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Method Article

Development and Validation of a Methodology for Establishing Obese Rat Models with Typical Fatty Pancreas

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DOI:

10.3791/69553

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November 11th, 2025

 ,  ,  ,  ,  ,  ,  ,  , 
1Key Laboratory of Acupuncture and Medicine Research of Ministry of Education, Nanjing University of Chinese Medicine, 2Department of Acupuncture and Moxibustion, Yangzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, 3Department of Specialty Medicine, Ohio University, 4Research Department, Swiss University of Traditional Chinese Medicine

Corresponding Authors: Youbing Xia <xybd1968@njucm.edu.cn>, Tiancheng Xu <xtc@njucm.edu.cn>

* These authors contributed equally

In This Article

Summary

This study offers a reproducible and standardized method in order to establish obese rat models that exhibit simple obesity, commonly characterized by fatty pancreas. It also includes detailed protocols for pancreatic tissue sampling and immunohistochemistry.

Abstract

Obesity has emerged as a predominant global health epidemic, with nearly half of the world's population now classified as overweight or obese. A key pathological feature in obese individuals is ectopic lipid deposition in non-adipose tissues, including the pancreas-a condition termed pancreatic steatosis or fatty pancreas. In high-fat diet (HFD)-induced rodent models, pancreatic steatosis consistently precedes hepatic steatosis, underscoring the particular susceptibility of the pancreas to lipid accumulation. This early involvement positions the pancreas as a critical organ for understanding metabolic dysregulation in obesity. Although diet-induced obese (DIO) rats recapitulate core aspects of human disease progression, well-characterized animal models that reliably exhibit this pancreatic phenotype remain scarce in the literature.

A significant methodological consideration in modeling human obesity is the timing of HFD introduction. Many existing models initiate HFD during the weaning period, which may introduce confounding developmental metabolic programming effects that do not fully reflect human obesity, which primarily develops post-developmentally. In contrast, the present study establishes a standardized protocol in which HFD is introduced post-weaning. This approach more accurately mimics the common human trajectory of obesity onset in adulthood, avoids early-life metabolic adaptations, and results in a more physiologically relevant progression toward pancreatic steatosis.

This research provides a detailed and reproducible framework for generating a rat model of obesity characterized by prominent fatty pancreas pathology. The methodology includes a 1-week acclimatization phase, a 14-week HFD induction period starting after weaning, and systematic tissue harvesting. Two tissue processing pathways are described: snap-freezing for molecular analyses (e.g., Western blotting) and paraformaldehyde perfusion-fixation for morphological evaluations (e.g., IHC, H&E). This model offers a robust platform for mechanistic investigations into obesity-associated pancreatic metabolic dysfunction.

Introduction

Simple obesity is defined as obesity that exists independently without other comorbidities, primarily attributed to energy imbalance resulting from unhealthy lifestyles, such as improper dietary patterns and insufficient physical activity1. Obesity significantly contributes to the rising incidence of non-communicable diseases among younger populations, characterized by high prevalence rates, early onset, and multisystem complications, positioning it as a critical global public health challenge2. Fatty pancreas is a condition marked by ectopic lipid deposition in this non-adipose organ during metabolic dysregulation. Fatty pancreas reflects a critical interplay between adipose tissue and pancreatic pathophysiology. Excessive pancreatic fat accumulation displaces normal acinar and islet tissues, inducing dual functional impairments in both exocrine and endocrine capacities3,4. This pathological process disrupts α/β-cell ratios, promotes β-cell dedifferentiation with subsequent loss of insulin secretory function5, and mediates direct lipotoxic damage to β-cells6. Concurrently, α-cell proliferation and dysregulated insulin release exacerbate glycemic instability, accelerating progression to type 2 diabetes mellitus (T2DM) and its associated complications7.

Investigating fatty pancreas pathogenesis reveals novel mechanisms underlying metabolic disease progression8. While prior obesity research emphasized hepatic steatosis, emerging evidence identifies pancreatic lipid deposition as an earlier event in metabolic dysregulation9. This paradigm shift enables the construction of a dual-organ pathology model for simple obesity, integrating pancreatic and hepatic steatosis10. Studying their cross-talk may elucidate critical pathways across the disease spectrum spanning lipid metabolism disorder, obesity, insulin resistance, and diabetes, offering fresh insights into disease mechanisms11.In the pathological process of the pancreas, the initial manifestation is significant deposition of pancreatic fat, accompanied by substantial expansion of pancreatic adipocytes. This is predominantly observed in the ventral aspect of the pancreatic head, neck, and body regions, and is primarily located within the interstitial areas between acinar and islet cells. Concurrently, chronic low-grade inflammation of the pancreas is present, while both endocrine and exocrine functions remain largely intact at this stage12,13. Extensive fatty infiltration further impairs pancreatic β-cell function through lipotoxicity and suppresses insulin activity. During this phase, acinar cells are progressively replaced by adipocytes, thereby contributing to the development of T2DM14,15. Although diabetes mellitus is not an independent risk factor for pancreatic cancer, patients with a history of acute pancreatitis and diabetes exhibit a significantly elevated risk of developing pancreatic cancer16.

Macroscopic anatomical analyses quantify pancreatic steatosis severity and infiltration patterns, while IHC evaluations at the microscopic level characterize acinar cell morphology, nuclear positioning, vacuolization frequency, and cellular population dynamics17. This multimodal approach, spanning gross anatomical observation to cellular-resolution IHC, enables comprehensive staging of pancreatic pathology across disease progression. The present study details a standardized protocol encompassing a 1-week acclimatization, a 14-week obesity induction in adult rats, and optimized pancreatic tissue processing for morphological analysis, establishing a robust framework for investigating steatosis-associated metabolic derangements.

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Protocol

The animal treatment procedure was approved by the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine (202006A016) and complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, ethical guidelines, guidance on the operation of the Animals (Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63 for the protection of animals used for scientific purposes. All experimental animals were anesthetized in accordance with approved rodent anesthesia procedures prior to dissection. The procedure was organized into three distinct components.

1. Establish an obese rat model

  1. Experimental animals and groups
    1. Select male Sprague-Dawley (SD) rats aged 4 weeks (80-120 g) from a strain sensitive to high-fat diet (HFD).
    2. Acclimate all rats to a maintenance diet for one week, then randomly divide them into two groups: the control group and the model group.
    3. Replace the maintenance diet with HFD for the model group after one week, while maintaining the control group on the original diet. Maintain both groups on their diets for 14 weeks. The HFD composition is detailed in Table 1, and the standard maintenance feed formulation is in Table 2.
  2. Feeding conditions and experimental parameters
    1. Environmental parameters: temperature 22 ± 2 °C, humidity 50 ± 10%, 12 h light/dark cycle, and free access to water and food.
  3. Monitoring indicators
    1. Record body weight and food intake weekly at a fixed time.
      NOTE: Doing it in the evening can minimize stress and align with diurnal/nocturnal activity patterns18.
    2. Plot a body weight growth curve using the recorded data.
    3. Define successful obesity modeling as when body weight exceeds 20% of the age-matched control group.
IngredientContent (g/kg)Major nutrients provided
Ordinary powdered feed365Basic nutritional supply of carbohydrates, Vegetable proteins, Fats, Vitamin complexes, Minerals, etc
Lard310Saturated fatty acids, Unsaturated fatty acids
Casein250Essential amino acids
Cholesterol10Cholesterol
Cellulose and mineral mixture60Fiber, Calcium, Phosphorus, Sodium, etc
DL-Methionine Methionine/Methionine Amino Acids3Essential amino acids
Yeast1B vitamins, β-glucan, etc
NaCl1NaCl 

Table 1: Special HFD formulation for establishing DIO in rat models. The table details the specific composition of the HFD used in this study for the model group, which was administered from the end of the acclimation period until tissue collection.

IngredientContentUnit
Moisture12.196%
Crude Protein (CP%)18.272%
Digestible Energy (DE)3.186Mcal/kg
Metabolizable Energy (ME)2.939Mcal/kg
Net Energy (NE)2.228Mcal/kg
Ether Extract (EE%)4.417%
Crude Fiber (CF%)3.677%
Calcium (Ca%)0.991%
Total Phosphorus0.681%
 (Total P%)
Non-Phytate Phosphorus0.19%
Salt0.407%
Lysine1.118%
Digestible Lysine0.813%
Methionine0.53%
Threonine0.779%
Tryptophan0.221%
Copper (Cu)22.06mg/kg
Iron (Fe)173.69mg/kg
Manganese (Mn)102.1176mg/kg
Zinc (Zn)60.3687mg/kg

Table 2: Standard maintenance feed formula in rats. The table presents the formulation of the standard maintenance feed, which was provided to all rats during the acclimation phase and to the control group throughout the entire study duration.

2. Tissue harvesting

  1. Preoperative preparation
    1. Fasting treatment: 12 h food restriction before euthanasia to minimize intestinal content interference with biochemical analyses.
    2. Anesthesia: Administer isoflurane via inhalation for deep anesthesia (induction concentration 4%, maintenance concentration 1.5-2%). The anesthesia machine was equipped with a vaporizer for isoflurane, and the anesthetic concentration was maintained at 1.5-2. As a reference for preparation, the physical consumption of liquid isoflurane was approximately 0.5 mL-0.8 mL per rat for the entire procedure.
    3. Monitor anesthesia depth: Continuously track respiratory rate and corneal reflexes. Maintain a sustained anesthetic state throughout the procedure to prevent postmortem pancreatic autolysis.
    4. Manage insufficient anesthesia: If anesthesia depth is inadequate or consciousness recurs, mmediately increase the concentration of isoflurane to supplement anesthesia and recheck vital signs before continuing.
      NOTE: Isoflurane anesthesia is the method compliant with the ethical requirements of the Experimental Animal Center of Nanjing University of Chinese Medicine. Additionally, the existing evidence suggests that isoflurane may offer a higher safety profile than ketamine in rodents19.
  2. Fresh tissue harvesting
    1. Skin disinfection: Spray 75% ethanol rapidly over the entire body (1-2 s) to prevent hair contamination of abdominal organs.
    2. Position fixation: Position the rat supine on the dissection table and fix all four limbs securely with tape.
    3. Laparotomy: Grasp the abdominal skin with forceps, and using scissors, make a transverse incision along the lower edge of the costal margin. Use blunt dissection to separate the subcutaneous fascia and connective tissue.
    4. Pancreas identification: Gently displace the bowel to the left to visualize the stomach, spleen, and duodenum. The spleen is attached to the tail of the pancreas, and the stomach is close to the pancreas.
    5. Rapid dissection: Gently elevate the spleen with forceps and isolate the pancreas by progressively cutting its attachments to the spleen, stomach, and duodenal mesentery. Due to its fragile texture, avoid direct clamping of pancreatic parenchyma and preserve pancreatic ducts/blood vessels to prevent tissue tearing.
    6. Tissue preservation: Rinse the dissected pancreas rapidly with ice-cold PBS (1× Phosphate buffered saline, PH 7.4) or saline to remove residual blood. Immediately transfer the pancreas to a 2 mL freezing tube, submerge in liquid nitrogen within 30 s, and store long-term at -80 °C.
    7. Euthanasia and carcass disposal Following tissue collection, euthanize all experimental rats by cervical dislocation. According to the regulations of the Experimental Animal Center of Nanjing University of Chinese Medicine, animal carcasses are classified as pathological biomedical waste (Waste Code: 831-003-01). Seal all animal carcasses in airtight bags according to regulations, and store them uniformly in designated freezers. Centralized, environmentally sound disposal will be conducted by the university. Under no circumstances should carcasses be discarded arbitrarily.
      NOTE: Avoid room temperature exposure to prevent enzymatic degradation of biomolecules20,21.
  3. Perfusion tissue harvesting (the remaining steps are the same)
    1. Expose the heart: Lift the rat's skin tissue with your left hand and open the thoracic cavity with your right hand to fully expose the heart.
      NOTE: A deep level of anesthesia must be induced prior to initiating the thoracotomy incision for the cardiac perfusion procedure.
    2. Prepare the left ventricle for perfusion: Make a small incision on the left ventricle (size matching the perfusion needle diameter) and incise the right auricle to allow blood outflow.
    3. Perfuse saline: Insert the perfusion needle gently into the left ventricle and slowly perfuse 100 mL of saline at a flow rate of 10 mL/min until the right atrial outflow becomes clear.
    4. Perfuse fixative: Switch to 200 mL of 4% paraformaldehyde fixative, flow rate: 5 mL/min.Perfuse each rat with 100 mL of saline, followed by 200 mL of 4% paraformaldehyde. This study used a commercially prepared PFA solution. Due to the instability of PFA in solution, with significant variability potential depending on preparation and freshness, the liquid solution must be protected from light and stored at 4 °C. Use an opened aliquot promptly to ensure efficacy. If prepared from powder, the solution should be freshly made immediately before use.
      NOTE: Wear gloves, goggles, and a lab coat; perform in a fume hood because paraformaldehyde is toxic. According to the regulations of the Experimental Animal Center of Nanjing University of Chinese Medicine, paraformaldehyde waste is classified as chemical biomedical waste (Waste Code: 831-004-01). After use, it must be stored in a dedicated waste container labeled with the biohazard symbol. Centralized disposal is carried out by the university. Under no circumstances shall it be discarded improperly.
    5. Confirm perfusion success: Check for limb rigidity indicating effective fixation.
    6. Place the pancreas in a 50 mL centrifuge tube containing 30 mL of 4% paraformaldehyde solution. Fix the tissue by storing the tube stationary in the dark for 2-4 h.
    7. Transfer the pancreas to a new 50 mL centrifuge tube containing 30 mL of 70% ethanol solution. Keep the tube stationary and protected from light for approximately 20-24 h22.
    8. Paraffin embedding: Embed the tissues in paraffin wax. Store the embedded tissues at 4 °C or -20 °C.
      NOTE: This study used paraffin sections. Frozen sections are a valid alternative, but antibody performance must be verified for the specific section type beforehand to ensure optimal results.

3. Immunohistochemistry (IHC) staining of pancreas

  1. Sectioning and mounting protocol
    1. Paraffin sections: Cut tissue sections at a thickness of 6 µm using a microtome and mount onto glass slides.
    2. Floatation method: Float the sections on a 40 °C tissue flotation bath to smooth wrinkles.
    3. Tissue collection: Mount sections onto adhesive-coated slides (2-3 per slide) and dry on an 80 °C slide warmer.
    4. Slide drying: Transfer slides with mounted sections to a 60 °C oven overnight after sectioning.
  2. Deparaffinization and rehydration protocol
    1. Place slides in a slide rack and immerse sequentially as follows: xylene I, 7 min; xylene II, 7 min; anhydrous ethanol, 5 min; 90% ethanol, 5 min; 85% ethanol, 5 min; 75% ethanol, 5 min; distilled water, 5 min; PBS, 5 min (three times).
      NOTE: Perform xylene steps in a fume hood with gloves and goggles. Dispose of xylene waste following the identical protocol specified for paraformaldehyde as step 2.3.4.
  3. Antigen retrieval protocol
    1. Preparation of sodium citrate solution: Prepare sodium citrate buffer (pH 6.0). We used a self-prepared sodium citrate buffer solution by dissolving one packet of the powder in 1 L of water.
    2. Microwave sequentially: Place the slides in a glass staining jar filled with sodium citrate buffer solution to cover the slides, then place the jar in the microwave. The samples were subjected to microwave treatment under the following sequence: medium heat (600 W) for 8 min, followed by standing without heating for 8 min, and finally low heat (300 W) for 7 min.
    3. Cool naturally to room temperature: Allow the container to cool naturally to room temperature.
    4. PBS: Rinse slides in PBS for 5 min for two times.
  4. Oven incubation
    1. H2O2: Apply 3% H2O2 to the tissue sections for 10 min to block endogenous peroxidase activity. Apply approximately 50 - 80 µL of H2O2 to each slide.
    2. PBS: Wash slides in PBS for 5 min three times.
    3. Strept-Avidin-Biotin-Peroxidase Complex (SABC): Apply SABC dropwise onto the tissue and stain for 30 min in a 37 °C incubator. Apply approximately 50 - 80 µL of SABC to each slide.
    4. PBS: Wash slides in PBS for 5 min three times.
  5. Color rendering
    1. 3,3'-diaminobenzidine (DAB): Apply DAB chromogenic solution to the tissue sections and monitor until brownish-yellow coloration develops. Stain the sections for approximately 1-5 min. Adjust the staining duration based on tissue specificity and ambient temperature. For pancreatic tissue, a staining time of 2 min at 22 °C is recommended.
    2. Rinse: Rinse slides immediately under a gentle stream of distilled water to stop the reaction.
    3. Immerse: Immerse slides in distilled water for 5 min to remove residual reagents.
  6. Hematoxylin staining
    1. Hematoxylin: Immerse slides in the hematoxylin for 1 min.Hematoxylin solution is prepared using pure water at a ratio of 56 mg/mL.
    2. Rinse: Rinse slides twice in distilled water (5 s per rinse).
    3. Hydrochloric Acid-Alcohol Differentiation: Differentiate the slides in the hydrochloric acid-alcohol differentiation solution for 1 s.
    4. Immerse: Immerse the slides in tap water for 10 min to develop stable blue nuclear staining.
  7. Dehydration: Dehydrate the slides sequentially through the following solutions: 70% ethanol, 80% ethanol, 90% ethanol, anhydrous ethanol, xylene I, and xylene II, for 3 minutes in each solution, respectively.
    NOTE: Perform xylene steps in a fume hood with gloves and goggles.
  8. Sealing
    1. Mount the sections with neutral balsam: Apply neutral balsam to the slide using a rubber bulb dropper. Cover with a coverslip. Avoid compressing the bulb forcefully to prevent bubble formation. Apply approximately 50 µL of neutral balsam to each slide.
    2. Air-dry: Place the slides in a fume hood overnight to allow the neutral resin to dry completely.

4. Western blotting (WB) analysis

  1. Decomposition of tissues
    1. Take 200 mg of rat pancreatic tissue, place it into a 2 mL grinding tube containing 1 mL of lysis buffer and 3 small steel balls. The lysis buffer is composed of complete Protease Inhibitor Cocktail and RIPA lysis buffer. Mixing ratio is 1 tablet of protease inhibitor: 50 mL RIPA lysis buffer.
    2. Grinding was performed using a ball mill with the following parameters: frequency: 30/s; time: 30 s; 10-15 times. Until no white particles are visible to the naked eye.
    3. Transfer the upper protein supernatant into a 1.5 mL micro-centrifuge tube, do not take any foam. Centrifuge it with 16900 x g 30min, 4 °C.
      NOTE: Homogenates for Western blotting analysis were prepared from the entire pancreas.
  2. Protein quantification: Quantify protein concentration using the Pierce BCA Protein Assay Kit according to the manufacturer's instructions. The mixing ratio for BCA working solution is Solution A: Solution B = 50:1.
  3. Protein electrophoresis:
    1. Add the prepared pancreatic protein samples (a volume of 6 µL was loaded into each lane) to sodium dodecyl sulfate-polyacrylamide gel-Young PAGE, Bis-Tirs, 10 × 8, for electrophoresis.
    2. Run at 70 V for 0.5 h, then run at 110 V for 1 h using Mini-PROTEAN Tetra System. Dilute the MOPS electrophoresis Running Buffer liquid 20×with pure water to 1× concentration.
  4. Membrane transfer
    1. Dilute the transfer concentrate eBlot L2 5× to 1× working concentration using pure water. This 1× transfer buffer can be reused up to 3 times. Dilute the balance concentrate eBlot L2 5× to 1× working concentration using pure water.
    2. Cut a PVDF membrane (Immobilon-P Transfer Membrane) to the appropriate size (e.g., 7.5 cm × 5.5 cm). The pore size is 0.45 um.
    3. Pour approximately 30 mL of methanol into an incubation box with 90 × 60 × 32 mm. Immerse the PVDF membrane in the Methanol for 5 s to activate it.
    4. Pour approximately 30 mL of the prepared 1x balance solution into another clean incubation box. Carefully remove the gel from the electrophoresis plates and place it into the balance solution.
    5. Transfer the activated PVDF membrane from the methanol and also place it into the incubation box containing the balance solution. Ensure the membrane is positioned beneath the gel for proper contact. Allow both to equilibrate.
    6. Following the blotting apparatus manufacturer's instructions, place the transfer "sandwich" in this order from up to bottom: sponge, gel, PVDF membrane, sponge. Ensure no air bubbles are trapped between layers. Correctly place the assembled transfer stack into the blotting apparatus chamber. Connect the power supply and run the transfer at standard mode for 15 min.
  5. Block
    1. Prepare two incubation boxes, one containing approximately 15 mL of TBST (Tris-buffered saline-Tween20), and the other with about 15 mL of 5% BSA blocking solution.
    2. Preparation of TBST solution: Dissolve one pouch of Tris Buffered Saline Powder in 2 L of pure water, then add 2 mL of Tween 20.
    3. Place the PVDF membrane into the TBST solution, ensuring the side that was in contact with the gel faces up.
    4. Wash the PVDF membrane on a shaker at 40 rpm at room temperature for 3-5 min.
    5. Transfer the PVDF membrane to the blocking buffer. Block it on a shaker at 20 rpm, at room temperature for 1 h.
  6. Primary antibody incubation
    1. Wash the PVDF membrane in an incubation box containing TBST, wash the membrane three times for 5 min each on a shaker at 40 rpm at room temperature.
    2. Incubate with primary antibody overnight (14-18 h) at 4 °C (1:40000, Vinculin Polyclonal antibody) in another antibody incubation box on a shaker at 20 rpm.
    3. Ensure that the antibody is diluted with 5% BSA blocking solution, prepared according to the ratio specified in section.
  7. Secondary antibody incubation
    1. Place the PVDF membrane in an incubation box containing 15 mL of TBST solution. Wash the membrane three times for 5 min each on a shaker at room temperature, agitating at 40 rpm.
    2. Prepare the secondary antibody by diluting Anti-rabbit IgG, HRP-linked Antibody at 1:2000 in 5% BSA blocking buffer.
    3. Incubate for 1 h at room temperature on a shaker at 20 rpm23.
    4. Wash the PVDF membrane three times with TBST solution for 5 min each, following the method described above.
  8. Detection
    1. Prepare the working substrate solution by mixing AccuWest ECL Plus, Raegent A and AccuWest ECL Plus, Raegent B at a 1:1 volume ratio directly in an incubation box. Perform all subsequent steps protected from light.
    2. Incubate the PVDF membrane in the solution for 5-10 s, then immediately detect immunoreactive bands using enhanced chemiluminescence (ECL) reagents-Vilber Fusion FX6 MINI. The typical exposure time ranges from 5 to 10 s.

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Results

We describe a reproducible protocol for establishing a simple obesity model that reliably exhibits fatty pancreas (Figure 1). The entire process, from the animal feeding and tissue collection to the subsequent molecular validation, is schematically summarized in Figure 1. The weekly weight changes of the model group (n = 5) and the control group (n = 5) were recorded, and there was no significant difference in the weight of the two groups before feeding with HFD...

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Discussion

As the primary driver of metabolic syndrome, uncomplicated obesity represents the initial manifestation of systemic lipid metabolism dysregulation. Without intervention, it may progress to T2DM and severe diabetic complications, underscoring the necessity of early metabolic correction30. Pancreatic steatosis (fatty pancreas), an early marker of ectopic lipid deposition in obesity, redefines the pancreas as a non-classical adipose organ, challenging the conventional adipo-hepatic axis paradigm. Thi...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This work was supported by the Project of Inheritors and Instructors for the Academic Experience Inheritance of the Seventh Batch of National Renowned Traditional Chinese Medicine Experts; The National Natural Science Foundation of China, Young Scientists Fund Project [grant number 82305376]; Project of Supporting Young Science and Technology Talents in Jiangsu Province in 2024 [grant number JSTJ-2024-380].

The flowcharts were produced using FigDraw, Biorender.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Material
1.5 mL micro-centrifuge tubebiosharp bioengineering co. ltdBS-15-MWB
2ml freezing tubeLABSELECTCV-002-200-EXWB
2ml grinding tubeWuhan Servicebio Technology Co., LtdHT-200-MWB
3% H2O2Booster Electronics Technology Co.,Ltd.20A23CIHC
4% paraformaldehydebiosharp bioengineering co. ltdBL539Aperfusion pancreas dissection operation
5% BSA solutionBiofroxx4240GR100IHC
50 mL centrifuge tube biosharp bioengineering co. ltdBS-500-Mtissue preservation
AccuWest ECL Plus, Raegent ANanjing Gene Easy Biotechnology Co., Ltd.Q201220WB
AccuWest ECL Plus, Raegent BNanjing Gene Easy Biotechnology Co., Ltd.Q201220WB
anhydrous ethanolShanghai SCR-Biotech Co., Ltd.10009257IHC
Anti-rabbit IgG, HRP-linked AntibodyCell Signaling Technology.Inc,7074WB
cOmplete Protease Inhibitor CocktailHoffmann-La Roche Ltd 82256300WB
coverslipCITOTEST LABWARE MANUFACTURING Co., LTD10212450CIHC
DAB chromogenic solutionBooster Electronics Technology Co.,Ltd.AR2017-1IHC
forcepsShenzhen RWD Life Science Co., Ltd.SP0007-Mdissection of SD rat
glass slides CITOTEST LABWARE MANUFACTURING Co., LTD188105IHC
glass staining jarShanghai Beyotime Biotechnology Co., Ltd.FG010IHC
HematoxylinShanghai Beyotime Biotechnology Co., Ltd.ST2067-20gIHC
high-fat dietSuzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.Table1feeding on experimental animals
hydrochloric acid alcoholShanghai Beyotime Biotechnology Co., Ltd.C0163MIHC
isofluraneShenzhen RWD Life Science Co., Ltd.R510-22-10anesthetized rats
methanol Biofroxx67-56-1WB
MOPS Running Buffer liquid 20×Nanjing Genscript Biotechnology Co., Ltd. C36842504WB
neutral balsamBeijing Solarbio Science & Technology Co., Ltd.G8590IHC
Neutral resinShanghai SCR-Biotech Co., Ltd.G8590IHC
PBS solutionJiangsu Keygen Biotech Corp., LtdKGL2206-500tissue preservation
Pierce BCA Protein Assay Kit Thermo Fisher Scientific Inc, Waltham, USA.ZB382867
Pierce BCA Protein Assay Kit hermo Fisher Scientific Inc, Waltham, USAZB382867WB
Protease Inhibitor CocktailHoffmann-La Roche Ltd 82256300
PVDF membrane (Immobilon-P Transfer Membrane, Millipore, 0000380346) Merck Millipore0000380346WB
RIPA lysis bufferbiosharp bioengineering co. ltdBL504A, 70096436WB
SABCBooster Electronics Technology Co.,Ltd.20C03AIHC
salineSHANXI SHENGAO ANIMAL PHARMACEUTICAL CO.,LTD. 270071460tissue preservation
SD male RatsJiangsu GemPharmatech Co., Ltd. D000017rats
sodium citrateMerck Life ScienceS1804IHC
standard maintenance feedSuzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.Table2feeding on experimental animals
the balance concentrate eBlot L2 5×Nanjing Genscript Biotechnology Co., Ltd. C97212410 WB
the transfer concentrate eBlot L2 5×Nanjing Genscript Biotechnology Co., Ltd. C97211504WB
Tris Buffered Saline, Powder biosharp bioengineering co. ltdBL602AWB
Tween 20BiofroxxEZ681A3EA8WB
urethaneSigma-Aldrich Co., Ltd.U2500-100Ganesthetized rats
Vinculin Polyclonal antibodyProteintech Group, Inc,26520-1-APWB
xyleneNanjing Reagent CorporationC0430530223IHC
Young PAGE, Bis-Tirs, 10×8Nanjing Genscript Biotechnology Co., Ltd. C33152505WB
Equipment
anesthesia machineShenzhen RWD Life Science Co., Ltd.Model R5805anesthetized rats
ball mill Retsch Technology GmbH - MicrotracMM400WB
CentrifugesEppendorf SE5418RWB
computerized biological tissue dryerZhejiang Jinhua Kedi Instrument Co.SC2013-G-KITIHC
enhanced chemiluminescence (ECL) reagents VILBER BIO IMAGING00170961WB
microtome Thermo Fisher Scientific Inc, Waltham, USA.HM325IHC
microwaveShanghai Boxun Industry Co., Ltd. 97000IHC
Mini-PROTEAN Tetra SystemBio-Rad Laboratories, Inc.1658004WB
ovenShanghai Jinghong Scientific Co., Ltd.DNP-9082IHC
pure waterSichuan UPT Ultra-Pure Technology Co., Ltd.ULUP-TEZIHC

References

  1. Wang, G., et al. Comparative effectiveness of massage combined with lifestyle intervention and lifestyle intervention alone for simple obesity: a systematic review and meta-analysis. Medicine. 104 (2), e41074(2025).
  2. World Obesity Federation. World Obesity Atlas 2024. , World Obesity Federation. London. (2024).
  3. Kromrey, M. L., et al. Pancreatic steatosis is associated with impaired exocrine pancreatic function. Invest Radiol. 54 (7), 403(2019).
  4. Miyake, H., et al. Association of fatty pancreas with pancreatic endocrine and exocrine function. PLoS One. 13 (12), e0209448(2018).
  5. López-Bermudo, L., et al. Contribution of liver and pancreatic islet crosstalk to β-cell function/dysfunction in the presence of fatty liver. Front Endocrinol (Lausanne). 13, 892672(2022).
  6. Vilas-Boas, E. A., Almeida, D. C., Roma, L. P., Ortis, F., Carpinelli, A. R. Lipotoxicity and β-cell failure in type 2 diabetes: oxidative stress linked to NADPH oxidase and ER stress. Cells. 10 (12), 3328(2021).
  7. Martínez, M. S., et al. The role of the α cell in the pathogenesis of diabetes: a world beyond the mirror. Int J Mol Sci. 22 (17), 9504(2021).
  8. Catanzaro, R., Cuffari, B., Italia, A., Marotta, F. Exploring the metabolic syndrome: nonalcoholic fatty pancreas disease. World J Gastroenterol. 22 (34), 7660-7675 (2016).
  9. Pinnick, K. E., et al. Pancreatic ectopic fat is characterized by adipocyte infiltration and altered lipid composition. Obesity. 16 (3), 522-530 (2008).
  10. Wang, Y. D., et al. New insight of obesity-associated NAFLD: dysregulated "crosstalk" between multi-organ and the liver. Genes Dis. 10 (3), 799-812 (2023).
  11. Savage, D. B., Petersen, K. F., Shulman, G. I. Disordered lipid metabolism and the pathogenesis of insulin resistance. Physiol Rev. 87 (2), 507-520 (2007).
  12. Shah, N., Rocha, J. P., Bhutiani, N., Omer, E. Nonalcoholic fatty pancreas disease. Nutr Clin Pract. 34 (S1), S49-S56 (2019).
  13. Chang, M. L. Fatty pancreas-centered metabolic basis of pancreatic adenocarcinoma: from obesity, diabetes and pancreatitis to oncogenesis. Biomedicines. 10 (3), 692(2022).
  14. Ou, H. Y., Wang, C. Y., Yang, Y. C., Chen, M. F., Chang, C. J. The association between nonalcoholic fatty pancreas disease and diabetes. PLoS One. 8 (5), e62561(2013).
  15. Yu, T. Y., Wang, C. Y. Impact of non-alcoholic fatty pancreas disease on glucose metabolism. J Diabetes Investig. 8 (6), 735-747 (2017).
  16. Petrov, M. S. Post-pancreatitis diabetes mellitus and excess intra-pancreatic fat deposition as harbingers of pancreatic cancer. World J Gastroenterol. 27 (17), 1936-1942 (2021).
  17. Mihoc, T., et al. Pancreatic morphology, immunology, and the pathogenesis of acute pancreatitis. Biomedicines. 12 (11), 2627(2024).
  18. Robinson-Junker, A. L., O'Hara, B. F., Gaskill, B. N. Out like a light? the effects of a diurnal husbandry schedule on mouse sleep and behavior. J Am Assoc Lab Anim Sci. 57 (2), 124-133 (2018).
  19. Virgilio, T., et al. Impact of prolonged isoflurane or ketamine-xylazine anesthesia with or without buprenorphine and oxygen on mouse vitals and immune responses. Lab Anim. 54, 1-8 (2025).
  20. Oh, S., Ahn, B., Lee, B. J., Nam, S. Y. Effect of humidity on postmortem changes in rats. J Vet Sci. 25 (3), e24(2024).
  21. Wu, C. T., et al. SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment. Cell Metab. 33 (8), 1565-1576.e5 (2021).
  22. Tremblay, C., Moullé, V., Zarrouki, B., Ghislain, J., Poitout, V. Immunofluorescent staining of pancreatic sections. protocols.io. , (2022).
  23. Liu, Y., et al. Electroacupuncture at ST25 mediated glial cells pruning of pancreatic TRPV1 neural synapse responds to neuropathy-associated beta cell dysfunction. Chin Med. 20 (1), 65(2025).
  24. Lu, M., et al. Electroacupuncture stimulation regulates adipose lipolysis via catecholamine signaling mediated by NLRP3 suppression in obese rats. Front Endocrinol. 12, 773127(2022).
  25. Lu, M., et al. Remodelling the inguinal adipose sensory system to switch on the furnace: electroacupuncture stimulation induces brown adipose thermogenesis. Diabetes Obes Metab. 26 (4), 1430-1442 (2024).
  26. Otsuka, N., Shimizu, K., Taniai, M., Tokushige, K. Risk factors for fatty pancreas and effects of fatty infiltration on pancreatic cancer. Front Physiol. 14, 1243983(2023).
  27. Zhang, X., Cui, Y., Fang, L., Li, F. Chronic high-fat diets induce oxide injuries and fibrogenesis of pancreatic cells in rats. Pancreas. 37 (3), e31(2008).
  28. Liu, Y., et al. Electroacupuncture at ST25 mediated glial cells pruning of pancreatic TRPV1 neural synapse responds to neuropathy-associated beta cell dysfunction. Chin Med. 20 (1), 65(2025).
  29. Liu, Y., et al. Neurophysiological mechanisms of electroacupuncture in regulating pancreatic function and adipose tissue expansion. World J Diabetes. 16 (5), 101354(2025).
  30. Jirawat, N., Srisuk, A. Obesity, metabolic syndrome, and cardiovascular disease: pathophysiological connections. Sci Acad J. 7 (1), 1-12 (2024).
  31. Fan, Y. H., et al. Inter-organ metabolic interaction networks in non-alcoholic fatty liver disease. Front Endocrinol. 15, 1494560(2025).
  32. Wang, Y. D., et al. New insight of obesity-associated NAFLD: dysregulated "crosstalk" between multi-organ and the liver. Genes Dis. 10 (3), 799-812 (2023).
  33. Zhang, X., Ji, X., Wang, Q., Li, J. Z. New insight into inter-organ crosstalk contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Protein Cell. 9 (2), 164-177 (2018).
  34. Zhang, C. L., Wang, J. J., Li, J. N., Yang, Y. Nonalcoholic fatty pancreas disease: an emerging clinical challenge. World J Clin Cases. 9 (23), 6624-6638 (2021).
  35. Corte, C. D., et al. Nonalcoholic fatty pancreas disease and nonalcoholic fatty liver disease: more than ectopic fat. Clin Endocrinol. 83 (5), 656-662 (2015).
  36. Saloman, J. L., et al. Animal models: challenges and opportunities to determine optimal experimental models of pancreatitis and pancreatic cancer. Pancreas. 48 (6), 759(2019).
  37. Hariri, N., Thibault, L. High-fat diet-induced obesity in animal models. Nutr Res Rev. 23 (2), 270-299 (2010).
  38. Lee, H. -Y., Min, K. -J. Dietary restriction and lipid metabolism: unveiling pathways to extended healthspan. Nutrients. 16 (24), 4424(2024).
  39. Rosini, T. C., Ramos da Silva, A. S., de Moraes, C. Diet-induced obesity: rodent model for the study of obesity-related disorders. Rev Assoc Med Bras (Engl Ed). 58 (3), 383-387 (2012).
  40. Uc, A., et al. Chronic pancreatitis in the 21st century - research challenges and opportunities: summary of a national institute of diabetes and digestive and kidney diseases workshop. Pancreas. 45 (10), 1365(2016).
  41. Fisher, W. E., et al. Standard operating procedures for biospecimen collection, processing, and storage: from the consortium for the study of chronic pancreatitis, diabetes, and pancreatic cancer. Pancreas. 47 (10), 1213(2018).
  42. Alexander, J., Chang, G. Q., Dourmashkin, J. T., Leibowitz, S. F. Distinct phenotypes of obesity-prone AKR/J, DBA2J and C57BL/6J mice compared to control strains. Int J Obes (Lond). 30 (1), 50-59 (2006).
  43. Nishikawa, S., Yasoshima, A., Doi, K., Nakayama, H., Uetsuka, K. Involvement of sex, strain and age factors in high-fat diet-induced obesity in C57BL/6J and BALB/cA mice. Exp Anim. 56 (4), 263-272 (2007).
  44. Levin, B. E., Dunn-Meynell, A. A., Balkan, B., Keesey, R. E. Selective breeding for diet-induced obesity and resistance in Sprague-Dawley rats. Am J Physiol. 273 (2 Pt 2), R725-R730 (1997).
  45. Kautzky-Willer, A., Harreiter, J., Pacini, G. Sex and gender differences in risk, pathophysiology and complications of type 2 diabetes mellitus. Endocr Rev. 37 (3), 278-316 (2016).
  46. Lee, M. J., Wu, Y., Fried, S. K. Adipose tissue heterogeneity: implication of depot differences in adipose tissue for obesity complications. Mol Aspects Med. 34 (1), 1-11 (2013).
  47. Geer, E. B., Shen, W. Gender differences in insulin resistance, body composition, and energy balance. Gender Med. 6 Suppl 1 (Suppl 1), 60-75 (2009).
  48. Nadal-Casellas, A., Proenza, A. M., Lladó, I., Gianotti, M. Sex-dependent differences in rat hepatic lipid accumulation and insulin sensitivity in response to diet-induced obesity. Biochem Cell Biol. 90 (2), 164-172 (2012).
  49. Stubbins, R. E., Holcomb, V. B., Hong, J., Núñez, N. P. Estrogen modulates abdominal adiposity and protects female mice from obesity and impaired glucose tolerance. Eur J Nutr. 51 (7), 861-870 (2012).
  50. Liu, Y., et al. Neuromechanical dimorphism of hypoglycemic effect of electroacupuncture. Neuroendocrinology. 113 (7), 679-691 (2023).
  51. Xu, T. C., Liu, Y., Yu, Z., Xu, B. Gut-targeted therapies for type 2 diabetes mellitus: a review. World J Clin Cases. 12 (1), 1-8 (2024).
  52. Xu, T., et al. Hypoglycemic effect of electroacupuncture at ST25 through neural regulation of the pancreatic intrinsic nervous system. Mol Neurobiol. 59 (1), 703-716 (2022).
  53. Feng, X., et al. Ferroptosis enhanced diabetic renal tubular injury via HIF-1α/HO-1 pathway in db/db mice. Front Endocrinol (Lausanne). 12, 626390(2021).
  54. Fang, X., et al. Beinaglutide shows significantly beneficial effects in diabetes/obesity-induced nonalcoholic steatohepatitis in ob/ob mouse model. Life Sci. 270, 118966(2021).
  55. Mao, T., et al. Semaglutide alters gut microbiota and improves NAFLD in db/db mice. Biochem Biophys Res Commun. 710, 149882(2024).
  56. Zeng, Z., et al. Bacillus amyloliquefaciens SC06 alleviates the obesity of ob/ob mice and improves their intestinal microbiota and bile acid metabolism. Food Funct. 13 (9), 5381-5395 (2022).
  57. Suriano, F., et al. Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: two sides of the same coin. Microbiome. 9 (1), 147(2021).
  58. Owada, Y., et al. Novel non-alcoholic steatohepatitis model with histopathological and insulin-resistant features. Pathol Int. 68 (1), 12-22 (2018).
  59. Flessa, C. M., et al. Genetic and diet-induced animal models for non-alcoholic fatty liver disease (NAFLD) research. Int J Mol Sci. 23 (24), 15791(2022).
  60. Liu, Y., et al. Electroacupuncture at ST25 mediated glial cells pruning of pancreatic TRPV1 neural synapse responds to neuropathy-associated beta cell dysfunction. Chin Med. 20 (1), 65(2025).
  61. Xu, T., et al. Hypoglycemic effect of electroacupuncture at ST25 through neural regulation of the pancreatic intrinsic nervous system. Mol Neurobiol. 59 (1), 703-716 (2022).
  62. Vimaleswaran, K. S., et al. Causal relationship between obesity and vitamin D status: bi-directional Mendelian randomization analysis of multiple cohorts. PLoS Med. 10 (2), e1001383(2013).
  63. Alvheim, A. R., et al. Dietary linoleic acid elevates endogenous 2-AG and anandamide and induces obesity. Obesity (Silver Spring). 20 (10), 1984-1994 (2012).
  64. Hintze, K. J., Benninghoff, A. D., Cho, C. E., Ward, R. E. Modeling the Western diet for preclinical investigations. Adv Nutr. 9 (3), 263-271 (2018).
  65. D, L. Obesity due to a high-fat diet in the rat and mouse. Nutr Dieta Eur Rev Nutr Diet. 9 (1), 27-42 (1967).
  66. Lemonnier, D., Tremolieres, J. Experimental obesity induced by high-fat diets. Probl Actuels Endocrinol Nutr. 7, 77-85 (1963).
  67. Samuel, V. T., et al. Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J Biol Chem. 279 (31), 32345-32353 (2004).
  68. Hu, S., et al. Dietary fat, but not protein or carbohydrate, regulates energy intake and causes adiposity in mice. Cell Metab. 28 (3), 415-431.e4 (2018).
  69. Goh, S. K., et al. Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering. Biomaterials. 34 (28), 6760-6772 (2013).
  70. Levin, B. E. Factors promoting and ameliorating the development of obesity. Physiol Behav. 86 (5), 633-639 (2005).
  71. Yuan, C. X., et al. Electroacupuncture alleviates diabetic peripheral neuropathy through modulating mitochondrial biogenesis and suppressing oxidative stress. World J Diabetes. 16 (2), 93130(2025).
  72. Liu, Y., et al. Neuromechanical dimorphism of hypoglycemic effect of electroacupuncture. Neuroendocrinology. 113 (7), 679-691 (2023).

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Pancreatic SteatosisHigh-Fat DietDiet-Induced ObesityLipid DepositionTissue HarvestingWestern BlottingParaformaldehyde FixationMetabolic Dysfunction
Development and Validation of a Methodology for Establishing Obese Rat Models with Typical Fatty Pancreas
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