Knockouts of SOD1 and GPX1 exert different impacts on murine islet function and pancreatic integrity

Abstract

Metabolic subtlety and clinical relevance of different forms of reactive oxygen species in diabetes remain unclear. Using single knockout of Cu,Zn-superoxide dismutase (SOD1(-/-)) or Se-glutathione peroxidase-1 (GPX1(-/-)) and their double-knockout (DKO) mouse models, we determined if elevating endogenously-derived superoxide and hydroperoxide exerted distinct impacts and mechanisms on body glucose homeostasis. Whereas the three knockout groups displayed decreased plasma insulin concentrations and islet β-cells mass, only SOD1(-/-) showed decreased body weight, increased blood glucose, and blocked glucose-stimulated insulin secretion. Null of SOD1 and GPX1 elevated respective islet superoxide and hydroperoxide production, and upregulated p53 phosphorylation. Knockout of SOD1 downregulated the foxhead box A2/pancreatic and duodenal homeobox 1 pathway in a superoxide-dependent fashion at epigenetic, mRNA, and protein levels in islets, but improved insulin signaling in liver and muscle. The SOD1(-/-) mice showed more apparent pancreatitis than the GPX1(-/-) mice that were more susceptible to the cerulein-induced amylase increase. Knockout of SOD1 impaired islet function, pancreas integrity, and body glucose homeostasis more than that of GPX1. Simultaneous ablation of both enzymes did not result in additive or aggravated metabolic outcomes.

FIG. 1.

Altered glucose homeostasis phenotypes in SOD1^-/-, GPX1^-/-, and DKO mice. (a) Verification of respective protein knockout in islets of SOD1^-/-, GPX1^-/-, and DKO mice by Western blot analysis. (b) Body weights of the four genotypes from 2 to 6 months of age. (c) Fasting blood glucose concentrations of the four genotypes at 3 months of age. (d) Fasting plasma (postmortem heart blood) insulin concentrations of the four genotypes at 3 months of age. (e) Glucose tolerance test at 4 months of age (GTT, 2 g/kg). (f): Insulin tolerance test at 4 months of age (ITT, 0.5 U/kg). All data are means ± SE (n = 5–6). Asterisks indicate differences (p ≤ 0.05) from the WT. Double asterisks indicate differences (p ≤ 0.05) from all other groups.

FIG. 2.

Islets physiology of in SOD1^-/-, GPX1^-/- and DKO mice. (a) Effects of knockouts of SOD1 and GPX1 on glucose-stimulated insulin secretion (GSIS, 1g/kg). (b) GSIS in cultured islets that (30 per sample) were incubated in 1 ml of Hanks buffered saline solution containing 2.8 (2.8 mM G) or 30 (30 mM G) mM glucose at 37°C for 1 h. (c) ATP content in cultured islets. (d) Representative images (n = 3 mice x three slides/genotype) of pancreatic β-cell mass shown by insulin staining (40X magnification) (upper panel) and quantification of β-cell mass (lower panel). Data represent mean ± SE; n = 4–6 for panels a, b, and c, and n = 9 for panel d. *Asterisks indicate significant differences (p < 0.05) from the respective WT within the same treatment, # indicates the treatment effect within the genotype (panel B), and ** indicates that the GPX1^-/- group was different (p < 0.05) from both WT and SOD1^-/- and DKO groups (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

Intracellular ROS Levels in SOD1^-/-, GPX1^-/-, and DKO mouse islets. (a) Islet peroxide levels (green fluorescence) after 5 h treatment with 5 mM and 25 mM glucose. (b) Islet superoxide levels (red fluorescence) after 5 h treatment without or with the superoxide generator: 200 μM HX and 50 mU/ml XO. (c) Islets superoxide levels after 5 h treatment with SOD1 inhibitor (100 μM DETC) and SOD1 mimic (10 μM CuDIPS). The images (40X magnification) shown were the representative of four sets of data (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Changes in gene and protein expressions of key regulators in SOD1^-/-, GPX1^-/-, and DKO mice. (a) Real time Q-PCR analysis of Foxa2, Pdx1, preproinsulin 1 (PPins1), and Ucp2 mRNA in islets (200 per sample/animal, n = 6 mice/genotype). Data are means ± SE (n = 6). Asterisks indicate differences from WT (p < 0.05). (b) Western blot analysis of Foxa2, Pdx1, and Ucp2 proteins in pancreas of the four genotypes. Lower panel: representative (n = 4) immunoblots of the three proteins separated by SDS-polyacrylamide gel (12%) electrophoresis (20–50 g protein/lane). Upper panel: the relative density of the protein bands quantified using the Alpha-Imager 2200 system (Alpha Innotech, San Leandro, CA), and normalized to that of ß-actin on the same membrane. Values are expressed as mean ± SE (n = 4), and the asterisk * indicates difference (p < 0.05) from the WT. (c) Western blot analysis of Foxa2 protein in islets (n = 200 per sample, n = three/treatment) in response to 1 mM DETC (a potent SOD inhibitor), 2 μM CuDIPS (a potent SOD mimic), or 1 μM–500 μU/ml HX/XO (superoxide generator). The values (n = 3) under the image represent the relative Foxa2 protein levels.

FIG. 5.

Epigenetic modifications of pdx1 promoter and its binding to Foxa2 in SOD1^-/-, GPX1^-/-, and DKO islets. Immunoprecipitation was carried out overnight with preimmune serum (negative control) or primary antibodies (all from Upstate Biotech/Millipore) to the acetylated histone 3 (H3) and trimethyl-histone 3 lysine 4 (H3K4). After protein elusion, the released DNA was extracted, precipitated, and resuspended in water. DNA sequences in the "input" and the immunoprecipitated samples were quantified relative to the WT by real-time Q-PCR. (a) H3 acetylation in the pdx1 promoter region. (b) H3K4 trimethylation in the pdx1 promoter region. (c) Binding of Foxa2 to the pdx1 promoter. The values are mean ± SE (n = 4) and expressed as relative to the WT. The asterisks* indicate difference (p < 0.05) from the WT.

FIG. 6.

Effects of knockouts of SOD1 and GPX1 on total and phosphorylated protein of p53 (P53 and P53^ser15) and p38 MAPK (P38 or Pp38) in islets (n = 200 per sample). Blots are representative images of three independent analyses, and values under respective bands are relative density (mean ± SE, n = 3). The asterisks* indicate difference (p < 0.05) from the WT.

FIG. 7.

Impacts of knockouts of SOD1 and GPX1 on liver and muscle insulin signaling. For determining total (WB) and phosphorylation (IP) of hepatic insulin receptor (β-subunit, IR_β IP), mice (n = 6) fasted overnight (8 h) were given an ip injection of insulin (5 U/kg) and killed to collect liver 3 min after the injection. After homogenization and immunoprecipiation with the anti-IR_β antibody, the precipitates were subjected to Western blot analysis using an antibody against phosphotyrosine (4G10). For determining Akt protein and phosphorylation (Thr308 and Ser473), liver and soleus muscle samples were collected 8 min after mice (fasted overnight for 8 h, n = 6) were given an ip injection of insulin (10 U/kg). For a given protein, the upper panel shows the representative images of six independent analyses, and the lower panel shows the relative density of protein bands (mean ± SE, n = 6). The asterisks* indicate difference (p < 0.05) from the WT.

FIG. 8.

Impacts of knockouts of SOD1 and GPX1 on pancreatic integrity and cerulein-induced amylase release. (a) Representative images (n = 4 mice x three slides per genotype by age) of pancreatic sections stained with hematoxylin and eosin (40X magnification). Left panel (2 months of age): WT, normal structures of insulin secreting islet, interstitial blood vessels and acini; GPX1^-/-, multifocal interstitial infiltration of lymphocytes and macrophages; SOD1^-/-, necrotizing pancreatitis, focally extensive; DKO, interstitial pancreatitis with individual acinar cell degeneration and necrosis. Right panel (14 months of age): WT, normal structures of insulin secreting islet and exocrine acinus; GPX1^-/-, perivascular and interstitial infiltration of neutrophils, macrophages and lymphocytes; SOD1^-/-, focal acinar nodular hyperplasia; DKO, interstitial pancreatitis with minimal acinar cell degeneration. (b) Cerulein-induced amylase release in plasma. Mice (3 months of age, n = 4–6/genotype) were given an ip injection of cerulein at dose of 50 mg/kg. Tail blood samples were collected at 0, 3, and 7 h after the injection for the assay of serum amylase activity. Values are mean ± SE (n = 4–6). The asterisks* indicate difference (p < 0.05) from the WT (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).