Bioscience Reports (2017) 37 BSR20170776

https://doi.org/10.1042/BSR20170776

Research Article

Impaired insulin/IGF-1 is responsible for diabetic

gastroparesis by damaging myenteric cholinergic
neurones and interstitial cells of Cajal
Shu Yang1,2,3 , Bo Wu1,2,3 , Haimei Sun1,2,3 , Tingyi Sun1,2,3 , Kai Han1 , Dandan Li1 , Fengqing Ji1,2,3 ,
Guoquan Zhang1 and Deshan Zhou1,2,3
1 Departmentof Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, P.R. China; 2 Beijing Key Laboratory of Cancer Invasion
and Metastasis Research, Beijing 100069, P.R. China; 3 Department of Histology and Embryology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, P.R.
China
Correspondence: Deshan Zhou ([email protected]) or Guoquan Zhang ([email protected])

Diabetic gastroparesis is a common complication of diabetes mellitus (DM) that is charac-

terized by decreased serum insulin and insulin-like growth factor-1 (IGF-1). Despite the fact
that insulin treatment not glycemic control potently accelerated gastric emptying in type 1
DM patients, the role of insulin/InsR and IGF-1/IGF-1R signaling in diabetic gastroparesis
remains incompletely elucidated. In the present study, type 1 DM mice were established
and treated with insulin or Voglibose for 8 weeks. The gastric emptying was delayed from
DM week 4 when the gastric InsR and IGF-1R were declined. Meanwhile, the gastric choline
acetyltransferase (ChAT) was significantly reduced and the myenteric cholinergic neurones
and their fibers were significantly diminished. The production of stem cell factor (SCF) was
dramatically repressed in the gastric smooth muscles in DM week 6. TWereafter, interstitial
cells of Cajal (ICC) were clearly lost and their networks were impaired in DM week 8. Signif-
icantly, compared with Voglibose, an 8-week treatment with insulin more efficiently delayed
diabetic gastroparesis development by protecting the myenteric cholinergic neurones and
ICC. In conclusion, diabetic gastroparesis was an aggressive process due to the succes-
sive damages of myenteric cholinergic neurones and ICC by impairing the insulin/InsR and
IGF-1/IGF-1R signaling. Insulin therapy in the early stage may delay diabetic gastroparesis.

Introduction
Diabetes mellitus (DM) is the leading dismetabolic chronic disease with a global prevalence approaching
400 million people [1]. Long-standing DM often results in systemic complications including cardiovas-
cular diseases, neuropathy, retinopathy, nephropathy, as well as gastroenteropathy. Diabetic gastroparesis,
characterized by early satiety, nausea, vomiting, postprandial fullness, abdominal pain, and delayed gas-
tric emptying without evidence of mechanical obstruction [2], occurs in approximately 50% of type 1 DM
patients and 30% of type 2 DM patients. Although the diabetic gastroparesis is not malignant, it inhibits
absorption of nutrients and oral antidiabetic medicine, which could interfere with glycemic control and
Received: 08 May 2017
lead to an inefficient treatment of DM. The pathogenesis of the diabetic gastroparesis is still under research
Revised: 14 September 2017 and it is believed to be multifactorial, e.g. hyperglycemia, enteric nervous system (ENS) injury, myopathy,
Accepted: 17 September 2017 loss of interstitial cells of Cajal (ICC) etc. [3-6]. Although there have been substantial advancement in our
understanding of the underlying cellular dysfunction of the diabetic gastroparesis, how the ENS and ICC
Accepted Manuscript Online:
20 September 2017 are damaged in diabetic gastroparesis remain poorly known.
Version of Record published: DM is characterized by a deficiency (type 1 DM) or resistance (type 2 DM) to insulin, apart from
27 October 2017 which, a decrease in insulin-like growth factor-1 (IGF-1) was documented in both type 1 and type 2

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diabetic patients [7,8]. Insulin and IGF-1 share structural homologies, so do their receptors, InsR and IGF-1R, respec-
tively. Thus there is a physiological and pharmacological cross-talk between insulin/InsR and IGF-1/IGF-1R systems
[9]. Evidence have shown that insulin/InsR and IGF-1/IGF-1R signaling were implicated in the development of ner-
vous system by promoting neuronal growth, survival, proliferation, and differentiation [10,11]. Impaired insulin/InsR
and IGF-1/IGF-1R signaling were closely related with diabetic peripheral neuropathy, which could be ameliorated or
prevented by insulin or IGF-1 treatment [11-13]. However, much less is known about the role of insulin/InsR and
IGF-1/IGF-1R signaling in the ENS and ICC during the development of the diabetic gastroparesis. A pilot trial on
patients with type 1 DM revealed that insulin treatment efficiently accelerated gastric emptying [14], suggesting a
potential role of insulin/InsR signaling in the diabetic gastroparesis.
Therefore, the present study investigated the alterations of the myenteric neurones and ICC within the gastric
wall of type 1 DM mouse model, in order to evaluate the effect of IGF-1/IGF-1R and insulin/InsR signaling on the
development of diabetic gastroparesis.

Materials and methods

Establishment of type 1 DM mouse model
Male BALB/c mice (6 weeks, 22–26 g) were purchased from the Animal Center of Capital Medical University (Bei-
jing, China). All mice were maintained in a temperature-controlled room (23 + ◦
− 1 C), with a constant 12 h-light/dark
cycle. Food and water were available ad libitum. Each mouse received a single intraperitoneal injection of Alloxan
monohydrate (200 mg/kg, Sigma–Aldrich) [15,16]. The littermates receiving the same dose of normal saline (NS)
were set as controls. Fasting intravenous blood glucose was measured by an Accu-Chek Active Complete blood glu-
cose monitor (Roche) 72 h later. The mice with blood glucose ≥11.1 mmol/l were considered as type 1 DM mice
and used in the present study. In total, 135 DM mice were successfully established and blood glucose was monitored
weekly. In DM week 2, 4, 6, and 8, mice were killed by an overdose of 4% chloral hydrate (0.02 ml/g). A separate group
of 60 DM mice daily received an intraperitoneal injection of insulin (0.04 U/g, Wanbang Biopharmaceuticals, China)
or oral antidiabetic drug of Voglibose (0.5 mg/mouse, Cisen Biopharmaceuticals, China) for 8 weeks. In addition,
15 DM mice daily received no).The subdivision of the total mice is indicated in Supplementary Table S1. All experi-
mental procedures were in accordance with the National Institutes of Health guide for the care and use of Laboratory
animals (NIH Publication number 8023, revised 1978) and approved by the Institutional Animal Care Committee
from Capital Medical University, Beijing, China.

ELISA
At the end of 2, 4, 6, and 8 weeks, 1.5 ml of blood was collected from eyes of each mouse anesthetized by 4% chloral
hydrate (0.01 ml/g). Serum insulin and IGF-1 were measured with corresponding ELISA Kit (Raybio technology) ac-
cording to the manufacturers’ instructions. The absorbance at 490 nm was read using a microplate reader (Multiskan
FC, Thermo Scientific). All experiments were repeated six times.

Gastric emptying
Gastric emptying was evaluated according to Song and Chen [17]. Briefly, each mouse was intragastrically admin-
istered with 0.2 ml of methylcellulose-Phenol Red solution. Thirty minutes later, the mouse was anesthetized by 4%
chloral hydrate (0.01 ml/g) and the stomach was removed. The gastric content was placed in 100 ml of 0.1 N NaOH
and settled for 1 h at 25◦ C. Afterward, 5 ml of supernatant was taken from the solution and put into 0.5 ml of 20%
trichloroacetic acid, then centrifuged at 3000 rpm for 30 min. The contents were then mixed with 4 ml of 0.5 N
NaOH. The absorbance was determined at a wavelength of 560 nm with a microplate reader (Multiskan FC). Gastric
emptying (%) = (Phenol Red absorbance – residual Phenol Red absorbance)/phenol Red absorbance × 100%.

Isometric tension recording

Gastric content was flushed out in precooled Kreb’s solution. Smooth muscle strip (2 mm × 10 mm) was prepared
and suspended vertically in an eight-channel organ bath filled with Kreb’s solution and oxygenated with 95% O2
and 5% CO2 at 37◦ C. One pole of the strip was fixed to an organ holder and the other pole was connected to an
isometric tonotransducer. Mechanical activity of the smooth muscle strip was recorded with a computer-aided data
acquisition system (Power lab biology signal recording system, AD Instruments). The initial tension was set as 1
g. The amplitude and frequency of the spontaneous contraction of the smooth muscle strip were analyzed by chart
software (AD Instruments).

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Western blotting
The gastric mucosa and submucosa were removed and the smooth muscles were homogenized in cold lysis buffer
(Applygen, China) and the supernatants were collected. After SDS/PAGE (10% gel), the proteins were transferred
on to PVDF membranes and blocked with 5% non-fat dry milk for 1 h at 25◦ C. The membranes were incubated
with primary rabbit anti-IGF-1R, goat anti-choline acetyltransferase (ChAT )mouse anti-SCF, or rat anti-KIT at 4◦ C
overnight, followed by incubation with corresponding HRP-conjugated secondary antibody for 1 h at 25◦ C. The
proteins were detected using ECL chemiluminescence (Thermo Scientific) and viewed in Fusion FX Vilber Lourmat
(France). A goat anti-GAPDH was used as an internal control in all the cases. The detailed antibodies are listed in
Supplementary Table S2.

Immunofluorescence staining
To obtain whole mount preparations, mouse stomach was inflated with 4% paraformaldehyde or 100% acetone for 2 h
and immersed in the same fixative for 12 h at 4◦ C. One sample of 0.5 cm × 0.5 cm was randomly cut from the gastric
corpus and antrum. The mucosa and submucosa were removed and the longitudinal smooth muscle layer containing
myenteric plexus was dissected. For cryosections, the stomach was opened along the gastric greater curvature and
embedded in optimal cutting temperature compound. Cryosections (8-μm thick) were cut with a cryostat (Leica,
CM3050S) and fixed with 4% PFA or 100% acetone for 30 min at 4◦ C.
The specimens were permeabilized with 0.3% Triton X-100 for 20 min. Non-specific binding sites were blocked
with 1% BSA for 30 min. The specimens were incubated with primary rabbit anti-IGF-1R, rabbit anti-InsR, goat
anti-ChAT, rabbit anti-S100, mouse anti-stem cell factor (SCF), or rat anti-KIT overnight at 4◦ C, followed by incuba-
tion with corresponding secondary antibody for 1 h at 25◦ C. The specimens were mounted with fluorescent mounting
medium containing DAPI (Zhongshan Jinqiao Biotechnology, China) and visualized by a fluorescence microscope
(Nikon, Ni) or confocal laser scanning microscope (Leica, TCS SP5). Specificity was verified by omitting the primary
antibody and by preabsorption with appropriate blocking peptide. The detailed antibodies are listed in Supplementary
Table S2.

Image analysis
Fifteen photographs were randomly taken per whole mount preparation stained by immunofluorescence based on
stereological rules at magnification of 20×. The target area of interest (AOI) was outlined and measured by Image
Pro-Plus Software 6.0 (Media Cybernetics, Silver Spring). To obtain the area density of the AOI, the total area of the
AOI was divided by the total area of the fields of view. To obtain the cell number density in the AOI, the total number
of the cells in the AOI was divided by the total area of the fields of view.

Statistics
Statistical analyses were performed with the SPSS 17.0 software. Data were expressed as the mean +
− S.E.M. and
compared using one-way ANOVA and LSD post hoc analysis. A 2P-value of 0.05 was adopted.

Results
Establishment of type 1 DM mouse model
In total, 155 mice were treated with Alloxan monohydrate. However, 20 mice died during the induction of DM by
Alloxan monohydrate treatment. Finally, 135 mice had profound hyperglycemia (Supplementary Table S3), indicating
the type 1 DM mouse model was successfully established. The body weight of the DM mice was less than that of
the controls (Supplementary Table S3), accompanied with several manifestations, such as polydipsia, polyphagia,
polyuria, and smell of ketone bodies in urine.

Diabetic gastroparesis occurred in DM week 4

To identify diabetic gastroparesis, the gastric emptying was measured at the end of DM week 2, 4, 6, and 8. In DM
week 2, the gastric emptying did not differ from that of the age-matched controls, while it was significantly delayed
from DM week 4 (*P<0.05, Figure 1A). The stomachs of the DM mice were evidently dilated owing to food retention
compared with control mice (Supplementary Figure S1). The delayed gastric emptying and food retention denoted
the occurrence of diabetic gastroparesis. The gastric emptying comes from spontaneous and rhythmic contractions
of gastric smooth muscles, so we recorded the contractions of gastric muscles by isometric tension recording. In DM
week 4, the amplitude of spontaneous contraction was significantly reduced while the frequency was significantly

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Figure 1. Gastric emptying was measured by the absorbance of Phenol Red solution before and after intragastric admin-
istration (n=5)
Amplitude and frequency of spontaneous contraction were recorded by isometric tension recording (n=5). (A) The gastric emptying
was significantly delayed from DM week 4 compared with the controls (n=5; *P<0.05). (B) The amplitude of spontaneous contraction
was significantly reduced in DM week 4 compared with the controls. Though the amplitude was gradually increased in DM week 6,
it could not completely regain the controls’ level (n=5; *P<0.05). (C) Despite the frequency of spontaneous contraction was overtly
increased in DM week 4, it was visibly decreased in the DM mice afterward compared with the controls (n=5; *P<0.05).

increased compared with the controls (*P<0.05; Figure 1B,C, Supplementary Figure S2). Afterward, the amplitude
and frequency were both markedly decreased in the DM mice (*P<0.05; Figure 1B,C, Supplementary Figure S2).

Gastric InsR and IGF-1R were decreased from DM week 4

Compared with the controls, the serum insulin and IGF-1 were significantly decreased from DM week 2 (*P<0.05;
Supplementary Table S4). Immunofluorescence staining showed that IGF-1R and InsR were coexpressed in the mus-
cularis mucosa, tunica muscularis, and myenteric plexus (Supplementary Figure S3). Considering the key role of the
ENS in the gastrointestinal motility, we paid special attention to the IGF-1R+ /InsR+ myenteric neurones and nerve
fibers in the ganglia. In DM week 2, there was no obvious change in the IGF-1R+ myenteric plexus or the IGF-1R
protein level compared with the controls (Figure 2A,B). The area densities of the IGF-1R+ myenteric neurones and

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Figure 2. Decreased gastric IGF-1R expression and damaged neuronal networks from DM week 4.
(A) IGF-1R+ myenteric plexuses including ganglion neurones (arrow) and nerve fibers (arrow head) were well exhibited via im-
munofluorescence staining. Neuronal networks were damaged from DM week 4 (n=5). (B) The protein level of IGF-1R in gastric
smooth muscles was significantly decreased from DM week 4 compared with the controls (n=5; *P<0.05). (C) The area density
of IGF-1R+ myenteric neurones in gastric corpus and antrum of DM mice were decreased from DM week 4 compared with the
controls (n=5; *P<0.05). (D) The area density of IGF-1R+ primary nerve fibers in gastric corpus and antrum of DM mice were also
diminished from DM week 4 compared with the controls (n=5; *P<0.05).

primary nerve fibers in the gastric corpus and antrum significantly shrunk from DM week 4 compared with controls
(*P<0.05, Figure 2C,D), when the protein level of IGF-1R in the gastric smooth muscles was significantly reduced
(*P<0.05, Figure 2B).

ChAT+ /IGF-1R+ /InsR+ neurones were reduced from DM week 4

We noticed that the IGF-1R+ /InsR+ cells in the myenteric ganglia were distinct, round or oval, exhibiting neurone-like
features. We further confirmed that most IGF-1R+ or InsR+ cells were ChAT+ cholinergic neurones in the myenteric
ganglia but not enteric glias by double-immunofluorescence staining, suggesting a possible role of IGF-1/IGF-1R
and insulin/InsR on the ChAT+ cholinergic neurones (Supplementary Figures S4 and S5). In accordance with the
weakened IGF-1/IGF-1R and insulin/InsR signaling, the ChAT+ cholinergic neurones decreased from DM week 4
(Figure 3A,B). Concomitantly, the protein level of ChAT in the stomach was significantly reduced (*P<0.05, Figure
3C). These results indicated that loss of ChAT+ cholinergic neurones in the gastric myenteric ganglia could be due to
the impaired IGF-1/IGF-1R and insulin/InsR systems and may be responsible for the dysfunction of gastric motility.

SCF production was lowered in DM week 6 and ICC was lost in DM week
8
SCF is mainly synthesized and secreted by the smooth muscles and myenteric neurones in the gastrointestinal tract.
Here, we showed that SCF was present in the gastric smooth muscles and myenteric neurones but not in ICC (Figure
4A), consistent with the distribution of IGF-1R and InsR (Supplementary Figure S3). The gastric SCF was clearly
decreased from DM week 6 (*P<0.05, Figure 4A,B). Whereafter, in DM week 8, the cellular networks of ICC were
deteriorated (Figure 5A) and the number densities of ICC-IM and ICC-MY were markedly decreased (Figure 5B,C),

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Figure 3. Dinimished ChAT+ neurons and gastric ChAT expression from DM week 4.
(A) ChAT was expressed in the neurones and nerve fibers within the myenteric plexuses. ChAT+ cholinergic neurones were distinct,
round or oval; and their processes formed neuronal networks. The immunofluorescence intensity for ChAT was gradually decreased
from DM week 4 (n=5). (B) The number density of ChAT+ cholinergic neurones in gastric corpus and antrum of DM mice was signif-
icantly diminished from the DM week 4 compared with the controls (n=5; *P<0.05). (C) The protein level of ChAT was significantly
reduced from DM week 4 compared with the controls (n=5; *P<0.05).

Figure 4. Reduced gastric SCF production from DM week 6.

(A) SCF (red) was expressed in the smooth muscles including circular muscles (CM) and longitudinal muscle (LM) and myenteric
plexuses but not in ICC (green). Immunoreactivity for SCF in the smooth muscles was much intensive than that in the myenteric
plexuses (arrow head), and the immunofluorescence intensity for SCF was clearly decreased in DM week 6 (n=5). (B) The protein
level of SCF in gastric smooth muscles was obviously reduced from DM week 6 (n=5; *P<0.05).

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Figure 5. Decreased ICCs and KIT expression in DM week 8.

(A) In the whole mount preparations, ICC-IM were fusiform bipolar cells; and ICC-MY had 2–4 slender processes which ramified to
form cellular networks. In DM week 8, ICC-IM were obviously lost when compared with the controls. Cellular networks of ICC-MY
were much more sparse in DM week 8 (n=5). (B) The number density of ICC-IM in gastric corpus and antrum of DM mice were
decreased in DM week 8 compared with the controls (n=5; *P<0.05). (C) The number density of ICC-MY in gastric corpus and
antrum of DM mice were decreased in DM week 8 compared with the controls (n=5; *P<0.05). (D) The protein level of KIT was not
significantly decreased until DM week 8 (n=5; *P<0.05).

parallel with the attenuated KIT (*P<0.05, Figure 5D). These results suggested that the impaired IGF-1/IGF-1R and
insulin/InsR systems damaged ICC by inhibiting SCF production instead of directly affecting ICC.

Insulin delayed the development of diabetic gastroparesis

It was recently proposed that the early usage of insulin could delay DM complications. Therefore, we treated the DM
mice with insulin or Voglibose for 8 weeks right after DM was induced. The delayed gastric emptying of the DM mice
was efficiently improved by the 8-week insulin administration (24.0%, *P<0.05) but not Voglibose compared with the
DM mice receiving NS (Figure 6A). The gastric IGF-1R in the insulin and Voglibose groups were evidently increased
compared with the NS group (*P<0.05; Figure 6B,C). The gastric ChAT was not decreased and the number of ChAT+
myenteric neurones was well preserved by the treatment of insulin or Voglibose (*P<0.05; Figure 6C–E). Notably,
insulin not Voglibose treatment was able to increase SCF production in the gastric smooth muscles (*P<0.05; Figure
6C). Moreover, only insulin treatment could partly prevent the loss of ICC-MY (28.8%, *P<0.05) and ICC-IM (48.3%,
*P<0.05) and their cellular networks (Figure 6F–I).

Discussion
In the present study, type 1 DM mouse model was successfully made by an injection of Alloxan monohydrate, which
resulted in apparent declines in the serum insulin by 62.2–78.9% and IGF-1 by 12.1–43.8% from as early as DM
week 2. Four weeks after the onset of the DM, the gastric emptying was delayed indicating a link between the re-
duced insulin/IGF-1 and diabetic gastroparesis. The amplitude of gastric muscle contraction was decreased but the
frequency of the gastric muscle contraction was increased in DM week 4, which may be due to compensation for the
gastric movement. Then, the amplitude and the frequency were reduced compared with controls.
The ENS plays a crucial role in regulating gastrointestinal movement. The number and morphometric changes
in the enteric neurones attributed to increased apoptosis, oxidative stress, and hyperglycemia likely participated in
the development of diabetic gastroparesis in mice, rats, and humans [18-20]. In the current study, the myenteric
neuropathy might arise as a consequence of reduced insulin/InsR and IGF-1/IGF-1R in DM mice because they are

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Figure 6. Effects of 8-week insulin or Voglibose treatment

(A) The gastric emptying of the DM mice treated with insulin was accelerated compared with that of the DM mice receiving NS (n=5;
*P<0.05); while the gastric emptying of the DM mice treated with Voglibose was not significantly improved. (B) The immunoreactive
intensity for IGF-1R in the myenteric plexuses of the insulin and Voglibose groups was obviously increased compared with the NS
group (n=10). (C) Compared with the controls, the protein levels of gastric IGF-1R, ChAT, and SCF were significantly decreased
in the DM mice that only received NS for 8 weeks (n=5; # P<0.05). The DM-associated decrease in IGF-1R and ChAT were clearly
restored by insulin or Voglibose compared with the NS group (n=5; *P< 0.05). However, only insulin had positive effect on the SCF
production (n=5; *P<0.05 compared with NS). (D) The immunoreactive intensity for ChAT in the myenteric plexus was increased
after insulin or Voglibose treatment (n=10). (E) The number density of ChAT+ myenteric neurones was well protected upon the
treatment of insulin or Voglibose compared with the NS group (n=10; *P<0.05). (F–I) Insulin partly recovered the count and cellular
networks of ICC-MY and ICC-IM that were labeled with KIT; while Voglibose had no obvious effect on ICC (n=10; *P<0.05).

considered as neurotrophic factors. Myenteric neuronal population comprises excitatory neurones, inhibitory neu-
rones, and interneurones. Previous studies have revealed an abnormality of inhibitory neuronal nitric oxide synthase
(nNOS) neurones in diabetic gastroparesis [21,22]. Here, we had a special interest in excitatory enteric neurones that
keep gastric motility balance in co-operation with inhibitory neurones. Our results showed that the InsR+ /IGF-1R+
neurones were mainly ChAT+ excitatory cholinergic neurones. In view of that, the insulin/InsR and IGF-1/IGF-1R
signaling play a key role in the neuronal survival and development, the decreased insulin/InsR and IGF-1/IGF-1R
might not fully maintain the ChAT+ cholinergic neurones viable. Consequently, the gastric ChAT and the number
density of ChAT+ myenteric cholinergic neurones were diminished from DM week 4, which aggravated the diabetic
gastroparesis progress.
Apart from the ENS, ICC also play a key role in the gastrointestinal motility as the pacemaker of spontaneous slow
waves within the gastrointestinal tract and the mediator of neuronal transmission from the ENS to gastrointestinal
smooth muscles. ICC are mainly distributed in the tunica muscularis (ICC-IM) and myenteric plexus (ICC-MY)
within the gastric wall. ICC-IM are spindle-shaped bipolar cells, and ICC-MY have 2–4 slender processes which

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ramify to form cellular networks. The depletion of ICC in the gastrointestinal tract is responsible for the diabetic gas-
troparesis both in type 1 and type 2 DM patients and mice [22-24]; and the loss of ICC in diabetic gastroparesis was
related with the deficiency of IGF-1 and/or insulin [25]. The progenitors of ICC (Kitlow CD44+ CD34+ InsR/IGF-1R+ )
could not develop mature cells (Kit+ CD44+ CD34− InsR/IGF-1R− ) in the absence of insulin and IGF-1; and the sur-
vival and function of ICC would not be well preserved either [6,26]. However, we failed to detect IGF-1R and InsR in
ICC [27]. These results raised a question: how IGF-1/insulin affects ICC? It is well known that the growth, survival,
and function of ICC are largely dependent on the activation of the membrane receptor KIT by its natural ligand SCF
[28,29]. Therefore, SCF deficiency may lead to the loss of ICC via impairing the KIT/SCF signaling pathway. As ex-
pected, in our DM mice, following the reduced insulin/InsR and IGF-1/IGF-1R signaling, the SCF production by the
gastric smooth muscles was clearly decreased in DM week 6, and ICC were significantly lost afterward (in DM week
8). The above evidence inferred that the protective effect of insulin and IGF-1 on ICC might be via indirect way of
facilitating SCF production. This hypothesis was supported by the fact that smooth muscles coexpressed InsR and
IGF-1R as well as SCF, and the SCF expression in the gastric smooth muscles was restrained by IGF-1R inhibitor [30].
Early usage of insulin has been favored in recent years to treat DM and its complications. Here, we treated the
DM mice with either insulin or Voglibose for 8 weeks. The gastric emptying was efficiently improved by insulin
treatment, while glycemic control by Voglibose failed in accelerating the gastric emptying, indicating that simple
glycemic control was unable to efficiently delay diabetic gastroparesis developing. A clinical trial by Russo et al. [14]
has consolidated the contribution of insulin to accelerate gastric emptying in long-standing type 1 DM; and they
thought it was owing to the insulin-induced hypoglycemia. In the present study, we suggested that the protective role
of insulin/InsR and IGF-1/IGF-1R signaling on the myenteric cholinergic neurones and ICC was also contributory.
Insulin treatment increased InsR and IGF-1R expressions in the myenteric plexuses and prevented the loss of myen-
teric cholinergic neurones. It was reported that insulin and IGF-1 increased ChAT activity in brain tissue and cultured
human central nervous system derived neuronal cells [31]. In addition to the findings in the central nervous system,
our results demonstrated that insulin was beneficial for the survival of the myenteric ChAT+ neurones. Besides, in-
sulin was helpful for the maintenance of ICC networks by stimulating SCF production. We also found that InsR and
IGF-1R were increased upon Voglibose treatment, so were the ChAT expression and myenteric cholinergic neurones.
A recent study revealed that Acarbose, an analogue of Voglibose, enhanced the level of hippocampal InsR in old
mice [32]. We presumed here that hyperglycemia might abate InsR and IGF-1R expressions in the myenteric ChAT+
neurones but the detailed mechanism needs to be further clarified. However, Voglibose was insufficient to increase
SCF production and hardly exhibited protection on ICC. There seemed to be a contradiction that Voglibose-induced
InsR and IGF-1R expression in the gastric wall did not promote SCF production. It might because insulin/InsR and
IGF-1/IGF-1R signaling are not the only factors that influence SCF production. For example, ghrelin was reported to
be capable of enhancing SCF expression in rats’ stomachs [33]. ICC gained a central place in the gastrointestinal motil-
ity because they integrate excitatory and inhibitory neurotransmission with intestinal slow-wave activity [34]. Once
ICC are dysfunctional, the neurotransmission of myenteric neurones will be retarded, which partly interprets that
simple glycemic control by Voglibose was unable to delay diabetic gastroparesis developing even if it well protected
myenteric ChAT+ neurones. For this reason, we believe that prophylactic insulin in DM patients not only because it
restores normoglycemia, but also preserves ICC.
In conclusion, we demonstrated that the myenteric ChAT+ excitatory neurones were lost with the decreased in-
sulin/InsR and IGF-1/IGF-1R signaling in the earlier stage of DM. Along with the further decrease in IGF-1/IGF-1R
and insulin/InsR, SCF production by the gastric smooth muscles was clearly repressed, resulting in loss of ICC and im-
pairment of their cellular networks in the latest DM stage. Significantly, compared with Voglibose, insulin treatment
effectively delayed diabetic gastroparesis from developing probably via retrieving insulin/InsR and IGF-1/IGF-1R sig-
naling that recovered the ChAT+ excitatory neurones and ICC. Therefore, we suggested prophylactic insulin therapy
may help to delay or even prevent the diabetic gastroparesis.

Author contribution
S.Y., B.W., H.S., F.J., T.S., K.H., D.L. and G.Z. performed the experiments. S.Y., G.Z., and D.Z. wrote the manuscript. S.Y. and D.Z.
gave suggestions on discussion and interpretation fn the data. D.Z. is the guarantor of this work and, as such, had full access to
all the data in the study and also takes responsibility for the integrity of the data and the accuracy of the data analysis.

Funding
This work was supported by the National Natural Science Foundation of China [grant numbers 81300285, 81572322, 31371220];
the Beijing Natural Science Foundation [grant numbers 7172021, 5172008, 5164029]; the Scientific Research Key Program of

c 2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons 9
Attribution License 4.0 (CC BY).
 Bioscience Reports (2017) 37 BSR20170776
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Beijing Municipal Commission of Education [grant number KM201610025002]; and the Project Supported by Beijing Training
Program Foundation for Young Talents [grant number 2015000020124G114].

Competing interests
The authors declare that there are no competing interests associated with the manuscript.

Abbreviations
AOI, area of interest; ChAT, choline acetyltransferase; DM, diabetes mellitus; ENS, enteric nervous system; HRP, horseradish
peroxidase; ICC, interstitial cell of Cajal; IGF-1, insulin-like growth factor-1; NS, normal saline; PFA, paraformaldehyde; SCF,
stem cell factor.

References
1 Nathan, D.M. (2015) Diabetes: advances in diagnosis and treatment. JAMA 314, 1052–1062
2 Tang, D.M. and Friedenberg, F.K. (2011) Gastroparesis: approach, diagnostic evaluation, and management. Dis. Mon 57, 74–101
3 Bharucha, A.E., Batey-Schaefer, B., Cleary, P.A., Murray, J.A., Cowie, C., Lorenzi, G. et al. (2015) Delayed gastric emptying is associated with early and
long-term hyperglycemia in type 1 diabetes mellitus. Gastroenterology 149, 330–339
4 James, A.N., Ryan, J.P., Crowell, M.D. and Parkman, H.P. (2004) Regional gastric contractility alterations in a diabetic gastroparesis mouse model:
effects of cholinergic and serotoninergic stimulation. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G612–G619
5 Ordög, T., Takayama, I., Cheung, W.K., Ward, S.M. and Sanders, K.M. (2000) Remodeling of networks of interstitial cells of Cajal in a murine model of
diabetic gastroparesis. Diabetes 49, 1731–1739
6 Horváth, V.J., Vittal, H., Lörincz, A., Chen, H., Almeida-Porada, G., Redelman, D. et al. (2006) Reduced stem cell factor links smooth myopathy and loss
of interstitial cells of cajal in murine diabetic gastroparesis. Gastroenterology 130, 759–770
7 Cortizo, A.M., Lee, P.D., Cédola, N.V., Jasper, H. and Gagliardino, J.J. (1998) Relationship between non-enzymatic glycosylation and changes in serum
insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in patients with type 2 diabetes mellitus. Acta Diabetol. 35, 85–90
8 AboElAsrar, M.A., Elbarbary, N.S., Elshennawy, D.E. and Omar, A.M. (2012) Insulin-like growth factor-1 cytokines cross-talk in type 1 diabetes mellitus:
relationship to microvascular complications and bone mineral density. Cytokine 59, 86–93
9 Butler, A.A. and LeRoith, D. (2001) Minireview: tissue-specific versus generalized gene targeting of the igf1 and igfr genes and their roles in insulin-like
growth factor physiology. Endocrinology 142, 1685–1688
10 Neff, N.T., Prevette, D., Houenou, L.J., Lewis, M.E., Glicksman, M.A., Yin, Q.W. et al. (1993) Insulin-like growth factors: putative muscle-derived trophic
agents that promote motoneuron survival. J. Neurobiol. 24, 1578–1588
11 Brussee, V., Cunningham, F.A. and Zochodne, D.W. (2004) Direct insulin signaling of neurons reverses diabetic neuropathy. Diabetes 53, 1824–1830
12 Ishii, D.N. and Lupien, S.B. (1995) Insulin-like growth factors protect against diabetic neuropathy: effects on sensory nerve regeneration in rats. J.
Neurosci. Res. 40, 138–144
13 Zhuang, H.X., Snyder, C.K., Pu, S.F. and Ishii, D.N. (1996) Insulin-like growth factors reverse or arrest diabetic neuropathy: effects on hyperalgesia and
impaired nerve regeneration in rats. Exp. Neurol. 140, 198–205
14 Russo, A., Stevens, J.E., Chen, R., Gentilcore, D., Burnet, R., Horowitz, M. et al. (2005) Insulin-induced hypoglycemia accelerates gastric emptying of
solids and liquids in long-standing type 1 diabetes. J. Clin. Endocrinol. Metab. 90, 4489–4495
15 Park, N.Y., Park, S.K. and Lim, Y. (2011) Long-term dietary antioxidant cocktail supplementation effectively reduces renal inflammation in diabetic mice.
Br. J. Nutr. 106, 1514–1521
16 Cai, S., Zhong, Y., Li, Y., Huang, J., Zhang, J., Luo, G. et al. (2013) Blockade of the formation of insoluble ubiquitinated protein aggregates by
EGCG3”Me in the alloxan-induced diabetic kidney. PLoS ONE 8, e75687
17 Song, G.Q. and Chen, J.D. (2007) Synchronized gastric electrical stimulation improves delayed gastric emptying in nonobese mice with diabetic
gastroparesis. J. Appl. Physiol. 103, 1560–1564
18 Wang, X.Y., Huizinga, J.D., Diamond, J. and Liu, L.W. (2009) Loss of intramuscular and submuscular interstitial of Cajal and associated enteric nerves is
related to decreased gastric emptying in streptozotocin-induced diabetes. Neurogastroenterol. Motil. 21, 1095–e92
19 Fregonesi, C.E., Miranda-Neto, M.H., Molinari, S.L. and Zanoni, J.N. (2001) Quantitative study of the myenteric plexus of the stomach of rats with
streptozotocin-induced diabetes. Arq. Neuropsiquiatr. 59, 50–53
20 Chandrasekharan, B. and Srinivasan, S. (2007) Diabetes and the enteric nervous system. Neurogastroenterol. Motil. 19, 951–960
21 Takahashi, T. (2003) Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J. Gastroenterol. 38, 421–430
22 He, C.L., Soffer, E.E., Ferris, C.D., Walsh, R.M., Szurszewski, J.H. and Farrugia, G. (2001) Loss of interstitial cells of cajal and inhibitory innervation in
insulin-dependent diabetes. Gastroenterology 121, 427–434
23 Yamamoto, T., Watabe, K., Nakahara, M., Ogiyama, H., Kiyohara, T., Tsutsui, S. et al. (2008) Disturbed gastrointestinal motility and decreased interstitial
cells of Cajal in diabetic db/db mice. J. Gastroenterol. Hepatol. 23, 660–667
24 Forster, J., Damjanov, I., Lin, Z., Sarosiek, I., Wetzel, P. and McCallum, R.W. (2005) Absence of the interstitial cells of Cajal in patients with gastroparesis
and correlation with clinical findings. J. Gastrointest. Surg. 9, 102–108
25 Horváth, V.J., Vittal, H. and Ordög, T. (2005) Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes-associated depletion of
interstitial cells of Cajal in the murine stomach. Diabetes 54, 1528–1533

10 c 2017 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons
Attribution License 4.0 (CC BY).
Bioscience Reports (2017) 37 BSR20170776
https://doi.org/10.1042/BSR20170776

26 Lorincz, A., Redelman, D., Horváth, V.J., Bardsley, M.R., Chen, H. and Ordög, T. (2008) Progenitors of interstitial cells of cajal in the postnatal murine
stomach. Gastroenterology 134, 1083–1093
27 Zhang, G., Yang, S., Li, X. and Zhou, D. (2014) Expression and possible role of IGF-IR in the mouse gastric myenteric plexus and smooth muscles. Acta
Histochem. 116, 788–794
28 Wu, J.J., Rothman, T.P. and Gershon, M.D. (2000) Development of the interstitial cells of Cajal: origin, Kit dependence and neuronal and nonneuronal
sources of Kit ligand. J. Neurosci. Res. 59, 384–401
29 Ward, S.M., Ordög, T., Bayguinov, J.R., Horowitz, B., Epperson, A., Shen, L. et al. (1999) Development of interstitial cells of Cajal and pacemaking in
mice lacking enteric nerves. Gastroenterology 117, 584–594
30 Ning, Y.J., Zhang, W., Cheng, J.F., Li, X.L., Wang, M.F. and Lin, L. (2010) Insulin-like growth factor 1 regulates expression of stem cell factor through
ERKMAPK signaling pathway in gastric smooth muscle cell. Zhonghua Yi Xue Za Zhi 90, 2402–2406
31 Rivera, E.J., Goldin, A., Fulmer, N., Tavares, R., Wands, J.R. and de la Monte, S.M. (2005) Insulin and insulin-like growth factor expression and function
deteriorate with progression of Alzheimer’s disease: link to brain reductions in acetylcholine. J. Alzheimers Dis. 8, 247–268
32 Yan, W.W., Chen, G.H., Wang, F., Tong, J.J. and Tao, F. (2015) Long-term acarbose administration alleviating the impairment of spatial learning and
memory in the SAMP8 mice was associated with alleviated reduction of insulin system and acetylated H4K8. Brain Res. 1603, 22–31
33 Iwasaki, E., Suzuki, H., Masaoka, T., Nishizawa, T., Hosoda, H., Kangawa, K. et al. (2012) Enhanced gastric ghrelin production and secretion in rats with
gastric outlet obstruction. Dig. Dis. Sci. 57, 858–864
34 Klein, S., Seidler, B., Kettenberger, A., Sibaev, A., Rohn, M., Feil, R. et al. (2013) Interstitial cells of Cajal integrate excitatory and inhibitory
neurotransmission with intestinal slow-wave activity. Nat. Commun. 4, 1630

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