Conditions: Type 2 Diabetes
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 23
Healthy volunteers: no

Brief summary:
This study aims to evaluate the mechanisms underlying the effect of incretin therapy on lipoprotein metabolism in subjects with type 2 diabetes and to study the effect of liraglutide on hepatic de novo lipogenesis.

Detailed description:
The well recognized dyslipidemia in people with type 2 diabetes consists of high fasting and non-fasting plasma triglycerides (TG), low high-density lipoprotein (HDL) -cholesterol and preponderance of small dense low-density lipoprotein (LDL) particles nominated as the atherogenic lipid triad. Humans are mostly in a postprandial rather than fasting state and therefore non-fasting TG values reflect more accurately the continuous exposure of arterial wall to triglyceride rich lipoproteins (TRLs) and more importantly, to substantial cholesterol load that these particles deliver.

Postprandial lipemia is highly prevalent even in type 2 diabetes patients with normal fasting TG concentrations. Intestinal overproduction of chylomicrons (CMs) and the structural protein apolipoprotein (apo)-B48 has been identified as an integral feature of postprandial lipemia in type 2 diabetes and insulin resistance. It is clinically important to elucidate the mechanism for delayed postprandial lipemia and the interactions between dysglycemia and dyslipidemia in type 2 diabetes patients.

Arms:
arm 1: Liraglutide subcutaneous injection once daily with following dose escalation:

liraglutide 0.6 mg once daily for one week; liraglutide 1.2 mg once daily for one week and thereafter liraglutide 1.8 mg once daily for 3.5 months. arm 2: Placebo subcutaneous injection once daily with following dose escalation:

placebo 0.1 ml once daily for one week; placebo 0.2 ml once daily for one week and thereafter placebo 0.3 ml once daily for 3.5 months.

Interventions: intervention 1: Liraglutide intervention 2: Placebo
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_SAP_000.pdf: 
1 of 9 
Protocol synopsis  
Date: 2014-2-24 
Version: 2 
 
The effect of Liraglutide treatment on postprandial chylomicron and VLDL kinetics, 
liver fat and de novo lipogenesis. 
 
A single-center randomized controlled study. 
 
UTN: U1111-1149-8774 
EudraCT: 2013-005075-40 
 
Principal investigators: Niina Matikainen1,2,3,4, Jan Borén5 
Other Investigators: Stephano Romeo5, Sanni Söderlund1,2,3, Martin Adiels5,Kirsi Pietiläinen2,3, 
Leonie-Helen Bogl 2,3,  and Marja-Riitta Taskinen1,2,3 
 
1 Heart and Lung Centre, Cardiovascular Research Unit, Helsinki University Central Hospital; 2Research 
Programs’ Unit, Diabetes & Obesity, University of Helsinki; 3Clinical Research Institute, Huch ltd. Biomedicum 
Helsinki, Finland; 4Department of endocrinology, Helsinki University Central Hospital, 5Sahlgrenska Academy 
at University of Gothenburg, Gothenburg, Sweden 
 
Background  
The substantial residual risk for cardiovascular disease (CVD) risk that persists in patients with optimal low-
density lipoprotein (LDL) cholesterol associates with the atherogenic dyslipidemia and postprandial lipemia, 
is a significant but clinically neglected CVD risk factor in particular in Type 2 diabetes (T2DM) patients [1]. 
The well recognized dyslipidemia in people with T2DM consists of high fasting and non-fasting plasma 
triglycerides (TG), low high-density lipoprotein (HDL) -cholesterol and preponderance of small dense LDL 
particles nominated as the atherogenic lipid triad. Recent studies consistently show that non-fasting 
triglyceride values are better associated with CVD risk and death than fasting triglyceride values. In addition, 
non-fasting triglycerides also predicted stroke risk [2]. Indeed, humans are mostly in a postprandial rather 
than fasting state and therefore non-fasting TG values reflect more accurately the continuous exposure of 
arterial wall to triglyceride rich lipoproteins (TRLs) and more importantly, to substantial cholesterol load that 
these particles deliver. In circulation TRLs undergo lipoprotein lipase (LPL)-induced lipolysis and remodeling 
with the formation of a spectrum of heterogeneous particles referred as TRL remnant particles with a 
progressive reduction of particle size that allows particles to penetrate the arterial wall. Although LDL 
cholesterol is recognized as the most important lipid factor in early stages of atherogenesis, postprandial 
TRLs may be relevant to later atherometabolic processes. TRL remnants contribute significantly to intimal 
cholesterol deposition as remnants contain more cholesterol per particle than LDL.  
Postprandial lipemia is highly prevalent even in T2DM patients with normal fasting TG concentrations. 
Intestinal overproduction of chylomicrons (CM s) and the structural protein apolipoprotein (apo)-B48 has 
been identified as an integral feature of postprandial lipemia in T2DM and insulin resistance [3,4]. Each CM 
particle contains one apo-B48 molecule that remains the integral part of CM remnant particles during the 
lipolysis of the particles by LPL. Consequently apo-B48 can be utilized in vivo to track the secretion and 
metabolic fate of intestinally derived CM particles [5]. CMs contribute significantly but not exclusively to the 
elevation of postprandial triglyceride levels. Hepatic very low-density lipoprotein (VLDL) particles are 
quantitatively the major component leading to increased number of remnant particles.  
We recently reported that elevation of plasma TG in abdominally obese dyslipidemic subjects at high CVD 
risk is due to dual metabolic defects; 1. overproduction of VLDL1 particles linked to increased liver fat and 
subcutaneous abdominal fat, and 2. impaired catabolic rate of VLDL1 particles associated with increases of 


2 of 9 
apo-CIII [6]. Our recent data suggest that the capacity of the lipolytic pathway is a determinant of both 
fasting and non-fasting triglyceride levels [7]. Therefore it is clinically important to elucidate the mechanism 
for delayed postprandial lipemia and the interactions between dysglycemia and dyslipidemia in T2DM 
subjects.  
Incretins are hormones released from the gut that respond to the ingested nutrients and potentiate glucose-
induced insulin secretion from the pancreatic β-cells. The most important incretins are glucagon-like 
peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which are rapidly 
inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4) [8-10]. Incretins are important in the regulation 
of postprandial glucose levels [11-13]. Importantly the incretin effect is strongly reduced or lost in T2 DM 
patients. Recently new approaches that targets incretin hormones including the GLP-1 receptor (GLP-1R) 
agonists and DPP4 inhibitors have been developed with a mechanism of action distinct from existing glucose 
lowering agents [10,14]. Incretin based therapies with GLP-1 agonists are accepted as an ideal add on 
therapy to intensify glucose control in combination therapy as they offer benefits like weight loss and no 
hypoglycemia [15]. 
Figure 1.Coordinated TRL metabolism 
during the fasting-to-fed transition. The 
intestine actively produces chylomicrons 
and secrets gut hormones, including 
GLP-1 and GLP-2. A shift of TRL 
metabolism from fasting to fed state 
involves 1) dietary fat absorption and 
chylomicron secretion, 2) decreased 
adipose tissue lipolysis and free fatty 
acid (FFA) flux to liver and intestine, with 
net FA uptake by adipose tissue, 3) 
increased chylomicron remnant uptake 
by the liver, 4) fatty acid “spillover” (i.e., 
FA released by intravascular lipolysis of 
TRL not taken up and stored in adipose 
tissue) and utilization for VLDL and 
possibly 
chylomicron 
synthesis, 
5) 
increased hepatic de novo lipogenesis 
(DNL), 6) increased liver lipid droplet storage, and 7) relative reduction in fatty acid (FA) oxidation vs. esterification 
of FA in liver. Altered hormonal secretion plays an important role in coordinating this transition, including glucose 
and GLP-1-stimulated insulin secretion and suppressed glucagon secretion from the pancreas. GLP-1 suppresses, 
GLP-2 stimulates, and insulin suppresses intestinal chylomicron secretion, whereas insulin and glucagon suppress 
hepatic VLDL secretion. The effect of GLP-1 on hepatic VLDL secretion is currently poorly characterized in humans. 
Emerging data indicate that incretins modulate not only glucose metabolism but also lipid metabolism via 
GLP-1 receptor activation. GLP-1 plays an important role in the assembly and secretion of chylomicrons 
(Figure 1) [16]. Pharmacological augmentation of GLP-1 receptor signaling by GLP-1 agonists or DPP-4 
inhibition reduces intestinal lipoprotein secretion in experimental studies, suggesting that DPP-4 inhibitors 
may ameliorate dyslipidemia and thus improve cardiovascular risk profile in patients with T2DM. [17]. 
Recently we reported that alogliptin (DPP-4 inhibitor) therapy over 16 weeks produced significant reductions 
in postprandial TG and TRL lipoproteins as well as fasting levels of TG, apo-B48 and RLP cholesterol indicating 
that burden of remnant particles is ameliorated in a randomized double-blind placebo controlled study [18]. 
Our data are in line with previous studies showing that vildagliptin, sitagliptin as well as exenatide, a GLP-1 
agonist, reduces postprandial lipemia in type 2 diabetic patients [19-21]. Recently exenatide was shown 
acutely to suppress intestinal lipoprotein production [22]. Likewise Liraglutide was reported to reduce 
postprandial responses of TG and apoB48 after a fat rich meal [23]. However long-term studies are needed 
to elucidate the lipid effects of incretin-based therapies. Thus, the available data suggest that the incretin-
based therapies have a class effect on intestinal lipid metabolism but there may be some difference in the 
efficacy to modulate lipid metabolism.  
Xiao C. et al. Am J Physiol Endocrinol Metab 301:E429-E446, 2011.


3 of 9 
Importantly, GLP-1 receptor expression was recently demonstrated in human hepatocytes and GLP-1 
treatment reduced VLDL production in mice fed a high-fat diet [24]. We reported that alogliptin not only 
reduced postprandial VLDL1 apo-B48 and TG incremental area under curve (iAUC) but also VLDL1 apo-B100 
iAUC. Recently GLP-1 receptor agonists have been reported to decrease hepatic de novo lipogenesis (DNL) 
linked with reduced VLDL production and hepatic steatosis in two different animal models [25,26]. 
Furthermore, we showed that vildagliptin reduced remnant-like lipoprotein particles with a concomitant 
increase in LDL size [27]. These findings suggest an improvement in the metabolism of the TRLs and decrease 
in atherogenic modification of LDL particles. Today, it is not known if incretin-based therapies improve 
reduce production rate of large VLDL particles or their clearance in humans.  
As abnormal concentrations of blood lipids can results from changes in the production, conversion or 
clearance of lipoprotein particle, static measurements of lipid levels do not reveal the underlying mechanism 
of these lipid disorders. Therefore it is necessary to confirm the effects of GLP-1 agonists on the kinetics of 
TG, apo-B48 and apo-B100 in the postprandial state using stable isotopes concomitantly with quantification 
of liver fat to demonstrate how GLP-1 system modulates lipoprotein metabolism in T2DM. Kinetic studies in 
humans provide an important and unique tool to quantitatively elucidate the dynamics of lipid metabolism 
providing estimates of lipoprotein secretion and clearance underlining the pathophysiology of lipid 
disorders. 
We have established and validated novel gas cromatography/mass spectrometry (GC/MS) methods to 
analyze the enrichment of combined load of stable isotopes; 2H5 glycerol, to track enrichments into VLDL-
TG, 2H3leucine incorporated to apo-B48 and apo-B100, 13C4palmitate a to track the contribution of dietary 
lipids to VLDL triglyceride secretion and 2H2O heavy water to quantitate DNL in the VLDL particles released 
by liver (Table 1, Figure 2).  
Table 1. Stable isotopes 
2H5-Glycerol Injected tracer that is esterified to triglycerides in the liver but not the intestine as it lacks 
glycerol kinase activity. It is thus only incorporated into VLDL–triglycerides. 
2H3-Leucine Injected tracer that is incorporated into apoB100 and apoB48. Chylomicrons secreted from the 
enterocytes contain apoB48, while VLDL secreted from the liver contain apoB100. 
13C4-Palmitate The dietary tracer [1,2,3,4-13C]-palmitate will be served in the meal to track the contribution 
of dietary lipids to VLDL–triglyceride secretion. 
2H2O (heavy water) Given orally. Incorporated into all fatty acids. Used to quantify the amount of fatty acids 
derived from liver de novo lipogenesis into VLDL1 and VLDL2. 
 
Figure 2.Outline of a kinetic study that includes three major steps: (i) clinical visit protocol, (ii) sample 
preparation/processing and enrichment analysis by GC/MS, and (iii) mathematical modeling. Subjects ingest 
heavy water and 13C4-Palmitate and receive infusions of 2H5-Glycerol and 2H3-Leucine (Table 1).After GC/MS-
detection of stable isotope enrichments in isolated lipoprotein fractions it is possible to mathematically follow the 
metabolism of both dietary and endogenous derived triglycerides, apoB-100 and apoB-48 and calculate the 
amount of de novo –lipogenesis in TRL fractions. 
Clinical visit
protocol
Sample 
preparation
by density gradient
ultracentrifugation
GC/ MS 
analysis
Mathematical
modelling
Clinical visit
protocol
Sample 
preparation
by density gradient
ultracentrifugation
GC/ MS 
analysis
Mathematical
modelling


4 of 9 
In the development of the model we have utilized the data from 5 subjects who have had a kinetic study (1) 
in the fasting state and (2) repeated together with an oral fat load test. We have established for the first 
time that our protocol allows us to follow the production, transfer and direct catabolism of hepatic (VLDL1 
and VLDL2) and intestinal (chylomicrons) lipoprotein particles and triglycerides in vivo, and to elucidate how 
DNL and dietary lipids contribute to postprandial TRLs. We also have demonstrated that our new non-steady 
state model allows the mathematical modeling of the data.  
RESEARCH HYPOTHESIS 
The aim of this study is to elucidate the mechanisms underlying the positive effect of incretin therapy on 
lipoprotein metabolism in type 2 diabetic subjects and for the first time establish the potential effect on liver 
DNL. Our study design allows differentiating the impact of weight loss dependent and independent effects 
of liraglutide on lipoprotein and carbohydrate metabolism. 
We hypothesize that:  
1. Reversal of the incretin defect by liraglutide will result in reduced production and/or improved 
clearance rate of chylomicrons and remnants 
2. GLP-1 receptor activation in the liver will be associated with decreased hepatic DNL, consequently, 
reduced production of large VLDL1 particles and decrease in liver fat content.  
3. Positive effects of liraglutide onTRL kinetics and liver fat are further potentiated with weight loss.  
TREATMENT  
The study will be single blinded into two groups matched for age, sex and BMI in order to achieve similar 
weight loss in liraglutide and placebo arms. In addition to a fixed dose of metformin (2 g/day) and simvastatin 
(20 mg/day) treatments, patients will be assigned to liraglutide therapy once daily and randomized to 
liraglutide group with uncontrolled diet or to placebo group with weight loss of 3.0 kg supervised by the 
study dietician for 4 months (Figure 3). Previous studies in type 2 diabetes show that the average weight 
loss with liragutide is 3-3.5 kg and stable weight is achieved within 3-3.5 months (LEAD1-6). The study cohort 
will comprise male and female (non-fertile or of child-bearing potential using a medically approved birth 
control method) with T2DM eligible for treatment with metformin or on stable dose of metformin.  
Figure 3.
Study design 
(Number of subjects = 13 in both groups, total 26 together)
Metformin 2 g and 
simvastatin 20 mg run-in
LIRA 1.8 mg, no weight  
intervention, n = 13
Placebo, weight  loss -3.0 
kg, n = 13
3.5 month LIRA 
treatment
Screening
- 1 month 
LIRA/Pbo 
dose escal 2 w
LIRA
0.6 -1.2 -
1.8 mg s.c.
or
Pbo
0.1-0.2-0.3 
ml s.c.
-Non-fasting kinetics
- OGTT
- Heparin test
- Liver and abdominal fat
Studies  repeated month 0 and 4:
= P- amyl-P, P-lipase measurement
LIRA =lLiraglutide, Pbo = Placebo
 
 
INCLUSION CRITERIA 


5 of 9 
Obese subjects with T2D and a large waist (waist > 88 cm in women and > 92 cm in men, BMI 27–40 kg/m2, 
triglycerides between 1.0 – 4.0 mmol/L and LDL < 4.5 mmol/l) treated with a lifestyle or metformin (any 
dose) (HbA1c 6.5-9% ) and aged 30 – 75 years will be included. Subjects may or may not use statins. Only 
subjects who sign an informed consent are included to the study.  
EXCLUSION CRITERIA 
● Type 1 diabetes 
● Apo E2/2 phenotype 
● ALT/AST > 3x ULN 
● GFR < 60 ml/min, clinically significant TSH outside normal range 
● Lipid-lowering drugs other than statins within 6 months 
● Treatment with pioglitazone, insulin, sulphonylureas, gliptines, glinides, SGLT-2 inhibitors or thiazide 
diuretics (at a dose of > 25 mg / day) within 6 months 
● Blood pressure > 160 mmHg systolic and/or > 105 diastolic 
● History of pancreatitis or stomach / other major bleeding, thyroid neoplasia, persistent 
hypothyroidism or persistent hyperthyroidism 
● Any medical condition that puts the patient in the risk of dehydration 
● Concurrent medical condition that may interfere with the interpretation of efficacy and safety data 
during the study.  
● Females of childbearing potential who are not using adequate contraceptive methods 
● Subjects who have experienced side-effects previously from GLP-1 agonists 
● Non-compliance or withdrawal of consent 
● Any information or clinical event decribed in liraglutide SPC that is a contraindication for the use 
of 
liraglutide 
(http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-
_Product_Information/human/001026/WC500050017.pdf) 
 
WHITHDRAWAL FROM THE STUDY 
● Withdrawal of consent 
● Clinical information/condition/adverse event that potentially endangers study subject/study results 
judged by the PI of the study 
● any information or clinical event decribed in liraglutide SPC that is a contraindication for the use 
of 
liraglutide 
(http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-
_Product_Information/human/001026/WC500050017.pdf) 
 
Restrictions during the study:  
● No aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) within 1 week before heparin tests 
(LPL / HL) and biopsies 
● No alcohol or strenuous exercise within 72 hours of each special test 
● Serum amylase and lipase is followed at screening, week 8 and end of liraglutide treatment period 
or at clinical suspicion of pancreatitis 
STUDY VISITS 
All subjects will undergo a screening visit. Before screening, the subjects may visit the study center for 
screening laboratory tests. After giving extensive oral and written information, a written informed consent 
form will be obtained from the subjects at the first visit before screening procedures. Subjects will be 
informed on all adverse events related to all the study procedures they will be submitted to both orally and 
in writing.  
Subjects eligible for the study will enter the run-in phase and treated with Metformin 2 g/day and 
Simvastatin 20 mg/day for 4 weeks. In the treatment period, subjects are randomized to Liragutide or 
placebo.To keep subjects blinded, Liraglutide and placebo will be visually identical and be packed. Liraglutide 
and placebo will be provided by Novo Nordisk A/S. Investigators are not blinded in order to advice subjects 


6 of 9 
in placebo group to lose weight 3 kg during the study. Dose will be escalated to Liraglutide 0.6 mg / placebo 
0.1 ml for one week, Liraglutide 1.2 mg/ placebo 0.2 ml one week and thereafter Liraglutide 1.8 mg / placebo 
0.3 ml s.c. daily injections.  
Thereafter the baseline visits will include an OGTT, heparin test, non-fasting kinetics and abdominal MRI 
investigation days. The investigational product (Liraglutide or placebo) will be given to the subjects 
together with detailed instructions about the usage and storage (Shelf life 30 months. After first use: 1 
month. Storage in a refrigerator (2°C - 8°C). Do not freeze. Store away from the freezer compartment.) 
(in +2- +8 degrees, opened product in +2-+29 degrees) of the product during these visits. After the study 
period of total 4 moths these investigations are repeated. In addition to the screening visit(s), the subjects 
will undergo 8 investigational visits. The subjects meets dietician after randomization. The follow-up about 
weight, diet and possible adverse events takes place with weekly phone calls/e-mail contacts by the study 
nurse/dietician/doctor. Subjects fill in dietary records for 3 days before and during the investigational drug.  
At week 8 after initiation of the treatment period a blood sample for safety is draw, weight is recorded and 
the patient meets dietician/study nurse if necessary. In case of unsuccessful weight loss, weight loss > 1.5 
kg/month or adverse events, an additional visit is arranged. The duration of the study is 1-1,5 month run in 
and 4 to 4,5 month investigational phase i.e. 5 to 6 months. The subjects will be followed by a phone 
call/e-mail/visit 2 weeks after the end of the treatment period. 
STUDY PROTOCOL: We will combine the novel kinetic protocol developed by us with several advanced 
analyses to elucidate how incretins modulate postprandial lipidemia and hepatic DNL in T2D subjects. To 
specifically elucidate the impact of a GLP-1 agonist on postprandial lipid metabolism, we will perform kinetic 
studies on all study subjects before and after the 4-months intervention with the GLP-1 agonist.
 
Figure 4.Detailed schedule for 
lipoprotein kinetic assessment. Red 
dots represent plasma samples for 
kinetic data (glycerol, leucine and 
palmitate 
enrichment 
in 
TRL 
fractions) and green arrows illustrate 
time-points for plasma incretin (GLP-
1, GIP), glucose and insulin sampling.
 
1.Kinetic study with stable isotopes combined with an oral fat load. This study is based on the novel 
protocol developed by us (Figure 4). A detailed description of analysis is given in Appendix A. 
Tracer labelling.On the evening of the first visit, 2H2O (2 g/kg) will be given between 18:00 and 22:00 to 
assess the contribution of de novo lipogenesis to fatty acids in VLDL (Figure 3).At 8:00 the following 
morning, the subjects will receive a bolus injection of 2H5-glycerol (500 mg)to assess the VLDL–triglyceride 
kinetics, and a bolus injection of 2H3-leucine (7 mg/kg) to assess VLDL–apoB100 and chylomicron–apoB48 
kinetics. The fat rich mixed meal (57 g fat, 63 g carbohydrates and 40 g protein, total 934 kcal) with 13C4-
palmitate tracers (1-2 g) is served 2 h after the infusions begins. 
Sampling and sample preparation. Blood samples will be taken at frequent time points as indicated by the 
red dots (Figure 4) and lipoprotein fractions (chylomicrons, VLDL1 and VLDL2) will be separated by density 
gradient ultracentrifugation (DGU). Concentrations of lipids and apolipoproteins (apo-B100, apo-B48) will 
be measured in whole plasma and in all fractions at each time point.  
20h    22h
6h        8h
20h
2H2O (3g/kg)
Test meal with
(1,2,3,4-13C4-) 
palmitate
Bonus injection of
(2H5-) glycerol and (2H3-) leucine
Plasma
VLDL1,
VLDL2,CM
20h    22h
6h        8h
20h
2H2O (3g/kg)
Test meal with
(1,2,3,4-13C4-) 
palmitate
Bonus injection of
(2H5-) glycerol and (2H3-) leucine
Plasma
VLDL1,
VLDL2,CM


7 of 9 
Quantification of lipoprotein fluxes. Enrichments of isotopes in lipids (TG, FAs) and in apo-B100 and apo-
B48 are determined by GC/MS [28] and/or by proteomics [29] at each time point (red dots). Hepatic DNL 
is calculated from enrichment of deuterated water ingested during the kinetic study at specified time 
points (0, 4 and 8 hrs.) [30]. We will use a novel multicompartmental model to describe the fluxes of 
materials combining apoB100 and apoB48 kinetics [7, 28, 29].  
 
2. Determination of liver, subcutaneous and intra-abdominal fat. Using standardized protocols, we will 
perform a proton magnetic resonance spectroscopy (MRS) to determine liver fat content and magnetic 
resonance imaging to determine subcutaneous abdominal and intra-abdominal fat. [31] 
3. Lipolytic enzymes, advanced lipid analysis, fat biopsies and genetic studies. We will analyze mass and 
enzyme activity of LPL and hepatic lipase after an injection of 75 IU/kg heparin. RLP-TG and RLP-cholesterol 
will be measured at 0, 4, and 8 hrs. A fasting sample is obtained for LDL and HDL size determination. 
4. Oral glucose tolerance test. An oral glucose tolerance test will be performed as indicated after overnight 
fast at screening and the end of the 4 months intervention with the GLP-1 agonist. Glucose and insulin will 
be measured at 0,5,10,30,60,90,120,180 and 240 minutes and modeling of β-cell function is performed [32]. 
Samples of markers of energy balance (FFA, β-OHB) will be taken at three time-points (0, 4, and 8hrs) after 
the oral fat load. 
OPTIONAL ANALYSES FOR THE FUTURE STUDIES 
1. Advanced lipid analysis, fat biopsies and genetic studies 
We will also utilize this protocol to collect material for optional analyses in the future not covered by this 
budget. We will perform lipidomics profiling of plasma and isolated lipoproteins by GC/MS technology to 
map lipids and proinflammatory lipid intermediates (with collaborators of principal instigators Matikainen 
and Borén). We will obtain needle biopsies of subcutaneous abdominal fat after fasting to measure mRNA 
levels of proinflammatory cytokines and biomarkers and to analyze lipids in adipose tissue. We will also take 
a blood sample for DNA extraction and identify common single nucleotide polymorphisms in genes that have 
been verified to play a role in lipid metabolism, insulin resistance, inflammation or oxidative stress  
2.Analysis of biomarkers during the oral fat load; apo-CIII, apo-A5, uric acid, inflammatory biomarkers 
(hsCRP, TNF-α, IL-6, SAA, IL-8, PON, MPO, MCP-1) and PAI-1 at three time-points (0, 4, and 8hrs) after the 
oral fat load. Spare samples of plasma/serum for future angiopoetin-like protein 4 and other lipid/metabolic 
parameters is stored at -80 degrees at all time-points during fat rich meal test and OGTT.  
3. Analysis of gut microbiome profile and urine microvesicles. Because obesity and diabetes may induce 
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 
before and after liraglutide treatment. A urinary sample for selective purification of nucleic acids and 
proteins that are potential markers of metabolic state and chronic diseases. 
STATISTICAL METHODS AND POWER 
The study continues until 13 subjects have completed in both arms. Subjects completing the protocol are 
included to the statistical analyses. Paired t-test (within group) and ancova (treatment effect) will be used. 
The number of subjects needed is based on previous studies and are in accordance with a recently published 
article showing that a 25% difference in VLDL kinetics can be detected with 9 subjects per each group [33]. 
PRIMARY OBJECTIVE 
1) To examine the effect of 16 weeks of treatment with liraglutide on triglycerides (TG) and apoB48 
production rates (PR) and/or fractional catabolic rates (FRC) in the lipoprotein fraction chylomicrons 
(Sf>400) in patients with T2DM treated with metformin. This will be fulfilled by kinetic modeling during a 
standardized fat-rich mixed-meal test combined with stable isotope administration. 


8 of 9 
2) To evaluate the for the first time in humans the effect of 16 weeks of treatment with liraglutide compared 
to baseline on hepatic DNL and liver fat content in patients with T2DM treated with metformin, assessed 
during a standardized fat-rich mixed meal test. 
SECONDARY OBJECTIVES  
To evaluate the effect of 16 weeks of treatment with liraglutide compared to baseline in patients with T2DM 
treated with metformin, assessed during a standardized fat-rich mixed-meal test combined with stable 
isotope administration on: 
- 
production rates (PR) of TG, apo-B48 and apo-B100 in the lipoprotein fractions VLDL1 (Sf 60–400) 
and VLDL2 (Sf 20–60)  
- 
fractional catabolic rates (FCRs) of TG, ap-B48 and apo-B100 in the lipoprotein fraction VLDL1 (Sf 60–
400) and VLDL2 (sf 20–60)  
- 
fasting and postprandial serum total TG, total cholesterol, and total apo-B, apo-B48, RLP-chol and 
RLP-TG, apo-CIII and apo-A5 as well as fasting LDL and HDL size. RLP-chol and RLP-TG are surrogate 
markers of remnants. Apo-CIII and apo-A5 are key regulators of the lipolytic cascade. 
- 
TRL clearance assessed as effect on post-heparin lipoprotein lipase (LPL) and hepatic lipase (HL) 
activities. LPL and HL are the key enzymes regulating lipolysis of TRLs and remnant removal by the 
liver. 
- 
fasting and postprandial glucose, insulin and C-peptide, assessed also during an OGTT. 
- 
fasting visceral andsubcutaneous fat depots assessed by MRS in both fasting and in postprandial 
period. 
SAFETY REPORTING 
Any adverse events will be reported to Finnish Medicines Agency (FIMEA) according to local procedures. All 
adverse events will be collected from the first study-related activity (from the signing of the informed 
consent) and in all following contacts with the study-subject through-out the project. This includes events 
from the first trial related activity after the subject has signed the informed consent, and until the post 
treatment follow-up period. The investigators will copy Novo Nordisk Finland Safety department when 
expediting to FIMEA any serious adverse reactions (SAR) or suspected unexpected serious adverse reactions 
(SUSAR) which occurred during the use of liraglutide in the study (appendix B). In addition, all pregnancies 
in trial subjects occurring during use of a Novo Nordisk Product will be reported to Novo Nordisk. The 
submission to Novo Nordisk Finland will be made within 15 days from the investigator’s first knowledge 
about a valid case. 
 
BUDGET 
The study protocol utilizes state-of the art methods to measure the dynamics of postprandial lipemia and 
extensive and complex assessments (including magnetic resonance imaging/spectroscopy, biochemical and 
molecular analyses) all of which need to be performed by skilled personnel. We have calculated an overall 
budget of approximately 574 000€. The budget does not include study medication (liraglutide). The study is 
financed by an independent grant from Novo Nordisk Ag and by other research grants. 
GENERAL CONSIDERATIONS 
The clinical part of the study will be conducted in the research facilities of Research Programs’ Unit, Diabetes 
& Obesity, University of Helsinki; Clinical Research Institute, Huch ltd. Biomedicum Helsinki, Finland. 
Laboratory work and analysis will be made in Helsinki and Gothenburg centers. The study protocol will be 
subjected to the consideration of the Medical Ethical committee of the Helsinki University Central Hospital 
and permission from the Finnish Medicines Agency (Fimea) will be obtained. The study will be conducted 
according to GCP and GLP standards.The subjects taking part to the study are insured by a patient 
insurance taken out by the Helsinki University Central Hospital. 


9 of 9 
Research responsibilities: Niina Matikainen, MD, PhD, Principal Investigator, responsible for clinical part 
including kinetic studies, associated laboratory work, biomarker analyses, data-analyses and  report, 
manuscript preparation, main Clinical Investigator, subject recruitment , adipose tissue biopsies, Kirsi 
Pietiläinen, MD, PhD adipose tissue biopsies and analyses, dietary counseling, data analyses, manuscript 
preparation; Marja-Riitta Taskinen, MD, PhD, responsible for clinical part including kinetic studies, 
associated laboratory work, biomarker analyses, data-analyses and  report, manuscript preparation;  Sanni 
Söderlund, MD, PhD, Clinical  Investigator, screening and recruitment of T2D subjects, kinetic studies, data 
analysis, manuscript preparation; Leonie Boegl, MS dietary counseling and analyses of dietary records; Niina 
Lundblom, MD, PhD and Antti Hakkarainen, MD  imaging and data analysis. 
Team at University of Gothenburg Sweden: Prof. Jan Borén, MD, PhD, Principal Investigator, Project leader 
for kinetic analyses, responsible for all GC/MS work and method validation, data analyses and manuscript 
preparation; Martin Adiels, PhD, responsible for multicompartmental modelling and statistics; Marcus 
Ståhlman, PhD, responsible for lipidomics platform and analysis; Prof. Fredrik Bäckhed, PhD, responsible 
for studies on microbiota 
TIME-TABLE AND PUBLICATION PLAN 
The ethical permission is obtained in January 2014. After permission, the screening will start in February 
2014 and recruitment will continue for a year. First subjects enter the run-in period in March-April 2014 and 
the intervention phase in May 2014. The clinical studies are expected to be completed in June 2015. The 
laboratory and mathematical analysis will be performed in 6 months and the preparation of the 
manuscript(s) will take additional 3-6 months and expected to be submitted to international high-quality 
medical journal(s) in Jan-Jun 2016. All publications will be sent for a courtesy review by Novo Nordisk at 
least 3 weeks prior to submission. Conference abstracts may be submitted before the completion of the 
whole study. The publication policy including the authors appearing in the publication(s) is decided by the 
principal investigators.The investigational data is collected and filed in Research Programs’ Unit, Diabetes & 
Obesity, University of Helsinki; Clinical Research Institute, Huch ltd. Biomedicum Helsinki, Finland according 
to GCP and ethical legislations. 
REFERENCES 
1.  
Chapman MJ, Ginsberg HN, Amarenco P, et al. Triglyceride-rich lipoproteins and high-density lipoprotein 
cholesterol in patients at high risk of cardiovascular disease: evidence and guidance for management. 
EurHeartJ. ; 2011;32:1345-1361. 
2.  
Varbo A, Nordestgaard BG, Tybjaerg-Hansen A, et al. Nonfasting triglycerides, cholesterol, and ischemic 
stroke in the general population. Ann Neurol. ; 2011;69:628-634. 
3.  
Matikainen N, Taskinen M. Postprandial triglyceride-rich lipoproteins in insulin resistance and Type 2 
diabetes. Future Lipidology. ; 2008;3:531-543. 
4.  
Adeli K, Lewis GF. Intestinal lipoprotein overproduction in insulin-resistant states.Curr Opin Lipidol. ; 
2008;19:221-228. 
5.  
Nakajima K, Nakano T, Tokita Y, et al. Postprandial lipoprotein metabolism: VLDL vs chylomicrons. 
ClinChimActa. ; 2011;412:1306-1318. 
6.  
Taskinen MR, Adiels M, Westerbacka J, et al. Dual metabolic defects are required to produce 
hypertriglyceridemia in obese subjects. ArteriosclerThrombVasc Biol. ; 2011;31:2144-2150. 
7.  
Adiels M, Matikainen N, Westerbacka J, et al. Postprandial accumulation of chylomicrons and chylomicron 
remnants is determined by the clearance capacity. Atherosclerosis. ; 2012. 
8.  
Ahren B. Dipeptidyl peptidase-4 inhibitors: clinical data and clinical implications. Diabetes Care. ; 
2007;30:1344-1350. 
9.  
Drucker DJ. Dipeptidyl peptidase-4 inhibition and the treatment of type 2 diabetes: preclinical biology and 
mechanisms of action. Diabetes Care. ; 2007;30:1335-1343. 
10.  
Phillips LK, Prins JB. Update on incretin hormones. Ann N Y AcadSci. ; 2011;1243:E55-74. 
11.  
Holst JJ, Vilsboll T, Deacon CF. Theincretin system and its role in type 2 diabetes mellitus. Mol Cell 
Endocrinol. ; 2009;297:127-136. 


10 of 9 
12.  
Farr S, Adeli K. Incretin-based therapies for treatment of postprandial dyslipidemia in insulin-resistant 
states. CurrOpinLipidol. ; 2012;23:56-61. 
13.  
Nauck MA. Incretin-based therapies for type 2 diabetes mellitus: properties, functions, and clinical 
implications. Am J Med. ; 2011;124:S3-18. 
14.  
Drucker DJ, Sherman SI, Gorelick FS, et al. Incretin-based therapies for the treatment of type 2 diabetes: 
evaluation of the risks and benefits. Diabetes Care. ; 2010;33:428-433. 
15.  
Meier JJ. GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus. Nat Rev 
Endocrinol. ; 2012;8:728-742. 
16.  
Xiao C, Lewis GF. Regulation of chylomicron production in humans.BiochimBiophysActa. ; 2011. 
17.  
Ussher JR, Drucker DJ. Cardiovascular biology of the incretin system.Endocr Rev. ; 2012;33:187-215. 
18.  
Eliasson B, Moller-Goede D, Eeg-Olofsson K, et al. Lowering of postprandial lipids in individuals with type 
2 diabetes treated with alogliptin and/or pioglitazone: a randomised double-blind placebo-controlled 
study. Diabetologia. ; 2012;55:915-925. 
19.  
Matikainen N, Manttari S, Schweizer A, et al. Vildagliptin therapy reduces postprandial intestinal 
triglyceride-rich lipoprotein particles in patients with type 2 diabetes. Diabetologia. ; 2006;49:2049-2057. 
20.  
Tremblay AJ, Lamarche B, Deacon CF, et al. Effect of sitagliptin therapy on postprandial lipoprotein levels 
in patients with type 2 diabetes. Diabetes ObesMetab. ; 2011;13:366-373. 
21.  
Bunck MC, Corner A, Eliasson B, et al. One-year treatment with exenatide vs. insulin glargine: effects on 
postprandial glycemia, lipid profiles, and oxidative stress. Atherosclerosis. ; 2010;212:223-229. 
22.  
Xiao C, Bandsma RH, Dash S, et al. Exenatide, a glucagon-like peptide-1 receptor agonist, acutely inhibits 
intestinal lipoprotein production in healthy humans. ArteriosclerThrombVasc Biol. ; 2012;32:1513-1519. 
23. 
Hermansen K., Baekdal A., During M. et al.Liraglutide suppresses postprandial triglyceride and 
apolipoprotein B48 elevations after a fat-rich meal in patients with type 2 diabetes: a randomized, double-
blind, placebo-controlled, cross-over trial. DiabObesMetab; 2013. 
24. 
Gupta NA, Mells J, Dunham RM, et al. Glucagon-like peptide-1 receptor is present on human hepatocytes 
and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling 
pathway. Hepatology. ; 2010;51:1584-1592. 
25.  
Parlevliet ET, Wang Y, Geerling JJ, et al. GLP-1 receptor activation inhibits VLDL production and reverses 
hepatic steatosis by decreasing hepatic lipogenesis in high-fat-fed APOE*3-Leiden mice. PLoS One. ; 
2012;7:e49152. 
26.  
Panjwani N, Mulvihill EE, Longuet C, et al. GLP-1 Receptor Activation Indirectly Reduces Hepatic Lipid 
Accumulation But Does Not Attenuate Development of Atherosclerosis in Diabetic Male ApoE-/- Mice. 
Endocrinology. ; 2013;154:127-139. 
27.  
Matikainen N, Taskinen MR. The effect of vildagliptin therapy on atherogenic postprandial remnant 
particles and LDL particle size in subjects with Type 2 diabetes. Diabet Med. ; 2012. 
28.  
Adiels M, Packard C, Caslake MJ, et al. A new combined multicompartmental model for 
apolipoprotein B-100 and triglyceride metabolism in VLDL subfractions.J Lipid Res 2005, 46:58-67. 
29. 
Lee AY, Yates NA, Ichetovkin M, et al. Measurement of fractional synthetic rates of multiple 
protein analytes by triple quadrupole mass spectrometry.Clin Chem 2012, 58:619-627. 
30. 
Matikainen N, Adiels M, Söderlund S, et al. Hepatic de novo lipogenesis and fat oxidation 
 
predict the postprandial response of triglyceride rich lipoprotein particles in healthy 
 
subjects. BBA Lipids, Submitted 2013. 
31.  
Lundbom J, Hakkarainen A, Fielding B, et al. Characterizing human adipose tissue lipids by long echo 
 
time (1)H-MRS in vivo at 1.5 Tesla: validation by gas chromatography. NMR Biomed. ; 2010. 
32 
Mari A, Pacini G, Murphy E, et al. A model-based method for assessing insulin sensitivity from the oral 
glucose tolerance test.Diabetes Care. ; 2001;24:539-548. 
33.  
Magkos F, Fabbrini E, Korenblat K, et al. Reproducibility of glucose, fatty acid and VLDL kinetics and multi-
organ insulin sensitivity in obese subjects with non-alcoholic fatty liver disease.Int J Obes (Lond). ; 
2011;35:1233-1240.