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A novel chemically modified analogue of xenin-25 exhibits improved glucose-lowering and insulin-releasing properties
Vadivel Parthsarathy, Nigel Irwin, Annie Hasib, Christine M. Martin, Stephen
McClean, Vikas K. Bhat, Ming T. Ng, Peter R. Flatt, Victor A. Gault
SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of
Ulster, Coleraine BT52 1SA, Northern Ireland, UK
Address correspondence and reprint requests to Professor Victor Gault
Email: [email protected]
Phone: ++44(0)-28-7012 3322
Fax: ++44(0)-28-7012 4965
Keywords: glucose homeostasis, gut hormone, insulin secretion, type 2 diabetes, xenin-25
ABSTRACT
Background: Xenin-25 is a K-cell derived gut peptide with insulin-releasing activity which is
rapidly degraded following release into the circulation. We hypothesized that substitution of
all naturally-occurring Lys and Arg residues with Gln would lead to prolonged enzyme
resistance and enhanced biological efficacy.
Methods: Peptide stability was assessed using murine plasma, in vitro insulin-releasing
actions evaluated in BRIN-BD11 cells and acute glucose-lowering and insulin-releasing
actions examined in high fat fed mice. For sub-chronic studies, a range of metabolic
parameters and pancreatic histology were assessed in high fat fed mice which had received
saline vehicle or xenin-25(gln) twice-daily for 21 days.
Results: In contrast to native xenin-25, xenin-25(gln) was resistant to plasma-mediated
degradation and significantly stimulated insulin secretion in BRIN-BD11 cells. Acute
administration of xenin-25(gln) in high fat fed mice significantly reduced blood glucose and
increased plasma insulin concentrations. Twice-daily administration of xenin-25(gln) in high
fat fed mice did not affect food intake, body weight or circulating insulin concentrations but
significantly decreased blood glucose from day 9 onwards. Furthermore, glucose tolerance,
glucose-mediated insulin secretion, insulin sensitivity and GIP-stimulated insulin-release
were significantly enhanced in xenin-25(gln)-treated mice. Pancreatic immunohistochemistry
revealed decreased alpha cell area with increased beta cell area and beta-to-alpha cell ratio in
xenin-25(gln)-treated mice. In addition, xenin-25(gln) exerted similar beneficial actions in
ob/ob mice as demonstrated by reduced blood glucose, superior glycaemic response and
glucose-mediated insulin release. Conclusions: xenin-25(gln) is resistant to plasma-mediated
degradation and exerts sustained and beneficial metabolic actions in high fat fed and ob/ob
mice.
General significance: Glutamine (gln)-modified analogues of xenin may represent an
attractive therapeutic approach for type 2 diabetes.
1. Introduction
Xenin-25 is a 25 amino acid mammalian peptide
(MLTKFETKSARVKGLSFHPKRPWIL) which has six identical C-terminal amino acids to
the amphibian octapeptide, xenopsin (EGKRPWIL) [1]. Although originally isolated from
human gastric mucosa, xenin-25 is present in several other organs and tissues including the
hypothalamus, liver, pancreas and stomach, indicating a broad range of biological actions [2].
Given the similarity between xenin-25 and neurotensin (ELYENKPRRPYIL), classical
studies were directed at probing the actions of xenin-25 on gastrointestinal function. This
early research demonstrated that xenin-25 increased gut motility, and inhibited both food
intake and gastric acid secretion [3,4]. More recent studies have demonstrated that xenin-25
can act as an independent insulinotropic agent [5] and a potentiator of gastric inhibitory
polypeptide (GIP)-induced insulin secretion [5,6,7]. Thus, xenin-25 represents a potential
therapeutic drug for patients with type 2 diabetes who exhibit defective insulin secretion,
including pancreatic beta cell GIP insensitivity [8].
As with many gut peptides, xenin-25, secreted from intestinal K-cells together with GIP
is rapidly metabolised by enzymes in the circulatory system [2,9]. Unlike the incretin peptides
glucagon-like peptide-1 (GLP-1) and GIP, dipeptidylpeptidase-4 (DPP-4) does not appear to
play a role in the degradation of xenin-25 [5]. Recent observations from our laboratory using
mass spectrometry has identified several degradation fragments including xenin 9-25, xenin
11-25, xenin 14-25 and xenin 18-25 [7]. Interestingly, xenin (9-25, 11-25 and 14-25) do not
appear to exhibit biological activity whereas the actions of xenin 18-25 are similar to native
xenin-25 [10,11]. Whilst precise mechanisms involved in xenin-25 metabolism are still not
fully understood, current thinking suggests a key role for serine protease-like enzymes [7].
Thus, given that xenin-25 contains six Lys or Arg residues, the enzyme trypsin poses a
significant threat to the long-term stability to the native form of the peptide [12]. Glutamine
(gln) amino acid substitutions have been shown to confer stability on alpha helical peptide
structures [13], therefore replacing Lys and Arg residues with Gln may be one strategy to
avoid trypsin degradation and provide greater stability to xenin-25.
In light of this observation, we hypothesized that substituting all six Lys and Arg residues
in xenin-25 (K4, K8, R11, K13, K20 and R21) with Gln residues would significantly enhance
stability and biological efficacy. Accordingly, we designed an analogue of xenin-25 (xenin-
25(gln)) and performed initial experiments to evaluate metabolic stability, in vitro insulin-
releasing actions and acute in vivo glucose-lowering properties. Given initial positive results,
we then assessed effects of twice-daily treatment with xenin-25(gln) on glycemic control,
insulin secretion, metabolic response to GIP, insulin sensitivity and islet morphology in
obese-insulin resistant high fat fed mice with additional observations using the ob/ob mouse
model of obesity-diabetes. The results provide the first experimental evidence that glutamine
(gln)-modified analogues of xenin may provide an effective means of treating type 2 diabetes.
2. Methods
2.1. Peptide synthesis and plasma stability
Native xenin-25 and xenin-25(gln) were obtained from GL Biochem Ltd. (Shanghai,
China; >95% purity). Peptide purity and identity were confirmed by in-house HPLC and
MALDI-ToF MS as described previously [7] and peptide characteristics are displayed in
Table 1. For plasma stability studies, peptides were incubated with murine plasma in the
presence of TEA-HCl buffer for 0, 2, 4, 8 and 24 hours and reaction products separated as
described previously [7]. HPLC peak area data was used to calculate percentage of intact
peptide remaining at each time point.
2.2. In vitro insulin secretion in BRIN-BD11 cells
Effects of native xenin-25 and xenin-25(gln) on in vitro insulin secretion were measured
using BRIN-BD11 cells whose characteristics have been described elsewhere [14]. In brief,
BRIN-BD11 cells were seeded (150,000 cells per well) into 24-well plates and allowed to
attach overnight at 37°C. Following a 40 min pre-incubation (1.1 mM glucose; 37oC) cells
were incubated (20 min; 37°C) in the presence of 5.6 or 16.7 mM glucose with a range of
peptide concentrations (10-12 to 10-6 M). After 20 min incubation, buffer was removed from
each well and aliquots (200 μl) stored at -20°C prior to measurement of insulin by
radioimmunoassay (RIA) as described previously [15].
2.3. Animals
Animal studies were carried out using male NIH Swiss mice (Harlan Laboratories UK
Ltd., Oxon, UK; 8-10 weeks old) and C57BL/6J ob/ob mice (Harlan Laboratories, UK Ltd.,
Oxon, UK; 10-12 weeks old). For high fat fed animals, mice were given free access to high
fat diet (45% AFE Fat; Product Code 824053; Special Diet Services, Witham, UK; total
energy 26.15 kJ/g) containing lard and soya oil for 70 days. High fat fed animals exhibited
progressive body weight gain (21.0 ± 0.6 versus 9.2 ± 1.0 g; p < 0.001) and hyperglycaemia
(12.0 ± 0.9 versus 0.8 ± 0.1 mM; p < 0.001) compared with age-matched controls on normal
laboratory chow. Ob/ob mice had free access to standard rodent chow (Teklad Global 18%
Protein Rodent Diet; Product Code 2018S; Harlan, UK; total energy 13.0 kJ/g). Animals were
housed in 12:12 h light/dark cycle (08:00–20:00 hours), remained on their respective diets for
duration of the study and had free access to drinking water. No adverse effects were observed
during experimental studies. Experiments were performed according to Principles of
Laboratory Animal Care (NIH publication no. 86-23, revised 1985) and UK Animals
Scientific Procedures Act 1986.
2.4. In vivo studies
Glucose and insulin concentrations were measured prior to and after injection of glucose
alone (18 mmol/kg body weight; ip) or in combination with xenin-25 or xenin-25(gln) (each
at 25 nmol/kg body weight; ip) in non-fasted high fat fed mice. Test solutions were
administered in a final volume of 5 ml/kg body weight. In the first series of sub-chronic in
vivo experiments, high fat fed mice received twice-daily injections (09:00 and 17:00 h) of
saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) for 21 days.
Peptide dose was chosen based on previous observations using xenin-based peptides [7]. Food
intake, body weight, circulating glucose and insulin concentrations were measured at regular
intervals. At the end of the study, glucose tolerance (18 mmol/kg body weight; ip; non-fasted
animals), insulin sensitivity (25 U/kg body weight; ip; non-fasted animals) and metabolic
response to native GIP (25 nmol/kg body weight; ip; fasted animals) tests were performed.
Pancreatic tissue was excised, weighed and stored for immunohistochemistry or processed for
measurement of insulin content following extraction with ice-cold acid ethanol [16]. In a
second series of experiments, ob/ob mice received twice-daily injections (09:00 and 17:00 h)
of saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) for a period
of 21 days and metabolic parameters were measured as described for high fat fed mice. All
test solutions were administered in a final volume of 5 ml/kg body weight.
2.5. Biochemical analyses
Blood samples were collected (at the time points indicated in the Figures) from the tail
vein of conscious mice into chilled fluoride/heparin micro-centrifuge tubes (Sarstedt,
Numbrecht, Germany) and centrifuged at 13,000 rpm for 3 min (Beckman Instruments,
Galway, Ireland). Blood glucose concentrations were measured using Ascencia Contour
Blood Glucose Meter (Bayer Healthcare, Newbury, UK) and plasma / pancreatic insulin
determined using a modified dextran-coated charcoal RIA [15].
2.6. Statistical analysis
Results were analyzed using GraphPad Prism version 5.0 (GraphPad Software Inc., CA,
USA) and data presented as the mean±S.E.M. Statistical analyses were performed using
students unpaired t-test. Where appropriate, data were compared using repeated measures or
one-way analysis of variance (ANOVA), followed by Student-Newman-Keuls post hoc test.
Incremental area under the curve (AUC) for plasma glucose and insulin and area above the
curve (AAC) for insulin sensitivity were calculated using GraphPad Prism. Groups of data
were considered to be significantly different if p < 0.05.
3. Results
3.1. Plasma stability and acute in vitro insulin secretion
As shown in Table 1, native xenin-25 was degraded by murine plasma resulting in a half-
life of less than 4 hours. In contrast, xenin-25(gln) remained completely intact and displayed
an in vitro half-life greater than 24 hours (Table 1). Native xenin-25 and xenin-25(gln)
stimulated (1.9 to 2.9 fold; p < 0.05 to p < 0.001) insulin secretion in a concentration-
dependent manner from BRIN-BD11 cells at both 5.6 and 16.7 mM glucose (basal values of
1.840.25 and 3.160.35 ng/1x106 cells/20 min, respectively; Fig. 1).
3.2. Acute in vivo glucose-lowering and insulin-releasing actions
Acute administration of xenin-25(gln) to high fat fed mice significantly improved glucose
tolerance and decreased blood glucose AUC0-60min concentrations compared to glucose given
alone (56% reduction; p < 0.001) or with native xenin-25 (38% reduction; p < 0.05) (Fig. 2A).
Correspondingly, plasma insulin concentrations were significantly increased at 15 min post-
injection and when calculated as AUC (3.4-fold; p < 0.05; Fig. 2B).
3.3. Twice-daily administration of xenin-25(gln) on body weight, food intake, circulating
glucose and insulin concentrations in high fat fed mice
Sub-chronic administration of xenin-25(gln) did not alter body weight (Fig. 3A) or food
intake (Fig. 3B) in high fat fed mice compared to control animals. Circulating blood glucose
concentrations were significantly reduced from day 9 onwards (34 to 40% reduction; p < 0.05
to p < 0.01) in xenin-25(gln) treated mice (Fig. 3C). Xenin-25(gln) did not alter plasma
insulin concentrations over the treatment period (Fig. 3D).
3.4. Twice-daily administration of xenin-25(gln) on glucose tolerance, insulin response to
glucose, insulin sensitivity and metabolic response to GIP in high fat fed mice
Administration of xenin-25(gln) for 21 days in high fat fed mice significantly decreased
(25 to 44% reduction; p < 0.05 to p < 0.001) glucose concentrations at 30, 60 and 105 minutes
following an intra-peritoneal glucose challenge (Fig. 4A). This was associated with a
significantly reduced glucose AUC value (54% reduction; p < 0.05) (Fig. 4A). Xenin-25(gln)-
treated mice also exhibited significantly elevated (1.6 to 1.7 fold; p < 0.05) overall glucose-
stimulated plasma insulin responses compared to high fat saline controls (Fig. 4B). As shown
in Fig. 4C, mice treated with xenin-25(gln) also displayed a significant improvement in
insulin sensitivity (24 to 28% reduction in blood glucose after insulin injection; p < 0.05).
This was accompanied by a marked increase in glucose AAC values (1.4-fold; p < 0.05; Fig.
4C). Xenin-25(gln) treatment also significantly decreased blood glucose concentrations (37%
reduction; p < 0.05) at 30 min after injection with native GIP together with the glucose load
(Fig. 5A). This improved glycaemic response was confirmed by a significantly reduced
glucose AUC value (43% reduction; p < 0.05; Fig. 5A). Xenin-25(gln) treated mice also
exhibited significantly enhanced (1.1-fold; p < 0.05) GIP-stimulated insulin release compared
to high fat saline controls (Fig. 5B).
3.5. Twice-daily administration of xenin-25(gln) on islet morphology and pancreatic
insulin content in high fat fed mice
Fig. 6A shows representative islet morphology of high fat fed saline control mice and
Fig. 6B illustrates islet morphology following 21 days treatment with xenin-25(gln) in high
fat fed mice. Xenin-25(gln) had no effect on the number of islets (Fig. 6C), islet area (Fig.
6D) or redistribution of islet size (Fig. 6H). However, xenin-25(gln)-treated mice exhibited
significantly reduced alpha cell area (62% reduction; p < 0.001; Fig. 6E) and increased beta
cell area (1.3-fold; p < 0.001; Fig. 6F) which was associated with a non-significant increase in
insulin content (data not shown). Furthermore, the beta cell to alpha cell ratio was
significantly increased (2.2-fold; p < 0.001; Fig. 6G).
3.6. Twice-daily administration of xenin-25(gln) on metabolic parameters in ob/ob mice
In ob/ob mice, xenin-25(gln) treatment had no effect on body weight (Fig. 7A), food
intake (Fig. 7B) and circulating plasma insulin concentrations (Fig. 7D) compared to normal
controls. However, glucose concentrations were significantly reduced from day 15 onwards
(34 to 45% reduction; p < 0.05 to p < 0.001) by xenin-25(gln) treatment (Fig. 7C). These mice
also displayed significantly decreased glucose concentrations in terms of both time-dependent
(27 to 32% reduction; p < 0.05 to p < 0.01) and overall AUC values (33% reduction; p < 0.05)
following an intra-peritoneal glucose challenge (Fig. 8A). Correspondingly, xenin-25(gln)-
treated mice exhibited significantly elevated (1.7 to 1.8-fold; p < 0.05) glucose-induced
insulin responses compared to saline controls (Fig. 8B). However, there was no improvement
in the glucose-lowering actions of exogenously administered insulin in xenin-25(gln)-treated
ob/ob mice (Fig. 8C).
4. DISCUSSION
Consistent with previous studies, native xenin-25 was rapidly degraded following
incubation with murine plasma exhibiting a half-life of between 2 and 4 hours [5]. In sharp
contrast, the modified peptide, xenin-25(gln), remained completely intact throughout the 24
hour plasma incubation indicating that substitution of the naturally-occurring Lys and Arg
residues with Gln protected against plasma-mediated degradation. The introduction of Gln
residues into peptide structures has been shown to protect against trypsin cleavage and
stabilization of alpha-helical stability [13]. However, further studies examining alpha helical
structure using circular dichroism and development of a specific assay to determine in vivo
biological half-life of xenin-25(gln) would be useful.
Xenin-25(gln) was observed to be equipotent to the native peptide with regards to in vitro
insulin secretion in a glucose-dependent manner in BRIN-BD11 cells. The actions of xenin-
25(gln) appear to be broadly similar to a previously characterised acylated xenin peptide [7],
suggesting that Gln substitutions did not adversely affect biological activity. Whilst the
mechanism of action of xenin-based peptides in islet cells is not fully understood and may be
distinct from xenin signalling in neural cells, we cannot rule out that xenin-25(gln) action in
islet cells may be specific and involve different signalling pathways from those associated
with the native peptide [6]. Furthermore, acute administration of xenin-25(gln) to high fat fed
mice resulted in a marked reduction in blood glucose and a significant increase in plasma
insulin concentrations. In contrast, the native peptide displayed only a moderate improvement
in glucose-lowering, which likely reflects rapid degradation and elimination [7] which is in
agreement with a lack of significant effects in type 2 diabetes [6]. Therefore, based on our
preliminary in vitro and in vivo data all further studies were conducted with the xenin-25
analogue, xenin-25(gln).
To assess the sub-chronic actions of xenin-25(gln) on metabolic control we employed
high fat fed mice. These mice displayed progressive weight gain, impaired glucose tolerance
and hyperglycaemia when compared to age-matched lean control animals on normal diet [16].
Twice-daily administration of xenin-25(gln) produced a marked improvement in glycemic
control both in terms of non-fasting circulating glucose concentrations and glucose tolerance.
Furthermore, xenin-25(gln) treatment elicited a significant increase in glucose-mediated
insulin secretion which is consistent with the action of xenin-25 as an independent stimulator
of insulin secretion [5,7]. Importantly, improvements in metabolic control were independent
of changes in body weight, however further detailed studies examining locomotor activity and
energy expenditure would be helpful. Given that xenin-25 has been shown to inhibit food
intake [17-20] and xenin-25 concentrations increase following a meal [21], it was interesting
to note that xenin-25(gln) did not affect food intake which could be due to the dose
administered in the present study and/or duration of the treatment period.
Improvements in glucose homeostasis following xenin-25(gln) treatment in high fat fed
mice were associated with enhanced hypoglycaemic action of exogenous insulin. Thus, xenin-
25(gln) serves not only to enhance beta cell function but improve insulin sensitivity. Whilst
not measured in the present study, effects of xenin-25(gln) on lipid profile, insulin signalling
proteins and circulating hormones such as glucagon and/or adipokines would be beneficial.
Indeed, a pyruvate or glycerol tolerance test could help provide important information on
hepatic glucose production following sustained xenin-25(gln) treatment. Previous studies
have shown that the bioactivity of native GIP is compromised in humans with type 2 diabetes
[22]. Interestingly, xenin-25(gln) treatment significantly potentiated the glucose-lowering and
insulinotropic response to native GIP indicating a marked improvement in GIP sensitivity.
Thus, whilst correction of hyperglycaemia has been shown to improve the insulinotropic
action of GIP [23], it is possible that part of the positive actions of xenin-25(gln) may well be
due to augmentation of GIP action [6,7]. Measurement of GIP receptor gene and protein
expression in islets following treatment with xenin-25(gln) mice would be useful in this
context. Immunohistochemistry revealed that mice treated with xenin-25(gln) exhibited
enhanced beta cell area which was associated with a non-significant tendency towards
increased insulin content. Interestingly, xenin-25(gln) treatment resulted in a significant
reduction in diabetes-induced expansion of the alpha cell which suggests a potentially useful
role for xenin in countering enhanced glucagon/glucagon secretion. Thus, high fat feeding is
known to induce alpha cell expansion in mice [add ref from letter – or perhaps find a better
ref??!]. These changes in alpha and beta cell area were not associated with any significant
changes in islet number, islet area or islet size distribution.
To further corroborate the anti-hyperglycaemic and insulinotropic effects of xenin-
25(gln) in diabetes, we performed an additional sub-chronic study using the genetically
obese-diabetic ob/ob mouse model. The ob/ob mouse presents with a very robust form of type
2 diabetes accompanied by hyperphagia, marked obesity, moderate hyperglycaemia, beta cell
dysfunction and severe hyperinsulinaemia [24]. Following twice-daily treatment with xenin-
25(gln), body weight and food intake were unchanged compared with control animals, as also
observed with high fat fed mice. Importantly, xenin-25(gln) significantly reduced non-fasting
blood glucose concentrations from day 15 onwards. Furthermore, glucose tolerance was
improved and this was accompanied by a significant increase in glucose-stimulated insulin
secretion thus confirming the long-acting insulinotropic properties of xenin-25(gln) in the
ob/ob model. In contrast to observations in high fat fed mice, improvement in glucose
homeostasis was independent of any change in insulin sensitivity which is likely due to the
severe insulin resistance in the ob/ob mutant [24]. We did not observe any adverse effects
following peptide administration.
In conclusion, the present study has demonstrated that the novel analogue, xenin-25(gln),
is resistant to plasma-mediated degradation with intact or enhanced in vitro and in vivo
biological activity. Furthermore, sub-chronic twice-daily administration of xenin-25(gln)
exerted beneficial effects on glycaemic control and insulin secretion in animal models of both
diet-induced and genetically inherited obesity-diabetes. These data highlight that xenin-
25(gln) could be positioned as a novel treatment for type 2 diabetes.
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Acknowledgements
This work was supported by the Department of Education and Learning (Northern Ireland)
and European Regional Development Fund/Invest Northern Ireland (ERDF/INI).
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Figure legendsFig. 1. Concentration-dependent effects of xenin-25 and xenin-25(gln) on insulin secretion in BRIN-BD11 cells at (A) 5.6 and (B) 16.7 mM glucose. BRIN-BD11 cells were incubated with a range of xenin peptide concentrations (10-12 to 10-6 M) for 20 min and insulin concentrations measured using radioimmunoassay. White bars, glucose alone; hatched bars, xenin-25; black bars, xenin-25(gln). Values are expressed as meanS.E.M. (n=8). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to respective glucose control.
Fig. 2. Acute actions of xenin-25 and xenin-25(gln) on (A) glucose and (B) insulin concentrations in high fat fed mice. Parameters were measured prior to (t=0) and 15, 30 and 60 min after administration of glucose alone (18 mmol/l kg body weight; ip) or in combination with xenin peptides (both at 25 nmol/kg body weight; ip). Glucose and insulin AUC values for 0-60 min post-injection are also included. White squares and white bars, glucose alone; black triangles and hatched bars, xenin-25; black circles and black bars, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05 and ***p < 0.001 compared to glucose alone. ∆p < 0.05 compared to native xenin-25.
Fig. 3. Effects of twice-daily administration of xenin-25(gln) on (A) body weight, (B) food intake, (C) glucose and (D) insulin concentrations in high fat fed mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. White circles, saline vehicle; black circles, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to saline vehicle.
Fig. 4. Effects of twice-daily administration of xenin-25(gln) on (A) glucose tolerance, (B) plasma insulin response to glucose and (C) insulin sensitivity in high fat fed mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. (A and B) Glucose and insulin concentrations were measured prior to and after administration of glucose (18 mmol/kg body weight; ip) in non-fasted mice. AUC values for 0-105 min post-injection are also included. (C) Glucose concentrations were measured prior to and after administration of insulin (25 U/kg body weight; ip) in non-fasted mice. AAC values for 0-60 min post-injection are also included. White circles and white bars, saline vehicle; black circles and black bars, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05 and ***p < 0.001 compared to saline vehicle.
Fig. 5. Effects of twice-daily administration of xenin-25(gln) on (A) glucose and (B) insulin responses to GIP in high fat fed mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. Glucose and insulin concentrations were measured prior to and after administration of glucose (18 mmol/kg body weight; ip) plus native GIP (25 nmol/kg body weight; ip) in fasted mice. AUC values for 0-105 min post-injection are also included. White circles and white bars, saline vehicle; black circles and black bars, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05 compared to saline vehicle.
Fig. 6. Effects of twice-daily administration of xenin-25(gln) on (A,B) islet morphology, (C) islet number, (D) islet area, (E) alpha cell area, (F) beta cell area, (G) beta cell/alpha cell ratio and (H) islet size distribution in high fat fed mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. White bars, saline vehicle; black bars, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. ***p < 0.001 compared to saline vehicle.
Fig. 7. Effects of twice-daily administration of xenin-25(gln) on (A) body weight, (B) food intake, (C) glucose and (D) insulin concentrations in ob/ob mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. White circles, saline vehicle; black circles, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to saline vehicle.
Fig. 8. Effects of twice-daily administration of xenin-25(gln) on (A) glucose tolerance, (B) plasma insulin response to glucose and (C) insulin sensitivity in ob/ob mice. Mice received saline vehicle (0.9% wt/vol; ip) or xenin-25(gln) (25 nmol/kg body weight; ip) twice-daily over 21 days. (A and B) Glucose and insulin concentrations were measured prior to and after administration of glucose (18 mmol/kg body weight; ip) in fasted mice. AUC values for 0-105 min post-injection are also included. (C) Glucose concentrations were measured prior to and after administration of insulin (25 U/kg body weight; ip). AAC values for 0-60 min post-injection are also included. White circles and white bars, saline vehicle; black circles and black bars, xenin-25(gln). Values are expressed as meanS.E.M. for 8 mice. *p < 0.05 and **p < 0.01 compared to saline vehicle.
Table 1 Peptide characteristics
PeptideModification
Lot Number HPLC retention time (min)
Experimental mass(Da)
Theoretical mass (Da)
Plasma stability (hours)
Xenin-25 None P110605- XZ055227 10.2 2969.5 2971.6 <4
Xenin-25(gln)K4, K8, R11, K13, K20 and
R21 replaced with QP110607-CL244613 12.0 2915.5 2915.4 >24
Table 1 - Peptide purity was assessed by HPLC using Phenomenex Kinetex 2.6u C-18 (150 x 4.6 mm) analytical column and retention times recorded. Purified peptides were spotted onto a stainless steel sample plate (in combination 10 mg/ml cyano-4-hydroxycinnamic acid in acetonitrile/ethanol) and applied to a Voyager-DE BioSpectrometry Workstation and molecular mass recorded. Peptides were incubated with murine plasma for 0, 2, 4, 8 and 24 hours and reaction products examined by HPLC with degradation calculated as percentage intact peptide.
Figure 1
Acells/ 20 min)
6Insulin secretion (ng/ 1 x 10
Peptide concentration (M)
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6Insulin secretion (ng/ 1 x 10
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0-6 10-8 10-10 10-12 10
Figure 2
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