Type 2 Diabetes Cell Models for Research

Type 2 diabetes (T2D) is a major global health challenge. According to the World Health Organization (WHO), the number of people with diabetes rose from 108 million in 1980 to 422 million in 2014, and in 2019, diabetes was the ninth leading cause of death, with an estimated 1.5 million deaths directly caused by the disease. T2D accounts for the vast majority of diabetes cases, characterized by insulin resistance and relative insulin deficiency. Key risk factors include obesity, physical inactivity, unhealthy diet, and genetic predisposition. The chronic hyperglycemia of T2D leads to long-term complications such as cardiovascular disease, kidney failure, neuropathy, and retinopathy, imposing a significant burden on individuals and healthcare systems. The prevalence of T2D continues to rise, emphasizing the urgent need for better therapeutic strategies and mechanistic understanding.

T2D is an ideal disease for mechanistic studies due to its complex interplay of genetic, environmental, and lifestyle factors. The availability of extensive public datasets, such as genome-wide association studies (GWAS) and transcriptomic profiles from pancreatic islets, provides a rich resource for identifying novel drug targets. However, many open questions remain, including the precise molecular mechanisms underlying beta-cell dysfunction, the contribution of specific genetic variants, and the development of drug resistance. Gene-edited cell models, particularly CRISPR-engineered isogenic lines, offer a powerful approach to dissect these mechanisms in a controlled environment, enabling functional validation of candidate genes and pathways.

While T2D is not a cancer, it involves several signaling pathways that are also implicated in cancer. Key pathways include:

• • Insulin signaling pathway: Insulin receptor (INSR) activation leads to PI3K/AKT and MAPK cascades, regulating glucose uptake and cell growth. Dysregulation contributes to insulin resistance.
• • AMPK pathway: AMP-activated protein kinase (AMPK) acts as a cellular energy sensor, promoting glucose uptake and fatty acid oxidation. Its activity is often reduced in T2D.
• • Inflammatory pathways: Chronic low-grade inflammation, involving NF-κB and JNK, impairs insulin signaling and promotes beta-cell dysfunction.
• • Wnt signaling: Wnt/β-catenin pathway plays a role in beta-cell proliferation and function; variants in TCF7L2, a key effector, are strongly associated with T2D risk.

Data derived from GWAS and ClinVar.

Key signaling networks in T2D include:

• • PI3K/AKT pathway: Insulin receptor activation recruits IRS-1, leading to PI3K activation and PIP3 production, which activates AKT. AKT promotes GLUT4 translocation and glycogen synthesis. In insulin resistance, this pathway is impaired.
• • MAPK/ERK pathway: Insulin also activates Ras-MAPK cascade, influencing gene expression and cell growth. Chronic hyperinsulinemia can overactivate this pathway, contributing to complications.
• • JNK pathway: Stress-activated JNK phosphorylates IRS-1 at serine residues, inhibiting insulin signaling. This is a key link between inflammation and insulin resistance.
• • Wnt/β-catenin pathway: TCF7L2, a transcription factor, interacts with β-catenin to regulate proglucagon and other genes. Variants in TCF7L2 impair beta-cell function.

Organoids derived from human pluripotent stem cells or adult pancreatic progenitors offer a more physiologically relevant model, recapitulating beta-cell function and allowing for long-term culture and genetic manipulation.

• • Genetic models: Ob/ob mice (leptin deficiency), db/db mice (leptin receptor mutation), and Zucker diabetic fatty rats are commonly used for T2D research.
• • Chemically induced models: Streptozotocin (STZ) injection selectively destroys beta-cells, inducing type 1-like diabetes, but can be used in combination with high-fat diet to model T2D.
• • High-fat diet (HFD) models: Feeding rodents a high-fat diet induces obesity, insulin resistance, and hyperglycemia, mimicking human T2D.
• • Genetically engineered mouse models (GEMM): Knockout or knock-in of specific genes (e.g., TCF7L2, IRS-1) allows for mechanistic studies.
• • Patient-derived xenografts (PDX): Not commonly used for T2D, but human islet transplantation into immunodeficient mice can be used to study human beta-cell function.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise genetic modifications, such as knockouts (KO) of specific genes or knock-in (KI) of disease-associated point mutations. These models are invaluable for studying the functional consequences of genetic variants in a controlled background. For example, a TCF7L2 knockout in INS-1 cells can reveal its role in insulin secretion, while a knock-in of the risk variant rs7903146 can assess its impact on gene expression. Commercially available, sequence-verified gene-edited cell lines accelerate research by providing ready-to-use models, eliminating the time and effort required for CRISPR engineering. These models are essential for target validation, drug screening, and functional genomics.

Gene-edited cells enable functional validation of genes identified in GWAS. For example, knocking out TCF7L2 in beta-cell lines can confirm its role in glucose-stimulated insulin secretion. Similarly, knock-in of a risk variant in SLC30A8 can assess its effect on zinc transport and insulin processing. These models allow researchers to study gene function in a specific cell type, providing mechanistic insights into disease pathogenesis.

Isogenic cell line pairs (wild-type vs. knockout) are powerful tools for drug screening. By comparing the response to a drug in the presence and absence of a specific gene, researchers can identify on-target effects and potential resistance mechanisms. For example, a PPARG knockout cell line can be used to test the efficacy of PPARγ agonists, which are used to improve insulin sensitivity. Additionally, gene-edited cells can be used to model drug resistance by introducing mutations that confer resistance to existing therapies, enabling the development of next-generation drugs.

CRISPR-based synthetic lethality screens can identify genes that are essential for cell survival when another gene is mutated. In T2D, such screens can reveal novel targets for beta-cell protection or regeneration. For example, knocking out a gene that is synthetically lethal with a T2D risk variant could identify a potential therapeutic target. Gene-edited cells also facilitate the discovery of biomarkers by enabling the study of gene expression changes associated with specific mutations, which can be used for patient stratification.