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. 2013 Aug;123(8):3305-16.
doi: 10.1172/JCI65390. Epub 2013 Jul 1.

Inactivation of specific β cell transcription factors in type 2 diabetes

Shuangli Guo, Chunhua Dai, Min Guo, Brandon Taylor, Jamie S Harmon, Maike Sander, R Paul Robertson, Alvin C Powers, Roland Stein

Inactivation of specific β cell transcription factors in type 2 diabetes

Shuangli Guo et al. J Clin Invest. 2013 Aug.

Abstract

Type 2 diabetes (T2DM) commonly arises from islet β cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with β cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of β cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key β cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet β cells restored nuclear MAFA, nuclear NKX6.1, and β cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human β cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet β cell function, with loss of MAFA (and/or MAFB) representing an early indicator of β cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

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Figures

Figure 1
Figure 1. H2O2 treatment induces MAFA dephosphorylation in βTC-3 cells but not in HeLa or αTC-6 cells.
(A) βTC-3 cells treated with H2O2. (B) βTC-3 cells incubated with 0.75 μM okadiac acid (O.A.) for 30 minutes before H2O2 addition. (C) Adenovirus-driven catalase decreases the effect of H2O2. (D and E) Transfected myc-MAFA is not dephosphorylated after H2O2 treatment in HeLa cells or αTC-6 cells. (F) βTC-3 cells were treated with (+) or without (–) 50 μM H2O2 for 90 minutes, and the whole-cell extract was separated by sucrose gradient ultracentrifugation (35). The MAFA DNA-binding species (fractions 3–7) was not detected after H2O2-induced dephosphorylation (data not shown and ref. 35). (A–F) MAFA protein was analyzed by immunoblotting.
Figure 2
Figure 2. MAFA is translocated to the cytoplasm after H2O2 treatment.
(A and B) Immunoblotting to determine MAFA levels in the cytoplasmic and nuclear fractions of βTC-3 cells. Subcellular marker controls: nuclear, p/CAF coactivator; cytoplasm, B55α regulatory subunit of PP2A. The immunosignal shows (C) MAFA, (D) FOXO1, (E–G) myc-MAFA, or (E) myc-MAFB levels in cells treated with H2O2 for 90 minutes. Yopro (blue) staining labels the nuclei. Notably, myc-MAFA is not cytoplasmic in non–β cell lines (F and G) after H2O2 treatment, although endogenous FOXO1 (D) is relocalized to the nucleus. P, phosphorylated; dP, dephosphorylated.
Figure 3
Figure 3. MAFA forms a covalent dimer species unable to bind DNA under oxidative stress conditions.
(A) MAFA and PDX1 DNA-binding activity in βTC-3 nuclear extract reactions conducted in the presence (+) and absence of 20 mM DTT. (B) Transfected myc-MAFA containing HeLa nuclear extracts prepared in the absence of DTT was analyzed in gel-shift (right panel: [+] 20 mM DTT) and immunoblotting assays (left panel: [+] 300 mM DTT). (C) The HeLa-produced myc-MAFA C277/C293A mutant does not lose gel-shift activity or form (MAFA)2 after H2O2 treatment, in contrast to WT or mutant C42/59/69S myc-MAFA. (D) Immunoblotting shows that only native MAFA (i.e., ~46 kD) levels and few, if any, other islet-enriched transcription factors were reduced in βTC-3 nuclear extract prepared in the absence of DTT. IB, immunoblotting; SS, antibody super-shifted complex.
Figure 4
Figure 4. NKX6.1 and PDX1 are also inactivated in H2O2-treated βTC-3 cells.
(A) Cytoplasmic and nuclear distribution of a variety of regulatory factors in βTC-3 cells treated with or without H2O2 for 90 minutes. The change in NKX6.1 and MAFA mobility and subcellular localization is shown by immunoblotting, with levels quantitated using NIH ImageJ software. Dephosphorylation causes MAFA (35) and NKX6.1 to run at a faster mobility on SDS-PAGE (see also Supplemental Figure 1C). (B) Immunostaining shows endogenous or myc-tagged transfected NKX6.1, ISL1, and/or NEUROD1 subcellular localization in control and treated cells. (C) Oxidative stress results in the specific loss of endogenous MAFA, NKX6.1, and PDX1 binding to target gene regulatory sequences in ChIP assays. hyperP, hyperphosphorylated.
Figure 5
Figure 5. Nuclear NKX6.1 levels are profoundly reduced in diabetic db/db islet β cells.
(A) Immunostaining for MAFA, ISL1, NEUROD1, PDX1, and insulin in 10-week-old WT and db/db mouse pancreas. (B) FOXO1, NKX6.1, and insulin immunosignals in WT and diabetic db/db pancreas. (C) Immunoblotting of MAFA and NKX6.1 in islet extracts prepared from 10-week-old WT and db/db mice (n = 3). Quantified MAFA, NKX6.1, and PAX6 levels were normalized to RBBP5. The relative change in each db/db mouse was compared with 3 controls. ***P < 0.001.
Figure 6
Figure 6. NKX6.1 and GLUT2 subcellular levels are rescued by transgenic Gpx1 expression in db/db islet β cells.
Immunostained images of (A) NKX6.1, PAX6, and (B) GLUT2 in 20-week-old WT, db/db, and Gpx1-db/db pancreas.
Figure 7
Figure 7. Islet β cell proliferation precedes the loss of MAFA and NKX6.1 in db/db islets of (A) MAFA, (B) NKX6.1, and (C) Ki67 in 4-, 6-, 8-, and 10-week-old WT and db/db pancreas.
Quantification of the percentage of insulin-positive (β) cells costaining for MAFA, NKX6.1, and Ki67 is shown (n = 4). ***P < 0.001.
Figure 8
Figure 8. MAFA, MAFB, NKX6.1, and PDX1 expression levels are compromised in human T2DM islet β cells.
(A and C) Real-time PCR analysis of normal human and T2DM human islets. (B) MAFA, PDX1, and NKX6.1 protein levels were reduced in human T2DM islets when compared with other islet-enriched transcription factors. #Predicted molecular weight of human HNF1α. (D) Reduction in MAFA, NKX6.1, PDX1, and MAFB in human T2DM islet insulin-positive cells by immunostaining. Quantification of the percentage of β cells containing nuclear MAFA, NKX6.1, PDX1, and MAFB is shown. *P < 0.10; **P < 0.01; ***P < 0.001.
Figure 9
Figure 9. Events leading up to islet β cell dysfunction in T2DM.
Adaptive responses: (I) β cell replication and (II) FOXO1 nuclear localization (41). Stress responses: (II) FOXO1 nuclear localization/inactivation (47) and sequential loss of (III) MAFA (and/or MAFB), and (IV) NKX6.1 (and/or PDX1) in response to hyperglycemia and oxidative stress. These circumstances lead to a decline in β cell function, also termed “the stunned β cell” (65).
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