, visceral obesity and inflammation independently of 
body weight changes.6 In addition, fructose has been reported to raise blood pressure when consumed as 
SSBs whereas glucose does not appear to have such deleterious effect.7 Thus, all sugars are not equal in 
respect to their adverse metabolic effects. Of note, fructose derived from fruits and high-fiber cereal 
products are considered beneficial.8 It is critically important to recognize the SSBs as the major source of 
not only extra calories but also of excess fructose. Excessive intake of SSB has turned out to be a serious 
public health problem and a restriction of SSBs should be a major target in the prevention against obesity, 
type 2 diabetes and CVD. To succeed in this fight it is critical to better understand the mechanisms by 
which excess fructose consumption contributes to the health hazards of obesity, particularly in the etiology 
of the related atherogenic dyslipidemia.  

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Dietary sugar intake and cardiometabolic health at the population level 
Recently, the association of blood lipid levels and consumption of added sugars was studied in the adult 
population in the National Health and Nutrition Examination Survey (NHANES) (n= 6113).9 In this American 
cross-sectional study higher fructose consumers had more unfavorable lipid levels, namely significantly 
lower HDL cholesterol, higher triglycerides and a high ratio of triglycerides to high density lipoproteins 
(HDL) whereas women also had higher low density lipoprotein (LDL) cholesterol levels. Likewise, in the 
Framingham study daily soft drink consumers had higher incidence of elevated triglycerides and low HDL 
cholesterol than non-consumers (relative risk: 1.22 and 1.22, respectively).10 
Several studies have consistently reported increased responses of fasting and postprandial triglyceride 
levels and 24 hr profiles to short term feeding of fructose as compared to glucose in both lean and obese 
subjects.2, 11 These perturbations directly lead to other lipid abnormalities including elevation of apo-B 
levels, accumulation of small dense LDL, and increased remnant lipoproteins, combined with reduced HDL 
cholesterol which all are components of the atherogenic lipid triad, a strong risk factor for CVD. 
Interestingly, the deleterious effect of fructose on lipid metabolism is directly linked to the daily intake; a 
fructose intake > 50 g/ day is associated with postprandial hyperlipemia whereas intake above 100 g/day 
also results in elevation of fasting serum triglycerides.5 Thus, fructose intake is directly linked to an 
atherogenic dyslipidemia. 
Metabolic consequences of fructose feeding 
Fructose is rapidly taken up by the hepatocytes from the portal circulation and found only in micromolar 
concentrations in the systemic circulation. Therefore, high fructose intake primarily alters hepatic glucose 
and lipid handling. The low glycemic index of fructose has been considered beneficial especially for diabetic 
subjects, but recent evidence suggest that the harms of added fructose outweigh its glycemic benefits. 
Although fructose does not stimulate acute insulin excursion like glucose, fructose has adverse effects on 
glucose metabolism and insulin sensitivity.12 In addition, fructose seems to trigger deleterious endocrine 
signals that are reflected in energy metabolism. 
Furthermore, fructose (in contrast to glucose) does not stimulate ghrelin secretion which together with low 
postprandial insulin response contributes to lower satiety and possibly overeating after fructose vs. 
glucose.13 Fructose is also associated with a blunted leptin response. In summary, fructose intake is 
linked with an endocrine profile potentially favoring extra energy intake and weight gain. 
POTENTIAL MECHANISMS UNDERLYING FRUCTOSE INDUCED HYPERTRIGLYCERIDEMIA  
Regulation of very density lipoprotein (VLDL) metabolism 
Serum triglyceride concentration reflects the balance between the secretion and removal of triglyceride-rich 
particles (TRLs). We have recently reported that overproduction of large VLDL1 particles is the hallmark of 
dyslipidemia in the metabolic syndrome and type 2 diabetes. We also reported that liver fat is one driving 
force that aggravates overproduction of VLDL particles in insulin resistance.14 Our results also provided the 
first in vivo evidence that insulin down regulates VLDL1 secretion in subjects with low liver fat while the 
suppression of VLDL1 secretion by insulin is hampered in subjects with high liver fat.14  
We recently reported that hypertriglyceridemia in abdominally obese subjects is due to dual metabolic 
defect, namely the combination of increased secretion and severely impaired clearance of triglyceride-rich 
VLDL1 particles. Increased liver and subcutaneous abdominal fat were linked to overproduction of VLDL1 
particles whereas increased plasma levels of apo-CIII associated with impaired catabolic rate of VLDL1 
particles in obese HTG compared to NTG subjects.15 The disturbed hepatic lipid metabolism is also critical 
in the pathogenesis of hepatic steatosis, and it has been suggested that the inability of the liver to secrete 
triglyceride-rich VLDL particles causes hepatic steatosis.16 This concept considers VLDL-secretion as a 
way to protect liver against excess lipid accumulation and lipotoxicity.  
In clinical practice, it has been suggested that the presence of an elevated waist circumference together 
with increased fasting triglyceride concentrations, the so-called hypertriglyceridemic waist phenotype, could 
represent a useful and simple screening approach to identify individuals likely to be characterized by 
excess ectopic fat deposition and the related dysmetabolic profile.17, 18 
Regulation of de novo lipogenesis (DNL) in the liver 
The liver X receptor (LXR) and the farnesoid receptor (FXR) play critical roles in the regulation of DNL and 
triglyceride synthesis via the transcription factors sterol response element-binding protein 1c (SREBP-1c) 
and carbohydrate response element-binding protein (ChREBP) that are major regulators of DNL via 
activation of fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD). The SREBP-1 is a master 
regulator of hepatic lipogenesis19 (Figure 1). The peroxisome proliferator-activator receptor gamma 

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coactivator-1 (PGC-1) coactivates SREBP-1c. ChREBP is also considered to be a hepatic carbohydrate 
sensor and it is activated by both glucose and fructose. An intriguing observation is that SREBP1-c seems 
also to be activated by fructose independently of insulin.20 Knockdown of PGC-1 resulted in reduced 
expression of SREBP-1, and downstream lipogenic genes in the liver prevent both fructose-induced 
hypertriglyceridemia and hepatic insulin resistance.19, 21 Collectively these actions mediate the lipogenic 
effects of fructose in the liver by enhancing its lipogenic potential and deposition of fat in the liver.12, 23 
Notably, DNL has been reported to be enhanced in subjects with NAFLD.24-26 
Fig 
1. 
Mechanisms 
for 
fructose-induced  
hepatic de novo lipogenesis 
The final step in triacylglycerol synthesis is regulated 
by diacylglycerol transferase-2 (DGAT2)24-25 that 
transfers FA from fatty acyl CoA to diacylglycerol 
(DAG). Knockdown of DGAT2 expression in vivo 
associates with reduction of liver fat.27-29 The 
accumulation of DAG in liver triggers the activation 
of protein kinase C (PKC) serine-threonine kinase. 
PKC is highly expressed in the liver with subsequent 
impairment of insulin signaling and is a link between 
insulin resistance and fatty liver.30 An intriguing 
observation is that DAGs comprised of unsaturated 
acyl chains are more potent activators of PKC than 
those containing saturated acylCoAs.31 Steatoryl-
CoA desaturase (SCD)-1 is a key enzyme 
converting saturated FAs to monosaturated FAs and 
it is reported to be a good surrogate marker for DNL.32 Recently hepatic DAG concentration measured from 
liver biopsies was reported to correlate with liver fat percent.33 
In addition to glycerolipids another candidate to interfere with insulin signaling in the liver is the 
accumulation of ceramides molecules of sphingolipids family.34 Ceramides not only interfere with insulin 
signaling but also interact with inflammation and stress signaling and mitochondrial function. Both increases 
of DAGs and ceramides are enhanced upon fatty acid oversupply and reflect a shift of fatty acyl CoAs 
between different metabolic pathways.35 To elucidate the role and impact of these lipids on metabolism 
request new technology utilizing specific platforms of ultra-performance liquid chromatography (UPLC)/ 
mass spectrometry (MS) based lipidomic analyses.36 In this protocol we will track the responses of major 
lipid components in TRL fraction to the dietary changes. In summary, the data support strongly the 
concept that the effects of glucose and fructose on lipid metabolism are not equal with respect to 
lipid metabolism and that hepatic insulin resistance covers not only glucose pathway but also 
pathways of lipid metabolism.  
Apo-CIII and VLDL assembly 
Apo-CIII is an apolipoprotein carried not only in TRLs and HDL particles but also in LDL in circulation.37, 38 
Several studies have shown that apo-CIII is a determinant of serum and VLDL triglycerides (TG) as well as 
of VLDL catabolic rate since it functions as an inhibitor of lipoprotein lipase (LPL), the rate limiting enzyme 
of TG lipolysis.39 Interestingly, VLDL apo-CIII production rate correlates closely with that of VLDL-TG in 
centrally obese men.40 Notably, the apo-CIII activity is influenced the dietary composition, and unsaturated 
fats have been shown to decrease apo-CIII as well as serum triglycerides 41. Recent data suggest that apo-
CIII is not only linked to the turnover of TRLs but may also be linked to the biosynthesis of VLDL particles.38, 
42 The observation that VLDL-apoCIII production is stimulated by plasma free fatty acids in humans43 
implicate that apo-CIII may enhance the production of large VLDL particles in the setting of excess TG 
availability as seen in NAFLD. This mechanism may also explain why carriers of specific apo-CIII variants 
(C-482™and T-455C) have elevated serum triglycerides and apo-CIII that associated with an increased 
prevalence of non-alcoholic fatty liver disease (NAFLD).44 Intriguingly, a novel mechanism has recently 
been indentified that links glucose and plasma TG metabolism via the regulation of apo-CIII expression by 
glucose (Caron S et al. submitted). This mechanism involves the transcription factors ChREBP and 
hepatocyte nuclear factor receptor α (HNF-4α). As ChREBP is activated by both glucose and fructose this 
is a novel mechanism explaining how fructose may enhance lipogenesis in the liver. In summary, 
emerging results clarify how glucose influences apo-CIII expression, but no data exists on the 
response of apo-CIII to fructose intake.  
 

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Genes influencing TRL metabolism 
Common genetic variations predisposing to dyslipidemia among subjects with and without type 2 diabetes 
include genes regulating lipid metabolism in the liver and VLDL clearance from circulation.45-47 Overall 
genetic variants seem to account for one-third of the susceptibility to high TG.48 Recently, allelic variants of 
the MLX interacting protein-like (MLXIPL) gene were reported to associate with high triglyceride levels. 
Interestingly, this gene controls hepatic energy metabolism, specifically lipogenesis from sugars through 
ChREBP.46-47, 49 Other genes regulating different steps in VLDL synthesis include microsomal triglyceride 
transfer protein (MTP), APO-B, glucokinase hexokinase 4 regulatory protein (GCKR), polypeptide N-
acetylgalactosaminyltransferase 2 (GALNT2) and DGAT2. Recent studies have found that common 
variants in the APO-CIII gene and palatin-like phospholipase domain containing 5 (PNPLA5) contributed to 
fatty liver.44, 50 APO-CIII and APO-A5 are a part of gene cluster on chromosome 11. Both APO-CIII, APO-
A5 and angiopoietin-like 3 (ANGPTL3) genes are important regulators of serum TG level by LPL mediated 
clearance of TRLs.51. Other genes regulating clearance capacity include LIPG (endothelial lipase), hepatic 
lipase (HL) and glycosylphosphatidylinositol (GPI)- anchored HDL-binding protein 1 (GPIHBP1). If fructose 
consumption results to more pronounced DNL, to storage of TAG in the liver and postprandial lipemia in 
carriers of these common variants has not been studied.  
This study aims to examine the diet-gene interactions by comparing the differences in dietary responses of 
postprandial lipemia between carriers and non-carriers of common variants of approximately 40 genes 
shown to associate with triglyceride metabolism (in particular variants in MLXIPL, GCKR, GALNT2, APOB, 
APOA5, LIPG, HL, LPL and ANGPTL3 as well as combined effects of many risk variants in all 40 genes).46 
We will further study expression quantitative trait loci (eOTLs) comparing mRNA transcript levels and their 
changes in response to glucose load in adipose tissue biopsies in hyper- vs. hyporesponders to fructose.  
Hepatic heparan-sulfate proteoglycans (HSPGs) are important to mediate TRL clearance by the liver, and 
recent studies have shown that changes in the composition of hepatic HSPGs resulted in profoundly 
reduced clearance of TLRs. HSPG synthesis is regulated by >35 genes, of which syndecan-1 and genes 
associated with initiation (exostosin: EXT1/2) or sulfation (N-deacetylase sulfotransferase: NDST1 as well 
as 2-O-sulfotransferase: HS2ST1) of HSPG have all been associated with TRL uptake in vitro. Degradation 
of HSPG is regulated by 2 enzymes; heparanase (HPSE) and sulfatase (SULF). In humans, little is known 
on the ‘metabolism’ of HSPGs in health or disease, whereas data on the effect of changes in HSPG 
metabolism on cardio-metabolic homeostasis is entirely unknown. We will therefore study the impact of 
genetic variations in HSPG-related genes and triglyceride metabolism. 
Fructose metabolism in the liver 
High fructose diets have been shown to induce insulin resistance and hypertriglyceridemia in several 
animal models.52 It should be recognized that the liver is the major site of fructose metabolism.53 
Importantly the metabolism of fructose differs fundamentally from that of glucose (Figure 2). The 
metabolism of fructose by fructokinase results in the release of three-carbon molecules substrates for DNL 
which are utilized for the synthesis of glycerol and free fatty acids. In contrast to glucose metabolism, 
fructose conversion to three carbon molecules occurs independently of regulation by insulin and there is no 
negative feedback via cell energy status to control this pathway resulting in increased DNL and 
consequently VLDL synthesis and secretion. 
Insulin action in the liver involves insulin 
receptor kinase which phosphorylates insulin 
receptor substrate-1 (IRS1) and IRS2 resulting 
in activation of PI3K (phosphatidylinositol 3-
kinase) 
and 
the 
protein-serine/threonine 
kinases Akt2. Activation of Akt2 enhances 
glycogen 
synthesis 
and 
inhibits 
gluconeogenesis. Defects in insulin signaling 
cascades have been observed in animal 
models 
on 
high 
fructose 
diet 
including 
increased 
activity 
of 
protein 
tyrosine 
phosphatase 1B (PTP-1B), phosphatase and 
tensin homolog deleted on chromosome ten 
(PTEN), 
SH2 
domain-containing 
inositol 
phosphatase (SHIP2) and c-Jun amino terminal 
kinase (JNK1) resulting in hepatic insulin 
resistance.12 
Fig 2. Metabolic pathways of fructose and 
glucose in the liver 

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High fructose feeding and DNL  
The available evidence that fructose increases DNL from animal models in particular from fructose fed 
golden hamster are plenty and convincing.52 The key question is whether fructose consumption increases 
DNL in vivo in man to the extent that is reflected in significant excess liver fat accumulation and VLDL 
overproduction resulting in atherogenic dyslipidemia and increased CVD risk. Presently, scientific evidence 
is limited because of small studies with short follow-up periods. Acute oral fructose load increased DNL in 
healthy men but not in women in one recent study.54 The recent data by Parks et al. confirmed that acute 
consumption of fructose enhanced lipogenesis and increased postprandial TG levels.55 In contrast, the 
contribution of newly synthesized fatty acids to VLDL-TG remained minimal after acute fructose load 
despite increases of postprandial TG levels in another study.56 A short term high fructose feeding (3 g/kg 
BW corresponding to 800-1000 extra calories per day) on top of isocaloric diet in lean healthy men 
increased hepatic DNL by six-fold and fasting triglycerides by 80%.57 Recently Stanhope et al.6 reported 
that fructose -sweetened beverages providing 25 % of daily energy intake over 10 weeks increased 
significantly DNL and postprandial TG response but not fasting TG levels. Subjects consuming fructose 
exhibited significant increase in visceral fat but no changes was seen in control subjects consuming 
glucose. Notably, both groups showed a small but significant weight gain. Fructose consumption, but not 
glucose consumption, was associated with significant decrease of insulin sensitivity and increases of 
fasting glucose and insulin levels. Overfeeding of fructose on top of isocaloric diet over 7 days lead to 
increased liver fat in healthy lean men.58 So far this is the only study suggesting that fructose may enhance 
fat deposition in the liver. However, it is still unknown whether this is due to calorie excess or fructose per 
se. In summary, acute fructose consumption in quantities similar to average American daily 
fructose intake stimulate DNL and enhance postprandial lipemia. The effect of chronic fructose 
intake and the direct contribution of DNL to liver fat, VLDL production and postprandial TG kinetics 
are not established in humans and research in this area is urgently needed.  
Fructose and inflammation 
Recently, fructose feeding has been reported to activate inflammatory pathways (Figure 3).59 Fructose 
feeding in rats is associated with increased inflammatory proteins and markers of oxidative stress.60 In two 
animal models fructose feeding was associated with increase of tumor necrosis factor α (TNFα) levels in 
plasma associated with activation of the proinflammatory transcription factor NF-B. Interestingly, TNFα 
has been reported to stimulate hepatic VLDL apo-B production accompanied by increases of MTP and 
hepatic insulin resistance.61 
Accumulation of TG in the liver leads to increased synthesis of the proinflammatory lipid family ceramide 
and reactive oxygen species (ROS) generation.62 Recently, fructose has been reported to increase 
ceramide concentration.63 In the liver accumulation of diacylglycerol (DAG) and generation of ROS 
activates protein kinase C delta (PKC-δ), IκB kinase (IKK-β) and NF-B pathways.64 IKK-β seems to act as 
a mediator of TNFα induced insulin resistance65, 66 IKK-β activation results in increased expression of sterol 
regulatory element-binding protein 1 (SREBP-1) and induction of DNL. Increased NF-B activation 
promotes JNK-1 activation via inhibition 
of serine phosphorylation of IRS-1 
resulting in defective insulin signaling 
and insulin resistance. Fructose has 
been also reported directly to activate 
JNK-1.66-68 
Given that fructose induces hepatic 
lipogenesis 
and 
associates 
with 
inflammatory processes it is plausible 
that these mechanism act in concert to 
further 
aggravate 
the 
lipogenesis 
pathway. To the best of our knowledge 
no 
data 
exist 
on 
markers 
of 
inflammation and oxidative stress in 
man during fructose feeding. 
Fig 3. Consequences of high fructose 
diet on lipid metabolism in the liver.  
Specific Aims 
Our hypothesis is that consumption of fructose-containing beverages aggravates postprandial lipemia by 
dual mechanisms; by an increased de novo lipogenesis reflected by increased liver fat content and VLDL 
production; and by a decrease in the clearance rate of TRLs linked to reduction in LPL activity. 

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This project addresses the following specific questions:  
1. 
Does a diet supplemented with fructose-containing beverages increase postprandial lipemia 
independently of body weight gain due to extra calories? 
2. 
Does a diet supplemented with fructose-containing beverages increase DNL, VLDL production and 
liver fat deposition more in high risk HTG obese subjects than in NTG obese individuals?  
3. 
Is the clearance of TRLs in the postprandial state impaired in response to a diet supplemented with 
fructose containing beverages?  
A. Is a potential clearance defect of TRLs linked to responses of apo-CIII and apo-A5 to a diet with 
high fructose? 
B. Are LPL mass and activity responses different in NTG vs. HTG subjects during fructose diet?  
4. 
Is blood pressure raised by a diet supplemented with fructose-containing beverages? 
5. 
Is the acute inflammatory response to a fructose-rich diet measured in the fasting state or the 
postprandial period exacerbated in obese subjects with HTG? 
6. 
Do common variants in genes affecting lipid metabolism (individually and/or when combined) modify 
the effect of a diet enriched in fructose-containing beverages on lipid metabolism (in particular 
hypertriglyceridemia), and gene expression in adipose tissue? 
7. 
Is gut microbiota modified by a diet enriched in fructose-containing beverages and are these 
changes linked to metabolic dysfunction?  
Study design, subjects and methods 
This international, multi-center, randomized study has four centers: Helsinki University, Finland; University 
of Gothenburg, Sweden; Napoli University, Italy; and Laval University, Canada. Each centre enrolls 32 
subjects (see footnote below).i Thus, total number of subjects will be n=128 (Figure 4).  
Study groups (n=16 in every 
centre) 
- Obese normolipidemic healthy 
men with big waist (>96 cm, BMI <40 
kg/m2), normal lipid values (TG <1.7 
mmol/l and LDL <3.5 mmol/l), and 
normal liver fat. 
 - Obese hypertriglyceridemic men 
with big waist (waist > 96 cm, BMI 
<40 kg/m2), hypertriglyceridemia (TG 
>1.7 mmol/l and LDL <3.5 mmol/l), 
with increased liver fat. 
Fig 4. Study design and numbers of 
subjects in each centre.  
Experimental diets 
Subject eligible for the study will be randomly assigned to one of the two diet groups for 12 weeks: 
 A) “Hypercaloric fructose diet group”  
Subjects will be instructed to drink 990 ml of a beverage sweetened with 7.6 % fructose per day while 
consuming a self-selected ad libitum (usual) diet. No advice is given to reduce the energy content of their 
usual diet. Subjects will be in positive energy balance and therefore subjected to a weight gain of up to 2.5 
kg in the 3 month study period. The subjects will be followed by the trained dietician 6 weeks after the study 
to ensure that the subjects return to a BW of maximum 1 kg above their baseline BW. 
                                                          
i Helsinki University plans to perform a kinetic study as an extension of the main study. The aim of the 
substudy is to elucidate the effects of fructose diet on lipoprotein metabolism using stable isotopes. This 
substudy replaces study visit 3 and is described on page 9.     
 

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B) “Isocaloric fructose diet group” 
Subjects will be instructed to drink 990 ml of a beverage sweetened with 7.6 % fructose per day while 
consuming an isocaloric diet. This group will be instructed by a trained dietitian to reduce their carbohydrate 
intake from other source by 75 g in order to remain in neutral energy balance. Subjects are required to 
keep their weight stable during the 3 months and their daily energy intake will be adjusted by the dietitian if 
the body weight deviates more than 1 kg at the weekly check-ups. 
Dietary protocol 
The beverages will be prepared as 7.6 % (w/w) solutions flavored with unsweetened drink mix. 750 ml of 
fructose sweetened beverage provides 75 g of fructose and 304 kcal corresponding to 15 % of energy 
intake for a person with a daily caloric intake of 2000 kcal. Subjects will be instructed to consume the 
beverages together with the main meals.  During traveling the fructose drink may be replaced by 
crystalline fructose (75 g, same brand in each center) during weeks 1-10 but not during the last two 
weeks of fructose consumption for maximum of 5 days during the whole study. 
Assessment of dietary intake 
To assess habitual food and beverage consumption, subjects will participate in a baseline evaluation that 
includes a dietary record on 3 consecutive days including 2 weekdays and 1 weekend day. The subjects 
will be carefully instructed not to change their  usual eating habits. Further, subjects are asked to repeat 
the 3-day food diaries at week 4, and 11 to obtain a total of 9-day diaries during the intervention period. 
The mean of the 6  9 days will be used to estimate energy and macronutrient intake from the diet during 
the intervention period. Participants are not required to weight foods but are asked to measure the 
volume of foods consumed with household measurements (cups, tablespoons) or to indicate the weight 
of the products. After completing the food records, participants will meet with the dietitian to review the 
food records for completeness. Energy and macronutrient intake will be calculated by the dietician using 
using local dietary programs, which is based on a national Finnish database for food composition.69 
Subjects carry a pedometer during the days of dietary recordings. 
Subjects are required to be in contact with the dietitian once per 1-3 weeks to control beverage supply, to 
monitor weight and compliance. The home scale is  checked  at the first visit.   
Power calculations  
Primary endpoints in the current study are 1) change in plasma triglyceride (TG) area under curve (AUC) 
between the NTG and HTG during fructose feeding (between groups) and 2) change in TG AUC in HTG 
subjects during isocaloric fructose vs. hypercaloric fructose feeding (within group). The calculations are 
based on following assumptions: TG AUC increases from 7 to 10.5 mmol/lhrs in normotriglyceridemic and 
from 7-9 to 14 mmol/lhrs in hypertriglyceridemic subjects during fructose. Standard deviation is assumed 
to be 4.3 based on previous studies in similar groups of patients. 1) To detect significant difference (>3.5 
mol/lhrs) between the groups (n=25) with unpaired t-test. 2) To detect significant increase (>3.5 mol/lhrs) 
within the group (n=15) with paired t-test. These calculations assume that alpha = 0.05 and power = 0.80 in 
all tests. 
Inclusion criteria  
Male subjects with body mass index (BMI) 27-40 kg/m2, waist >96 cm will be recruited. The weight must 
have been stable (+- 1 kg during the last 6 monts). Each participant will undergo a physical examination 
and laboratory tests to exclude hepatic diseases (other than hepatic steatosis) and renal, thyroid and 
hematological abnormalities. Subjects with total cholesterol <6.2 mmol/l, serum triglycerides <5.0 mmol/L, 
BMI <40 kg/m2, non-smoking and regular daily alcohol consumption less than 2 doses (i.e., 5 g pure 
alcohol, maximum alcohol consumption is 2 portions per day or 14 portions per week) are included to the 
study.  Any lipid medication (indicated for primary prevention) or other medication known to affect lipid 
levels is discontinued at least a month before the screening visit and reintroduced after the completion of 
the study. Type 2 diabetes is excluded with an oral glucose tolerance test. 
Exclusion criteria: Age < 20 years or >65 years, BMI or lipid levels outside the inclusion range, smoking, 
alcohol consumption over 2 doses/day, type 2 diabetes, cardiovascular disease, hormonal therapy, any 
chronic disease requiring medication except for controlled hypertension.  

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The reasons for focusing on males are two-fold: previous studies demonstrate an effect of gender to 
fructose feeding.70 Furthermore, as there are major gender differences in body fat distribution, visceral and 
ectopic fat deposition (women having, on average less visceral and liver fat than men), the study size in a 
study with both men and women would have been forced to be significantly larger. This wouldn’t be 
compatible with the budget limitations for this grant application. However, the gender difference to fructose 
feeding is exceedingly interesting and our goal is to perform a follow-up study with both males and females. 
That study will be designed by the results from this study.   
Study visits The number of visits is about 3-5 depending on the local circumstances to allocate imaging 
studies; the kinetic study will have a separate schedule for visits. 
Please see specific instructions in excel: Clinical data files_040612 and Fructose_sample 
handling_fatload_ogtt_heparin_TIMEPOINTS_040612 and Fructose Fat load  bleeding schedule 
040612. 
Record all results of patients included to study in: Fructose_data_040612. 
1.  
Screening  
Subjects will undergo physical examination (cardiorespiratory status, blood pressure, BMI, waist-hip-ratio 
and recording of their medical history. A blood sample is drawn after an overnight fast.  
1.1  
The screening measures: serum triglycerides, LDL cholesterol and HDL cholesterol, blood count, 
creatinine, sodium, potassium, thyroid stimulating hormone, glucose, and liver enzymes.  
1.2  
An oral glucose tolerance test (see below). 
Studies 2-5 for screen positives are performed twice: before and at the end of the three month diet 
period. 
2.  
Lipolytic enzymes and genetic studies 
2.1  
Subjects will receive 75 IU/kg heparin i.v. to determine lipoprotein lipase and hepatic lipase masses 
and activities.71  
2.2  
A blood sample for DNA extraction is drawn. Single nucleotide polymorphisms (SNPs) in genes 
involved in regulation of lipid metabolism, insulin resistance, inflammation or oxidative stress will be 
analyzed using Taqman and Sequenom methods. In addition, among hyper- and hyporesponders to 
fructose, expression quantitative trait loci (eOTL) analysis will be performed comparing mRNA 
transcript levels and their changes during the intervention from adipose tissue biopsies and relating 
this to genetic variation. 
2.3  
Since diet may lead to low-grade inflammation and insulin resistance due to changes in gut microbial 
community, the subjects will also provide a stool sample (using specific tubes and instructions) for 
analysis of gut microbiota profile (future analysis) and answer a health questionnaire. A blood 
sample for lipopolysaccharide (LPS) and angiopoietin-related protein 4 (Angptl4/fait) analyses is 
drawn for future analyses.72-74 
2.4 
The questionnaire of physical activity is done twice: before fructose period and during weeks 10-12 
3.  
Oral fat load study 
After a 12-h fast, subjects will receive a mixed meal consisting of bread, butter, cheese, low fat milk and tea 
or coffee (63 g carbohydrates, 56 g fat (P/S ratio 0.11), 39.9 g protein) in the morning. Blood samples are 
drawn before and up to 8 hours after the meal. During this time only water will be served ad libitum and the 
subjects will remain physically inactive. 
To study de novo lipogenesis during postprandial period the subjects will receive deuterated water (2H2O) 2 
g per kg body weight. In the evening before the oral fat load study a blood sample (background sample)  
(Taken DURING PRECEEDING week before: see sample handling) will be drawn. The subjects will sip 
deuterated water (2 dl) between 18.00 and 22 hrs together with 5 dl of mineral water.  
3.1  
Blood 
samples 
(For 
all 
sampling: 
please 
see 
Exel: 
Fructose_sample 
handling_fatload_ogtt_heparin_TIMEPOINTS_040612 and Fructose Fat load  bleeding schedule 
040612) are taken the night before (background serum sample) for DNL analysis and before and 
during the fat load. Lipoprotein fractions will be isolated using density gradient ultracentrifugation.75 

Research plan, ethical committee/ Version 7/26.08.2013 
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Concentrations of lipids, apolipoproteins, other biochemical variables will be performed in whole 
plasma and/or chylomicron (Sf > 400), VLDL1 (Sf 60-400) and VLDL2 (Sf 20-60) fractions. Analysis 
will include: 
3.1.1  Triglycerides, cholesterol, apo-B100, apo-B48, markers of energy balance (FFA, β-OHB), glucose 
 
and insulin are studied in PLASMA.  
3.1.2  Enrichment of deuterated water to measure DNL in TRL- fractions are measured.76  
3.1.3  Apo-CIII, apo-A5, uric acid, markers of inflammation (high-sensitive CRP, TNF-α, interleukin-6) are 
measured. Samples for other biomarkers (serum amyloid A [SAA], IL-8, paraoxonase [PON1], 
myeloperoxidase [MPO], monocyte chemotactic protein-1 [MCP-1]), markers of coagulation activity 
(plasminogen activator inhibitor-1) and incretins (glucagon-like peptide-1 [GLP-1] and  gastric 
inhibitory polypeptide  [GIP]) are collected for future analyses. 
3.1.4  Lipidomics profiling utilizing the state of the art GC/MS technology to map a large and 
comprehensive spectrum of lipid and lipid intermediate species including SCD desaturation index 
(i.e., the ratio of 16:1n-7/16:0 that is used as an index of SCD enzyme activity) and their responses 
to diets in serum and VLDL fractions are measured at two time points.77  
3.1.5  Fasting samples: apo-B, apo-AI, apo-AII, adiponectin, ghrelin, leptin, LDL size and HDL size.  
3.1.6 Needle fat biopsies from subcutaneous abdominal fat are taken fasting and 4 hrs postprandially. 
Adipose tissue mRNA levels will be profiled to detect which genes are upregulated vs. 
downregulated in response to fructose and how this relates to genetic variation.  
3.1.7  Four spare samples (two plasma and two serum samples) of each time-point.  
3.1.8  In fasting state and during the postprandial period (0 and 4 hrs)78, 79 blood pressure measures will be 
performed using a standardized system (BP Tru 200 manometer) and heart rate variability will be 
tested by ECG recording. 
4.  
Determination of liver, subcutaneous and intra-abdominal fat 
On a separate visit the amount of liver, subcutaneous and visceral fat are determined. Proton magnetic 
resonance spectroscopy is performed with a 1.5 T whole-body device80 to determine liver fat content. 
Magnetic resonance imaging is used to determine subcutaneous abdominal and intra-abdominal fat as 
previously described.80 A standardized protocol will be used at all centers.  
5.  
Oral glucose tolerance test 
An 
oral 
glucose 
tolerance 
test 
(For 
all 
sampling: 
please 
see 
Excel: 
Fructose_sample 
handling_fatload_ogtt_heparin_TIMEPOINTS_040612) will be performed in the morning after an overnight 
fast at screening as well at the end of the 3 month diet period. Sampling time will be at 0, 5, 10, 30, 60, 90, 
120, 180 and 240 minutes for determination of glucose, insulin, C-peptide and gut incretins to allow 
modeling oral glucose insulin sensitivity index (OGIS).81 
SUMMARY:  
1.  
Measurements of routine laboratory lipid and biomedical parameters will be centralized and workload 
allocated to the expertise of the centers (Helsinki, Laval, Gothenburg, and Naples). 
2.  
DNL measurements and lipidomic analyses will be done in Gothenburg (Prof. Jan Borén). 
3.  
SNP genotyping by Taqman and Sequenom and adipose tissue mRNA profiling by Affymetric 
microarray will be performed in Malmö (Prof. Marju Orho-Melander). 
4. 
Studies of microbiota will be performed in Gothenburg (Dr. Fredrik Bäckhed). 
Substudy (in Helsinki) 
Kinetic protocol visit (replacing study visit 3)  
This study will replace study visit 3 and is performed before and at 3 months after the diet period. The aim 
of the study is to elucidate the effects of fructose diet on triglyceride and apo-B non-fasting kinetics using 
stable isotopes. We will use d3-leusine to study apo-B kinetics and d5-glycerol to study triglyceride 
kinetics.82 Simultaneously, hepatic DNL is calculated from enrichment of deuterated water ingested during 
the kinetic study. The study outline is shown in figures 5 and 6. Study subjects will consume 2H2O during 
the kinetic protocol as described in visit 3. At 8:00 the subjects will receive a bolus injection of 2H3-leucine (7 
mg/kg) and 2H5-glycerol (500 mg) as in our previous kinetic protocol.83 The subjects will be served an oral 
fat load described in Visit 3. Urine samples of minimum 100 ml will be collected in the morning of 

Research plan, ethical committee/ Version 7/26.08.2013 
Page 10 
ApoB 48 (b)
FA (e)
TG (e)
MAG
LIVER
Leucine (b)
Glycerol (a)
ApoB 100 (b)
FA (c,d,e)
TG (a,c,d,e)
NEFA (c,e)
CM
(b,e)
FA (e)
FA (e)
Glycerol (a)
Leucine (b)
NEFA (c)
TG (e)
DNL (d)
2H2O
VLDL 
(a,b,c,d,e)
INFUSION
kinetic protocol visit and 4 hours after serving the mixed meal. The urine samples will be used to 
measure the microvesicles containing multiple protein and nucleic acid classes (mRNA, miRNA, 
siRNA).  
Dietary triglyceride tracers will be administered through test meal served as outlined in Figure 6 and 
containing 1,2,3,4-13C16 –palmitate (1-2 g). Blood samples are drawn according to the figure 6 (red dots). 
Other biochemical, physiological, lipid and apolipoprotein analysis are done as described in visit 3. 
 
 
 
 
 
 
 
 
 
Fig 5. Summary on TRLs metabolism 
 
 
 
Fig 6. Study design 
Kinetic modeling 
Times series data from enrichments of plasma leucine, leucine in apo-B100 from VLDL1 and VLDL2, and 
glycerol from triglycerides from VLDL1 and VLDL2 are used as input to the kinetic model together with pool 
size measurements to simultaneously determine the kinetics of VLDL-triglycerides and apo-B100.83 
Novelty and importance  
This study provides a multidisciplinary state-of-the-art program to elucidate the deleterious effects of 
fructose on metabolism using a novel multidisciplinary approach in which clinical studies and molecular 
medicine are combined. The participating investigators, who all are at the scientific forefront in their 
respective fields, have different but highly complementary knowledge and technical competence. This 
enables us to perform innovative and cutting-edge science to dissect how fructose influences dyslipidemia, 
insulin resistance and inflammation utilizing novel imaging technology, lipidomics and genomics. The study 
focuses on abdominally obese subjects with hypertriglyceridemia to furthering evidence that these 
individuals are not only prone to type 2 diabetes but also to the development of related cardiometabolic 
abnormalities. The study is therefore designed to identify fructose mediated effects that are independent of 
weight gain. Overall, this research program has relevance to our understanding of factors involved in the 
development of type 2 diabetes and related metabolic complications including CVD risk factors.  
Lipoprotein metabolism is a complex system in which abnormal concentrations of various lipoprotein 
particles can result from alterations in their rates of production, conversion and/or catabolism. Traditional 
methods that measure plasma lipoprotein concentrations only provide static estimates of lipoprotein 
metabolism and hence limited mechanistic information. By contrast, we will use tracers labeled with stable 
isotopes, which provide us with a powerful and cutting-edge tool for probing lipid and lipoprotein kinetics in 
vivo and furthering our understanding of the pathogenesis of dyslipoproteinemia. Thus, the combination of 
stable isotopes, studies of postprandial lipid metabolism and measurements of hepatic DNL will for the first 
time allow us to explore the dynamic changes leading to both postprandial accumulation of chylomicrons 
and elevated VLDL secretion. The great synergism and excellence of the network guarantees success of 
the proposed study.  
The microvesicles measured in urine samples are rapidly emerging as valuable, non-invasive ways 
to identify biomarkers of kidney metabolism. Consumption of fructose leads to increasing urate 
concentrations in plasma. The measurement of  urine microvesicles offers a unique opportunity to 
study the effect of fructose consumption on novel biomarkers of kidney metabolism.  
 
 

Research plan, ethical committee/ Version 7/26.08.2013 
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Collaborative partners  
 Helsinki University Central Hospital, Biomedicum, Helsinki, Finland. Principal Investigator Marja-Riitta 
Taskinen, MD, PhD, professor. Team members: Niina Matikainen, MD, PhD, scientist, endocrinologist, 
coordinator of the clinical studies; Kirsi Pietiläinen, MD, PhD, senior lecturer, internist and nutritionist; 
Leonie Bogl, PhD, nutritionist; and Nina Lundbom, MD, PhD, senior lecturer, radiologist and imaging 
expert.  
 Quebec Heart and Lung Institute (QHLI), Quebec, Canada. Jean-Pierre Després, PhD, professor, local 
PI. Team members: Natalie Alméras, PhD, the coordinator of clinical studies with great expertise in 
metabolic studies; Eric Larose, MD, chief of the Cardiovascular Imaging Center and responsible for the 
imaging; and Caroline Rhéaume, MD, PhD, a primary care physician and scientist responsible for the 
medical supervision of the subjects. 
 Sahlgrenska Academy at University of Gothenburg and Sahlgrenska University Hospital, Gothenburg, 
Sweden. Jan Borén, MD, PhD. professor, the local PI. Team members: Björn Eliasson, MD, PhD, 
adjunct professor, clinical studies; Fredrik Bäckhed, PhD, lecturer, studies of microbiota and its 
metabolic consequences; Martin Adiels, PhD, scientist, modeling and biostatistics and Marcus 
Ståhlman, PhD, scientist, advanced lipidomics analysis. 
 University of Naples, Federico II, and Faculty of Medicine, Naples, Italy. Angela A. Rivellese, MD, 
associate professor in Internal Medicine, local PI. Team members: Gabriele Riccardi, MD, professor in 
endocrinology; Giovanni Annuzzi, MD; and Lidia Patti, biologist. 
 Lund University Diabetes Center, Malmö, Sweden. Marju Orho-Melander, PhD, professor of genetic 
epidemiology, local PI. 
 Urine microvesicle analysis (Helsinki substudy): Harry Holthöfer MD, PhD, Professor, Director, 
National Centre for Sensor Research/Bioanalytical Sciences, Dublin City University, Ireland.  
 
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