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Abstract
Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic β-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-βH1 cells, a human β-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-βH1 cells for large-scale siRNA screening to identify candidate genes regulating β-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional β-cell mass occurring in T2D.
Highlights
Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide [1]. It is a leading cause of death due to its co-morbidities such as kidney failure, heart attack, or stroke [2]. β-cell insufficiency, the failure of β-cells to meet increased demand for insulin caused by insulin resistance, leads to the development of overt T2D
To increase assay throughput for screening we developed an automated protocol for EndoC-βH1 culture and the glucose-stimulated insulin secretion (GSIS) assay in a 384-well format
Therein, EndoC-βH1 responded with 2–3-fold and 5–6-fold induction of secreted insulin when stimulated with 20 mM glucose and 20 mM glucose + IBMX, respectively (Figure 1A)
Summary
Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide [1]. It is a leading cause of death due to its co-morbidities such as kidney failure, heart attack, or stroke [2]. The regulation of glucose-stimulated insulin secretion (GSIS) is complex and the underlying molecular mechanisms are still not fully understood. Identifying novel regulators of insulin secretion could lead to the development of new therapeutic interventions for T2D. Most studies investigating cellular mechanisms regulating insulin secretion utilize non-human models such as mouse or rat β-cells [5,6,7]. Molecular and functional differences between rodents and human β-cells argue for the use of human-derived in vitro
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