Novel glucagon- and OXM-based peptides acting through glucagon and GLP-1 receptors with body weight reduction and anti-diabetic properties

Article abstract of DOI:10.1016/j.bioorg.2019.103538

Oxyntomodulin (OXM) is an endogenous gastrointestinal hormone, which activates both the Glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR). However, OXM has shortcomings including poor GLP-1R agonism to control glycemia, short half-life and others. Inspired from the sequence relationship between OXM and glucagon, in this study, we introduced different C-terminus residues of GLP-1, exenatide and OXM to glucagon to get a series of hybrid peptides with enhanced GLP-1R activation. The formed glucagon-exenatide hybrid peptide shows higher GLP-1R activation properties than OXM. Then the peptides based on the glucagon-exenatide hybrid peptide were coupled with fatty acid side chains to prolong their half-lives. As a result, the most potent compound 16a could stimulate insulin secretion and maintain blood glucose in normal level for ~42.6 h in diabetic mice. 16a exhibited reduced HbA1c level in diabetic mice, lowered body weight significantly in obesity mice on chronic treatment assay. 16a, combined efficient GCGR/GLP-1R activity, is potential as novel treatment for obesity and diabetes. This finding provides new insights into balancing GLP-1/GCGR potency of glucagon-exenatide hybrid peptide and is helpful for discovery of novel anti-diabetic and bodyweight-reducing drugs.

Full text of DOI:10.1016/j.bioorg.2019.103538

Journal Pre-proofs
Novel glucagon- and OXM-based peptides acting through glucagon and
GLP-1 receptors with body weight reduction and anti-diabetic properties
Xingguang Cai, Chengye Li, Jie Zhou, Yuxuan Dai, Yosefa Avraham, Lidan
Sun, Chunxia Liu, Jiayi Tong, Yao Wang, Xinzhou Bi, Liang He, Wenlong
Huang, Hai Qian
PII:
S0045-2068(19)30787-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.103538
YBIOO 103538
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 May 2019
5 December 2019
21 December 2019
Please cite this article as: X. Cai, C. Li, J. Zhou, Y. Dai, Y. Avraham, L. Sun, C. Liu, J. Tong, Y. Wang, X. Bi, L.
He, W. Huang, H. Qian, Novel glucagon- and OXM-based peptides acting through glucagon and GLP-1
receptors with body weight reduction and anti-diabetic properties, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.103538
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Novel glucagon- and OXM-based peptides acting through glucagon and GLP-1
receptors with body weight reduction and anti-diabetic properties
Xingguang Cai 1, #, Chengye Li1, #, Jie Zhou 1, #, Yuxuan Dai , Yosefa Avraham , Lidan Sun , Chunxia Liu , Jiayi
1
2
1
1
Tong , Yao Wang , Xinzhou Bi , Liang He , Wenlong Huang 1,4, Hai Qian 1,4,
3
3
1
1
*
1
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, P. R. China
2
Department of Human Nutrition and Metabolism, Braun School of Public Health, Faculty of Medicine, Hebrew
University, Jerusalem, Israel
3
Zhongda Hospital, Southeast University, Nanjing 210009, China
4
Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, P.R. China
#
These authors contributed equally to this work.
*
Corresponding Author
H.Q.: phone, +86-25-83271051; fax, +86-25-83271051; e-mail, qianhai24@163.com.
1

ABSTRACT
Oxyntomodulin (OXM) is an endogenous gastrointestinal hormone, which activates both the
Glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR). However, OXM has
shortcomings including poor GLP-1R agonism to control glycemia, short half-life and others. Inspired
from the sequence relationship between OXM and glucagon, in this study, we introduced different C-
terminus residues of GLP-1, exenatide and OXM to glucagon to get a series of hybrid peptides with
enhanced GLP-1R activation. The formed glucagon-exenatide hybrid peptide shows higher GLP-1R
activation properties than OXM. Then the peptides based on the glucagon-exenatide hybrid peptide
were coupled with fatty acid side chains to prolong their half-lives. As a result, the most potent
compound 16a could stimulate insulin secretion and maintain blood glucose in normal level for ~42.6
h in diabetic mice. 16a exhibited reduced HbA1c level in diabetic mice, lowered body weight
significantly in obesity mice on chronic treatment assay. 16a, combined efficient GCGR/GLP-1R
activity, is potential as novel treatment for obesity and diabetes. This finding provides new insights
into balancing GLP-1/GCGR potency of glucagon-exenatide hybrid peptide and is helpful for
discovery of novel anti-diabetic and bodyweight-reducing drugs.
Key Words: obesity, diabetes, long-acting, glucagon, OXM analogs
2

1
. Introduction
1
Obesity is highly associated with type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and
2
all-cause mortality in people with body mass index (BMI) ≥ 35 kg/m compared to those with normal
2
body weight . Glucagon like peptide-1(GLP-1, Peptide 1) analog is one of the few medications which
3
are available for good management of obesity and related comorbidities, like diabetes . GLP-1 as the
mostly studied incretin, clinically, is a gut hormone that enhances the secretion of insulin after nutrient
ingestion. The functions of GLP-1 include delaying gastric emptying, stimulating insulin secretion and
mediating satiety in central nervous system (CNS). These actions are beneficial to individuals with
4
obesity and type 2 diabetes . However, its function in reducing body weight is not prominent.
Exenatide (Peptide 2), known as the first approved GLP-1R agonist, is a synthetic version of
exendin-4 which was found in the saliva of the Gila monster. It has only 53% amino acid sequence
5
homology with human GLP-1 . In addition to the amino acid differences throughout the sequence
compared to human GLP-1, exendin-4 contains a C-terminal extension (Cex) sequence that greatly
6
, 7
improves its biophysical and metabolic stability compared to human GLP-1 . The effect of exenatide
was tested in DMGO trials8-10. Which showed that exenatide could reduce the HbA1c by
approximately 1.0-1.2% and decrease the fasting plasma glucose by about 1.0-1.4 mmol/L compared
8
to placebo . Glucagon (Peptide 3) is a well-established insulin counter hormone, which is processed
1
1
from proglucagon, the same biosynthetic precursor as GLP-1 and Oxyntomodulin (OXM, Peptide 4) .
Recent reports demonstrated that glucagon has beneficial effects on increasing energy expenditure.
Additionally, glucagon has both catabolic and thermogenic actions12-14
.
Diseases like obesity and diabetes are concurrent and heterogeneous etiology. Improved efficacy
in the treatment of such diseases could be achieved by targeting multiple biochemical pathways within
a single therapy15-17. However, physical combinations of independent medications normally would
complicate the processes of drug development and clinical practice. Therefore, a single molecule with
1
8
appropriate balanced activities at multiple receptors is the best option . OXM, for instance, binds to
and fully activates both the GLP-1 receptor(GLP-1R) and the glucagon receptor(GCGR) in vitro with
1
9
similar potencies . Interestingly, OXM is composed of the entire 29 amino acid sequence of glucagon,
along with an octapeptide extension to the C-terminus. In a one-month study, an average weight loss
of 2.3 kg on patients with overweight and obesity was observed after three times daily preprandial
subcutaneous administration of OXM, while an average weight loss of 0.5 kg was observed in the
3

2
0
placebo group .
However, OXM is not the most potent agonist on GLP-1R or GCGR, and OXM could slightly
2
1
reduce the blood glucose level . Similar to GLP-1, endogenous OXM is quickly degraded (about 12
2
2
minutes) in vivo due to N-terminal degradation by DPP-IV . Currently, a few candidates are in
preclinical or clinical research development. MEDI0382 is in phase II clinical development at
MedImmune. It was accepted by once-daily injection. MEDI0382 consist of 30 amino acids based on
2
3
modified OXM, with gamma-glutamate and palmitic acid coupled at lysine residue . Phase II results
showed marked reductions in fasting plasma glucose and postprandial glucose from baseline with no
increase in hypoglycemia. Body weight was significantly reduced after injected by MEDI0382, with a
2
4
weight loss of -3.8 kg from baseline compared to -1.7 kg with placebo . Evers et al. designed a series
of dual agonists based on exenatide to adjust the selectivity ratio of GLP-1/glucagon. Anti-obese
activity was studied on the selected peptide as well as Liraglutide which were dosed at 50 μg/kg twice
2
5
daily over 33 days. A significant decrease of body weight by 29.1% was observed .
In this paper, we proposed a novel approach to overcome the disadvantages of OXM including
weak activation of GLP-1 and short half-life. Inspired from the sequence connection between OXM
and glucagon, C-terminus residues of GLP-1, Exenatide and OXM were introduced to the glucagon
C-terminus. Further modification and fatty acid side chain conjugation were also performed to prolong
the half-life of the selected hybrid peptide. In our previous work, it was proved that the fatty acid side
chain attached at position 16 had a positive effect on stabilizing plasma glucose and lowering body
2
6
weight . In this study, the activation of GLP-1R/GCGR and intra-peritoneal glucose tolerance test
was performed and the influences on body weight changes were observed. Since fatty liver diseases
may be associated with diabetes, biomarkers of liver diseases like ALT and AST were detected.
Compound 16a was identified as the most potent GLP-1/GCGR agonist which could stabilize plasma
glucose level, lower body weight of diet-induced-obesity (DIO) mice and have long-term beneficial
effects on streptozotocin (STZ)-induced diabetic mice.
2
2
. RESULTS
.1 Design of C-terminus hybrid peptides and receptors activation assay
According to the relationship between OXM and glucagon, we hypothesized that introducing
different C-terminus extensions to glucagon may result in peptides with different agonism ratio on
4

GLP-1R and GCGR. cAMP was chosen to prove the activation of the two receptors by our compounds
Figure 1, Table 1). Native OXM activated GCGR at an effective concentration (EC ) of 6.1±0.2 pM
(
5
0
and GLP-1R at an EC of 15.7±0.2 pM. Compared to OXM, most of the OXM analogues had
5
0
improved potency on GLP-1R and maintained activity on GCGR, indicating that the C-terminus
modification method was feasible. The octapeptide of OXM was substituted by part of its sequences,
GLP-1 and Exenatide to afford compounds 5, 6 and 7 respectively. Truncated peptide 5 showed 1.86-
fold weaker GCGR agonism than native OXM. Compound 7 showed a slight increase in GLP-1R
potency and little reduced potency at GCGR compared to OXM. Substitution of three amino acid
residues of glucagon C-terminus to exenatide residue led to compound 8. It exhibited great GLP-1R
activation and moderate GCGR potency, which was nearly comparable to native OXM. This indicates
that the C-terminus of glucagon could be rationally modified to further improve the relative ratio of
2
7
GLP-1R/GCGR co-agonism, although a degree of conservation for both receptors was observed .
Compound 10 showed a 2.06-fold increase in GLP-1R potency compared with OXM.
Figure 1. Structures of compounds.
Table 1. EC of OXM and OXM analogues
5
0
Peptides
mGLP1R (pM) mGCGR (pM) Peptides
mGLP1R (pM) mGCGR (pM)
1
2
3
4
5
(GLP-1)
4.3±0.4
>1000
6
7.8±0.2**##
4.2±0.2**##
7.4±0.6**##
12.6±0.7**##
1.9±0.1**##
8.4±0.2**
(Exenatide) 5.8±0.6
(Glucagon) 34.9±0.4
>1000
7
7.2±0.5
1.1±0.5
6.1±0.2
11.4±0.3**
8
8.8±0.5**
24.1±0.5**
14.4±0.9**
(OXM)
15.7±0.2
15.8±0.2##
9
10
Results are expressed as mean ± SD, *P<0.05, **P<0.01 vs OXM, # P<0.05, ## P<0.01 vs Exenatide.
5

2
.2 Preliminary activity assessment of hybrid peptides in Kunming mice
The activity of the hybrid peptides on feeding was monitored in normal fasted Kunming mice. As
shown in Figure 2A, most of the peptides could reduce food intake in the first hour after administration
compared with vehicle. Peptides 6, 7, 10 had a greater anorectic effect than other peptides. Among all
peptides, no significant difference in cumulative food intake over up to 24 h was observed (data not
shown), which indicates that the duration of peptides was less than 24 h. To further evaluate chronic
lowering effects of body weight, administration of hybrid peptides at 25 nmol/kg once per day to
Kunming mice for two weeks was conducted (Figure 2B). After two weeks treatment of 7, 9 and 10,
the average body weight gain was lower than that in the vehicle treatment group (P<0.05), indicating
good weight control potency of these analogues. Particularly, the least body weight increase was
observed in mice groups treated with 7 and 10 versus vehicle-treated mice (P<0.01, 2.36±0.23g for 10,
2
.66±0.32g for 7, 3.88±0.16g for vehicle (saline), respectively).
To evaluate whether the administration of hybrid peptides could improve systemic response to
glucose, IPGTT was performed to Kunming mice. Most OXM analogues exhibited good
hypoglycemic effects. Especially, after 7 and 10 were administrated, plasma glucose levels decreased
significantly(p<0.001) at 15, 30 and 60 min (Figure 2C).
6

Figure 2. (A) Cumulative food intake(g) in normal fasted Kunming mice for 8h with 25 nmol/kg
hybrid peptides (B) Body weight change with 25 nmol/kg once per day to Kunming mice for two
weeks (C) Glucose-lowering effect of control(saline), OXM, 7 and 10 using IPGTT in Kunming
mice.
2
.3 Energy balance and physiological measurements in DIO mice
Peptides 7 and 10 were administered at 25 nmol/kg intraperitoneally once daily for five days
(Figure 3). The injections of 10 led to body weight decrease in DIO mice by 1.3±0.2 g, while increased
body weight of 1.75±0.13 g was shown in the vehicle group (P<0.01, n=5 per group) (Figure 3A). For
-1
7
and 10, energy expenditure was also increased compared to vehicle (18.44±3.46 kcal/(kg.h )
-1
-1
and16.20±3.03 kcal/(kg.h ) compared to 15.47±2.22 kcal/(kg.h ), P <0.05). They had no significant
-1
difference with OXM (17.5±3.67 kcal/(kg.h )), and the respiratory quotient tended to decrease slightly
(Figure 3B, C). However, the increased energy expenditure was not associated with a change in
spontaneous physical activity–induced thermogenesis since locomotor activity did not differ between
treatment groups and controls (Figure 3D), which suggested that 7 and 10 reduced weight through
7

appetite suppression and energy expenditure.
Figure 3. One-week treatment of diet-induced obese mice with OXM, 7 and 10: (A) dynamic body
weight change, (B) energy expenditure, (C) respiratory quotient, (D) locomotor activity.
2
.4 One-month therapy on DIO mice by 7 and 10
In the follow-up experiment, we tested the effect of intraperitoneal injections of 7 or 10 (25 nmol/
kg) every other day for one month. The injections decreased the body weight of DIO mice by 0.78 ±
.22 g for 7 and 1.49±0.36 g for 10, respectively. In contrast, in the vehicle group, the body weight of
0
DIO mice was increased by 1.66±0.32 g (P<0.01) (Figure 4A). Decreased white adipose content was
observed in 7- and 10-treated DIO mice. The white adipose tissue (WAT) cell number in the unit area
8

was associated with body weight changes. The more cells counted; the smaller white adipose cells are.
The results are as follows:34.4±3.36 for 7, 64.4±4.23 for 10, 33.6±5.96 for OXM, and 25.4±3.44 for
controls, P < 0.001 (Table 2).
The IPGTT performed on day 1 and 30 revealed that the glucose tolerance ability was
significantly improved in both groups (Figure 4B, C). Furthermore, basal blood glucose level was
normalized by chronic treatment with 10, and this result was comparable with that using OXM (Figure
4
D).
Several other metabolic parameters in plasma were also improved by chronic treatment with the
peptides (Table 2). Increases in adiponectin and leptin as well as decreases in insulin levels correlated
with decreased adiposity were observed at the end of the study in each treatment group. Reduced
cholesterol and decreased hepatic lipid droplets relative to vehicle treatment group were also noted,
especially for mice treated with 10. Nevertheless, these peptides had no significant effects on
triglycerides levels.
9

Figure 4. (A)Body weight change of DIO mice for one month. (B-E) One-month treatment of DIO
mice with OXM, 7 and 10: (B) IPGTT results in mice at day 1. (C) IPGTT results in mice at day 30.
(D)AUC of IPGTT results at day 1. (E)AUC of IPGTT results at day 30.
Table 2. Metabolic parameters in plasma at the end of the chronic study
White adipose Insulin
Leptin
(ng/ml)
Adiponectin Cholesterol Triglycerides
Compound
cell numbe
(ng/ml)
(ug/ml)
(mmol/l)
(mmol/l)
Control
OXM
25.44±3.44
33.60±5.90
13.33±0.45 1.9±0.07
0.89±0.38
4.33±0.19
3.82±0.56
0.84±0.12
1.04±0.21
8.96±0.13* 1.64±0.007 1.15±0.05*
1
0

7
1
34.40±3.36
10.34±0.23* 3.26±0.36* 1.60±0.11** 3.06±0.05* 0.81±0.23
0
64.40±4.63*** 9.40±0.35* 2.66±0.39* 1.94±0.09*** 3.78±0.30* 0.81±0.14
Results are expressed as mean ± SD, *P<0.05，**P<0.01, ***P<0.001,significantly different from vehicle.
2
.5 Optimization of long-acting effect of hybrid peptides
As demonstrated above, compound 10 displayed favorable GLP-1/GCGR activation, and in vivo
study showed that compound 10 could improve glucose tolerance and decrease body weight in
Kunming mice. Compound 10 also increased energy expenditure and decreased the respiratory
quotient. In the long-period therapy on DIO mice, compound 10 lowered the body weight significantly,
improved glucose tolerance and several other metabolic parameters in plasma. Therefore, compound
1
0 shows good prospects as exenatide C-terminal sequence hybrid peptide. Doyle et al. investigated
the in vivo biological activity of exenatide (1–30) and also found that exenatide (1–30), the C terminal-
truncated exendin peptide, had a reduced affinity for GLP-1R and a lessened ability to increase
2
8
intracellular cAMP in insulinoma cells .
To prolong the acting effect of glucagon-exenatide hybrid peptides, we tried to raise the
homologies compared with Exenatide by changing a series of amino acids in the middle of the peptides
(Figure 5). Ser in position 2 was substituted with Gly to prevent rapid inactivation by the metabolic
2
9
enzymes like DPP-IV . Meanwhile, in compound 11-16, amino acid residues located at
1
4,15,17,19,21 and 24 were substituted respectively by the residues of Exenatide in the same location.
Position 16 was replaced by Cys for coupling hybrid peptides with fatty chains in the next step to yield
compound 11a-16a. As shown in Table 3, most hybrid peptides preserved their GLP-1R and GCGR
activation ability, only compound 11 had a 19.06-fold decrease in GLP-1R activation and 12.46-fold
decrease in GCGR activation compared with OXM. Compounds 13 to 16 showed good GLP-1R
activation ability. It was demonstrated that the C-terminus sequences of Exenatide had positive effect
on raising GLP-1R activation in hybrid peptides without resulting in drastic reduction in GCGR
activation. It was reported that the fatty acid chain would interrupt the interaction force between
OXM/GLP-1 analogues and GLP-1R/GCGR in many studies 30, 31. However, in this work, we found a
1
1

well-marked elevation on GLP-1/GCGR activation after fatty acid was introduced in each hybrid
peptide. The activation on GLP-1R of compound 13a had a 3.65-fold increase compared with that of
compound 13, while that of compound 16a had a 10.95-fold increase compared with that of compound
1
6. The most potent agonist on GLP-1/GCGR in this group was compound 16a that could activate
GLP-1R with an EC of 0.20±0.5 pM and activate GCGR with an EC of 2.70 ± 0.4 pM. Therefore,
5
0
50
the potential of compound 16a for further research is optimistic.
Figure 5. Structures of compounds
Table 3. EC of OXM and OXM analogues
5
0
peptides
mGLP1R (pM)
mGCGR (pM)
16.5±0.6**
peptides
10b
mGLP1R (pM)
5.4±0.6**
mGCGR (pM)
12.3±1.1**
1
1
1
1
1
0a
.9±0.7**
6
1
1
1
1
1
33.2±1.5**##
102.5±1.2**
11a
12a
13a
14a
52.2±0.8**##
27.4±0.6
2
2.4±1.1**##
3.5±0.8**
1.6±0.3**
7.6±1.7
3
5.4±0.6##
22.5±1.7**
2.3±0.5**##
128.0±1.9**
19.3±1.8**
4
9.5±1.5**##
62.5±1.2**
11.4±0.9**##
1
2

1
1
5
6
.9±0.9**
22.1±0.4**
15a
16a
2.5±0.4**##
6
3
3.4±0.6
.8±0.4**
13.6±1.3**
0.3±0.1**##
2.5±0.3*
Results are expressed as mean ± SD, *P<0.05, **P<0.01 vs OXM, # P<0.05, ## P<0.01 vs Exenatide.
2
.6 Preliminary activity assessment and hypoglycemic test
Traditional IPGTT has a limitation that it could not evaluate the long-acting plasma glucose
lowering effect. Therefore, we ameliorate the procedure by injecting the compound intraperitoneally
in Kunming mice and loading the glucose 24 hours later. After the glucose was injected, the plasma
glucose was measured at 0, 15, 30, 60 and 120 min. As shown in Figure 6A, the activation abilities of
compound 13a, 15a and 16a were superior in IGPTT while the others including Exenatide and
Liraglutide almost lost their activation. Afterwards, compound 11a-16a, OXM and saline were
administrated once a day for 2 weeks (Figure 6B), and compound 16a exhibited good slimming effect,
thus it was chosen for further studies.
Plasma and compounds were incubated over 96 h for evaluating in vitro stability of long-acting
compounds 11a-16a. As illustrated in Figure 6C, the in vitro half-life of most compounds was
prolonged compared with exenatide and liraglutide. More specifically, the plasma half-life of
compound 16a is up to 71.79 h.
The hypoglycemic effects of compound 16a were assessed via intraperitoneal injection in STZ-
induced diabetic mice. Normal blood glucose level was set as no more than 8.35 mmol/L (150 mg/dL).
Euglycemic durations under this value were calculated and considered as a practical indication for
3
2
antidiabetic treatment . As shown in Figure 6D-E, saline-treated control mice exhibited a
hyperglycemic state (average > 25 mmol/L) while those injected with Exenatide and Liraglutide (25
nmol/kg) rapidly maintained normal blood glucose level for nearly 3.8 h and 12.4 h, respectively. At
a dose of 25 nmol/kg, the time required to rebound to a glucose level of 8.35 mmol/L was ~42.6 h in
1
6a-treated mice which was much longer than that in Exenatide-treated mice and better than that in
Liraglutide-treated mice. The AUC0–60 h values of 16a were 2.63 times lower than those of Exenatide
and 1.93 times lower than those of Liraglutide.
The in vivo glucoregulatory and insulin tropic activities of 16a were examined in SD rats by
IPGTT. After intraperitoneal injection of 16a, Liraglutide or Exenatide at -30 min, a glucose challenge
of 10 g/kg was loaded at 0 min, then the mean blood glucose levels at 15, 30, 45, 60, 90, 120 and 180
1
3

min were measured. A significant increase in the glucose tolerance patterns was observed. As shown
in Figure 7, after a glucose challenge, the mean blood glucose level in the saline-treated group rapidly
increased to 16.94 ± 3.6 mmol/L at 30 min, while in those injected with Liraglutide, Exenatide and
1
6a, the level dramatically reduced to ~7.0, ~6.4, and ~6.9 mmol/L at the same time point, respectively.
Moreover, the glucose AUC of Liraglutide, Exenatide and 16a were significantly lower than that of
the saline-treated group. The decreases in plasma glucose came with the increases in plasma insulin
concentration which was consistent with the GLP-1-dependent mechanism. Similar time courses for
plasma insulin concentrations in 16a-, Liraglutide- or Exenatide-treated group were observed and all
plasma insulin concentrations from 15 to 60 min time points were significantly greater than those of
the control group. In particular, 16a showed comparable promoting insulin secretion ability than
Liraglutide and Exenatide (p < 0.05). The insulin tropic activities and glucose-lowering abilities of 16a
were also comparable with those of Liraglutide and Exenatide.
1
4

Figure 6. (A) IPGTT result of 13a, 15a and 16a. (B) Body weight change after 2 weeks of treatment
by 11a-16a. (C)Plasma half-life of compounds
Glucose-lowering and stabilizing effect of Liraglutide, Exenatide and 16a as determined by
hypoglycemic duration test in nonfasted diabetic mice. (D) Time-course average blood glucose levels
of diabetic mice after an i.p. injection of Liraglutide, Exenatide or 16a (25 nmol/kg). Times depict
hypoglycemic duration rebound to 8.35 mmol/L. (E) Hypoglycemic effects of Liraglutide, Exenatide
and 16a based on the calculated glucose AUC0-60h values.
Figure 7. In vivo biological activity tests of 16a, Liraglutide and Exenatide (25 nmol/kg)i.p. injected
into SD rats; glucose was taken orally (10 g/kg). (A) Insulinotropic activities and calculated insulin
AUC0−180min (B) of Liraglutide, Exenatide and 16a. (C) The glucose-lowering effects and calculated
plasma glucose AUC0−180min (D) of Liraglutide, Exenatide and 16a.
2
.7 Chronic in vivo studies on STZ-induced diabetic mice
To prove in vivo activity and potential therapeutic efficiency towards diabetes, compound 16a,
Liraglutide, Exenatide and saline were chronically administrated to STZ-induced diabetic mice every
day. As shown in Figure 8A, before the treatment, fasting blood glucose level in diabetic mice was
1
5

~20.0 mmol/L. After 21 days, it increased to ~22.0 mmol/L in saline-treated group, while treatment
by compound 16a lowered the fasting blood glucose toward the normal levels. Furthermore, HbA1c
was a more sensitive index of diabetes control than blood glucose level. HbA1c was a more sensitive
index of diabetes control than blood glucose level. HbA1c was generated from non-enzymatic
irreversible glycation and thus it could be regarded as an indirect indicator of cumulative blood glucose
4
2
concentrations . As shown in Figure 8B, initially, HbA1c was well matched in all diabetic groups.
During the 21-day treatment, diabetic mice daily injected with 16a exhibited a marked reduction on
HbA1c, while it was high in the control group. The HbA1c reduction caused by the administration of
1
6a is indicative of the promising role of 16a in clinical studies. To determine whether long-term
treatment by 16a could improve glucose tolerance or not, IPGTT test was performed before and after
treatment (day 0 and day 24). At the beginning of treatment, few differences were observed between
mice in each group. However, at the end the treatment, 16a-, Liraglutide- and Exenatide-treated mice
showed rapid blood glucose reductions, and it returned to baseline at 120 min after glucose
administration, whereas saline-treated mice showed a typical diabetic pattern of slow reduction in the
hyperglycemic state at 120 min (Figure 8C). The blood glucose AUC of 16a was significantly lower
than that of the saline-treated diabetic mice (p < 0.05) (Figure 8D).
Compound 16a improved liver regeneration in STZ-induced diabetic mice, and regeneration ratio
of liver corrected by body weight was analyzed at the end of the 3-week treatment of saline, Liraglutide,
Exenatide or 16a. Saline-treated diabetic mice had a low ratio of liver weight/body weight compared
to the normal mice (Figure 8E,). Compound 16a-treated mice had a significant increase in liver
weight/body weight ratio. Meanwhile, serum ALT and AST were assayed on a Beckman Coulter
Chemistry Analyzer. As shown in Figure 8F-G, ALT and AST increased in saline-treated mice, while
treatment by compound 16a reduced both ALT and AST levels. To evaluate the effect of compound
1
6a on the morphology of pancreatic islets in STZ-induced diabetic mice. The number of insulin-
positive beta cells were reduced in saline-treated mice compared with that in normal mice. As shown
in Figure 8H, 16a-treated mice had a significant increase in the number of insulin-positive beta cells
than saline-treated mice.
1
6

Figure 8. The effects of chronically administered 16a, Liraglutide and Exenatide in diabetic mice.
A) Fasted plasma glucose measured every five days. (B) HbA1C measured at day 24. (C) IPGTT
(
results in mice at day 24. (D) AUC of IPGTT test results in mice at day 24. E) Regeneration ratio of
liver corrected by body weight was analyzed at the end of the 4-week treatment of saline,
Liraglutide, Exenatide or 16a. Effects of 16a on serum biomarkers examined at the end of the 4-
1
7

week treatment. (H) Representative images of pancreatic insulin-positive β cells after treatment.
.8 Chronic in vivo studies on DIO mice
2
During the 4-week treatment course, saline-treated mice did not inhibit cumulative food and water
intake (Figure 9A-B), while compound 16a reduced cumulative food and water intake by up to 49.6%
and 42.1%, respectively. Noticeably, after 4-week treatment (Figure 9C), compound 16a lowered
body weight of DIO mice significantly (~4.3 g). To further confirm the appetite suppression effect, an
acute in vivo study was conducted (Figure 9D,), and compound 16a could inhibit food intake by up to
7
0.2% at 6 h after administration. In addition, in the saline-treated DIO mice, total cholesterol (TC)
and triglyceride (TG) level were higher than those in the lean mice. However,compound 16a reduced
TC and TG to a normal level after treatment. Two important adipocytokines secreted from white
adipose tissue, the serum levels of leptin and adiponectin, were also altered after treatment by 16a.
Circulating leptin concentration in DIO mice was significantly higher than that in LFD controls (p <
0
.05). In contrast, administration of 16a significantly (p < 0.05) reduced serum leptin level to a near
normal value of 2.3 ± 0.2 ng/ml. As shown in Figure 9I, compound 16a-treated mice had a significant
increase in the number of insulin-positive beta cells than saline-treated mice.
1
8

Figure 9. Effects of 16a in DIO mice. (A) Time course for chronic effect of 16a administered for 4
weeks in DIO mice on cumulative food intake. (B) Time course for chronic effect of 16a
administered for 4 weeks in DIO mice on cumulative water intake. (C) Time course of 16a effect on
changes in body weight. (D) Time course of acute effect on food intake in fasted mice with
intraperitoneal doses of 16a. Effects of 16a TC (E), TG (F), leptin (G), adipocytokines (H) examined
at the end of the 4-week treatment. (H) Representative images of pancreatic insulin-positive β cells
after treatment.
3
. CONCLUSIONS
In summary, six hybrid peptides based on glucagon were designed and synthesized. The
1
9

glucagon-exenatide hybrid peptide, compound 10 had better receptor activation and glucose tolerance
abilities. The intraperitoneal injections of 10 every other day for one month decreased the body weight
of DIO mice significantly. A series of fatty acid attached compounds were designed and synthesized
to prolong the half-life of compound 10. A well-marked elevation was found on GLP-1/GCGR
activation after fatty acid was introduced. In hypoglycemic test, the most potent GLP-1/GCGR
agonism, compound 16a, maintained normal blood glucose level for nearly 42.6 h in the STZ-induced
diabetic mice. Moreover, compound 16a showed comparable promoting insulin secretion ability to
Exenatide and Liraglutide. The insulin tropic activities and glucose-lowering abilities of 16a were
comparable with those of Liraglutide and Exenatide. During the 21-days treatment, the HbA1c in the
diabetic mice daily injected with 16a exhibited a marked reduction, while it continued to be high in
the control group. Furthermore, compound 16a improved liver regeneration in the STZ-induced
diabetic mice. In the 4-week treatment on DIO mice, compound 16a reduced cumulative food and
water intake and lowered body weight of DIO mice significantly. Compound 16a with good receptor
activation ability, plasma glucose regulator properties, body weight lowering effect, long-term
beneficial effects and most importantly, long-acting antidiabetic properties, could be potentially useful
for the development of OXM analogues.
4
4
. Materials and Methods
.1 Chemistry
Fmoc Rink amide-MBHA resin, Fmoc-protected amino acids, Liraglutide, Exenatide, and Gly8-
GLP-1(7−36)-NH were purchased fromGL Biochem (Shanghai, China); HPLC grade acetonitrile and
2
methanol were purchased from Merck (Darmstadt, Germany); cAMP dynamic kit was purchased from
Cisbio (Bedford, MA); rat insulin ELISA kit was purchased from Millipore (Billerica, MA); HbA1c
kit was purchased from Glycosal (Deeside, UK). All other reagents, unless otherwise indicated, were
obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA) and used as received. Microwave
irradiation procedures were performed in a Discover focused single mode microwave synthesis system
(CEM, NC), which generated continuous irradiation at 2450 mHz. HPLC purification were performed
on a Shimadzu LC-8A preparative RP-HPLC system. Purity verification and ESI mass spectra were
obtained on a Waters ACQUITY UPLC system (Milford, MA). Sprague−Dawley (SD) rats (male,
2
00−250 g) were purchased from the Comparative Medical Center of Yangzhou University (Jiangsu,
2
0

China). Kunming mice, ob/ob mice and wild-type nondiabetic C57BL/6 littermate (male and female,
−8 weeks old) were obtained from the Model Animal Research Center of Nanjing University (Jiangsu,
6
China). Animals were housed in groups of three (rat) and six (mice) in cages under controlled
temperature (22 ± 2 °C) and relative air humidity (set point 50%) with a 12 h light:12 h dark cycle.
Tap water and standard laboratory chow were provided ad libitum throughout the study. High fat diet
(D12492; 60% fat, 20% protein and 20% carbohydrate; 5.24 kcal/g) and low fat diet (D12450B; 10%
fat, 20% protein and 70% carbohydrate; 3.85 kcal/g) were supplied by Research Diets (New Brunswick,
NJ, USA). All animal experimental protocols adhered to the Guide for the Care and Use of Laboratory
Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1986).
4
.1.1 General synthetic route and HPLC purification of peptides
Peptides were prepared using methodology called solid phase peptide synthesis (SPPS). Briefly,
Fmoc Rink amide-MBHA resin (0.01 mmol) was deprotected with 25% piperidine in 5 mL of DMF
for 4 min under microwave irradiation (10 W). A mixed solution of 0.025 mmol of first amino acid,
0
.030 mmol of HBTU, 0.030 mmol of HOBt, and 0.050 mmol of DIPEA dissolved in 2.5 mL of DMF
was added after washing three times with DMF. The mixture was bubbled with N for 10 min under
2
microwave irradiation (31 W) and washed three times with DMF. Deprotection and coupling were
repeated with the relevant Fmoc-protected amino acids, and peptides were cleaved with 7 mL of
reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 4 h at room temperature. Then,
peptides were precipitated by addition of 50 mL of cold ethyl ether. After centrifugation, the crude
peptides were purified using a Shimadzu preparative RP-HPLC: samples were injected into a
Shimadzu C18 reverse phase column (5 μm, 340 mm × 28 mm) equilibrated in water with 0.1% TFA
and purified using a linear gradient from 30% to 75% acetonitrile with 0.1% TFA over 30 min at a
flow rate of 6.0 mL/min with UV detection at 214 nm. The molecular mass of the purified peptides
was confirmed by LC−MS.
4
.1.2 General synthetic route of fatty chain-maleimide
Hexadecan-1-amine (482.6 mg, 2mmol) and furan-2, 5-dione (235.5 mg, 2.4 mmol) were
dissolved in acetic acid, stirred at 120 ℃ for 8 h. The reaction was cooled to room temperature, and
solvent was removed by reduced pressure condense. The crude solid was purified by column
chromatography over silica gel using light petroleum/ethyl acetate (1:3, v/v) to give the final
2
1

1
compound (579.6 mg, yield 90.0%), m.p. 103–105 ℃. H NMR (DMSO-d , 300 MHz): d 7.01 (s, 2H,
6
COCH CHCO), 3.38 (t, 2H, J = 6.2 Hz, NCH ), 1.49–1.45 (m, 2H, NCH CH (CH ) CH ), 1.23 (s,
5
2
2
2
2 13
3
2
6H, NCH CH (CH ) CH ), 0.85 (t, 3H, J = 6.4 Hz, NCH CH (CH ) CH ); ESI-MS: m/z Calcd for
2 2 2 13 3 2 2 2 13 3
+
C H NO [M+H] 322.0 found.
2
0
35
2
4
.1.3 General synthetic route of conjugates
Cysteine altered peptide (5 mmol) was conjugated with fatty chain-maleimide (12 mmol) in 5 mL
of 0.05 mol/L sodium phosphate buffer (pH 7.0). The reaction mixture was stirred at 20 ℃ under N2
for 1.5 h until UPLC showed completion. The analytical condition was as follows: C18 column (5 mm,
1
50 mm × 4.6 mm); a linear gradient of mobile phase 20–80% B in 20 min (mobile phase A: water
with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 1 mL/min, and
ultraviolet (UV) detection at 214 nm was utilized. The crude conjugate was purified on Shimadzu
preparative RP-HPLC: Shimadzu C18 reversed-phase column (5 mm, 340 mm × 28 mm), a linear
gradient of mobile phase 40–90% B (mobile phase A: water with 0.1% TFA, mobile phase B:
acetonitrile with 0.1% TFA) in 30 min at a flow rate of 6.0 mL/min, and ultraviolet (UV) detection at
2
14 nm. The molecular mass of the purified conjugates was confirmed by UPLC–MS.
4
4
.2 Pharmacology
.2.1 GLP-1 and glucagon receptor activation assay
a) Construction of GLP-1 receptor retroviral expression plasmid. A large number of retroviral
vector pBABE-puro plasmid with puromycin resistance and pMSCV-sulfuric acid GLP-1 receptor
plasmid containing GLP-1 receptor with full-length cDNA sequence were prepared. The primers of
GLP-1 receptor gene were synthesized by Shanghai Shenggong Biology Co. Ltd., and the ends of the
receptor gene were digested with EcoRI and NcoI respectively. The target gene sized 1.7 kb was
amplified by PCR, and the template was the pMSCV- sulfuric acid GLP-1 receptor plasmid
preservative. The PCR product was digested with the restriction endonuclease EcoRI and NcoI, and
then the TaKaRa DNA Ligation Kit (TaKaRa Biotechnology Co. Ltd. Dalian) was used to connect the
PCR target gene and the pBABE-puro empty vector for the product named pBABE-HPA. The
recombinant was transformed into Escherichia coli DH5α competent cells, cultured in LB solid
medium containing Ampicillin and selected for positive clones of Ampicillin. Transfection could be
identified by PCR, restriction endonuclease digestion and sequencing.
2
2

b) Preparation of retrovirus. 24 h before transfection, Phoenix 293 cells were cultured in plate.
5
Trypsin digests Phoenix 293 cells and seeded into 6-well plates in 2 x 10 cells/well, incubated to about
7
0-80% when cultured to about 1 mL of serum-free antibiotic Opti-MEM medium. Plasmid DNA (2
μl) and 5 μL of Lipofectamin 2000 Reagent were diluted with 130 μL of serum-free antibiotic Opti-
MEM medium, respectively, standing for 5 min before mixing. The mixed solution was added to a 6-
well cell culture plate. The complete medium was replaced by the orifice place after 4-6 h to continue
training for 48 h. After the virus was produced in Phoenix 293 cells, the virus supernatant was collected
by centrifugation and protected from light at 4 ° C after filtration using a 0.45 μm pore size filter.
c) Establishment of stable expression of cell lines. Normal HEK293 cells in the six-well plate, 2
5
×
10 cells each hole, cultured 24h, was added with 2 mL of each virus supernatant and polybrene
(final concentration of 6 μg/mL). The cells were cultured in the incubator (32℃) after 30 min of
centrifugation with 1000 rpm at room temperature. Five hours later, the medium was replaced to
normal, which was cultured at 37℃ for48 h. In order to obtain a stable virus-infected cell line, 0.45
μg/mL of puromycin was added to the platelet for one week.
d) Human embryonic kidney (HEK293) cells transfected with either the human GLP-1R or the
human GCGR, were used to assess effects on cAMP production. Two hours before the test started,
cells were plated in 96-well half area with solid black microplates. The conjugates were solubilized in
DMSO and further diluted in medium containing 0.1% BSA fraction V (Genview Scientific, Florida,
USA). The maximum final DMSO concentration in the assay was 0.1%. The resulting solution was
added to cells which was incubated for 20 min. It was then assayed for cAMP using the kit with
homogenous time-resolved fluorescence technology (Cisbio, Bedford, MA, USA) tested by an
Envision 2104 Multilabel Reader according to the manufacturer’s instructions. The potency of the
conjugates (EC50 values) was calculated by sigmoidal curve fitting using GraphPad Prism version 5.0
(GraphPad, San Diego, CA, USA).
4
.2.2 Plasma stability test
Plasma were obtained from adult male Sprague-Dawley (SD) rats and compounds were then
incubated with plasma over 96 h. Samples from each time points underwent solid-phase extraction and
the resulting extract was analyzed by LC-MS/MS to assess the profile of plasma degradation. In the in
2
3

vitro stability test, the initial concentration in rat plasma of lipid chain modified conjugates 11a-16a,
and the positive controls (Exenatide and liraglutide) were 1000 ng/mL at 37 ℃. At 0, 1, 2, 4, 6, 8, 12,
2
4, 36, 48, 72 and 96 h time points, 100 μL of mixture was aliquoted, extracted on an Oasis HLB 96-
well plate (Waters, Milford, MA,USA) and analyzed by LC-MS/MS. The signal of test articles was
detected by multiple reaction monitoring with the use of electrospray ionization mass spectrometry on
a Sciex API-4000and Turbo Ionspray (Applied Biosystems, Foster City, CA, USA). The condition of
reverse phase liquid chromatographic separation was as aforementioned.
4
.2.3 Glucose tolerance test in normal Kunming mice
Male Kunming mice (8 weeks, male, 6-8 per group) were acclimatized to laboratory conditions
for at least one week. For the determination of glucose tolerance, the mice were subjected to 12 h
fasting. At 0.5 h prior to the oral glucose loading by intubation (18 mmol/kg glucose in 0.9% w/v
saline), mice were administered with OXM or OXM analogues (25 nmol/kg in 0.9% w/v saline)
intraperitoneally. Tail blood glucose levels were measured by using the tail cut method and blood
glucose strips (San Nuo Changsha, Changsha, China) at-30, 0, 15, 30, 60, 90 and 120 min.
4
.2.4 Acute feeding study and body weight change in normal ICR mice
For acute feeding study, mice (9 weeks, male, 6-8 per group) were fasted for 8 h preceding the
injection. All peptides were dissolved in 0.9% saline. After intraperitoneal injection of OXM
analogues (25 nmol/kg) and 0.9% saline as control at 9 o’clock AM, animals were returned to their
home cages containing a preweighed amount of food that was reweighed at 0.5, 1, 2, 4, 8, and 24 h
after injection.
For body weight change study, mice (10 weeks, male, 6-8 per group) were assigned to treatment groups
based on their body weight. The mice were fasted for 12 h preceding the injection. All peptides were
dissolved in 0.9% saline. OXM analogues (25 nmol/kg) and 0.9% saline as control were
intraperitoneally injected once daily at 9 o’clock AM for two weeks. Body weight and food
consumption were measured every day at the same time.
4
.2.5 Energy balance physiology measurements
C57Bl/6 mice (8 weeks) were singly housed and fed on either standard chow (D12491: 20% kcal
from fat; Research Diets) or high fat diet (D12492: 60% kcal from fat; Research Diets) in a 12 h
2
4

light/12 h dark cycle for 14 weeks with free access to food and water. Mice (22 weeks) were
randomized and distributed to test groups according to body weight and body composition. Energy
intake and expenditure, as well as home-cage activity, were assessed by using a combined indirect
calorimetry system (TSE Systems) in DIO mice (22 weeks, male, 6 per group, body weight ~40g). O2
consumption and CO production were measured every 39 min for a total of 120h (including 24h of
2
adaptation) to determine the respiratory quotient and energy expenditure. Food intake was determined
continuously for 120 h at the same time. Home-cage locomotor activity was determined using a
multidimensional infrared light beam system with beams scanning the bottom and top levels of the
cage and activity being expressed as beam breaks
4
.2.6 Chronic treatment on DIO mice by 7 and 10
DIO mice (23 weeks, male, 6 per group, body weight ~40g, diet D12492) were administered
intraperitoneally with 7 (25 nmol/kg), 10 (25 nmol/kg), control (OXM (25 nmol/kg), Exenatide(25
nmol/kg) or 0.9% saline as single injection every other day at 9 o’clock in the morning for 30 days.
Body weight, food consumption and water intake were measured every day.
4
.2.7 Hypoglycemic efficacies test
Hypoglycemic efficacies of compounds were evaluated on the STZ-induced diabetic mice using
3
0
a modified previously described method . Kunming mice received a single intraperitoneal injection
of compound at 25 nmol/kg body weight intraperitoneally with free access to food and water. At 0, 1,
2
, 3, 4, 6, 8, 12, 16, 20, 24, 30, 36, 48, 60 h, the second drop of blood was drawn from a tail vein to
determine the blood glucose levels by blood glucose monitor. Moreover, the hypoglycemic durations
were also calculated with the blood glucose level <8.35 nmol/L (150 mg/dL).
4
.2.8 Glucoregulatory and insulin secretion assay
The glucoregulatory and insulin secretion assays were carried out in accordance with a previous
method. Briefly, overnight fasted (12 h) SD rats (n = 6/group, 200–250 g) were orally administrated
with 16a, Exenatide and Liraglutide (25 nmol/kg) with saline used as negative control. Male SD rats
were administered saline, Exenatide, Liraglutide or 16a half hour prior to loading of oral glucose (10
g/kg) (the time point was set as 0 min). At 30, 0, 15, 30, 45, 60, 90, 120 and 180 min, blood sample
was collected from the cut tip of the tail vein to measure the blood glucose level using a blood glucose
2
5

monitor. Meanwhile, blood samples (0.1 ml) were collected in EDTA-containing microcentrifuge
tubes from the lateral tail vein at the same aforementioned time point. Plasma samples were then
obtained by centrifugation (1000 rpm, 15 min) and assayed for insulin levels using a Rat Insulin ELISA
kit (Millipore, Billerica, MA, USA).
4
.2.9 Chronic in vivo studies on STZ-induced diabetic mice
Male Kunming mice (6–8 weeks old, weighing 18–22 g) were injected with STZ intraperitoneally
(
40 mg/kg/day) for 5 consecutive days after fasted overnight. Five days after STZ injection, blood
glucose was measured to validate diabetic hyperglycemia. Mice with fasting blood glucose level of
1.1 mM or higher were assigned to groups with matched body weight and were injected
intraperitoneally with 25 nmol/kg Exenatide, Liraglutide, or 16a once daily for 3 weeks. On days 0,
, 10, 15, and 20, overnight fasting blood samples were obtained from the tail vein to measure blood
1
5
glucose levels. At the end of the study, mice were sacrificed and blood samples were collected to
determine the levels of HbA1c by Beckman Coulter Chemistry Analyzer (Tokyo, Japan). At the end
of the study, blood samples were collected via arteria carotis under anaesthesia by sodium
pentobarbital and sera separated subsequently for further analyses.
4
.2.10 Chronic in vivo studies on DIO mice
C57BL/6 mice (6 weeks) were maintained on prescribed DIO for 12 weeks. They were injected
intraperitoneally once daily with 25 nmol/kg of Exenatide, Liraglutide or 16a, respectively with
saline as control after randomly assigned to treatment groups (n = 6) with matched body weight.
C57BL/6 mice fed with LFD (n = 6/group) were used to index responses to normal values. Water
consumption, food intake, and body weight were measured daily. At the end of the study, blood
samples were collected via arteria carotis under anaesthesia by sodium pentobarbital and sera separated
subsequently for further analyses.
4
.2.11 Data analysis and statistical assessment
Data were analyzed using Prism version 5 software (GraphPad, San Diego, CA, USA). General
effects were tested using a 1-way ANOVA with Tukey’s multiple-comparison post hoc test. Data
throughout are stated as mean ± SD. p < 0.05 was considered as significant.
2
6

Acknowledgements
This study was supported by grants from National Natural Science Foundation of China (81872734,
8
1570734 &81673299).
Conflict of interest
The authors declare no competing financial interest.
Abbreviations
AUC, area under the curve; cAMP, cyclic adenosine monophosphate; DIO, diet induced obese; EC50,
half maximally at an effective concentration; Ex-4, exenatide; GCG, glucagon; GCGR, glucagon
receptor; GLP-1, glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; HFD, high fat
diet; HPLC, high-performance liquid chromatography; i.p, intraperitoneal administration; LC−MS,
liquid chromatography−tandem mass spectrometry; LFD, low fat diet; NA, not active; NT, not test;
OXM, oxyntomodulin; TFA, trifluoroacetic acid; WAT, white adipose tissue.
2
7

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