Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets
Karl Bacos, Alexander Perfilyev, Alexandros Karagiannopoulos, Elaine Cowan, Jones K. Ofori, Ludivine Bertonnier-Brouty, Tina Rönn, Andreas Lindqvist, Cheng Luan, Sabrina Ruhrmann, Mtakai Ngara, Åsa Nilsson, Sevda Gheibi, Claire L. Lyons, Jens O. Lagerstedt, Mohammad Barghouth, Jonathan L.S. Esguerra, Petr Volkov, Malin Fex, Hindrik Mulder, Nils Wierup, Ulrika Krus, Isabella Artner, Lena Eliasson, Rashmi B. Prasad, Luis Rodrigo Cataldo, Charlotte Ling
Karl Bacos, Alexander Perfilyev, Alexandros Karagiannopoulos, Elaine Cowan, Jones K. Ofori, Ludivine Bertonnier-Brouty, Tina Rönn, Andreas Lindqvist, Cheng Luan, Sabrina Ruhrmann, Mtakai Ngara, Åsa Nilsson, Sevda Gheibi, Claire L. Lyons, Jens O. Lagerstedt, Mohammad Barghouth, Jonathan L.S. Esguerra, Petr Volkov, Malin Fex, Hindrik Mulder, Nils Wierup, Ulrika Krus, Isabella Artner, Lena Eliasson, Rashmi B. Prasad, Luis Rodrigo Cataldo, Charlotte Ling
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Research Article Endocrinology Metabolism

Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets

Abstract

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing β cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to β cell dysfunction in T2D pathophysiology.

Authors

Karl Bacos, Alexander Perfilyev, Alexandros Karagiannopoulos, Elaine Cowan, Jones K. Ofori, Ludivine Bertonnier-Brouty, Tina Rönn, Andreas Lindqvist, Cheng Luan, Sabrina Ruhrmann, Mtakai Ngara, Åsa Nilsson, Sevda Gheibi, Claire L. Lyons, Jens O. Lagerstedt, Mohammad Barghouth, Jonathan L.S. Esguerra, Petr Volkov, Malin Fex, Hindrik Mulder, Nils Wierup, Ulrika Krus, Isabella Artner, Lena Eliasson, Rashmi B. Prasad, Luis Rodrigo Cataldo, Charlotte Ling

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Figure 5

Increased expression of Pax5 results in perturbed mitochondrial activity.

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Increased expression of Pax5 results in perturbed mitochondrial activity...
(A) Immunohistochemical staining of human pancreas sections (n = 5 ND; n = 4 T2D) showing increased PAX5 (green) expression in T2D pancreas sections. Most of the expression was confined to β cells, as evidenced by costaining with insulin (red). Nuclei are stained with DAPI (blue). Representative images are shown (original magnification, ×40). (B) Pax5 overexpression blunted GSIS but increased secretion stimulated by elevated K+ (n = 6). (C) OCR in clonal β cells overexpressing GFP or Pax5. The OCR was measured at 2.8 mM glucose (basal respiration) and then after sequential addition of 16.7 mM glucose (glucose-stimulated respiration), 5 μM oligomycin (inhibits ATP synthase), 4 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and 1 μM rotenone/antimycin A (electron transport chain inhibitors) (n = 4). (D–F) Respiratory response to addition of high glucose (change in respiration compared with basal glucose, D), glucose-stimulated respiration (respiration at high glucose, E), and maximal OCR (respiration after mitochondrial uncoupling, F) (n = 4). (G) PercevalHR trace on INS1 β cells stimulated with 2.8 and 16.7 mM glucose, and after addition of oligomycin (n = 148, pcDNA3.1; n = 213, Pax5). (H and I) Pax5-overexpressing INS1 β cells (n = 213) exhibited a significantly lower increase of the ATP/ADP ratio when glucose was raised to 16.7 mM (average signal between 94 and 354 seconds compared with average signal between 0 and 75 seconds, H), and a greater drop in the ATP/ADP ratio after addition of oligomycin (average signal between 409 and 567 seconds compared with average signal between 94 and 354 seconds, I), when compared with pcDNA3.1-transfected cells (n = 148). (J–O) Levels of citrate synthase and subunits of complex I–V of the electron transport chain (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, paired t test (B–F and J–O) and 2-tailed, unpaired t test (G–I). Box-and-whisker plots show the median, 25th and 75th percentiles, and minimum and maximum values.