AIMS/HYPOTHESIS: In type 1 diabetes, the counterregulatory glucagon response to low plasma glucose is impaired. The resulting increased risk of hypoglycaemia necessitates novel strategies to ameliorate alpha cell impairment. Here, we aimed to establish an in vitro type 1 diabetes-like model of alpha cell impairment using standardised reaggregated human islet microtissues (MTs) exposed to proinflammatory cytokines. Additionally, we investigated the therapeutic potential of incretin receptor agonists in improving alpha cell responses to low glucose. METHODS: Human islet MTs were exposed to proinflammatory cytokines (IL-1β, IFN-γ and TNF-α) for 1 day (short-term) and 6 days (long-term). Alpha cell function was assessed by sequential glucose-dependent secretion assays at 2.8 and 16.7 mmol/l glucose, followed by glucagon measurements. Additional evaluations included ATP content, caspase-3/7 activity, chemokine secretion and content of islet transcription factors (aristaless-related homeobox [ARX] and NK6 homeobox 1 [NKX6.1]) and hormones. The effects of incretin receptor agonist treatment (glucose-dependent insulinotropic polypeptide [GIP] analogue [D-Ala]-GIP with or without the glucagon-like peptide 1 [GLP-1] receptor agonist liraglutide) alongside or after cytokine exposure were also investigated, focusing on low-glucose-dependent glucagon secretion. RESULTS: Short-term cytokine exposure increased glucagon secretion at both 2.8 and 16.7 mmol/l glucose in islet MTs. In contrast, long-term cytokine exposure caused dose-dependent suppression of glucagon secretion at 2.8 mmol/l glucose, resembling a type 1 diabetes phenotype. Long-term cytokine exposure also diminished insulin and somatostatin secretion, reduced ATP content, increased caspase 3/7 activity and decreased islet content of ARX, NKX6.1, glucagon and insulin. Despite cytokine-induced impairment, alpha cells partially retained secretory capacity to L-arginine stimulation. Treatment with incretin receptor agonists during long-term cytokine exposure did not prevent alpha cell impairment. However, acute treatment with [D-Ala]-GIP with or without liraglutide, or with the single-molecule dual agonist tirzepatide, after cytokine exposure partially restored glucagon secretion at low glucose. CONCLUSIONS/INTERPRETATION: Long-term cytokine exposure of human islet MTs created a type 1 diabetes-like phenotype with impaired low-glucose-induced glucagon secretion. This cytokine-induced alpha cell impairment was partially restored by [D-Ala]-GIP with or without liraglutide, or by tirzepatide.