Name: Development and Validation of a Methodology for Establishing Obese Rat Models with Typical Fatty Pancreas (Video) | JoVE Journal
Uploaded: 2025-11-11T15:00:00.000+00:00
Duration: 3 min 7 s
Description: Learn a reproducible method to establish obese rat models with fatty pancreas for obesity research. This protocol includes pancreatic tissue sampling and immunohistochemistry techniques.

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5 minutes

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.

The authors have no conflicts of interest to disclose.

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...

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 &#945;/&#946;-cell ratios, promotes &#946;-cell dedifferentiation with subsequent loss of insulin secretory function5, and mediates direct lipotoxic damage to &#946;-cells6. Concurrently, &#945;-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 &#946;-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.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL micro-centrifuge tube</td><td>biosharp bioengineering co. ltd</td><td>BS-15-M</td><td>WB</td></tr><tr><td>2ml freezing tube</td><td>LABSELECT</td><td>CV-002-200-EX</td><td>WB</td></tr><tr><td>2ml grinding tube</td><td>Wuhan Servicebio Technology Co., Ltd</td><td>HT-200-M</td><td>WB</td></tr><tr><td>3% H2O2</td><td>Booster Electronics Technology Co.,Ltd.</td><td>20A23C</td><td>IHC</td></tr><tr><td>4% paraformaldehyde</td><td>biosharp bioengineering co. ltd</td><td>BL539A</td><td>perfusion pancreas dissection operation</td></tr><tr><td>5% BSA solution</td><td>Biofroxx</td><td>4240GR100</td><td>IHC</td></tr><tr><td>50 mL centrifuge tube&amp;nbsp;</td><td>biosharp bioengineering co. ltd</td><td>BS-500-M</td><td>tissue preservation</td></tr><tr><td>AccuWest ECL Plus, Raegent A</td><td>Nanjing Gene Easy Biotechnology Co., Ltd.</td><td>Q201220</td><td>WB</td></tr><tr><td>AccuWest ECL Plus, Raegent B</td><td>Nanjing Gene Easy Biotechnology Co., Ltd.</td><td>Q201220</td><td>WB</td></tr><tr><td>anhydrous ethanol</td><td>Shanghai SCR-Biotech Co., Ltd.</td><td>10009257</td><td>IHC</td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling Technology.Inc,</td><td>7074</td><td>WB</td></tr><tr><td>cOmplete Protease Inhibitor Cocktail</td><td>Hoffmann-La Roche Ltd&amp;nbsp;</td><td>82256300</td><td>WB</td></tr><tr><td>coverslip</td><td>CITOTEST LABWARE MANUFACTURING Co., LTD</td><td>10212450C</td><td>IHC</td></tr><tr><td>DAB chromogenic solution</td><td>Booster Electronics Technology Co.,Ltd.</td><td>AR2017-1</td><td>IHC</td></tr><tr><td>forceps</td><td>Shenzhen RWD Life Science Co., Ltd.</td><td>SP0007-M</td><td>dissection of SD rat</td></tr><tr><td>glass slides&amp;nbsp;</td><td>CITOTEST LABWARE MANUFACTURING Co., LTD</td><td>188105</td><td>IHC</td></tr><tr><td>glass staining jar</td><td>Shanghai Beyotime Biotechnology Co., Ltd.</td><td>FG010</td><td>IHC</td></tr><tr><td>Hematoxylin</td><td>Shanghai Beyotime Biotechnology Co., Ltd.</td><td>ST2067-20g</td><td>IHC</td></tr><tr><td>high-fat diet</td><td>Suzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.</td><td>Table1</td><td>feeding on experimental animals</td></tr><tr><td>hydrochloric acid alcohol</td><td>Shanghai Beyotime Biotechnology Co., Ltd.</td><td>C0163M</td><td>IHC</td></tr><tr><td>isoflurane</td><td>Shenzhen RWD Life Science Co., Ltd.</td><td>R510-22-10</td><td>anesthetized rats</td></tr><tr><td>methanol&amp;nbsp;</td><td>Biofroxx</td><td>67-56-1</td><td>WB</td></tr><tr><td>MOPS Running Buffer liquid 20&amp;times;</td><td>Nanjing Genscript Biotechnology Co., Ltd.&amp;nbsp;</td><td>C36842504</td><td>WB</td></tr><tr><td>neutral balsam</td><td>Beijing Solarbio Science &amp;amp; Technology Co., Ltd.</td><td>G8590</td><td>IHC</td></tr><tr><td>Neutral resin</td><td>Shanghai SCR-Biotech Co., Ltd.</td><td>G8590</td><td>IHC</td></tr><tr><td>PBS solution</td><td>Jiangsu Keygen Biotech Corp., Ltd</td><td>KGL2206-500</td><td>tissue preservation</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Fisher Scientific Inc, Waltham, USA.</td><td>ZB382867</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>hermo Fisher Scientific Inc, Waltham, USA</td><td>ZB382867</td><td>WB</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Hoffmann-La Roche Ltd&amp;nbsp;</td><td>82256300</td><td></td></tr><tr><td>PVDF membrane (Immobilon-P Transfer Membrane, Millipore, 0000380346)&amp;nbsp;</td><td>Merck Millipore</td><td>0000380346</td><td>WB</td></tr><tr><td>RIPA lysis buffer</td><td>biosharp bioengineering co. ltd</td><td>BL504A, 70096436</td><td>WB</td></tr><tr><td>SABC</td><td>Booster Electronics Technology Co.,Ltd.</td><td>20C03A</td><td>IHC</td></tr><tr><td>saline</td><td>SHANXI SHENGAO ANIMAL PHARMACEUTICAL CO.,LTD.&amp;nbsp;</td><td>270071460</td><td>tissue preservation</td></tr><tr><td>SD male Rats</td><td>Jiangsu GemPharmatech Co., Ltd.&amp;nbsp;</td><td>D000017</td><td>rats</td></tr><tr><td>sodium citrate</td><td>Merck Life Science</td><td>S1804</td><td>IHC</td></tr><tr><td>standard maintenance feed</td><td>Suzhou Shuangshi Laboratory Animal Feed Technology Co., Ltd.</td><td>Table2</td><td>feeding on experimental animals</td></tr><tr><td>the balance concentrate eBlot L2 5&amp;times;</td><td>Nanjing Genscript Biotechnology Co., Ltd.&amp;nbsp;</td><td>C97212410&amp;nbsp;</td><td>WB</td></tr><tr><td>the transfer concentrate eBlot L2 5&amp;times;</td><td>Nanjing Genscript Biotechnology Co., Ltd.&amp;nbsp;</td><td>C97211504</td><td>WB</td></tr><tr><td>Tris Buffered Saline, Powder&amp;nbsp;</td><td>biosharp bioengineering co. ltd</td><td>BL602A</td><td>WB</td></tr><tr><td>Tween 20</td><td>Biofroxx</td><td>EZ681A3EA8</td><td>WB</td></tr><tr><td>urethane</td><td>Sigma-Aldrich Co., Ltd.</td><td>U2500-100G</td><td>anesthetized rats</td></tr><tr><td>Vinculin Polyclonal antibody</td><td>Proteintech Group, Inc,</td><td>26520-1-AP</td><td>WB</td></tr><tr><td>xylene</td><td>Nanjing Reagent Corporation</td><td>C0430530223</td><td>IHC</td></tr><tr><td>Young PAGE, Bis-Tirs, 10&amp;times;8</td><td>Nanjing Genscript Biotechnology Co., Ltd.&amp;nbsp;</td><td>C33152505</td><td>WB</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anesthesia machine</td><td>Shenzhen RWD Life Science Co., Ltd.</td><td>Model R5805</td><td>anesthetized rats</td></tr><tr><td>ball mill&amp;nbsp;</td><td>Retsch Technology GmbH - Microtrac</td><td>MM400</td><td>WB</td></tr><tr><td>Centrifuges</td><td>Eppendorf SE</td><td>5418R</td><td>WB</td></tr><tr><td>computerized biological tissue dryer</td><td>Zhejiang Jinhua Kedi Instrument Co.</td><td>SC2013-G-KIT</td><td>IHC</td></tr><tr><td>enhanced chemiluminescence (ECL) reagents&amp;nbsp;</td><td>VILBER BIO IMAGING</td><td>00170961</td><td>WB</td></tr><tr><td>microtome&amp;nbsp;</td><td>Thermo Fisher Scientific Inc, Waltham, USA.</td><td>HM325</td><td>IHC</td></tr><tr><td>microwave</td><td>Shanghai Boxun Industry Co., Ltd.&amp;nbsp;</td><td>97000</td><td>IHC</td></tr><tr><td>Mini-PROTEAN Tetra System</td><td>Bio-Rad Laboratories, Inc.</td><td>1658004</td><td>WB</td></tr><tr><td>oven</td><td>Shanghai Jinghong Scientific Co., Ltd.</td><td>DNP-9082</td><td>IHC</td></tr><tr><td>pure water</td><td>Sichuan UPT Ultra-Pure Technology Co., Ltd.</td><td>ULUP-TEZ</td><td>IHC</td></tr></tbody></table>

development and validation of a methodology for establishing obese rat models with typical fatty pancreas

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

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&#38;E). This model offers a robust platform for mechanistic investigations into obesity-associated pancreatic metabolic dysfunction.

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...

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.

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 ...

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Learn a reproducible method to establish obese rat models with fatty pancreas for obesity research. This protocol includes pancreatic tissue sampling and immunohistochemistry techniques.

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

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