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Eduvest – Journal of Universal Studies Volume 2 Number 12,
December, 2022 p- ISSN 2775-3735, e-ISSN 2775-3727 |
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EFFECT OF SLEEVE GASTRECTOMY WITH
PANCREATIC OMENTOPLASTY ON PANCREATIC VEGF INTENSITY IN NON-OBESITY DIABETES
MELLITUS RATS |
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Mughni Abdul, Chemy
Wiryawan, Reza Tri Sutrisno,
Vito Mahendra, Dimas Erlangga Universitas Diponegoro,
Indonesia |
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ABSTRACT |
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Diabetes
mellitus is one of the most common non-communicable diseases that that is
still growing today especially diabetes mellitus type 2 (DMT2). Diabetes in
non-obese patients is an important problem to solve because it tends to be
worse than DM in obese patients. Chronic hyperglycemia can damage body
tissues through several mechanisms like polyol pathway, hexosamine pathway,
activation of protein kinase C (PKC) and advance glycation end products
(AGEs). This pathway can damage the pancreas so that insulin cannot be
secreted and worsen the condition of diabetes. Sleeve gastrectomy procedure
can secrete hormones such as insulin, GLP-1, PYY (Peptide YY) dan PP
(Pancreatic polypeptide) which play a role in the regulation of glucose in
the blood. Omentoplasty can play a role in cell regeneration, especially
Langerhans β cells so that insulin can be produced adequately. Objective
to evaluate the effect of Sleeve Gastrectomy and Pancreas Omentoplasty on
Pancreatic VEGF Intensity in non-obese diabetes mellitus rats. Methods
True experimental study with "post-test only control design" on 18
rats with Diabetes Mellitus was divided into 3 groups: K (control), P1
(Sleeve Gastrectomy), P2 (Sleeve Gastrectomy + Omentoplasty). 10 days after
procedure, we evaluated the VEGF Intensity using immunohistochemistry.
Statistical analysis with One Way ANOVA and Post Hoc LSD. Result Pancreatic
VEGF increased in P1 and P2. In all groups in pancreatic VEGF levels were
statistically significant (p = <0.01). Conclusion Sleeve Gastrectomy and
Pancreas Omentoplasty increased pancreatic VEGF in non-obese rats with
diabetes mellitus. |
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KEYWORDS |
Diabetes Mellitus, Sleeve Gastrectomy, Omentoplasty, Pancreatic VEGF |
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This
work is licensed under a Creative Commons Attribution-ShareAlike
4.0 International |
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INTRODUCTION
Diabetes
mellitus is a chronic condition due to an increase in blood glucose levels due
to a lack of the insulin hormone in the body, or the insulin hormone does not
work effectively. Based on the International Diabetes Federation (IDF) as many
as 463 million people in the world have diabetes mellitus and Indonesia is
ranked 7th (10.7 million people).(Atlas, 2019)
There are 2
types of diabetes mellitus and 90% of cases of diabetes mellitus are type 2
diabetes mellitus (DMT2). The risk factors for DMT2 can be influenced by a
combination of genetic factors associated with insulin secretion failure,
insulin resistance and environmental factors such as obesity, overeating,
stress and lack of exercise. Obesity is considered a frequently recognized
factor in the pathogenesis of DMT2. However, in Asian countries the majority of
the body mass index (BMI) population is included in the non-obese category
which makes it possible that non-obese diabetes is a concern for public health
policy and has a worse prognosis than diabetes with obesity (Kashima, Inoue,
Matsumoto, & Akimoto, 2015),(Ozougwu, 2013)
Chronic
hyperglycemia causes tissue damage, especially insulin target tissue. The
mechanism of tissue damage in DMT2 is through the polyol pathway, hexosamine
pathway, activation of protein kinase C (PKC) and advance glycation end
products (AGEs). In the PKC pathway, intracellular hyperglycemia causes an increase
in the synthesis of diacylglycerol which causes the expression of PKC in cells
to also increase which will change various gene expressions as a whole and
damage blood vessels (Decroli, 2019). One of the target organs damaged is
the pancreas, so it will worsen the condition of DMT2 due to Langerhans cells
cannot secrete insulin.
The current therapeutic
management of DMT2 focuses on preventing and managing complications, not on
radical therapy, because they regard DMT2 as a chronic progressive and
irreversible disease. 92.7% of patients with diabetes mellitus have
uncontrolled blood glucose levels. Bariatric surgery, in obese patients,
results in resolution of diabetes in more than 90% of patients. The underlying
mechanism is thought to be due to significant weight loss and calorie
restriction. However, a large scale study with 608 patients followed for 14
years showed that resolution of diabetes occurred before significant weight
loss occurred and DMT2 therapy was continued when the patient was obese.
Surgical therapy has attracted attention as a therapy for non-obese DMT2
patients. Surgery has been reported to control blood glucose levels by reducing
insulin requirements.(Baskota, Li, Dhakal,
Liu, & Tian, 2015)
Sleeve
Gastrectomy (SG) procedure affects the
absorption of nutrients and secretion of metabolic hormones such as insulin,
GLP-1, PYY (Peptide YY) and PP (Pancreatic polypeptide) which can significantly
control blood glucose as a therapy for DMT2.(Huang, Ding, Fu,
& Cai, 2019) Omentoplasty is a surgical procedure where the omentum major is used to cover
defects, improve arterial or portal venous circulation, absorb effusions, or
increase lymphatic drainage.(Pai, 2019) The omentum is an organ that moves around the peritoneal cavity and
plays a role in controlling infection and contaminants. The omentum also
functions as an anti-inflammatory, increasing revascularization and tissue
regeneration.(Di Nicola, 2019) Omentoplasty has succeeded in providing a significant
output in cases of hydatidiform cysts on the liver that omentoplasty can
help the healing process and absorb serosal fluid.(Muftuoglu, Koksal,
& Topaloglu, 2005) Pancreatic omentoplasty is expected to increase the
vascularization of Langerhans cells so that it can increase secretion insulin.
Insulin can act as an anti-inflammatory agent. So it is expected that insulin secretion
by Langerhans cells which increases can suppress the inflammatory process in
the body which will eventually decrease insulin resistance.(Sun, Li, & Gao,
2014)
There has been
no study linking the combination of Sleeve Gastrectomy and Pancreatic
Omentoplasty on pancreatic histology in non-obese DMT2 patients. This study
will evaluate pancreatic VEGF levels in non-obese DMT2 subjects who underwent Sleeve
Gastrectomy and Pancreatic Omentoplasty and then analyze the
correlation between the two procedures.
RESEARCH METHOD
Animals
The
subjects of this study used 18 male Sprague-Dawley rats, aged 6-8 weeks,
weighing 170-200 grams, obtained and treated at the Integrated Research and
Testing Laboratory (LPPT), Gadjah Mada University, Yogyakarta. All rats were
adapted for 1 week to laboratory conditions before the study started. Rats were
given a diet according to the standard.
Induction materials
Rats
were injected with 230 mg/kgBW Nicotinamide (NA) intraperitoneally 15 minutes
before administration of a single dose of Streptozotocin (STZ) 65 mg/kgWB
intraperitoneally. On the 3rd day after induction, the rats without fasting
were taken from the infraorbital vein. Serum glucose levels were measured.
Glucose level ≥ 200 mg/dL is categorized as Diabetes Mellitus.
Sleeve Gastrectomy
This
procedure was performed after the rats were anesthetized using an intramuscular
injection of ketamine hydrochloride 20 mg/kgWB. The abdominal cavity was opened
with a left oblique subcostal incision. The rat's stomach was injected with
saline solution to increase the volume to make it easier to measure and cut above
the greater curvature until 50% of the stomach volume remained. To minimize
bleeding, clamp before the cutting line. Then closed the remaining stitches
with PGA 5.0 thread.
Pancreas Omentoplasty
The
rat pancreas was identified as a small tapered lump located between the
duodenum and spleen. Histologically, cells were found predominantly in the tail
of the pancreas, attaching to the spleen. The tail of the pancreas was closed
with a pedicled-omentum, and the sheath was closed using one loose suture with
PGA 5.0 suture, to avoid injury to the splenic vessels. Close the abdominal
cavity with simple sutures using PGA 3.0 suture.
Experimental Design
After the rats were diagnosed with diabetes mellitus, the rats were
randomly divided into 3 groups. Controls (Group K), Sleeve Gastrectomy (Group
P1), Sleeve Gastrectomy and Prancreas Omentoplasty (Group P2). Body weight and
serum glucose levels were measured on one day before surgery, day 5 and day 10
post procedure. Pancreatic VEGF levels were measured on day 10. Serum glucose
levels were obtained with the Glucose GOD FS Kit (DiaSys). VEGF levels were
measured using immunohistochemistry (IHC).
On day 10, the rats were terminated and the pancreas was extracted.
Paraffin blocks were fabricated and analyzed at the Laboratory of Anatomical
Pathology, Faculty of Medicine, Sebelas Maret University, Surakarta. Each
sample was cut to a thickness of about 4 microns and stained with
Immunohistochemistry (IHC) anti-Insulin monoclonal antibody. VEGF levels were
calculated based on pancreatic -cells with the intensity of the percentage of
all cells stained brown in the islets of Langerhans from a significant area
with 400x microscopic magnification view in 5 different view planes, in one
paraffin block. All samples were confirmed by 2 pathologists.
Figure 2
Sleeve gastrectomy procedure. A: identification of the stomach, B:
removal of part of the stomach and suturing, C: closure of the left oblique
subcostal incision of the abdomen
Statistic analysis
Body weight and
serum glucose levels were presented descriptively in the form of mean, SD, and
number of sample tables for each group. VEGF levels are presented descriptively
in the form of numbers and percentages. VEGF levels on day 10 post-procedure data
were tested for normality, followed by the One Way ANOVA. The
significance level was limited to p 0.05 with a 95% confidence interval. Data
were analyzed with SPSS 20.0 software for Windows.
RESULT AND DISCUSSION
Figure 4
Pancreatic VEGF intensity. A: weak intensity (control), B: moderate
intensity (SG), C: strong intensity (SG+Omentoplasty)
Sampel characteristics
18 rats that were still alive until the 10th day after treatment
were analyzed. Characteristics of sample body weight and serum glucose level
are presented in table 1 and table 2.
Table 1
Weight data (grams)
|
Day |
Group |
N |
Mean±SD |
Median |
|
-1 |
K P1 P2 |
6 6 6 |
177,17±5,64 173,83±3,06 174,67±4,63 |
176,50 174,00 175,50 |
|
5 |
K P1 P2 |
6 6 6 |
160,67±4,50 157,17±2,79 158,17±4,45 |
159,50 157,00 159,50 |
|
10 |
K P1 P2 |
6 6 6 |
144,17±6,34 139,33±3,14 142,83±6,24 |
143,00 140,50 143,50 |
The mean body weight was almost the
same between all groups on the same measurement day. Weight loss was observed
on day 5 and day 10 post-procedure, with the lightest weight on day 10.
Table 2
Serum glucose level data (mg/dL)
|
Day |
Group |
N |
Mean±SD |
Median |
|
-1 |
K P1 P2 |
6 6 6 |
266,54±3,99 264,53±3,23 262,61±3,05 |
264,59 264,21 263,00 |
|
5 |
K P1 P2 |
6 6 6 |
271,48±2,27 193,66±13,61 179,13±5,51 |
271,49 190,67 180,91 |
|
10 |
K P1 P2 |
6 6 6 |
274,76±2,81 174,44±7,85 160,51±4,41 |
274,32 177,65 159,41 |
Mean serum glucose levels were
similar in all groups at pre-procedure 1 day. over time, the observed serum
glucose levels showed different results between all groups. The P2 (SG +
Omentoplasty) group had the best mean serum glucose improvement among the other
groups, on day 10 post-procedure (160.51±4.41).
Pancreatic VEGF Level
Table 3
Pancreatic VEGF level data
|
Group |
Pancreatic
VEGF Level |
N |
p |
||
|
Weak |
Medium |
Strong |
|||
|
K |
6 |
0 |
0 |
6 |
0.421* |
|
P1 |
0 |
5 |
1 |
6 |
0.820* |
|
P2 |
0 |
1 |
5 |
6 |
0.421* |
|
Total |
6 |
6 |
6 |
18 |
|
Notes: * Normal data (p > 0.05);
Shapiro-wilk
Strong-intensity pancreatic VEGF
levels were found to be higher in P2 (SG + Omentoplasty) compared to P1 (SG)
and controls. All data are normal, test the hypothesis using the One Way
ANOVA test.
Table 4
One Way ANOVA Test Pancreatic VEGF Levels
|
Groups |
Pancreatic
VEGF Level |
N |
p |
||
|
Weak |
Medium |
Strong |
|||
|
K |
6 |
0 |
0 |
6 |
<0.01* |
|
P1 |
0 |
5 |
1 |
6 |
|
|
P2 |
0 |
1 |
5 |
6 |
|
|
Total |
6 |
6 |
6 |
18 |
|
Note: * Significant (p < 0.05)
Based on the One Way ANOVA
test, the differences in all groups in pancreatic VEGF levels were
statistically significant (p = <0.01). The Post Hoc test was performed to
analyze the significance between each group.
Table 5
Post Hoc LSD Test Pancreatic VEGF Level
|
Group |
P1 |
P2 |
|
K P1 |
<0.01* - |
<0.01* 0.003* |
Note: * Significant (p < 0.05)
From the Post Hoc LSD test result,
P2 group was statistically significant difference compared to P1 and K group (p
< 0.01). P1 group also has significant difference, compared to K group (p
< 0.01).
Discussion
In
this study, of 18 Sprague-Dawley rats induced by STZ-NA, all were
confirmed to be diabetic. Streptozotocin (STZ) is an antibiotic derived from
Streptomyces achromogenes and a structural derivative of glucosamine from
nitrosourea, has a selective toxic effect on pancreatic cells and causes
diabetes. Streptozotocin is taken up by cells via the glucose transporter
GLUT-2 and increases the production of free radicals causing DNA damage,
followed by activation of the core enzyme poly(ADP-ribose) synthetase, which is
involved in DNA repair. This leads to media depletion of the nuclear
poly(ADP-ribose) synthetase enzyme, nicotinamide adenine dinucleotide (NAD),
leading to decreased insulin synthesis in cells and cell death. This
streptozotocin-induced cytotoxicity is overcome with nicotinamide, a component of
NAD that inhibits poly(ADP-ribose) synthetase.(Ghasemi, Khalifi, &
Jedi, 2014)
Diabetes,
if not treated properly, increases the risk of premature death. Globally, 11.3%
of deaths are caused by diabetes. The IDF estimates that approximately 4.2
million adults died from diabetes and complications from diabetes in 2019.(IDF Diabetes Atlas 9th
edition, 2019) In
this study, we did not provide any medication or diet that could improve the
glucose status of rats. SG and Omentoplasty are the only intervening treatments
of diabetes.
The
American Society for Metabolic and Bariatric Surgery (ASMBS), Diabetes Surgery
Summit 2nd Consensus Guideline (DSS-II), and the International Diabetes
Federation (IDF) have agreed that bariatric procedures: Adjustable Gastric
Banding (AGB), Sleeve Gastrectomy (SG), Rouxen-Y Gastric Bypass (RYGB),
Biliopancreatic Diversion (BPD), and Biliopancreatic Diversion with Duodenal
Switch (BPD-DS), as diabetes surgeries can be used. Findings from comparative
studies and systematic reviews suggest the following magnitude of benefits and
risks among metabolic surgical procedures: BPDDS>RYGB>SG>AGB. More
extensive switching procedures are generally associated with greater weight
loss, greater metabolic benefit, but with a higher risk of surgical
complications and malnutrition.(Buchwald & Buchwald,
2019)
Currently,
more than 95% of bariatric procedures performed worldwide in obese and DMT2
patients are SG and RYGB. The RYGB procedure is the first definitive procedure
to demonstrate resolution of type 2 diabetes prior to weight loss. However, in
some studies, RYGB had twice the postoperative complication rate compared to
SG. On the other hand, shorter operative time, absence of gastrointestinal
anastomoses, retained pylorus, and unchanged intestinal absorption tract,
contribute to the safety of the SG procedure. SG will also be a better choice
in patients with high surgical risk.(Buchwald & Buchwald,
2019)
According
to this study, Sleeve Gastrectomy procedure improved serum glucose status
(mean±SD 174.44±7.85 compared to control mean±SD 274.76±2.81). This shows that
after the SG procedure, theoretically there will be some changes in the body's
metabolism, including increased levels of hormones such as Glucagon-like
Peptide-1 (GLP-1). The “Hindgut” hypothesis states that the presence of
undigested food in the distal small intestine stimulates the secretion of
“incretin” substances, which ultimately determine the normalization of serum
glucose, increase insulin production, and decrease insulin resistance. GLP-1 is
an incretin hormone secreted by enteroendocrine L cells in the small intestine
that is associated with stimulating β cell
growth, reducing apoptosis and, increasing β cell
mass in rats.(Huang et al., 2019),(Buchwald & Buchwald,
2019)
In
this study also used a combination of SG and Pancreatic Omentoplasty which
showed an increase in pancreatic VEGF levels had statistically significant (p =
<0.01) compared to control. Meanwhile, if the SG + Pancreas omentoplasty was
compared with the SG group, the VEGF level have a significant result (p =
0.003).
Omental
adipocytes are the main source of VEGF protein. VEGF can induce angiogenesis
and neovascularization. In experimental animals it was reported that the
omental vascular anastomosis with the intestinal wall could form as early as
the third day after surgery. In addition, hypoxia can lead to increased VEGF
expression in omental adipocytes. Omentoplasty can protect the disrupted
anastomosis by providing a biological space to prevent leakage and a source of
granulation tissue and neovasculature for wound repair.(Tuo et al., 2020)
Based
on this study, after SG and Omentoplasty, pancreatic VEGF levels were higher
than controls. That is, the SG procedure and Omentoplasty have sufficient
effect in resolution in non-obese rats with DMT2, especially in repairing
Langerhans cells thereby increasing insulin secretion and decreasing insulin
resistance.
There
are some limitations to this study, namely that induction from animal models of
diabetes may not reflect the true pathogenesis of Diabetes Mellitus and the
absence of long-term sample outcomes (evaluating relapse, partial or complete
remission of diabetes). However, this study shows that sleeve gastrectomy and
pancreatic omentoplasty are useful procedures for treating diabetes mellitus in
non-obese rats. Future research is needed to make it applicable to humans
CONCLUSION
Sleeve Gastrectomy and Pancreas Omentoplasty
can improve type 2 Diabetes Mellitus status in non-obese rat subjects. SG +
pancreatic omentoplasty increases pancreatic VEGF levels.
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