
==== Front
STAR Protoc
STAR Protoc
STAR Protocols
2666-1667
Elsevier

S2666-1667(24)00441-6
10.1016/j.xpro.2024.103276
103276
Protocol
Protocol for producing a rat model of non-obese prediabetes using a mild hypercaloric diet approach
Dwaib Haneen S. haneen.dwaib@paluniv.edu.ps
14∗
El-Yazbi Ahemd F. ayazbi@aiu.edu.eg
235∗∗
1 Department of Clinical Nutrition and Dietetics, Faculty of Allied Medical Sciences, Palestine Ahliya University, Bethlehem 1041, Palestine
2 Research & Innovation Hub, Alamein International University, Alamein 51718, Egypt
3 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
∗ Corresponding author haneen.dwaib@paluniv.edu.ps
∗∗ Corresponding author ayazbi@aiu.edu.eg
4 Technical contact

5 Lead contact

21 8 2024
20 9 2024
21 8 2024
5 3 103276© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Metabolic disease complications pose a significant health risk due to their early development, making their diagnosis and radical therapy a considerable challenge. Here, we present a protocol for producing a rat model of non-obese prediabetes characterized by hyperinsulinemia, normoglycemia, and normal body weight. We describe steps for inducing the model in Sprague-Dawley (SD) male and ovariectomized female rats by free feeding on a mild hypercaloric diet. This protocol offers a potential model of metabolically unhealthy lean individuals.

For complete details on the use and execution of this protocol, please refer to Elkhatib et al.1 and Dwaib et al.2

Graphical abstract

Highlights

• Protocol using mild hypercaloric (MHC) diet to induce an early stage of prediabetes

• Instructions to perform ventral bilateral ovariectomy

• Details on panel for measuring cardiometabolic parameters in prediabetes

• Guide to studying hallmarks of adipose dysfunction and morphology

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Metabolic disease complications pose a significant health risk due to their early development, making their diagnosis and radical therapy a considerable challenge. Here, we present a protocol for producing a rat model of non-obese prediabetes characterized by hyperinsulinemia, normoglycemia, and normal body weight. We describe steps for inducing the model in Sprague-Dawley (SD) male and ovariectomized female rats by free feeding on a mild hypercaloric diet. This protocol offers a potential model of metabolically unhealthy lean individuals.

Subject areas

Health Sciences
Metabolism
Model Organisms
==== Body
pmcBefore you begin

1. Equipment, supplies and chemicals required for preparation of diet:a. A sufficient amount of standard chow for rodent growth AIN-933 or equivalent. As a general guideline for daily chow consumption, 5 g/100 g body weight should be budgeted, and then adjusted up or down accordingly.

b. A sufficient amount of food-grade fructose.

c. A sufficient amount of plant-based fat (hydrogenated vegetable oils). Typical commercial products offer 5% saturated fatty acids and 9 kcal/g.

d. Laboratory-grade sodium chloride.

e. Phosphate-free vitamin/mineral mix.

f. Two-liter flask, baking sheets, microwave oven, a hot air oven, and a refrigerator.

2. A sufficient number of Sprague-Dawley (SD) rats (4–5 weeks old) based on the sample size calculations of the study protocol. Researchers should make their best efforts to minimize the number of animals used for the study (replacement, reduction, and refining).

3. Supplies and chemicals for surgery and other procedures:a. Pharmaceutical-grade ketamine and veterinary-grade xylazine.

b. small animal veterinary set.

c. Alcohol and antiseptic solution.

d. Hair clipper.

e. Disposable syringes, needles, Eppendorf vials of different volumes as required.

4. Equipment, supplies and chemicals required for blood analysis:a. Rat restrainers.

b. Disposable syringes, needles, Eppendorf vials of different volumes as required.

c. Cooling Centrifuge.

d. −80°C freezer.

5. LF10 Minispec Nuclear Magnetic Resonance (NMR) machine (Bruker, MA, USA) for body composition analysis.

6. CODA tail-cuff High Throughput Monitor (Kent Scientific, Torrington, CT) for non-invasive blood pressure measurement.

7. SonixTouch Q+ ultrasound (BK ultrasound, Peabody, MA) and water-based lubricant gel for echocardiography.

8. Accu-Chek glucometer (Roche Diagnostics, Basel, Switzerland) or a similar device, together with its strips, for fasting and random blood glucose levels.

9. A1Chek meter (Biosense Technologies Private Limited, India) or a similar device together with its strips, for measuring glycosylated hemoglobin A1c blood levels.

Institutional permissions

Readers are required to obtain the necessary approvals from the relevant institutional committee before commencing their research protocol. In this protocol we obtained the necessary approvals from the relevant Institutional Animal Care and Use Committee (IACUC) at the American University of Beirut.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Rabbit monoclonal anti-uncoupling protein 1 (UCP1) (dilution 1/1,000 )	Cell Signaling Technology, Danvers, MA	14670S	
Rabbit polyclonal anti-interleukin-1β (IL-1β) (dilution 1/500 )	Abcam, Cambridge, UK	ab205924	
Rabbit monoclonal anti-GAPDH (dilution 1/1,000 )	Abcam, Cambridge, UK	ab181602	
Rabbit polyclonal anti-dynamin-related protein 1 (DRP1) (dilution 1/500)	Cell Signaling Technology, Danvers, MA	8570S	
Rabbit polyclonal anti-hypoxia inducible factor 1α (HIF1-α)(dilution 1/500)	Abcam, Cambridge, UK	ab228649	
	
Critical commercial assays	
	
Insulin ELISA kits	Thermo Fisher Scientific, Waltham, MA	#ERINS	
	
Other	
	
Normal chow	Teklad Rodent Diets, Madison, WI	ENVIGO++++	
LF10 minispec nuclear magnetic resonance machine	Bruker, MA, USA	N/A	
CODA high-throughput monitor	Kent Scientific, Torrington, CT	N/A	
SonixTouch Q+ ultrasound	BK Ultrasound, Peabody, MA	N/A	
Accu-Chek glucometer	Roche Diagnostics, Basel, Switzerland	N/A	
A1Chek meter	Biosense Technologies Private Limited, India	N/A	

Step-by-step method details

Mild-hyper caloric (MHC) diet preparation

Timing: 12–18 h

1. Prepare 1.102 mol/L fructose water.a. Place 400 g fructose in a 2 L flask.

b. Add tap water to a total volume of 2 L.

c. Stir until completely dissolved.

2. Soak 1300 g of standard chow overnight with the previously prepared fructose water.

3. Add 2.1 g of NaCl, and 70 g phosphate-free vitamin/mineral mix.

4. In the following day, add 300 g of melted plant-based fat (melt in the microwave for 1–2 min).

5. Mix well until fat is equally distributed in the soaked chow.

6. Spread in baking trays equally and dry in the oven for 4 h at 180°C.

7. After cooling down at room temperature, the mix should be shaped into equal small balls 15–25 g each.

8. Store in the refrigerator till the time of use in plastic or any convenient container that is well closed at temperature 4–6°C.

Note: When prepared in this manner, the MHC diet will offer 4.5 Kcal/g vs. 3.8 Kcal/g for the AIN-93-based standard chow as measured by bomb calorimetry.2

Animals and design

Timing: 12–24 weeks

9. Randomly divide 4–5 weeks SD male and female rats into different dietary groups.

10. Free feeding of either standard chow or MHC for 12 or 24 weeks (short and long protocols respectively), depending on the study.

11. Calculate daily and cumulative caloric intake of individually housed rats based on the measurement of the weight of chow consumed on a daily basis.

12. Do the required experiments on week 12 or 24, then euthanized the animals as per the approved protocol, Figure 1.Figure 1 Protocol timeline

This figure gives a visual presentation of the experiments required for the short and long protocol (12 and 24 weeks respectively) at each time point. Routine data collected include plasma samples, random and fasting blood glucose levels, body weight, body composition analysis, non-invasive blood pressure measurements (using the CODA machine), echocardiography and food intake. On the day of Sacrifice, in addition to all the previous measurements invasive hemodynamic recordings are measured using the vasoactive method.4

Bilateral ovariectomy

Timing: 1–2 h

Only rats following the long protocol (24 weeks) will be subjected to this procedure to comprehensively understand the sexual dimorphism in this animal model, Figure 2.13. Weigh female SD rats fed for 12 weeks on either NC or MHC.

14. Give anesthesia via the intraperitoneal route, a mix of ketamine and xylazine (1.5 mg/mL/kg of ketamine followed by 0.035 mg/mL/kg of xylazine).

15. While waiting for the animal to be fully sedated, prepare the setup for the surgery.a. Sanitize the bench with ethanol or antiseptic solution.

b. Prepare the surgical pad on the bench.

c. Set the animal on a supine position on the ventral side.

d. Shave ∼ 1 cm of dorsal hair in the sacral area.

e. Make a small incision (∼ 0.4 cm) with a sharp sterile scalpel, until you reach the pelvic cavity.

f. Open the incision with a small sterile retractor.

g. Using 2 sterile small forceps try to reach the left ovary, clip the ovary with one forceps, excise the ovary without the surrounding fat (periovarian pad), then ligate underneath the forceps using absorbable thread, to prevent any possible bleeding.

h. Repeat step (g) with the other ovary.

i. Remove the retractor.

j. Close the wound using sterile needle and silk sutures.

k. Move the animal to its cage underneath a heating bulb until fully awake.

16. Post-surgery, place rats into their perspective individual cages and following the same pre-op diet for another 12 weeks.

Figure 2 Prediabetes in ovariectomized female rats

After 12 weeks of either mild hyper caloric diet (MHC) or normal chow (NC) free feeding female rats are subjected to bilateral ovariectomy, then proceed with their respective pre-op diet. Only female rats on MHC developed the prediabetic metabolic phenotype.

Body composition analysis (BCA)

Timing: 10–15 min for each rat

To measure the impact of MHC diet on body compositions rather than crude weight.17. This measurement is collected every 4 weeks.

18. Weigh rats on same day of BCA.

19. Place rats in special restrainers to ensure minimal movement.

20. Insert rat in the restrainer into the LF10 Minispec Nuclear Magnetic Resonance (NMR) machine (Bruker, MA, USA) after logging the rat information.

21. The machine takes 5–10 min to finish the analysis.

22. Remove rat from the restrainer and into its cage.

Tail cuff blood pressure measurement

Timing: 25–45 min

To monitor the changes in gross hemodynamics in response to MHC feeding.23. Weigh rats are on the same day of blood pressure measurement and echocardiography.

24. Sedate rats using ketamine (1.5 mg/mL/kg).

25. Place rats in restrainer with their tails out.

26. Rest rats in restrainers on the heating pad.

27. Place the cuffs of the CODA tail-cuff High Throughput Monitor (Kent Scientific, Torrington, CT)on the tails according to the manufacturer’s instructions.

28. Wait 5–7 min until rats are stable and well adjusted, start the session that will measure blood pressure 15 times.

29. Export the data and shut down the system according to the manufacturer’s instructions.

30. Remove the rats from the restrainers and prepare them for the Echocardiography.

31. Please note that the timing stated is for 6–8 rats and it may differ based on the channels and restrainers available.

Echocardiography (echo)

Timing: 15–30 min for each rat

To assess the cardiac physiological and morphological changes posed by MHC diet.32. After the tail-cuff measurement and based on the rat weight, give xylazine (0.35 mg/mL/kg) to induce full anesthesia.

33. Shave the trunk area of rats with a clean razor or shaving machine, around the heart.

34. Position rats on the echo plane and tape the four limbs into it.

35. Apply gel to the shaved area.

36. Make echo recordings along the parasternal long axis M- and B-modes using SonixTouch Q+ ultrasound (BK ultrasound, Peabody, MA).

Blood serum analyses

Timing: 1–2 h depending on the sample size

To profile the changes of several parameters between groups such as cholesterol, fasting blood glucose.37. Withdraw 0.5–1 mL of blood from the tail vein of the animal on week 8 for short protocol and on week 12 for the long one.

38. Put animals in a restrainer with their tails out, heat animals’ tails using heating bulb.

39. After 10 min sedate each animal individually by isoflurane.

40. Disinfect the tail with 70% alcohol.

41. Withdraw blood with a 1 mL syringe and 28 gauge needle, insert the needle with a 45 degrees angle into the tail vein of the animal.

42. Transfer the collected blood to a 2 mL Eppendorf and then centrifuge for 5 min for 3600 rpm, 4°C.

43. Collect the Serum using a pipette to a new Eppendorf and store in −80°C.

44. On the day of sacrifice, week 12 or 24, collect 6–8 mL of blood from the catheter inserted into the carotid artery, after the invasive hemodynamic experiment.

45. Centrifuge the blood for 10 min for 3600 rpm, 4°C.

46. Transfer the serum into few 1.5 mL Eppendorf vials and store at −80°C.

Fasting and random blood glucose

Timing: 1–3 min for each rat

47. Measure random blood glucose levels (RBG) at baseline and every 4 weeks.

48. Put rats in restrainers.

49. Insert a strip in the Accu-Chek glucometer (Roche Diagnostics, Basel, Switzerland).

50. Hold the tail of the animal in one hand, and with the other puncture the lateral tail vein using 28-gauge needle.

51. Place One drop of blood on the strip and the record the reading.

52. Measure fasting blood glucose level at baseline, every 4 weeks, and on the day of sacrifice. Use the same steps (48–51), however, subject rats are to 12 h fasting prior the measurement.

Intraperitoneal glucose tolerance test

Timing: 2–3 h for each rat

53. One week before the sacrifice, subject rats to 12 h fasting.

54. Prepare a 20% glucose in saline solution (GS).

55. Weigh rats and measure fasting blood glucose (use the same steps as in the previous section).

56. Inject 1 mL GS/100 g of body weight in the peritoneal cavity (in the lower right quadrant of the abdomen).

57. Measure blood glucose level at different time points 15, 30, 60 and 120 min (following the steps in the previous section).

58. After the last time point, return rats to the respective cages and resume their preassigned treatment and diet protocol.

Hemoglobin A1C (HbA1C) measurement

Timing: 3–8 min for each rat

59. Measure HbA1C level on the day of sacrifice.

60. Put rats in restrainers.

61. Hold the tail of the animal in one hand, and with the other puncture the lateral tail vein using 28-gauge needle.

62. Place one drop of blood on the strip of the A1Chek meter ( Biosense technologies private limited, India) and follow the steps per the manufacturer’s protocol.

63. Record the results.

Serum insulin

Timing: 4–8 h for every 96-well plate kit

To test insulin resistance and identify prediabetic rats based on it.64. Measure Insulin level in the sera using ELISA kits (#ERINS) according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA).

Homeostatic model assessment of insulin resistance (HOMA-IR)

Timing: 10–15 min for all rats

65. Log the results of fasting blood glucose and fasting serum insulin from the day of sacrifice into the mathematical equation: fasting insulin (microU/L) × fasting glucose (nmol/L)/22.5.

Note: Make sure that the units fit the equation.

Hallmarks of adipose tissue dysfunction

Timing: Depends on the number of samples and gels to be run

66. Adipose tissue dysfunction is characterized by increase in the inflammatory marker interleukin 1- ß (IL1- ß), hypoxia marker hypoxia inducible factor-1α (HIF1-α), mitochondria fission dynamin related protein 1 (DRP1) and uncoupling protein 1 (UCP1).

67. If needed, changes in the above markers can be detected by western blotting carried out as described previously.1,2

68. Homogenize on ice samples of Perivascular adipose tissue (PVAT) of the thoracic aorta, epididymal, periovarian and infra-scapular adipose tissue , and separate the protein extracts by SDS-polyacrylamide gel electrophoresis.

69. Blot the proteins to nitrocellulose membranes and incubate in primary antibodies (1:500 for rabbit polyclonal anti-IL-1 ß, 1:1000 for rabbit monoclonal anti-GAPDH rabbit polyclonal anti-hypoxia inducible factor 1α (HIF1-α), Abcam, Cambridge, UK, and rabbit polyclonal anti-uncoupling protein 1 (UCP1)and anti-dynamin related protein 1 (DRP1), Cell signaling, Danvers, MA) overnight at 4°C.

70. Wash the membranes with 0.02% TBST (Tris-buffered saline with 0.1% Tween 20) and incubated for 1 h at room temperature in 1:40,000 biotinylated conjugated goat anti-rabbit Ig.

71. Then wash the membranes and incubate for 30 min at room temperature with 1:200,000 HRP-conjugated streptavidin (Abcam, Cambridge, UK).

72. After two washes with 0.02% TBST (5 min) and two washes with TBS (5 min), expose the blots to Clarity Western ECL substrate (Bio-Rad, Hercules, California) for 5 min and then detect by Chemidoc imaging system (BioRad, Hercules, CA).

73. Preform densitometric analysis of the protein bands using ImageJ software (Fiji).

Adipocyte size

Timing: Depends on the number of samples and slides to be done

74. Perform serial sectioning and staining of formalin fixed thoracic PVAT, epididymal, periovarian and infra-scapular adipose simultaneously for accurate comparison as previously described.2

75. Use hematoxylin and eosin (H&E) staining to compare adipocyte size in different adipose depots across treatments.

76. Take Images using OLYMPUS CX41 light microscope (Olympus, Shinjuku, Tokyo, Japan).

77. Assess adipocyte expansion, adipocyte size distribution, and the number of adipocytes per image area using the plugin Adiposoft on ImageJ (Fiji) as described previously.5

Expected outcomes

Over the course of 12–24 weeks, MHC-fed rats consume an additional 5000-10,000 kcal. This is not accompanied by an increase in total body weight, fasting and/or random blood glucose levels, HbA1c levels, glucose tolerance, and non-invasive blood pressure.1,2,4,6,7All MHC-fed rats show an increased fat/lean ration and a reduced metabolic efficiency (weight gained/Kcal consumed), however only male and ovariectomized female rats show increased serum insulin levels and insulin resistance (HOMA-IR values), compared to intact females. At least in male rats, MHC feeding leads to a consistent rise in blood lipid levels including LDL-cholesterol and triglycerides.1,7,8

The discordance among the blood glucose, HbA1c, and glucose tolerance values on the one hand and serum insulin levels on the other hand ascertains the early prediabetic phenotype, and potentially offers an animal model of the metabolically unhealthy lean state. Further metabolic stress leads to a gradual rise in serum glucose levels.1 The early metabolic impairment in male and ovariectomized female rats is associated with a localized inflammation of certain visceral adipose depots including perivascular and perirenal adipose depots,1,7 which involves adipocyte hypertrophy. Although other visceral adipose depots, including retroperitoneal and gonadal adipose, and brown infra-scapular adipose tissues also show varying degrees of adipocyte hypertrophy, only the former tissues manifest clear signs of inflammation owing to their increased susceptibility to hypoxia with both sufficient hypertrophy and upregulation of UCP-1.2

The local inflammatory milieu in these depots spills to the neighboring tissues in a paracrine manner leading to functional and structural changes in the heart,4 blood vessels,1,6 and kidneys.7 Nevertheless, remote functional and structural disorders were observed under these circumstances as well, including autonomic dysfunction,4 increased cerebrovascular tone, and cognitive impairment.8

Most structural and functional deficits observed were ameliorated upon pharmacological treatment with anti-inflammatory agents or non-pharmacological therapeutic interventions to correct the metabolic dysfunction. Inhibition of UCP-1 activity improved the observed phenotype.2

Quantification and statistical analysis

The metabolic and morphometric measures listed in this protocol as a requirement to follow up on the development of the prediabetic model typically yield normally distributed continuous variables. As such, the appropriate parametric statistical tests are used and chosen based on the experimental design.

Limitations

This protocol has been studied using SD rats, using other animal breeds may not yield the expected outcomes. Moreover, this model depends on MHC diet, hence, accurate preparation of this diet is crucial, consistently using the same ingredients and measurements. Since It is a chronic exposure model, both long and short protocol need time to develop. Finally, this model only represents prediabetes prognosis in male and ovariectomized female rats, so it is inapplicable to intact female rats as they were resilient to insulin resistant and metabolic damage.

Troubleshooting

Problem 1

The MHC diet should be prepared following the exact recipe. However, the drying step poses a major challenge, since the moisture content needs to be adjusted to reduce mold formation and to maintain the consistency of the calorific value. On the other hand, over drying could lead to charring or caramelization of the sugar content, producing a consistency that was not palatable for rats.

Potential solution

• The drying step should be optimized in several trials at different temperatures depending on the efficiency and power of the available oven.

• The resultant pellets should be checked for consistency and hardness.

• A storage trial under standard laboratory conditions is recommended to test for mold formation.

• It is paramount to regularly monitor daily caloric intake and chow composition and calorific value using bomb calorimetry during the optimization phase.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by Ahmed F. El-Yazbi, ayazbi@aiu.edu.eg.

Technical contact

Haneen S. Dwaib (haneen.dwaib@paluniv.edu.ps).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate code or analyze datasets.

Acknowledgments

This work was supported by grants from the 10.13039/100007688 AUB Medical Practice Plan (320148 ), the President Collaborative Research Stimulus, and the Science, Technology and Innovation Funding Authority grant number 45912 to A.F.E.-Y. Figures were generated using www.Biorender.com.

Author contributions

Both H.S.D. and A.F.E.-Y. contributed to the writing of the first draft and the review of the final form.

Declaration of interests

The authors declare no competing interests.
==== Refs
References

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