Abstract 2

INTRODUCTION

  • Type 1 Diabetes Mellitus (T1D)

    • Result of autoimmune-mediated destruction of insulin-producing beta cells in the pancreas.

    • No cure exists; reliance on experimental model systems for therapeutic identification.

  • Main Experimental Models for T1D:

    • Nonobese Diabetic Mouse (NOD)

    • Beta Cell Destruction Models:

    • Utilization of chemical agents such as Alloxan and Streptozotocin (STZ).

  • Streptozotocin (STZ):

    • An alkylating agent selectively cytotoxic to beta cells.

    • Induces DNA damage, oxidative stress, and apoptosis.

    • Results in pancreatic islet inflammation (insulitis) and insulin deficiency.

    • Mimics features of human T1D.

  • Mechanisms of STZ-Induced Beta Cell Mass Loss:

    • Complete mechanisms remain incompletely understood.

    • Single high dose (≥150 mg/kg) of STZ:

    • Causes near-complete necrotic ablation of beta cells within 24 hours.

    • Results in hyperglycemia within 48 hours.

    • Multiple administrations of lower doses (e.g., 35 mg/kg, 55 mg/kg):

    • Induces gradual loss of beta cell mass and delayed hyperglycemia; represents a more human disease progression.

    • Preferred model to study immune infiltration and beta cell dysfunction.

    • Variability in the chosen low dose, with commonly used ranges including 30 mg/kg to 55 mg/kg.

  • Study Objective:

    • Compare impacts of 55 mg/kg and 35 mg/kg multiple low-dose STZ on metabolic, morphological, functional, and transcriptomic progression of beta cell dysfunction in rodents as models for T1D.

RESULTS

  • Differences in Disease Progression:

    • 55 mg/kg-treated animals become glucose intolerant within 3 days post-STZ.

    • 35 mg/kg-treated animals maintain glucose tolerance for several days more despite similar beta cell mass loss.

  • Insulin Secretion:

    • Confirmed via hyperglycemic clamps (gold standard for in vivo insulin secretion quantification).

    • 35 mg/kg-treated mice partially retain glucose-stimulated insulin secretion longer than 55 mg/kg-treated mice.

  • Gene Expression Analysis:

    • Significant differences in gene expression between treatments, including dose-dependent shifts in gene alterations and pathways.

METHODS

Animal Studies

  • Animal Welfare Compliance:

    • All procedures adhered to City of Hope Institutional Animal Care and Use Committee protocols.

  • Mice Used:

    • Male C57BL/6J mice; STZ shows varying effectiveness in females.

    • Mice housed in ventilated cages with light/dark cycles; maintained on standard rodent diet.

  • Administration of STZ:

    • STZ administered intraperitoneally over 5 consecutive days at doses of 35 mg/kg or 55 mg/kg.

    • Saline administered to control groups (34 mice as vehicle control).

Glucose Tolerance Tests

  • Conducted on 10-12 week old male mice after a 5-hour fast with 1.5 g/kg glucose delivered intraperitoneally.

  • Blood glucose measured using AlphaTRAK 2 glucometer.

Beta Cell Mass Quantification

  • Organ Handling:

    • Mice euthanized post CO2 asphyxiation and pancreata fixed in formalin.


  • Histological Processing:

    • Pancreas processed into blocks for sectioning.

    • Immunohistochemistry (IHC) performed to label insulin.

Hyperglycemic Clamps

  • Surgical catheters implanted into carotid artery and jugular vein under anesthesia.

  • Gold standard methodology to assess in vivo beta cell function.

  • Blood glucose monitored; insulin levels measured during clamp procedure.

Islet Isolation and RNA Sequencing

  • Islet Isolation:

    • Freshly prepared collagenase and protease solution utilized for islet isolation.

    • Cultured islets overnight in ATP-supplemented media.

  • RNA Sequencing:

    • Isolated RNA from 75-100 healthy islets; library preparation included enrichment for mRNA.

Data Analysis

  • Preprocessing of RNA-seq reads and differential gene expression analysis using DESeq2 and HTSeq.

  • Adjusted P values < 0.1 deemed significantly differentially expressed.

  • Gene set enrichment analysis using relevant R packages.

DISCUSSION

  • Importance of STZ-Induced Diabetes Models:

    • Crucial for examining functional beta cell mass loss and evaluating intervention strategies.

    • Demonstrated significant differences in disease progression depending on STZ dosage.

    • 35 mg/kg STZ animals showed sustained beta cell function longer despite equivalent beta cell mass loss compared to 55 mg/kg STZ-treated animals, which had a faster decline in insulin secretion.

  • Implications for Future Research:

    • Need for standardized dosing of STZ for reproducibility in diabetes research.

    • Further investigation into the mechanistic differences influencing both first- and second-phase insulin secretion and their clinical relevance is essential.

  • Limitations:

    • Focus on male C57BL/6J mice, recognizing strain and sex differences in STZ response.

    • Findings as potential basis for future studies elucidating mechanisms behind STZ-induced diabetes.