Regulatory Effect of Liraglutide on Endoplasmic Reticulum Stress in Non-alcoholic Fatty Pancreas Disease

Background The pathophysiology and treatment of non-alcoholic fatty pancreas disease (NAFPD) have not been well established. Thus, the aim is to prove hypotheses that endoplasmic reticulum (ER)stress may take part in the pathogenesis of NAFPD. Methods The mice were randomly grouped into normal-group, model-group and experimental-group. The normal-group was fed with regular-diet, while model-group and experimental-group were fed with high-fat-diet for 16 weeks. After the NAFPD model was established successfully, the experimental-group began to give liraglutide 0.6 mg·kg -1 ·d -1 for 4 weeks. The normal-group and model-group were received saline only, total pancreatectomy was performed and pancreas samples were divided for western-blot and immunohistochemical, to detect the expression of GRP78PERKeLF2αATF4 CASPASE12 and CHOP. Results Western-blot and immunohistochemistry both revealed that the expression of above proteins of model-group were significantly increased compared with normal-group and were also obviously increased compared with experimental-group. (all P ≤0.05). The weight of body and the pancreas of the model-group was markedly higher than that of normal-group, and was also dramatically higher than that of experimental-group (all P ≤0.01). Conclusion Those results show ER stress may be one of the pathogenesis of NAFPD and liraglutide have the effect on regulating ER stress in NAFPD. Those findings have great significance in figuring out the pathophysiology of NAFPD.


Background
The high calorie intake in today's society could cause ectopic fat infiltration. The excessive fat could be accumulated in different visceral organs, for instance, liver and pancreas, [1] leading to nonalcoholic fatty liver disease (NAFLD) and/or non-alcoholic fatty pancreas disease (NAFPD). NAFPD is range from the ectopic adipose infiltration in pancreatic cells to pancreatic steatosis and inflammation, eventually resulting in fibrosis, that is akin to process of NAFLD. [2] Similar in pathophysiology to how NAFLD causes liver cancer, NAFPD may lead to the pancreatic carcinoma, and may also be associated withβ-cell dysfunction, insulin resistance, type 2 diabetes(T2DM),NAFLD, severity of acute pancreatitis, and pancreaticoduodenal leakage. What's more, NAFPD has instructive significance in the treatment and prognosis of the above diseases. It may also be one of the early intervention indications of metabolic syndrome and T2DM. However, on the contrary to NAFLD the pathophysiology and treatment of NAFPD have not been well established yet. It is urgent to find a treatment for NAFPD.
Glucagon like peptide-1 (GLP-1), a gastroenteric hormone excreted by the intestinal cell , [3] stimulates the secretion of glucose-dependent insulin to regulate blood glucose, [3,4] playing a role in losing weight and improving the NAFLD as well. [5]Liraglutide is an agonist of the GLP-1 receptor and, thereby, occupying a certain position in the treatment of NAFLD.
Innumerable pathological conditions,including excessive accumulation of unfolded proteins in endoplasmic reticulum (ER) and saturated fatty acids [6]in cells could initiate the non-homeostasis of ER and the ER stress. [7]The cellular reaction to ER stress is to activate an signaling cascade known as the unfolded protein response (UPR) pathway [8]which enables the cell to reduce unfolded protein load in ER and restores homeostasis. [9] However the long-term ER stress or acute ER stress could cause chronic activation of UPR or full mobilization of UPR which induces an apoptotic procedure. [10] Cao J et al reported that the suppression of UPR could significantly inhibit apoptosis of hepatocytes in NAFLD. [6]Surveys such as that conducted by Na Ao et al have shown that Liraglutide could decrease the expression of GRP78 PERK CHOP and Caspase12 in mice of NAFLD and improve liver histology.
[11] Sandberg MB et al founded that the of ER stress may take a part in the pathogenesis of NAFPD. [7] Given that the liver and pancreas have similar embryological origins [2]and ectopic fat deposition, [1]it is also plausible, as recommended for NAFLD, that GLP-1 could improve NAFPD by inhibiting UPR.
Thus, the aim of our research is to prove hypotheses that ER stress may take part in the pathogenesis of NAFPD and the inhibition of GLP-1 on ER stress may improve NAFPD.

Animal modeling and grouping
Five-week-old SPF male C57BL/6 mice were grouped into normal-group, model-group and experimental-group. After 16-week feeding high-fat-diet and confirming the successful establishment of NAFPD model in the model-group (n = 10) and experimental-group (n = 10) , while the normalgroup was fed with regular-diet. Then the experimental-group began to give liraglutide 0.6 mg·kg -1 ·d -1 for 4 weeks. The normal-group and model-group were received saline only.

Euthanasia and Experimental specimens:
After the intraperitoneal injection and weigh daily in the 4-week period and an overnight fast with water allowed ad libitum, the mice were sedated with an isoflurane-soaked gauze placed in a 2000 cm 3 glass jar. They were then anesthetized with an intraperitoneal injection of xylazine (15 mg/kg) and ketamine (50mg/kg) and were sacrificed by cervical dislocation. After that ,the total pancreatectomy was executed and pancreatic tissues were weighed and then split up for western-blot and immunohistochemistry.
2.3 Western-blot was carried out to discover the expression of GRP78 PERK eLF2α ATF4 CASPASE12 and CHOP in pancreatic tissue.
The tissue was cut into fine fragments, and the PMSF was added in the proportion of 100-250 μl lysate per 20mg tissue, and the homogenizer homogenized until completely cracked. After pyrolysis, the samples were centrifuged at 4 ℃ for 12000 rpm for 5 min, and the protein was quantified with BCA working solution. According to the quantitative results of protein, the required protein was added with appropriate amount of sample buffer, and then centrifuged to take the sample after boiling water bath for 5 min. The prepared PAGE gel was put into the electrophoresis cell, added with proper amount of electrophoresis buffer, and then sealed with 5% skimmed milk powder. According to the instructions: rabbit Anti-GRP78 BiP antibody(ab21685) 1: 2000, rabbit Anti-PERK antibody (ab192591) 2.4 Immunohistochemistry (IHC) was carried out to discover the expression of GRP78 PERK eLF2α ATF4 CASPASE12 and CHOP.
The pancreatic tissue of 0.2cm-0.3cm was fixed with 10% neutral formalin for 48 h and then waxed at about 58 ℃. After wax soaking, it was sliced and mounted with silicified glass, then roasted at 60 ℃ for 1 h, immediately rinsed with xylene 10 min with ethanol of different concentrations from high to low (100%-30%), rinsed with tap water for 5 min, at a time, and fresh 3% H 2 O 2 was equipped with distilled water. After sealing at room temperature for 10 min, rinse with distilled water for 3 minutes each time. The slices were immersed in 0.01m hydrochloric acid buffer, microwave was adjusted to 98 ℃-100 ℃ to boil, cooled for 5 min, and repeated twice, the slices were naturally cooled to room temperature, and washed with PBS for 3 times for 5 min each time. The excess liquid was removed by sealing solution at room temperature for 20 min, and incubated at 4 ℃ with first antibody (GRP78

Impact of liraglutide on the ER stress in pancreas of mice with NAFPD
Western-blot analysis displayed that the expression of GRP78 PERK eLF2α ATF4 CASPASE12 and CHOP in pancreatic tissue of the normal group, compared with the model group, was significantly decreased(all P ≤0.05) ( Fig.1 and Table 1).At the same time, immunohistochemical analysis also showed that the expression of those proteins in the normal group was significantly decreased than that in the model group (all P≤0.05) ( Fig.2 and Table 2).Those results suggest that ER stress does take part in the pathogenesis of NAFPD, specially the PERK-eIF2α-ATF4 pathway CHOP pathway and Caspase12 pathway .
Using western-blot and immunohistochemistry, we found that Liraglutide have the effect on inhibiting ER stress in NAFPD especially the PERK-eIF2α-ATF4 pathway CHOP pathway and Caspase12 pathway.
After the intervention, the expression of above proteins in the model group was founded to be markedly increased than those in the experimental group by western blot and immunohistochemistry (all P ≤0.05) (as shown in Fig.1 Fig.2 Table 1 and Table 2).

Impact of liraglutide on the weight of pancreas of mice with NAFPD
To prove the effect of Liraglutide on losing weight of the pancreas and the body of mice with NAFPD, the weight of the pancreas and the body of mice on the last day were weighed. The weight of body of the normal group the model group and the experimental group was (29.10 ±2.37), (36.10 ±2.73), (28.50 ±1.58) g, respectively. Compared with the normal group, the weight of the model group was notably higher(P≤0.01). At the same time, the weight of the experimental group was notably decrease than that of the model group (P≤0.01) (as shown in Fig. 3). The weight of pancreas of the normal group the model group and the experimental group was (0.11 ±0.01), (0.14 ±0.01), (0.12 ±0.01) g, respectively. Compared with the normal group, the weight of pancreas of the model group was dramatically higher(P≤0.01). Meanwhile, the weight of pancreas of the experimental group was notably decrease than that of the model group (P≤0.01) (as shown in Fig. 4). Those results showed that liraglutide could loss the weight of the pancreas and the body of mice with NAFPD. Discussion 1.ER stress may be part of the cause of NAFPD UPR,a self-protective response, mainly includes three branches: PERK-eIF2α-ATF4 pathway, IRE1αpathway and ATF6 pathway. [12] Upon the non-stressed circumstance the these transducers of signaling pathways of UPR are combine with the BiP/GRP78,which suppress these signaling pathways.
[12] When non-homeostasis of the ER take place, BiP/GRP78 will be separated from these transducers, leading to the activation of signaling pathways. However the long-term ER stress or acute ER stress could cause chronic activation of UPR or full mobilization of UPR which upregulates the expression of CHOP (also known as DDIT3) pathway, Caspase12 pathway and IRE1α-ASK1-JNK pathway and induces an apoptotic procedure. [10]Caspase12, a member of the Caspase family, is the only one in the family could be motivated by URP [13] and subsequently activates the rest of the Caspase family (such as Caspase9, Caspase3, etc.) to promote apoptosis. [13]Elida Lai et al reported that together with others, [6,14] the PERK-eIF2α-ATF4 pathway takes a significant position about mediating fat-induced ER stress and pancreatic cell apoptosis. [15] Elizabeth Karaskov et al also showed that the palmitate could increase the expression of the CHOP transcription factor. [14] Here we report that the expression of GRP78, PERK,eIF2α,ATF4 Caspase-12 and CHOP in pancreatic tissue of the model group was higher than that of the normal group, which indicates that ER stress may be part of the cause of NAFPD,especially the PERK-eIF2α-ATF4 CHOP and Caspase12 pathway.
Those findings have great significance in figuring out the pathophysiology of NAFPD, and have great value in the treatment plan for NAFPD.

Liraglutide have the effect on regulating the signal pathway of ER stress in pancreatic cell of NAFPD mice
Here we found that the expression of PERK-eIF2α-ATF4-related factors in the experimental group was decreased ,compared with the model group,illustrates that liraglutide have the effect on inhibiting ER stress in NAFPD especially the PERK-eIF2α-ATF4 pathway CHOP pathway and Caspase12 pathway.
However, the mechanistic relevance of the finding is also unclear, since these could be protective, deleterious or just a bystander effect.
Although, Na Ao et al showed that liraglutide could decrease the expression of GRP78 PERK CHOP and Caspase12 in mice of NAFLD and improve liver histology. [11] Meanwhile, many studies have confirmed that the effect of drugs on inhibiting the expression of ER stress pathway protein could improve the liver histology of NAFLD. [16][17][18] The PERK-eIF2α-ATF4 signaling pathway takes a significant position in mediating fat-induced ER stress and pancreatic cell apoptosis. [6,14,15]Given that the liver and pancreas have similar embryological origins [2]and ectopic fat deposition [1] it is also plausible, as recommended for NAFLD, that GLP-1 could improve NAFPD by inhibiting UPR, however,the straight evidence of improvement of NAFPD was not displayed in present study. Hence, further studies are needed.

Liraglutide has effect on weight loss of the pancreas and the body of NAFPD mice
Obesity is considered to be associated with NAFPD, recent study suggests that obesity is an independent risk factor. [2] Animal experiments have shown that the accumulation of pancreatic interlobular or pancreatic intralobular fat, accompanied by inflammation and fibrosis, can be found in mice of long-term high-fat chow, leading to the impairment of islets and pancreatic acinar and the destruction of islet cell structure.
[19] The pancreatic fat capacity and the amount of intaking fatty acid were markedly decreased in 7 of 10 cases of weight-loss surgery, that were all diagnosed with NAFPD, [20] which suggests that weight loss treatment may be used to treat NAFPD, such as liraglutide. [5] Some studies have shown that liraglutide can reduce the weight of mice fed on high-fat diet, inhibit ER stress, and improve steatosis. [13]Our study reveals that liraglutide not only could reduce the weight but also decrease the weight pancreas in NAFPD mice, and suggest that, together with the above research, the weight loss effect of liraglutide may helpful to improve the fat infiltration of NAFPD.
One of the shortcomings of this study is the absence of the straight evidence of improvement of NAFPD.

Conclusion
In conclusion, the ER stress may be part of the cause of NAFPD,especially the PERK-eIF2α-ATF4 CHOP and Caspase12 pathway. and Liraglutide does repress the ER stress and could lighten the pancreas of NAFPD mice which may improve NAFPD. Hence, further studies are needed.  Comparison of the expression of GRP78 p-PERK eLF2α ATF4 CASPASE12 and CHOP of three groups after treatment with liraglutide in experimental group for 4 weeks(`x±s ) .Compared with model group, * P ≤0.05 ** P ≤0.01 Compared with Experimental group # P ≤0.05 ### P≤0.001. GRP78: Glucose-regulatedprotein78;PERK:R-like endoplasmic reticulum kinase;EIF2α:eukaryotic translation initiation factor 2α; ATF4:activating transcription factor4;Caspase12:cysteme aspartate specific protease 12;CHOP:C/EBP-homologousprotein; Table 2 the expression of proteins of three groups after the intervene (IHC) Figure 3 Values are means ± SEM n= 10 mice per group. ** P≤0.01 vs. Normal-group / Modelgroup; ## P ≤0.01 vs. Experimental-group / Model-group.

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