Conditions: Type 2 Diabetes
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 40
Healthy volunteers: no

Brief summary:
This is a single center, prospective randomized double blind, parallel and placebo controlled study to evaluate oxidative stress and inflammation before and after treatment with linagliptin for 12 weeks. We will also, testing whether Linagliptin is an insulin sensitizer.

Detailed description:
This is a single center, prospective randomized double blind, parallel and placebo-controlled study to evaluate oxidative stress and inflammation before and after treatment with linagliptin for 12 weeks. The study enrolls type 2 patients with diabetes.

Arms:
arm 1: Patients will be treated for 12 weeks with placebo once daily arm 2: Patients will be treated for 12 weeks with Linagliptin 5mg once daily.

Interventions: intervention 1: Linagliptin intervention 2: Placebo
Intervention descriptions: intervention 1: 5 mg daily oral tablets of linagliptin intervention 2: matching placebo giving orally daily

Protocol document:
Prot_SAP_000.pdf: 
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1
THE EFFECT OF TRADJENTA ® (LINAGLIPTIN) ON 
INFLAMMATION, OXIDATIVE STRESS AND INSULIN 
RESISTANCE IN OBESE TYPE 2 DIABETIC SUBJECTS 
 
 
RESEARCH PROTOCOL 
Division of Endocrinology, Diabetes and Metabolism 
State University of New York at Buffalo and Kaleida Health 
115 Flint Road 
Williamsville, NY 14221 
 
 
INVESTIGATOR: 
Paresh Dandona, MBBS, PhD 
 
Co Investigators:    Antoine Makdissi, MD 
 
            Husam Ghanim, PhD 
 
            Ajay Chaudhuri, MBBS, MRCP 
 
 
Version:  
May 22, 2014 
 
 
 
Correspondence To: 
Paresh Dandona, M.B.B.S., Ph.D., F.R.C.P, F.A.C.P, F.A.C.C 
Director, Diabetes-Endocrinology Center of Western NY 
Chief of Endocrinology, SUNY at Buffalo 
115 Flint road, Williamsville, NY 14221 
Telephone: (716) 626-7970 
Fax: (716) 626-7990 
 
E-mail: pdandona@kaleidahealth.org 
 


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Over the past decade, following the initial suggestion that pharmacological 'harnessing' of the in vivo 
stability of GLP-1 may be a novel approach to the management of type 2 diabetes(1), two separate 
strategies to overcome the inherent drawbacks in using native GLP-1 have been investigated by academic 
laboratories and the pharmaceutical industry alike (2, 3). The outcome of this research has been the design 
of long-acting, DPP IV-resistant analogs, and/or derivatives of GLP-1, suitable for exogenous 
administration, and the development of inhibitors of DPP IV to enhance the endogenously released 
hormone. 
 
Linaglitpin and other DPP-IV inhibitors reduce blood glucose concentrations by stimulating c-AMP 
dependent insulin release from the  cell, suppressing post prandial glucagon increase and by slowing 
gastric emptying. Linagliptin is a potent, long- acting, competitive, reversible inhibitor of DPP IV. 
Linagliptin once daily increases postprandial plasma intact GLP-1 levels by 1.5-to 3-fold compared with 
placebo. Linagliptin also decreases postprandial glucagon levels in patients with type 2 diabetes during 
12 weeks of therapy, thereby lowering fasting glucose concentrations and reducing glucose excursions 
after a meal. Linagliptin has been studied as a monotherapy in drug- naïve patients with type 2 diabetes, 
and in combination with other glucose lowering medications. It is not yet known if linagliptin is anti-
inflammatory. 
 
Obesity, diabetes and inflammation  
Both obesity and diabetes have been shown to be associated with oxidative stress (4-7) and inflammation 
(8-12). One possible reason why obesity and type 2 diabetes are associated with oxidative stress and 
inflammation is the state of insulin resistance. This is due to the fact that 1) resistance is associated with 
the presence of pro-inflammatory factors including cytokines (13); 2) insulin has been shown to exert an 
anti-inflammatory and anti-oxidant effects at the cellular and molecular level both, in vitro, and in vivo 
(14-17). A low dose infusion of insulin (2 IU/h) has been shown to reduce ROS generation by mononuclear 
cells (MNC), suppress NADPH oxidase expression and intranuclear NFB binding, induce IB 
expression and suppress plasma ICAM-1 and MCP-1 concentrations (17). It also suppresses intranuclear 
Egr-1, plasma tissue factor (TF), PAI-1 and MCP-1 concentrations (14). An interruption of insulin signal 
transduction would prevent the anti-inflammatory effect of insulin from being exerted. The anti-
inflammatory and pro-fibrinolytic effects of insulin have also been confirmed in the setting of acute ST 
segment elevation myocardial infarction (18).  We have previously demonstrated that macronutrient intake 
(glucose, protein and lipids) induce ROS generation and cause oxidative stress in humans. Glucose or a 
fast food mixed meal intake cause an increase in NFB and a fall in total cellular IB, the two cardinal 
indices of inflammation at the cellular level (19-21). Mixed fast food meal also induces an increase in C-
reactive protein (CRP) (19).  Glucose intake has further been shown to increase the ratio of phosphorylated 
IB to native non-phosphorylated IB and to induce an increase in IB kinase (IKK) and IKK. At 
a cellular level, the changes that occur in the MNC following a glucose challenge are very similar to those 
which occur after endotoxin challenge.  
It is possible that obesity is a pro-oxidative and pro-inflammatory state resulting from chronically 
increased macronutrient intake. An increasing amount of epidemiologic data show that persistent, low-
grade inflammation is an independent predictor of ischemic heart disease (22, 23), stroke (24, 25), diabetes 
(26, 27), and all-cause mortality (28, 29). In addition to these epidemiologic findings, experimental 
evidence shows that markers of chronic inflammation, such as pro-inflammatory cytokines IL-6 and TNF-
 and the acute-phase reactant CRP, play a direct role in the etiology of atherosclerosis and insulin 
resistance (30, 31). Diet-induced weight loss decreases concentrations of CRP, IL-6, and TNF-  (8, 32-
37). It is also relevant that caloric restriction in the obese and a fast in normal subjects result in a reduction 


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3
in oxidative stress and inflammatory mediators (5, 8, 38) In this regard, it is interesting to note that TNF-
 and IL-1 have been shown to reduce hypothalamic GnRH and LH secretion in animals and in vitro.(39, 
40) It is therefore possible that inflammatory cytokines may partly mediate hypogonadotrophic 
hypogonadism (HH) of obesity and type 2 diabetes. 
  
Measuring Inflammation and Oxidative Stress 
Inflammation at the cellular level can be described as an increase in the pro-inflammatory transcription 
factor, NFB, in the nucleus and with a concomitant decrease in its inhibitors IB and/or IB. NFB 
is a heterodimer, and usually consists of two proteins, a p65 (RelA) subunit and a p50 subunit. In the basal 
state NFB is bound to its inhibitor protein IB, which restricts NFB to the cytoplasm. Stimulation of 
cells by cytokines like TNF or IL-1 or endotoxin results in phosphorylation of IB, unbinding of NFB 
from IB and activation of NFB with its subsequent translocation into the nucleus (41, 42). This in turn 
induces the transcription of pro-inflammatory cytokines (such as TNF, IL-1 and IL-6), adhesion 
molecules (ICAM, VCAM), chemokines (such as MCP-1 and MIF), adhesion molecules, 
metalloproteinases (MMP-1, and MMP-9) and many other genes that regulate transcription, apoptosis and 
cell proliferation. 
  Oxidative stress can be defined as an increase in ROS generation. The term ROS is a collective term that 
includes not only oxygen-centered radicals such as superoxide (O·2−) and hydroxyl (OH), but also some 
non-radical derivative of oxygen such as hydrogen peroxide (H2O2), singlet oxygen and hypochlorous 
acid (HOCL). A critical balance exists between generation and detoxification of ROS in respiring cells of 
normal individuals. The various risk factors for atherosclerosis, including hyperlipidemia, hypertension, 
and diabetes, have in common the generation of oxidative stress. ROS are able to break off cell membrane 
proteins, fuse membrane lipid and proteins, hardening the cell membrane and exposing genetic material 
in the nucleus, leaving the DNA open for mutation or destruction. They may damage the endothelium of 
blood vessels, thereby leading to atherosclerosis. ROS-mediated lipid peroxidation is a cardinal step in 
the initiation of atherosclerosis (43, 44).  NFB is one of the transcription factors that may be controlled 
by the redox status of the cell (45). NADPH oxidase complex, composed of cytoplasmic (p47phox p67phox, 
small GTP-binding p21rac) and plasma membrane components (heterodimer of gp91 & p22phox)  is a key 
enzyme involved in the production of ROS.(46)  
Lipid Peroxidation and Protein Oxidation: Lipid peroxidation is an autocatalytic free radical-mediated 
chemical mechanism in which polyunsaturated fatty acids (PUFA) undergo oxidation to form lipid 
hydroperoxide (LHP) (47, 48). Levels of 15-isoprostane F2 in urine are useful for the non-invasive 
assessment of oxidant stress in vivo (49). There is evidence that oxidative modification of proteins may 
be physiologically important, serving as a “marking” step for initiating protein degradation. Oxidation of 
proteins by microsomal oxidases and metal-catalyzed oxidation treatment is known to produce carbonyl 
modifications of amino acids (50). Phe reacts with the hydroxyl radical (OH) to form the hydroxylated 
products ortho-, meta-, and para-tyrosine (o-, m-, and p-tyr). The aromatic hydroxylation of Phe can be 
used to detect OH formation and is considered as a marker of protein oxidation (51).  
 
Incretins and Inflammation 
 Use of exenatide in type 2 diabetes patients on insulin has shown very promising and exciting results. We 
studied the effect of exenatide in an observational study in our clinic patients (52). We found that in 
addition to improvement in glycemic control over 12 weeks, levels of CRP fell by 30% in patients. A 
reduction of 25-30% in fast acting insulin requirements were also observed in patients along with a 
significant weight loss. This study, for the first time, showed the potential anti-inflammatory action of 
synthetic incretins. We then did a randomized prospective placebo controlled blinded trial in patients with 


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4
type 2 diabetes(53). Type 2 diabetic patients on insulin received exenatide or placebo for 12 weeks. There 
was a significant reduction in reactive oxygen species generation and nuclear factor-κB binding by 22 and 
26%, respectively after use of exenatide. The mRNA expression of TNFα, IL-1β, JNK-1, TLR-2, TLR-4, 
and SOCS-3 in mononuclear cells also decreased by 16-31%. We also showed that a single injection of 
exenatide has an anti-inflammatory effect.  
 
DPP-IV and inflammation 
Recent advances in the understanding of DPP IV have provided new insights into the link between 
inflammation and DPP IV. It has been found to have a role in activation and function of T cells. DPP IV 
serves as a target of efficient enzymatic inhibitors which induce autocrine production of TGF- β1 and 
subsequent suppression of T cell proliferation and cytokine release (54). In vivo, targeting DPP IV has 
provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, 
an animal model of multiple sclerosis.  It has been suggested that DPP IV inhibition of pathogenic T cells 
represents a novel and efficient therapy for autoimmune disease of the central nervous system by a 
mechanism that involves an active TGF-β1 mediated anti-inflammatory effect at the site of pathology. 
  We investigated the anti-inflammatory effects of sitagliptin in insulin naïve patients with type 2 
diabetes. Twenty-two patients with type 2 diabetes were randomized to receive either 100 mg daily of 
sitagliptin or placebo for 12wk (55). Fasting glucagon-like peptide-1 concentrations increased 
significantly, whereas the mRNA expression in mononuclear cell of CD26, the proinflammatory cytokine, 
TNFα, the receptor for endotoxin, Toll-like receptor (TLR)-4, TLR-2, and proinflammatory kinases, c-
Jun N-terminal kinase-1 and inhibitory-κB kinase (IKKβ), and that of the chemokine receptor CCR-2 fell 
significantly after 12 wk of sitagliptin. TLR-2, IKKβ, CCR-2, and CD26 expression and nuclear factor-
κB binding also fell after a single dose of sitagliptin. There was a fall in protein expression of c-Jun N-
terminal kinase-1, IKKβ, and TLR-4 and in plasma concentrations of C-reactive protein, IL-6, and free 
fatty acids after 12 wk of sitagliptin. These effects are consistent with a potent and rapid anti-inflammatory 
effect of sitagliptin and may potentially contribute to the inhibition of atherosclerosis. The suppression of 
CD26 expression suggests that sitagliptin may inhibit the synthesis of DPP-IV in addition to inhibiting its 
action. This contributes to lower GLP-1 concentrations in plasma after such meals. Therefore, it would be 
extremely interesting to determine what effect linagliptin has on GLP-1, GIP, and c-peptide 
concentrations. 
 
Insulin Sensitization by Linagliptin 
  As mentioned above, sitagliptin exerts a potent and rapid anti-inflammatory effect as reflected in the 
suppression of ROS generation and intranuclear NFκB binding(55). In association with this action, we 
also observed the suppression of the expression of SOCS-3, a protein whose expression is induced by the 
pro-inflammatory cytokines, TNFα, IL-1β and IL-6 and which interferes with insulin signal transduction. 
This interference occurs at the IRS-1 level. The expression of SOCS-3 has been shown to be increased in 
the obese and is related to BMI and HOMA-IR, NFκB activity and TNFα expression(56). Furthermore, 
SOCS-3 is also induced by a high fat high carbohydrate meal in parallel with the increased expression of 
TNFα and IL-1β (57). More recently, it has been shown that weight loss following gastric bypass surgery 
is associated with a reduction not only in inflammatory indices but also that of SOCS-3 expression (58). 
Protein phosphotyrosine phosphatase-1B (PTP-1B) is another key protein which dephosphorylates 
proteins and interferes with insulin and leptin signaling and is also induced by the HFHC meal (59, 60). 
Indeed, a recent study has shown that Sitagliptin treatment for 6 weeks in patients with type 2 diabetes 
increases insulin sensitivity by 10% (measured as oral glucose insulin sensitivity during a meal test) (61). 
The same study also showed that Sitagliptin decreases endogenous glucose production in fasting as well 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
5
as post-prandial state by ~5%.While the effect of Sitagliptin on endogenous glucose output is probably 
caused by glucagon suppression, the mechanism underlying its insulin sensitizing effect is not known. 
  On the basis of the above, we suggest that sitagliptin and other DPP-IV inhibitors like linagliptin could 
potentially increase insulin signal transduction and increase insulin sensitivity by suppressing 
inflammatory mediators of insulin resistance including SOCS-3 and PTP-1B. 
  Therefore, in a study directed to investigate the potential anti-inflammatory effect of linagliptin, we 
would like to also test whether linagliptin exerts an insulin sensitizing effect. The methodology used will 
be based on the measurement of insulin sensitivity by hyperinsulinemic euglycemic clamps before and 
after the institution of linagliptin treatment in patients with type 2 diabetes.  
      
Rationale for study 
Linaglitpin has been shown to result in better glycemic control in type II diabetes patients. As described 
above, obesity and diabetes are states of increased inflammation and are also major risk factors for 
atherosclerotic disease. In view of the data suggesting that DPP-IV is a pro-inflammatory mediator and 
that its inhibition may suppress autoimmune inflammatory mechanisms, it is likely that linagiptin may 
exert an anti-inflammatory effect in the human. The increase in GLP-1 concentrations may also exert an 
anti-inflammatory effect similar to that observed with exenatide. Indeed, our investigations on the anti-
inflammatory effects of sitagliptin, the first DPP-IV inhibitor to be licensed, have demonstrated profound 
evidence to this effect. Our findings are summarized under preliminary data below. 
Preliminary data: 
In a study on obese type 2 diabetics, the administration of sitagliptin 100 mg daily for 12 weeks resulted 
in several novel observations. There was an inhibition of DPP-IV enzyme by 90% within 2 hours of the 
first dose. In addition, there was a suppression of CD26 expression. CD26 is a pro-inflammatory mediator 
expressed on the cell membranes and is the cellular analog of the DPP-IV enzyme. Thus, sitagliptin and 
possibly other DPP-IV inhibitors may also inhibit the expression and synthesis of DPP-IV. In addition, 
there was a reduction in plasma concentrations of FFA, CRP and IL-6; a reduction in the expression of 
CCR-2, IKKβ, JNK-1, TNFα (see figures below).  
 
.
Time
0h
2h
4h
6h
2W
4W
8W
12W
DPP-IV Activity (RFU/sec)
0
1
2
3
4
5
6
7
8
9
10
11
Sitaglibtin
Placebo
*#
*#
*#
*#
*#
*#
*#
Time (Weeks)
0
2
4
8
12
GLP-1  Plasma Concentration (% Change)
6
7
8
9
10
11
12
13
14
15
Placebo
Sitagliptin
*#
*#
*
 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
6
Time (Weeks)
0
2
4
8
12
CD26 mRNA expression (% Change)
65
70
75
80
85
90
95
100
105
110
115
120
125
Placebo
Sitagliptin
*#
*#
 
 
Time (Weeks)
0
2
4
8
12
JNK-1 mRNA expression (% Change)
60
70
80
90
100
110
120
Placebo
Sitagliptin
*#
*#
 
Time (Weeks)
0
2
4
8
12
TNF mRNA expression (% Change)
40
50
60
70
80
90
100
110
120
130
Placebo
Sitagliptin
*#
*#
*#
 
Time (Weeks)
0
2
4
8
12
TLR4 mRNA expression (% Change)
50
60
70
80
90
100
110
120
130
Placebo
Sitaglibtin
*#
*#
Time (Weeks)
0
2
4
8
12
CCR2 mRNA expression (% Change)
60
70
80
90
100
110
120
Placebo
Sitagliptin
*#
*#
 
Figure 1: Change in DPP-IV activity (A), 
GLP-1 Concentrations (B) and CD26 
mRNA in MNC (C) following placebo and 
Sitagliptin 100mg daily for 12 weeks in type 
2 diabetic subjects.  N=12 each, data are 
presented as mean±SE. (*, **:P<0.05 by 
RMANOVA (compared to baseline) in 
sitagliptin and placebo groups, respectively, 
#:P<0.05 
by 
Two-way 
RMANOVA 
compared to control groups. 


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7
Time (Weeks)
0
2
4
8
12
IKK mRNA expression (% Change)
60
70
80
90
100
110
120
130
Placebo
Sitaglibtin
*#
*#
  
Time (Weeks)
0
2
4
8
12
CRP Plasma Concentration (% Change)
60
65
70
75
80
85
90
95
100
105
110
115
120
Placebo
Sitagliptin
*#
*#
*#
Time (Weeks)
0
2
4
8
12
FFA Plasma Concentration (mM)
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Placebo
Sitagliptin
*#
*#
*#
 
Time (Weeks)
0
2
4
8
12
IL-6  Plasma Concentration (% Change)
60
65
70
75
80
85
90
95
100
105
110
115
120
Placebo
Sitagliptin
*#
*#
*#
  
 
 
 
 
 
Figure 2: Percent Change in mRNA 
expression of JNK-1(A), TNF-, (B) 
TLR-4 (C), CCR-2 (D) and IKK- (E) in 
MNC following placebo and Sitagliptin 
100mg daily for 12 weeks in type 2 
diabetic subjects.  N=12 each, data are 
presented as mean±SE. (*, **:P<0.05 by 
RMANOVA (compared to baseline) in 
sitagliptin 
and 
placebo 
groups, 
respectively, #:P<0.05 by Two-way 
RMANOVA 
compared 
to 
control 
groups. 
Figure 3: Change in CRP (A), FFA (B) 
and IL-6 (C) concentrations following 
placebo and Sitagliptin 100mg daily for 
12 weeks in type 2 diabetic subjects.  
N=12 each, data are presented as 
mean±SE. 
(*, 
**:P<0.05 
by 
RMANOVA (compared to baseline) in 
sitagliptin 
and 
placebo 
groups, 
respectively, #:P<0.05 by Two-way 
RMANOVA 
compared 
to 
control 
groups. 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
8
 
Time (Weeks)
0
4
8
12
JNK-1 protein in MNC (% Change)
50
60
70
80
90
100
110
120
130
Placebo
Sitagliptin
*#
*#
 
 
Figure 4: Representative Western blot and percent change in protein levels of JNK-1 after 12 wk of placebo and 
sitagliptin (100 mg/d) treatment in MNC of obese type 2 diabetic subjects (n _ 10 and 12, respectively). Data are 
presented as mean ±SE. *, P <0.05 by RMANOVA (compared with baseline); #, P <0.05 by two-way 
RMANOVA compared with placebo groups. 
 
 
Study Hypothesis and Aims 
 
Hypothesis 1: Linagliptin treatment suppresses basal and meal induced oxidative and inflammatory stress 
in mononuclear cells (MNC) and adipose tissue of obese patients with type 2 diabetes.  
Aim 1.1: To compare reactive oxygen species (ROS) generation by MNC, protein expression of p47phox 
subunit of NADPH oxidase, in MNC’s and adipose tissue prior to and following 12 weeks of linagliptin 
or placebo.  
Aim 1.2: To compare intranuclear NFκB binding and the expression of JNK-1, TLR-4, TLR-2, CD26 and 
TNFα in mononuclear cells (MNC)  and adipose tissue before and following 12 weeks of treatment with 
linagliptin or placebo. 
Aim 1.3: To compare plasma hs-CRP and IL-6 concentrations and oxidized lipids in plasma (TBARS) 
and in urine (F2-isoprostane) of obese type 2 diabetic patients prior to and following 12 weeks of 
linagliptin or placebo.  
Aim 1.4: To compare post prandial (after a HFHC meal) changes in ROS generation, NFκB binding and 
the expression of JNK-1, TLR4, TLR-2 IL-1 and TNFα in MNC and adipose tissue prior to and following 
12 weeks of linagliptin and placebo. 
 
Hypothesis 2: Linagliptin acutely suppresses oxidative and inflammatory stress in mononuclear cells 
(MNC) of obese patients with type 2 diabetes. 
Aim 2.1: to study the effect of single dose of linagliptin or placebo on oxidative stress and inflammatory 
mediators listed in Aim 1.1, 1.2 and 1.3 in MNC from obese type 2 diabetic patients. 
 
Hypothesis 3: Linagliptin is an insulin sensitizer 
Aim 3.1: To compare the glucose uptake induced by hyperinsulinemic-euglycemic (HE) clamp prior to 
and following 12 weeks of linagliptin or placebo.  
Aim 3.2: To compare the endogenous glucose output and suppression of endogenous glucose production 
by insulin during HE clamp prior to and following 12 weeks of linagliptin or placebo. 


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9
Aim 3.3: To compare mRNA and protein expression of SOCS-3 and PTP-1B, two important insulin 
resistance inducers, in MNC and adipose tissue prior to and following 12 weeks of linagliptin or placebo. 
 
Hypothesis 4: Linagliptin increases post-prandial GLP-1, GIP and c-peptide concentrations. 
Aim 4.1: To measure GLP-1, GIP and c-peptide concentrations following HFHC meal prior to and 
following 12 weeks of linagliptin or placebo. 
 
 
RESEARCH DESIGN AND METHODS 
 
RESEARCH DESIGN: 
This is a prospective, randomized, placebo -controlled, double blind trial. The study will be 
conducted at the Diabetes – Endocrinology Center of Western New York under the direction of Dr. 
Paresh Dandona, M.D.  
 
STUDY POPULATION: 
Forty type 2 diabetic patients in the age group of 20 – 80 years will be recruited into the study. An 
informed consent will be obtained. They will be randomized into 2 groups: placebo and 5 mg/day 
linagliptin for 12 weeks 
 
INCLUSION CRITERIA: 
 
▪ Age 20-80 years inclusive. 
▪ Type 2 diabetes 
▪ BMI ≥30 kg/m2 
▪ Subjects on statins, ACE inhibitors, thiazolidenediones and antioxidants will be allowed as long 
as they are on stable doses of these compounds and the dosage in not changed during the course 
of study. 
▪ HbA1c ≤ 7.5% 
 
EXCLUSION CRITERIA: 
▪ Use of GLP-1 agonists or DPP-IV therapy in the last 3 months. 
▪ History of Pancreatitis. 
▪ Risk for pancreatitis, i.e., history of gallstones, alcohol abuse, and hypertriglyceridemia. 
▪ Subjects who are taking antiplatelet or anticoagulant medications will be excluded from the fat 
biopsy portion of the study. 
▪ Coronary event or procedure (myocardial infarction, unstable angina, coronary artery bypass, 
surgery or coronary angioplasty) in the previous four weeks 
▪ Hepatic disease (liver function tests more than 3 times the upper limit of normal) 
▪ Renal impairment (serum eGFR <30 ml/min) 
▪ Any other life-threatening, non-cardiac disease 
▪ Uncontrolled hypertension (BP > 160/100 mm of Hg) 
▪ Congestive Heart Failure class III or IV. 
▪ Use of an investigational agent or therapeutic regimen within 30 days of study 


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10
▪ Participation in any other concurrent clinical trial  
 
 
 
STUDY DESIGN: 
 
This study requires 8 visits to the clinic: a screening visit (week -1), a baseline visits (weeks 0, 1 and 2) 
and then follow up at 2, 4 and 12 weeks after starting the drug. Each visit will consist of blood draw. 
Subjects will be required to bring in a 24 hour urine sample on weeks 0, 4 and 12. 
After the screening visit, subjects will be randomized into 2 groups. The first group will be started on 5 
mg linagliptin (supplied by Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI)) daily for 12 weeks. 
Fructosamine testing will be done at baseline, 4 and 12 weeks (and HbA1c at 0 and 12 weeks) to assess 
glycemic control. The second group will serve as a control group and will be placed on a placebo (mainly 
mannitol and corn starch, provided by BIPI in similar shape and color as the active drug) for 12 weeks. 
Blood samples will be obtained as described for patients on linagliptin. Each blood draw will consist of 
45 ml of which 35 ml is collected in EDTA tubes and 10 ml in no-additive tubes. A single dose effect will 
be investigated at the first day of drug or placebo in 24 subjects (12 from each group) only and blood will 
be collected at 2, 4 and 6 hr following the first dose. The effect of a standard high-fat high- carbohydrate 
meal (HFHC) will be investigated on those 12 subjects from each group at baseline and at 12 weeks.  
 
 
Figure 1: Study Design.   
 
STUDY PROCEDURES BY VISIT: 
Screening Day  
Each subject will complete the following procedures and meet the inclusion and exclusion criteria prior 
to participating in the study.  
1. Medical History 
2. Physical Exam 
3. Informed consent 
4. Baseline lab draws to measure CBC, SMA, lipid profile, fructosamine and HbA1C 
 
Weeks: 
A, B, C 
BC 
UC 
FB 
BC 
UC 
FB 
Groups assigned 
Single dose effect  
Meal Challenge, HE 
clamp. 
2 
4 
12 
-1 
 
BC 
    BC 
    UC 
   
      
BC –Blood Collection 
UC – Urine Collection 
FB- Fat Biopsy 
Meal Challenge, 
HE clamp.  
Screening & 
Physical Exam 
 


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11
Visit 1: Week -2 to Week 0 
Visit 1A: The participants will undergo HE clamp. They will be advised to bring 24 hour urine at this visit 
and research blood will be collected. All patients will have visit A. Visits B and C will be done only in 24 
patients (12 in drug group and 12 in placebo group). 
Visit 1B: The effect of a standard high-fat high- carbohydrate meal (HFHC) will be investigated in 24 
subjects.   Fat biopsy will be done before and after the meal.  
Visit 1C (in 24 subjects): A single dose (5 mg or placebo) effect will be studied in 24 subjects with blood 
collections at 2, 4 and 6 hr following the first dose. 
 
Visits A, B and C will be separated by one week each. All patients will be randomized to drug or placebo 
groups on visit 1A but only patients who are not participating in visit 1B and 1C will start the drug or 
placebo at the end of visit 1A (considered week 0). Patients who are participating in visits 1B and 1C will 
start the drug or placebo at the beginning of visit 1C (considered week 0). . Patient will return 2 weeks 
after starting drug or placebo. The drug will be started after these visits have been completed. Patient will 
return after 2 weeks. 
 
Visit 2: Week 2 
The participants will have their finger stick blood glucose records reviewed and blood will be drawn for 
labs. A container for 24 hour urine collection will be given. They will be advised to start collecting urine 
24 hours prior to their next visit. 
 
Visit 3: Week 4 
The participants will have their finger stick blood glucose records reviewed and blood will be drawn for 
labs. 24 hour urine will be collected. They will be advised to start collecting urine 24 hours prior to their 
next visit. 
 
Visit 4: Week 12 
 
Visit 4A: The participants will have their finger stick blood glucose records reviewed. They will be advised 
to bring 24 hour urine at this visit and research blood will be collected. The participants will undergo HE 
clamp. All patients will have visit 4A.  
Visit 4B: The effect of the HFHC meal will be investigated in 24 subjects who participated in visit 1B. Fat 
biopsy will be done before and after the meal.  
Visits 4A and 4B will be separated by 2-5 days. 
 
Compliance 
The study coordinator will maintain a log of the entire study drug dispensed and returned.  
Storage conditions:  stored at room temperature in a locked cabinet. 
 
Drug accountability: A master drug accountability log will be completed on arrival of supplies, Patient 
specific drug tracking logs will be completed including the following information:  Date and amount of 
drug dispensed, initials of person dispensing study drug, date and amount of drug returned initials of 


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12
person receiving returned study drug. 
 
 
Meal Challenge description 
Subjects will arrive after having fasted overnight (10 hr) around 8-8:30AM. Subcutaneous fat biopsy will 
be done. The subjects will eat a meal that consists of 900 kcal of HFHC breakfast (egg muffin and sausage 
muffin sandwiches and two hash browns which contain 81g carbohydrates, 51g fat and 32 g protein).  
Meal must be consumed within 15 minutes.  Blood samples will be obtained at baseline (before the meal) 
and at the following time points after the meal is consumed: 15min, 30min, 45min, 1hr, 75min, 90min, 
105min, 2hr, 3hr, and 5 hr.  Samples will measure fasting and post meal glucose, insulin, glucagon, GLP-
1, GIP, c-peptide in plasma, and indices of inflammatory stress, specifically intra-nuclear NFĸB, 
expression of TNFα, and JNK-1 in MNC.  Blood will be collected from an intravenous cannula in a 
superficial forearm vein. Samples at 15, 30, 45, 75, 90, 105, and 120 min will each be 5mL. Samples at 
baseline (0h) and 1hr, 3hr and 5hr are 35mL each. The total amount of blood drawn during this meal 
challenge is 175mL. Fat biopsy will be repeated after 5 hours. Samples for GLP-1, GIP and c-peptide 
measurement will be collected in DPP-IV inhibitor and protease inhibitors containing tubes. 
 
 
 LABORATORY PROCEDURES  
 
1. MNC isolation: Blood samples (30-40ml) is collected in Na-EDTA and carefully layered on 
Lympholyte medium (Cedarlane Laboratories, Hornby, ON) according to manufacturer’s instructions. 
Samples are centrifuged and two bands separate out at the top of the RBC pellet. The MNC band was 
harvested and washed twice with Hank's balanced salt solution (HBSS). This method provides yields 
greater than 95% MNC preparation. 
2. ROS generation measurement by chemiluminescence: Five hundred L of MNC (2 x 105 cells) are 
delivered into a Chronolog LumiAggregometer cuvette. Luminol is then added, followed by 1.0 L 
of 10 mM formylmethionyl leucinyl phenylalanine (fMLP).  Chemiluminescence is recorded for 15 
minutes. Our method, developed independently(62, 63), is similar to that published by Tosi and 
Hamedani (64). In this assay system, the release of superoxide radical as measured by 
chemiluminescence, has been shown to be linearly correlated with that measured by the 
ferricytochrome C method.  The interassay coefficient of variation of this assay is 8 %. We have 
further established that the biological variation in reactive oxygen species generation in normal 
subjects is approximately 6 % for readings obtained 1 to 2 weeks apart. Similarly, the variation in 
reactive oxygen species generation in the obese over a period of 1 to 2 weeks is less than 8 %. 
3. Quantification of IKKβ, JNK-1, TNFα, IL-6, IL-1, TLR4, TLR2, SOCS-3, PTP-1B, CCR-2 and 
CD-26 Expression: The mRNA expression is measured in MNC and adipose tissue by RT-PCR: 
Total RNA was isolated using commercially available RNAqueous®-4PCR Kit (Ambion, Austin, 
TX). One µg of total RNA is reverse transcribed using Advantage RT-for-PCR Kit (Clontech, CA). 
Real Time RT-PCR was performed using Stratagene Mx3000P QPCR System (La Jolla, CA), 
Sybergreen master mix (Qiagen, CA) and gene specific primers (Life Technologies, MD).The 
specificity and the size of the PCR products are tested by adding a melt curve at the end of the 
amplifications and by running it on a 2% agarose gel. All values were normalized to the expression 
of a group of housekeeping genes including actin, ubiquitin C and cyclophilin A. 
4.   Western blotting: MNC and adipose tissue total cell lysates are prepared by suspending the cells in 
150 μL of lysis buffer (10 mM Tris-HCl, 150mM NaCl, 0.2 % Triton X and proteases and phospatases 


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13
inhibitors). After 30 min incubation on ice, samples are centrifuged at 12000 Xg for 10 min, 
supernatants are collected and total protein concentrations are determined. Sixty μg of total cell lysate 
are boiled in 2X SDS buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, 0.06 % bromophenol blue 
and 200 mM DTT), proteins separated by SDS-PAGE and then transferred to PVDF membrane. 
Polyclonal or monoclonal antibodies against p47phox, JNK-1, IKK-, SOCS-3, PTP-1B and TLR-4, 
(Abcam, Cambridge, MA) and actin (Santa Cruz Biotechnology, CA) will be used and the 
membranes are developed using super signal, chemiluminescence reagent (Pierce Chemical, IL).  
Densitometry is performed using molecular analyst software (Biorad, CA) and all values are 
corrected for loading with actin. 
5. NFB DNA binding: Nuclear NFkB DNA binding activity is measured by electrophoretic mobility 
shift assay (EMSA). Nuclear extract is prepared from MNC and fat tissue by high salt extraction as 
described by Andrews et al. Protein concentrations are determined using BCA protein assay (Pierce, 
IL). NFkB gel retardation assay is performed using double-stranded oligonucleotide consensus 
sequence for the NFkB-binding site (Santa Cruz Biotechnology, CA) radiolabeled with -P32 by T4 
kinase. Seven and a half µg of the nuclear extract are mixed with the incubation buffer (10 mM Tris 
(pH 7.5), 100 mM NaCl, 1 mM DTT, 1mM EDTA, 5% glycerol) containing 0.5µg poly dI-dC and 
0.2 mg/mL salmon sperm DNA to a final volume of 20 mL and the mixture is preincubated on ice 
for 15 min. Labeled oligonucleotides (60,000 cpm) are added, and the mixture is incubated at room 
temperature for 20 min. Samples are then applied to wells of 6% nondenaturing polyacrylamide gel. 
The gel is dried under vacuum and exposed to x-ray film. Densitometry is performed using Bio-Rad 
molecular analyst software (Hercules, CA). The specificity of the bands is confirmed by supershifting 
these bands with specific antibodies against Rel-A (p65) and p50 (Santa Cruz Biotechnology, CA) 
and by competition with cold oligonucleotides. Alternativley, binding to DNA can be performed 
using TransAM NFB (p65) kit (Active motive, Carlsbad, CA) on NE. 
6. Plasma glucose, insulin and FFAs measurements: Insulin levels are determined using an ELISA kit 
from Diagnostic Systems Laboratories Inc. (Webster, TX). Glucose levels are measured in plasma 
by YSI 2300 STAT Plus glucose analyzer (Yellow Springs, Ohio). FFAs levels are measured by a 
colorimetric assay (Roche, Richmond, VA) Active GLP-1 will be measured using ELISA kit  
(Millipore, MA) from samples collected in DPP-IV inhibitor (Millipore, MA) to prevent GLP-1 
degradation. GLP-1 assay sensitivity is 1.5pM. 
7.   15-isoprostane F2t (also known as 8-epi-PGF2α or 8-iso-PGF2α) in urine samples and TBARS will 
be measured by an ELISA kit from Oxford Biomedical Research (Oxford, MI). 
8.   Plasma concentrations of inflammatory cytokines: Concentrations of IL-6 and TNF are measured 
in plasma using sensitive and high sensitive ELISA kits (R&D Systems, MN). ELISA will be used 
to measure C-reactive protein (American Diagnostica Inc., CT) and DPPIV activity (reported as 
relative fluorescence units/sec (RFU/sec).  
9.  Measurement of whole body glucose uptake and endogenous glucose production by HE clamp:  
Subjects will come to the clinical research center after an overnight 10 hour fast. The patient’s blood 
sugar before the clamp is started should be ≤10mmol/L. Patients with blood sugar >10mmol/l will be 
re-scheduled. Oral hypoglycemic medications will be held on the morning of HE clamp. Long acting 
insulin will be held the night before the clamp. HE clamp will be done using radioactive glucose (3-
3H-glucose) as previously described (65, 66). Subjects will arrive at 8 AM for HE clamp with a prime-
continuous infusion of 3-3H-glucose. The 3-3H glucose prime will be 20 Ci x (fasting plasma 
glucose/100); the continuous infusion will be given at the rate of 0.20 Ci/min for 420 minutes (180 
minutes for isotopic equilibration and 240 minutes during the insulin clamp). Insulin clamp will be 
started at 180 min with a priming dose of short acting human insulin given over 10 minutes and then 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
14
an infusion at the rate of 80mU/m2. 20% glucose infused through the cephalic vein will be started at 
5 minutes and glucose infusion rate will be titrated to maintain blood glucose concentration at the 
fasting glucose level in the obese and at 5.5mmol/l in T2D. Plasma samples for titrated glucose will 
be obtained at 10 minute intervals starting 30 minutes before the clamp, to quantitate rates of whole 
body glucose disposal and suppression of endogenous (primarily hepatic) glucose production. Blood 
sample will be collected for insulin and free fatty acids (FFA) concentrations every 30 minutes during 
the clamp. Calculations will be done using Steele’s equation(67). 
Calculations: Under steady-state postabsorptive conditions, the rate of endogenous glucose 
appearance (Ra) will be calculated as the 3[3H] glucose infusion rate (disintegrations per minute per 
minute) divided by the steady-state plasma 3[3H] glucose-specific activity (disintegrations per minute 
per milligram). During the euglycemic insulin clamp, the rate of Ra will be calculated using Steele’s 
equation, using a distribution volume of 250 ml/kg. Endogenous (primarily hepatic) glucose 
production (EGP) will be calculated by subtracting the mean exogenous glucose infusion rate during 
the last 60 minutes (after steady state has been achieved) from Ra. The rate of insulin-mediated total 
body glucose disposal (Rd) will be determined by adding the rate of residual EGP to the exogenous 
glucose infusion rate. Insulin sensitivity will be expressed as Rd/lean body mass. 
10. Fat aspiration procedure: Subjects who are taking antiplatelet or anticoagulant medications will be 
excluded from the fat biopsy portion of the study. Subcutaneous fat tissue aspiration will be 
performed on abdomen at a 10 cm distance from umbilicus. The skin will first be prepared and a 
sterile drape will be placed around the appropriate area. Under local anesthesia, 500mg-3g of fat 
tissue will be obtained. The adipose tissue will be centrifuged to remove blood and fluid 
contaminants. The upper adipose tissue will be collected into a separate sterile tube, washed twice 
with cold sterile Phosphate Buffered Saline (PBS) and centrifuged to remove the PBS. Total RNA, 
nuclear extracts and total cell lysates will be prepared from the adipose tissue.  
 
 
DATA ANALYSIS  
 
This is a prospective study to evaluate oxidative stress before and after treatment with linagliptin. The 
similarities between the study groups, baseline values for subject’s demographics will be compared 
using appropriate parametric tests. Transformations of the data on order to meet statistical assumptions 
may be considered. All statistical analysis will be carried out using SPSS software (SPSS Inc, Chicago, 
Illinois). Comparison of each endpoint will be made using an analysis of covariance model, with 
baseline medications, change in BMI and change in HbA1c as covariates. The primary endpoint of the 
study is to detect a change in JNK-1 protein in MNC before and after linagliptin use as compared to 
placebo. We have chosen this as the primary endpoint because JNK-1 is an inflammatory mediator 
that also interferes with insulin signaling. Our prior data with sitagliptin show a 24% fall (with SD of 
24%) in JNK-1 following 12 weeks of treatment in type 2 diabetics(55). There was a 6% non-
significant increase in JNK-1 protein in the placebo group in that study. Conservatively estimating a 
difference in change in JNK-1 cellular concentrations of 24% among the two groups, a sample size of 
20 patients per group (assuming a drop-out rate of 15%) should provide adequate power ( = 0.2) to 
detect a significant difference ( = 0.05), provided the standard deviation of the residuals is not equal 
to or greater than the mean difference (see table below). 
 
 
 


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15
Sample Size Estimate 
 
Justification of drop-out rate: The analysis will adjust for only one baseline covariate: baseline 
medications. Two other covariates are change in BMI (which is not expected with linagliptin or 
placebo) and change in HbA1c. Sulfonylureas, metformin and subcutaneous insulin are not likely to 
induce insulin sensitization or decrease inflammation. Subjects currently on GLP-1 agonists or DPP-
IV inhibitors will be excluded from the study. Thiazolidinediones can induce insulin sensitization as 
well as decrease inflammation. The use of thiazolidinediones should be equally distributed among the 
two groups. Less than 20% of our research subjects ore on thiazolidinediones. Therefore it is likely 
that adjustment for thiazolidinediones should decrease sample size by <5%. Based on this justification 
and on our previous experience in diabetes trials, we have estimated that our drop-out rate will be 15% 
of 20 patients (3 patients per group). 
 
The secondary endpoints for the study will be comparison of the relative change from baseline in a) 
inflammatory mediators in serum and adipose tissue, b) single dose effect of linagliptin on 
inflammation and oxidative stress, c) HFHC induced inflammation in serum and adipose tissue and d) 
insulin sensitivity and hepatic glucose output; before and after using Linagliptin for 12 weeks.  
 
FACILITIES 
 
LABORATORY: The Diabetes-Endocrinology Center of Western New York Research Laboratories at 
SUNY at Buffalo, contain 6 laboratories with space totaling approximately 1600 Qs ft designated for 
research purposes. These labs are dedicated for the P.I. research. There is a cold room, dark room and four 
labs have fume hoods. In addition, six offices (600 Qs ft) are attached to the labs for lab staff. 
 
CLINICAL:  The clinical activities of Diabetes/Endocrinology center of Western New York, founded 
by the P.I. 16 years ago, have grown to dominate the diabetes-metabolism scene in upstate New York. 
Approximately 2,000 new patients referral are seen every year while approximately 3,000 patients with 
diabetes are under our long term care. Over 500 patients have been started on insulin pumps and currently 
4 patients are started on insulin pumps every month. Four satellite centers have been started covering 
various areas in our region where our team provides care fulfilling an important need. The unit employs 
35 people including 6 attending physicians, RNs, CDEs, RDs and research staff. The center is the hub of 
the endocrinology fellowship program at the university (3 fellows/year). The P.I. is the director of this 
program. There are nine examination rooms in the center and the clinical unit is provided with 3 CRC 
beds which are utilized for prolonged intensive investigative protocols. The center is the main base for the 
training of the university fellowship program in endocrinology; P.I. is the head of the division of 
endocrinology at SUNY Buffalo. 
 
MAJOR EQUIPMENT: Real time PCR Cepheid Smart Cycler, A Shimadzu RF-1501 
spectroflurometer; a Shimadzu BioSpec-1601UV spectrophotometer; a Shimadzu LC-10 model HPLC 
with diode array, autosampler, UV-Vis/fluorescence and electron capture detectors and data processing 
Difference in the 
mean % change 
from baseline 
  
5% 
10% 
15% 
20% 
10% 
5 
17 
36 
62 
15% 
3 
8 
17 
28 
20% 
3 
5 
10 
17 


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16
module; Savant Speed vacuum; a Sorvall Super T21 high speed refrigerated centrifuge and a CR312 
centrifuge, each with a variety of fixed angle and swinging bucket rotors; two Fisher Model Automatic 
water jacketed CO2 incubator for cell cultures; Elzon 180 cell counter; a Vibracell VC40 ultrasonic tissue 
disrupter with microtip probe; three Olympus inverted microscopes, each with a camera attached to it; 
several sizes and types of gel apparatuses and power supplies for performing 1D and 2D PAGE and 
separation of RNA/DNA; UVP (Epi-Chem darkroom) gel documentation system that detects 
chemiluminescenc/fluorescence/UV; Bio-Rad gel dryer apparatus; hybridization oven; Ericomp thermal 
cycler; UV crosslinker; Tecan Fluostar microplate reader; Packard cyclone phosphoimager; two Chrono-
log Lumi-aggregometers; Nichols institute system luminometer; Millipore Ultrapure water purification 
system; Wallac 1409 beta counter; a variety of water baths, vortex mixers, stirrors, hotplates and 
sonicators. 
 
 


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17
Assessment of adverse events 
Definitions of adverse events 
Adverse event 
An adverse event (AE) is defined as any untoward medical occurrence, including an exacerbation of a 
pre-existing condition, in a patient in a clinical investigation who received a pharmaceutical product. The 
event does not necessarily have to have a causal relationship with this treatment. 
Serious adverse event 
A serious adverse event (SAE) is defined as any AE which results in death, is immediately life-threatening, 
results in persistent or significant disability / incapacity, requires or prolongs patient hospitalisation, is a 
congenital anomaly / birth defect, or is to be deemed serious for any other reason if it is an important 
medical event when based upon appropriate medical judgement which may jeopardise the patient and may 
require medical or surgical intervention to prevent one of the other outcomes listed in the above 
definitions. 
Patients may be hospitalised for administrative or social reasons during the study (e.g. days on which 
infusion takes place, long distance from home to site,). These and other hospitalisations planned at the 
beginning of the study do not need to be reported as a SAE in case they have been reported at screening 
visit in the source data and have been performed as planned. 
 
Adverse Events of Special Interest 
Based on regulatory recommendation that is based on the experience with other compounds 
in the DPP-4 inhibitor class, the following events should be defined as adverse events of special interest. 
•  Hypersensitivity reactions such as angioedema, angioedema-like events, and 
anaphylaxis 
•  Skin lesions such as exfoliative dermatitis, and exfoliative rash 
• Renal adverse events (e.g. renal failure, >=2x increase of creatinine from baseline) 
• Pancreatitis 
Hepatic adverse events such as hepatitis, liver injury, and increased liver enzymes. Hepatic injury is 
defined by the following alterations of liver parameters: For patients with normal liver function (ALT, 
AST and bilirubin within normal limits) at baseline an elevation of AST and/or ALT >3 fold ULN 
combined with an elevation of bilirubin >2 fold ULN measured in the same blood draw sample.  
 
Refer to the appendix 1 for a list of prefered terms (PT’s) that are considered as AEs of Special 
Interest. 
 
Protocol-specified significant events are to be reported in an expedited manner similar to Serious 
Adverse Events, even if they do not meet any of the seriousness criteria. 
 
 
Intensity of adverse event 
The intensity of the AE should be judged based on the following: 


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• Mild: Awareness of sign(s) or symptom(s) which is/are easily tolerated 
• Moderate: Enough discomfort to cause interference with usual activity 
• Severe: Incapacitating or causing inability to work or to perform usual activities 
 
Causal relationship of adverse event 
Medical judgment should be used to determine the relationship, considering all relevant factors, including 
pattern of reaction, temporal relationship, de-challenge or re-challenge, confounding factors such as 
concomitant medication, concomitant diseases and relevant history. Assessment of causal relationship 
should be recorded in the case report forms.  
Yes: 
There is a reasonable causal relationship between the investigational product  
administered 
and the AE.  
No: 
There is no reasonable causal relationship between the investigational product  
administered 
and the AE.  
 
Worsening of the underlying disease or other pre-existing conditions 
Worsening of the underlying disease or of other pre-existing conditions will be recorded as an (S)AE in 
the (e)CRF. 
Changes in vital signs, ECG, physical examination, and laboratory test results 
Changes in vital signs, ECG, physical examination and laboratory test results will be recorded as an (S)AE 
in the (e)CRF , if they are judged clinically relevant by the investigator. 
Responsibilities for SAE reporting 
The Sponsor shall report (i.e., from signing the informed consent onwards through the trialdefined follow-
up period)  all SAEs and non-serious AEs occurring at the same time and/or which are medically related 
to the SAE and Adverse Events of Special Interest by fax or other secure method to the BI Unique Entry 
Point in accordance with timeline specified in the pharmcovigilance agreement 
 
For each adverse event, the investigator will provide the onset date, end date, intensity, treatment required, 
outcome, seriousness, and action taken with the investigational drug. The investigator will determine the 
expectedness of the investigational drug to the AEs as defined in the Listed Adverse Events section of the 
Boehringer Ingelheim’s (BI’s) Investigator Brochure for the Product or BI Drug Information e.g. 
Summary of Product Characteristics (SmPC) or Product Information (PI) for the authorised Study Drug 
provided by BI. 
 


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Appendix 1 
 
Following preferred terms are considered as 
Adverse Event of Special Interest 
Hepatic events 
Acute graft versus host disease in liver 
Acute hepatic failure 
Alanine aminotransferase abnormal 
Alanine aminotransferase increased 
Ammonia abnormal 
Ammonia increased 
Ascites 
Ascites 
Aspartate aminotransferase abnormal 
Aspartate aminotransferase increased 
Asterixis 
Autoimmune hepatitis 
Bacterascites 
Bacterascites 
Bile output abnormal 
Bile output decreased 
Biliary cirrhosis 
Biliary cirrhosis primary 
Biliary fibrosis 
Bilirubin conjugated abnormal 
Bilirubin conjugated increased 
Bilirubin excretion disorder 
Biopsy liver abnormal 
Blood bilirubin abnormal 
Blood bilirubin increased 
Blood bilirubin unconjugated increased 
Caput medusae 
Child-Pugh-Turcotte score increased 
Cholaemia 
Cholestasis 
Cholestatic liver injury 
Cholestatic liver injury 
Cholestatic pruritus 
Chronic hepatic failure 
Chronic hepatitis 
Coma hepatic 
Cryptogenic cirrhosis 
Cytolytic hepatitis 
Duodenal varices 
Foetor hepaticus 
Galactose elimination capacity test abnormal 


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20
Galactose elimination capacity test decreased 
Gamma-glutamyltransferase abnormal 
Gamma-glutamyltransferase increased 
Gastric varices 
Gastric varices haemorrhage 
Guanase increased 
Hepaplastin abnormal 
Hepaplastin decreased 
Hepatectomy 
Hepatic artery flow decreased 
Hepatic atrophy 
Hepatic calcification 
Hepatic cirrhosis 
Hepatic congestion 
Hepatic encephalopathy 
Hepatic encephalopathy prophylaxis 
Hepatic enzyme abnormal 
Hepatic enzyme decreased 
Hepatic enzyme increased 
Hepatic failure 
Hepatic fibrosis 
Hepatic function abnormal 
Hepatic hydrothorax 
Hepatic hydrothorax 
Hepatic infiltration eosinophilic 
Hepatic lesion 
Hepatic mass 
Hepatic necrosis 
Hepatic pain 
Hepatic sequestration 
Hepatic steatosis 
Hepatic vascular resistance increased 
Hepatitis 
Hepatitis acute 
Hepatitis cholestatic 
Hepatitis cholestatic 
Hepatitis chronic active 
Hepatitis chronic persistent 
Hepatitis fulminant 
Hepatitis fulminant 
Hepatitis toxic 
Hepatobiliary disease 
Hepatobiliary scan abnormal 
Hepatocellular foamy cell syndrome 
Hepatocellular injury 
Hepatomegaly 


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21
Hepatopulmonary syndrome 
Hepatorenal failure 
Hepatorenal syndrome 
Hepatosplenomegaly 
Hepatotoxicity 
Hyperammonaemia 
Hyperbilirubinaemia 
Hyperbilirubinaemia 
Hypercholia 
Hypertransaminasaemia 
Icterus index increased 
Ischaemic hepatitis 
Jaundice 
Jaundice cholestatic 
Jaundice hepatocellular 
Kayser-Fleischer ring 
Liver and small intestine transplant 
Liver disorder 
Liver function test abnormal 
Liver induration 
Liver injury 
Liver operation 
Liver palpable subcostal 
Liver scan abnormal 
Liver tenderness 
Liver transplant 
Lupoid hepatic cirrhosis 
Lupus hepatitis 
Mitochondrial aspartate aminotransferase increased 
Mixed liver injury 
Mixed liver injury 
Molar ratio of total branched-chain amino acid to tyrosine 
Nodular regenerative hyperplasia 
Ocular icterus 
Oedema due to hepatic disease 
Oedema due to hepatic disease 
Oesophageal varices haemorrhage 
Perihepatic discomfort 
Portal hypertension 
Portal hypertensive enteropathy 
Portal hypertensive gastropathy 
Portal triaditis 
Portopulmonary hypertension 
Radiation hepatitis 
Renal and liver transplant 
Retrograde portal vein flow 


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22
Retrograde portal vein flow 
Reye's syndrome 
Subacute hepatic failure 
Total bile acids increased 
Transaminases abnormal 
Transaminases increased 
Ultrasound liver abnormal 
Urine bilirubin increased 
Urobilin urine present 
Varices oesophageal 
X-ray hepatobiliary abnormal 
 
 
 
Following preferred terms are considered as 
Adverse Event of Special Interest 
Hypersensitivity reactions 
Allergic bronchitis 
Allergic cough 
Allergic oedema 
Allergic respiratory disease 
Allergic respiratory symptom 
Alveolitis allergic 
Anaphylactic reaction 
Anaphylactic shock 
Anaphylactic transfusion reaction 
Anaphylactoid reaction 
Anaphylactoid shock 
Angioedema 
Bronchial hyperreactivity 
Bronchospasm 
Circulatory collapse 
Circumoral oedema 
Conjunctival oedema 
Corneal oedema 
Cough 
Drug eruption 
Drug hypersensitivity 
Epiglottic oedema 
Erythema 
Eye oedema 
Eye swelling 
Eyelid oedema 
Face oedema 
First use syndrome 
Gingival oedema 


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23
Gingival swelling 
Gleich's syndrome 
Hereditary angioedema 
Hypersensitivity 
Idiopathic urticaria 
Kounis syndrome 
Laryngeal oedema 
Laryngotracheal oedema 
Lip oedema 
Lip swelling 
Oculorespiratory syndrome 
Oedema mouth 
Oropharyngeal swelling 
Palatal oedema 
Periorbital oedema 
Pharyngeal oedema 
Pruritus 
Pruritus allergic 
Pruritus generalised 
Rash 
Rash erythematous 
Rash generalised 
Rash pruritic 
Scleral oedema 
Shock 
Small bowel angioedema 
Swelling face 
Swollen tongue 
Tongue oedema 
Toxic skin eruption 
Tracheal oedema 
Type I hypersensitivity 
Type I hypersensitivity 
Urticaria 
Urticaria 
Urticaria cholinergic 
Urticaria chronic 
Urticaria papular 
Urticaria papular 
Wheezing 
 
 
Following preferred terms are considered as 
Adverse Event of Special Interest 
Pancreatitis 
Cullen's sign 


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24
Hereditary pancreatitis 
Ischaemic pancreatitis 
Oedematous pancreatitis 
Pancreatic abscess 
Pancreatic necrosis 
Pancreatic phlegmon 
Pancreatic pseudocyst 
Pancreatic pseudocyst drainage 
Pancreatitis 
Pancreatitis acute 
Pancreatitis chronic 
Pancreatic haemorrhage 
Pancreatitis haemorrhagic 
Pancreatitis necrotising 
Pancreatitis relapsing 
Pancreatorenal syndrome 
 
 
 
Following preferred terms are considered as 
Adverse Event of Special Interest 
Renal events 
 
Acute phosphate nephropathy 
Acute prerenal failure 
Anuria 
Azotaemia 
Continuous haemodiafiltration 
Dialysis 
Haemodialysis 
Neonatal anuria 
Nephropathy toxic 
Oliguria 
Peritoneal dialysis 
Renal failure 
Renal failure acute 
Renal failure neonatal 
Renal impairment 
Renal impairment neonatal 
 
 
 
Following preferred terms are considered as 
Adverse Event of Special Interest 
Skin lesions 


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25
Acute generalised exanthematous pustulosis 
Cutaneous vasculitis 
Dermatitis bullous 
Dermatitis exfoliative 
Dermatitis exfoliative generalised 
Drug eruption 
Epidermal necrosis 
Erythema multiforme 
Exfoliative rash 
Lip exfoliation 
Mucocutaneous ulceration 
Mucosal exfoliation 
Oral mucosal exfoliation 
Penile exfoliation 
Skin erosion 
Skin exfoliation 
Skin necrosis 
Stevens-Johnson syndrome 
Toxic epidermal necrolysis 
Toxic skin eruption 
Vaginal exfoliation 
Vaginal ulceration 
Vulval ulceration 
Vulvovaginal ulceration 
 
 
 


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26
REFERENCES: 
 
1. Deacon, C.F., Nauck, M.A., Toft-Nielsen, M., Pridal, L., Willms, B., and Holst, J.J. 1995. Both 
subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the 
NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes 44:1126-1131. 
2. Deacon, C.F., Ahren, B., and Holst, J.J. 2004. Inhibitors of dipeptidyl peptidase IV: a novel approach 
for the prevention and treatment of Type 2 diabetes? Expert Opin Investig Drugs 13:1091-1102. 
3. Deacon, C.F. 2004. Therapeutic strategies based on glucagon-like peptide 1. Diabetes 53:2181-2189. 
4. Dandona, P., Thusu, K., Cook, S., Snyder, B., Makowski, J., Armstrong, D., and Nicotera, T. 1996. 
Oxidative damage to DNA in diabetes mellitus. Lancet 347:444-445. 
5. Dandona, P., Mohanty, P., Ghanim, H., Aljada, A., Browne, R., Hamouda, W., Prabhala, A., Afzal, 
A., and Garg, R. 2001. The suppressive effect of dietary restriction and weight loss in the obese on the 
generation of reactive oxygen species by leukocytes, lipid peroxidation, and protein carbonylation. J 
Clin Endocrinol Metab 86:355-362. 
6. Higdon, J.V., and Frei, B. 2003. Obesity and oxidative stress: a direct link to CVD? Arterioscler 
Thromb Vasc Biol 23:365-367. 
7. Vincent, H.K., Powers, S.K., Stewart, D.J., Shanely, R.A., Demirel, H., and Naito, H. 1999. Obesity 
is associated with increased myocardial oxidative stress. Int J Obes Relat Metab Disord 23:67-74. 
8. Dandona, P., Weinstock, R., Thusu, K., Abdel-Rahman, E., Aljada, A., and Wadden, T. 1998. Tumor 
necrosis factor-alpha in sera of obese patients: fall with weight loss. J Clin Endocrinol Metab 83:2907-
2910. 
9. Hotamisligil, G.S., Shargill, N.S., and Spiegelman, B.M. 1993. Adipose expression of tumor necrosis 
factor-alpha: direct role in obesity-linked insulin resistance. Science 259:87-91. 
10. Katsuki, A., Sumida, Y., Murashima, S., Murata, K., Takarada, Y., Ito, K., Fujii, M., Tsuchihashi, K., 
Goto, H., Nakatani, K., et al. 1998. Serum levels of tumor necrosis factor-alpha are increased in obese 
patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 83:859-862. 
11. Madrid, L.V., Mayo, M.W., Reuther, J.Y., and Baldwin, A.S., Jr. 2001. Akt stimulates the 
transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B 
kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276:18934-18940. 
12. Davi, G., Guagnano, M.T., Ciabattoni, G., Basili, S., Falco, A., Marinopiccoli, M., Nutini, M., Sensi, 
S., and Patrono, C. 2002. Platelet activation in obese women: role of inflammation and oxidant stress. 
Jama 288:2008-2014. 
13. Dandona, P., Aljada, A., and Bandyopadhyay, A. 2004. Inflammation: the link between insulin 
resistance, obesity and diabetes. Trends Immunol 25:4-7. 
14. Aljada, A., Ghanim, H., Mohanty, P., Kapur, N., and Dandona, P. 2002. Insulin inhibits the pro-
inflammatory transcription factor early growth response gene-1 (Egr)-1 expression in mononuclear 
cells (MNC) and reduces plasma tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) 
concentrations. J Clin Endocrinol Metab 87:1419-1422. 
15. Aljada, A., Ghanim, H., Saadeh, R., and Dandona, P. 2001. Insulin Inhibits NFkappaB and MCP-1 
Expression in Human Aortic Endothelial Cells. J Clin Endocrinol Metab 86:450-453. 
16. Aljada, A., Saadeh, R., Assian, E., Ghanim, H., and Dandona, P. 2000. Insulin inhibits the expression 
of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric 
oxide. J Clin Endocrinol Metab 85:2572-2575. 
17. Dandona, P., Aljada, A., Mohanty, P., Ghanim, H., Hamouda, W., Assian, E., and Ahmad, S. 2001. 
Insulin inhibits intranuclear nuclear factor kappaB and stimulates IkappaB in mononuclear cells in 
obese subjects: evidence for an anti-inflammatory effect? J Clin Endocrinol Metab 86:3257-3265. 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
27
18. Chaudhuri, A., Janicke, D., Wilson, M.F., Tripathy, D., Garg, R., Bandyopadhyay, A., Calieri, J., 
Hoffmeyer, D., Syed, T., Ghanim, H., et al. 2004. Anti-inflammatory and profibrinolytic effect of 
insulin in acute ST-segment-elevation myocardial infarction. Circulation 109:849-854. 
19. Aljada, A., Mohanty, P., Ghanim, H., Abdo, T., Tripathy, D., Chaudhuri, A., and Dandona, P. 2004. 
Increase in intranuclear nuclear factor kappaB and decrease in inhibitor kappaB in mononuclear cells 
after a mixed meal: evidence for a proinflammatory effect. Am J Clin Nutr 79:682-690. 
20. Mohanty, P., Ghanim, H., Hamouda, W., Aljada, A., Garg, R., and Dandona, P. 2002. Both lipid and 
protein intakes stimulate increased generation of reactive oxygen species by polymorphonuclear 
leukocytes and mononuclear cells. Am J Clin Nutr 75:767-772. 
21. Aljada, A., Ghanim, H., Mohanty, P., Assian, E., and Dandona, P. 2000. Glucose intake stimulates 
intranuclear NFkB and p47phox in mononuclear cells. ENDO '2000, the 82nd Annual Meeting of the 
Endocrine Society. 
22. Ridker, P.M. 2001. High-sensitivity C-reactive protein: potential adjunct for global risk assessment in 
the primary prevention of cardiovascular disease. Circulation 103:1813-1818. 
23. Ridker, P.M., Rifai, N., Stampfer, M.J., and Hennekens, C.H. 2000. Plasma concentration of 
interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 
101:1767-1772. 
24. Elkind, M.S., Cheng, J., Boden-Albala, B., Rundek, T., Thomas, J., Chen, H., Rabbani, L.E., and 
Sacco, R.L. 2002. Tumor necrosis factor receptor levels are associated with carotid atherosclerosis. 
Stroke 33:31-37. 
25. Rost, N.S., Wolf, P.A., Kase, C.S., Kelly-Hayes, M., Silbershatz, H., Massaro, J.M., D'Agostino, R.B., 
Franzblau, C., and Wilson, P.W. 2001. Plasma concentration of C-reactive protein and risk of ischemic 
stroke and transient ischemic attack: the Framingham study. Stroke 32:2575-2579. 
26. Pradhan, A.D., Manson, J.E., Rifai, N., Buring, J.E., and Ridker, P.M. 2001. C-reactive protein, 
interleukin 6, and risk of developing type 2 diabetes mellitus. Jama 286:327-334. 
27. Schmidt, M.I., Duncan, B.B., Sharrett, A.R., Lindberg, G., Savage, P.J., Offenbacher, S., Azambuja, 
M.I., Tracy, R.P., and Heiss, G. 1999. Markers of inflammation and prediction of diabetes mellitus in 
adults (Atherosclerosis Risk in Communities study): a cohort study. Lancet 353:1649-1652. 
28. Harris, T.B., Ferrucci, L., Tracy, R.P., Corti, M.C., Wacholder, S., Ettinger, W.H., Jr., Heimovitz, H., 
Cohen, H.J., and Wallace, R. 1999. Associations of elevated interleukin-6 and C-reactive protein 
levels with mortality in the elderly. Am J Med 106:506-512. 
29. Strandberg, T.E., and Tilvis, R.S. 2000. C-reactive protein, cardiovascular risk factors, and mortality 
in a prospective study in the elderly. Arterioscler Thromb Vasc Biol 20:1057-1060. 
30. Cermak, J., Key, N.S., Bach, R.R., Balla, J., Jacob, H.S., and Vercellotti, G.M. 1993. C-reactive 
protein induces human peripheral blood monocytes to synthesize tissue factor. Blood 82:513-520. 
31. Robbie, L., and Libby, P. 2001. Inflammation and atherothrombosis. Ann N Y Acad Sci 947:167-179; 
discussion 179-180. 
32. Ziccardi, P., Nappo, F., Giugliano, G., Esposito, K., Marfella, R., Cioffi, M., D'Andrea, F., Molinari, 
A.M., and Giugliano, D. 2002. Reduction of inflammatory cytokine concentrations and improvement 
of endothelial functions in obese women after weight loss over one year. Circulation 105:804-809. 
33. Zahorska-Markiewicz, B., Janowska, J., Olszanecka-Glinianowicz, M., and Zurakowski, A. 2000. 
Serum concentrations of TNF-alpha and soluble TNF-alpha receptors in obesity. Int J Obes Relat 
Metab Disord 24:1392-1395. 
34. Heilbronn, L.K., Noakes, M., and Clifton, P.M. 2001. Energy restriction and weight loss on very-low-
fat diets reduce C-reactive protein concentrations in obese, healthy women. Arterioscler Thromb Vasc 
Biol 21:968-970. 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
28
35. Bastard, J.P., Jardel, C., Bruckert, E., Blondy, P., Capeau, J., Laville, M., Vidal, H., and Hainque, B. 
2000. Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese 
women after weight loss. J Clin Endocrinol Metab 85:3338-3342. 
36. Abad, L.W., Schmitz, H.R., Parker, R., and Roubenoff, R. 2002. Cytokine responses differ by 
compartment and wasting status in patients with HIV infection and healthy controls. Cytokine 18:286-
293. 
37. Nicklas, B.J., Ambrosius, W., Messier, S.P., Miller, G.D., Penninx, B.W., Loeser, R.F., Palla, S., 
Bleecker, E., and Pahor, M. 2004. Diet-induced weight loss, exercise, and chronic inflammation in 
older, obese adults: a randomized controlled clinical trial. Am J Clin Nutr 79:544-551. 
38. Dandona, P., Mohanty, P., Hamouda, W., Ghanim, H., Aljada, A., Garg, R., and Kumar, V. 2001. 
Inhibitory effect of a two day fast on reactive oxygen species (ROS) generation by leucocytes and 
plasma ortho-tyrosine and meta-tyrosine concentrations. J Clin Endocrinol Metab 86:2899-2902. 
39. Watanobe, H., and Hayakawa, Y. 2003. Hypothalamic interleukin-1 beta and tumor necrosis factor-
alpha, but not interleukin-6, mediate the endotoxin-induced suppression of the reproductive axis in 
rats. Endocrinology 144:4868-4875. 
40. Russell, S.H., Small, C.J., Stanley, S.A., Franks, S., Ghatei, M.A., and Bloom, S.R. 2001. The in vitro 
role of tumour necrosis factor-alpha and interleukin-6 in the hypothalamic-pituitary gonadal axis. J 
Neuroendocrinol 13:296-301. 
41. Baeuerle, P.A., and Baltimore, D. 1996. NF-kappa B: ten years after. Cell 87:13-20. 
42. Baldwin, A.S., Jr. 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu 
Rev Immunol 14:649-683. 
43. Halliwell, B. 1994. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? 
Lancet 344:721-724. 
44. McCord, J.M. 2000. The evolution of free radicals and oxidative stress. Am J Med 108:652-659. 
45. Barnes, P.J., and Karin, M. 1997. Nuclear factor-kappaB: a pivotal transcription factor in chronic 
inflammatory diseases. N Engl J Med 336:1066-1071. 
46. Leusen, J.H., Verhoeven, A.J., and Roos, D. 1996. Interactions between the components of the human 
NADPH oxidase: intrigues in the phox family. J Lab Clin Med 128:461-476. 
47. Yagi, K. 1982. Lipid peroxides in biology and medicine. New York and London:: Academic Press. 
48. Armstrong, D., Abdella, N., Salman, A., Miller, N., Rahman, E.A., and Bojancyzk, M. 1992. 
Relationship of lipid peroxides to diabetic complications. Comparison with conventional laboratory 
tests. J Diabetes Complications 6:116-122. 
49. Roberts, L.J., and Morrow, J.D. 2000. Measurement of F(2)-isoprostanes as an index of oxidative 
stress in vivo. Free Radic Biol Med 28:505-513. 
50. Stadtman, E.R., and Oliver, C.N. 1991. Metal-catalyzed oxidation of proteins. Physiological 
consequences. J Biol Chem 266:2005-2008. 
51. Blount, B.C., and Duncan, M.W. 1997. Trace quantification of the oxidative damage products, meta- 
and ortho-tyrosine, in biological samples by gas chromatography-electron capture negative ionization 
mass spectrometry. Anal Biochem 244:270-276. 
52. Viswanathan, P., Chaudhuri, A., Bhatia, R., Al-Atrash, F., Mohanty, P., and Dandona, P. 2007. 
Exenatide therapy in obese patients with type 2 diabetes mellitus treated with insulin. Endocr Pract 
13:444-450. 
53. Chaudhuri, A., Ghanim, H., Vora, M., Sia, C.L., Korzeniewski, K., Dhindsa, S., Makdissi, A., and 
Dandona, P. 2012. Exenatide exerts a potent antiinflammatory effect. J Clin Endocrinol Metab 97:198-
207. 


v5 1 13; v12 9 13, v 3 14, v4 14, v5 22 14 
29
54. Reinhold, D., Biton, A., Pieper, S., Lendeckel, U., Faust, J., Neubert, K., Bank, U., Tager, M., 
Ansorge, S., and Brocke, S. 2006. Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N 
(APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation. Int 
Immunopharmacol 6:1935-1942. 
55. Makdissi, A., Ghanim, H., Vora, M., Green, K., Abuaysheh, S., Chaudhuri, A., Dhindsa, S., and 
Dandona, P. 2012. Sitagliptin Exerts an Antinflammatory Action. J Clin Endocrinol Metab. 
56. Ghanim, H., Aljada, A., Daoud, N., Deopurkar, R., Chaudhuri, A., and Dandona, P. 2007. Role of 
inflammatory mediators in the suppression of insulin receptor phosphorylation in circulating 
mononuclear cells of obese subjects. Diabetologia 50:278-285. 
57. Ghanim, H., Abuaysheh, S., Sia, C.L., Korzeniewski, K., Chaudhuri, A., Fernandez-Real, J.M., and 
Dandona, P. 2009. Increase in plasma endotoxin concentrations and the expression of Toll-like 
receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-
carbohydrate meal: implications for insulin resistance. Diabetes Care 32:2281-2287. 
58. Monte, S.V., Caruana, J.A., Ghanim, H., Sia, C.L., Korzeniewski, K., Schentag, J.J., and Dandona, P. 
2012. Reduction in endotoxemia, oxidative and inflammatory stress, and insulin resistance after Roux-
en-Y gastric bypass surgery in patients with morbid obesity and type 2 diabetes mellitus. Surgery 
151:587-593. 
59. Zabolotny, J.M., Bence-Hanulec, K.K., Stricker-Krongrad, A., Haj, F., Wang, Y., Minokoshi, Y., Kim, 
Y.B., Elmquist, J.K., Tartaglia, L.A., Kahn, B.B., et al. 2002. PTP1B regulates leptin signal 
transduction in vivo. Dev Cell 2:489-495. 
60. Ghanim, H., Sia, C.L., Abuaysheh, S., Korzeniewski, K., Patnaik, P., Marumganti, A., Chaudhuri, A., 
and Dandona, P. 2010. An antiinflammatory and reactive oxygen species suppressive effects of an 
extract of Polygonum cuspidatum containing resveratrol. J Clin Endocrinol Metab 95:E1-8. 
61. Muscelli, E., Casolaro, A., Gastaldelli, A., Mari, A., Seghieri, G., Astiarraga, B., Chen, Y., Alba, M., 
Holst, J., and Ferrannini, E. 2012. Mechanisms for the antihyperglycemic effect of sitagliptin in 
patients with type 2 diabetes. J Clin Endocrinol Metab 97:2818-2826. 
62. Dandona, P., Suri, M., Hamouda, W., Aljada, A., Kumbkarni, Y., and Thusu, K. 1999. 
Hydrocortisone-induced inhibition of reactive oxygen species by polymorphonuclear neutrophils. Crit 
Care Med 27:2442-2444. 
63. Dandona, P., Thusu, K., Hafeez, R., Abdel-Rahman, E., and Chaudhuri, A. 1998. Effect of 
hydrocortisone on oxygen free radical generation by mononuclear cells. Metabolism 47:788-791. 
64. Tosi, M.F., and Hamedani, A. 1992. A rapid, specific assay for superoxide release from phagocytes in 
small volumes of whole blood. Am J Clin Pathol 97:566-573. 
65. DeFronzo, R.A., Tobin, J.D., and Andres, R. 1979. Glucose clamp technique: a method for quantifying 
insulin secretion and resistance. Am J Physiol 237:E214-223. 
66. Bajaj, M., Suraamornkul, S., Piper, P., Hardies, L.J., Glass, L., Cersosimo, E., Pratipanawatr, T., 
Miyazaki, Y., and DeFronzo, R.A. 2004. Decreased plasma adiponectin concentrations are closely 
related to hepatic fat content and hepatic insulin resistance in pioglitazone-treated type 2 diabetic 
patients. J Clin Endocrinol Metab 89:200-206. 
67. Steele, R. 1959. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann 
N Y Acad Sci 82:420-430.