The small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human β-cell line and loss of function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction.

No abstract

Insulin resistance in mice typically does not manifest as diabetes due to multiple compensatory mechanisms. Here, we present a novel digenic model of type 2 diabetes in mice heterozygous for a null allele of the insulin receptor and an N-ethyl-N-nitrosourea-induced alternative splice mutation in the regulatory protein phosphatase 2A (PP2A) subunit PPP2R2A. Inheritance of either allele independently results in insulin resistance but not overt diabetes. Doubly heterozygous mice exhibit progressive hyperglycemia, hyperinsulinemia, and impaired glucose tolerance from 12 weeks of age without significant increase in body weight. Alternative splicing of Ppp2r2a decreased PPP2R2A protein levels. This reduction in PPP2R2A containing PP2A phosphatase holoenzyme was associated with decreased serine/threonine protein kinase AKT protein levels. Ultimately, reduced insulin-stimulated phosphorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 cells knocked down using Ppp2r2a small interfering RNAs. Altered AKT signaling and expression of gluconeogenic genes in the fed state contributed to an insulin resistance and hyperglycemia phenotype. This model demonstrates how genetic changes with individually small phenotypic effects interact to cause diabetes and how differences in expression of hypomorphic alleles of PPP2R2A and potentially other regulatory proteins have deleterious effects and may therefore be relevant in determining diabetes risk.Type 2 diabetes is a complex disease where cellular resistance to insulin combined with a failure in b-cell compensation results in the development of the disease. Underlying this process are multiple genetic and environmental factors that interact to determine susceptibility risk. However, there are relatively few examples of patients with diabetes whose disease can be demonstrated to be due to the interaction of mutations in two or more genes. One of these is due to heterozygous mutations in two unlinked genes, peroxisome proliferator-activated receptor g (PPARG) and protein phosphatase 1, regulatory (inhibitor) subunit 3A (PPP1R3A), expressed in adipocytes and skeletal muscle, respectively, resulting in severe insulin resistance and lipodystrophy (1). A second example is haploinsufficiency for the insulin receptor (IR) in combination with chimerin 2 (CHN2), a GTPase-activating protein, that results in insulin resistance and deficiency in intrauterine growth (2). In this latter example, the CHN2 mutation implicates a novel gene in insulin signaling and its regulation of metabolism and growth (2). Although there are other examples of doubly heterozygous individuals with diabetes, e.g., in the maturity-onset diabetes of the young HNF1A and HNF4A genes, it is unclear how these impact the severity of disease (3). In a mouse model, a digenic insulin resistance phenotype has been described whereby 40% of mice heterozygous for both IR and insulin receptor substrate 1 (IRS-1) null alleles develop overt diabetes at 4-6 months of age, demonstra...

Objective β-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. Methods We performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. Results Bulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell: β-cell contacts, increased extracellular matrix (ECM) deposition, and TGFβ-dependent islet fibrosis. We found that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2 , which encodes a transcriptional repressor of the EMT transcription factor Zeb2 . Following compromised β-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of β-cells through the induction of Zeb2 . We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. Conclusions Our study indicates that miR-7-mediated β-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating β-cell dysfunction and fibrosis in T2D.