Dual Opposing Roles of Metallothionein Overexpression in C57BL/6J Mouse Pancreatic β-Cells

<div><p>Background</p><p>Growing evidence indicates that oxidative stress (OS), a persistent state of excess amounts of reactive oxygen species (ROS) along with reactive nitrogen species (RNS), plays an important role in insulin resistance, diabetic complications, and dysfunction of pancreatic β-cells. Pancreatic β-cells contain exceptionally low levels of antioxidant enzymes, rendering them susceptible to ROS-induced damage. Induction of antioxidants has been proposed to be a way for protecting β-cells against oxidative stress. Compared to other antioxidants that act against particular β-cell damages, metallothionein (MT) is the most effective in protecting β-cells from several oxidative stressors including nitric oxide, peroxynitrite, hydrogen peroxide, superoxide and streptozotocin (STZ). We hypothesized that MT overexpression in pancreatic β-cells would preserve β-cell function in C57BL/6J mice, an animal model susceptible to high fat diet-induced obesity and type 2 diabetes.</p><p>Research Design and Methods</p><p>The pancreatic β-cell specific MT overexpression was transferred to C57BL/6J background by backcrossing. We studied transgenic MT (MT-tg) mice and wild-type (WT) littermates at 8 weeks and 18 weeks of age. Several tests were performed to evaluate the function of islets, including STZ <i>in vivo</i> treatment, intraperitoneal glucose tolerance tests (IPGTT) and plasma insulin levels during IPGTT, pancreatic and islet insulin content measurement, insulin secretion, and islet morphology assessment. Gene expression in islets was performed by quantitative real-time PCR and PCR array analysis. Protein levels in pancreatic sections were evaluated by using immunohistochemistry.</p><p>Results</p><p>The transgenic MT protein was highly expressed in pancreatic islets. MT-tg overexpression significantly protected mice from acute STZ-induced ROS at 8 weeks of age; unexpectedly, however, MT-tg impaired glucose stimulated insulin secretion (GSIS) and promoted the development of diabetes. Pancreatic β-cell function was significantly impaired, and islet morphology was also abnormal in MT-tg mice, and more severe damage was detected in males. The unique gene expression pattern and abnormal protein levels were observed in MT-tg islets.</p><p>Conclusions</p><p>MT overexpression protected β-cells from acute STZ-induced ROS damages at young age, whereas it impaired GSIS and promoted the development of diabetes in adult C57BL/6J mice, and more severe damage was found in males.</p></div

MT overexpression in pancreatic islets of transgenic C57BL/6J mice.

<p>MT (red) and insulin (green) immunostaining of islets of MT-tg mice and its control littermates is indicated in different colors. MT staining coincided with insulin staining is indicated by overlaid images.</p

Glucose tolerance tests (GTT) and plasma insulin levels during GTT in 8- and 18-week-old MT-tg and WT mice.

<p><b>(</b>A, C) 8-week old MT-tg (n = 10) and WT (n = 14) male mice; (B, D) 8-week old MT-tg (n = 8) and WT (n = 10) female mice; (E, G) 18-week old MT-tg (n = 5) and WT (n = 5) male mice; (F, H) 18-week old MT-tg (n = 8) and WT (n = 10) female mice. * p<0.05, ** p<0.01 <i>vs</i> WT.</p

Relative mRNA levels in islets of MT-tg and WT male mice.

<p><b>(</b>A) using regular RT-PCR, the value of each gene was normalized to <i>Actb</i> and <i>Rpl13a</i>. (B) using PCR Array, the value of each gene was normalized to <i>Gusb</i>, <i>Hprt1</i>, <i>Hsp90ab1</i> and <i>Actb</i>. The gene expression level in MT-tg islets was presented as fold relative to WT (set as 1). Data are mean ±SEM. n values represent the number of mice. * p <0.05, ** p <0.001, ^† p = 0.053 <i>vs</i> WT.</p

Blood glucose levels in MT-tg and WT mice after <i>in vivo</i> STZ treatment.

<p>(A) MT-tg (n = 8) and WT (n = 7) male mice; (B) MT-tg (n = 13) and WT (n = 21) female mice. * p<0.05, ** p<0.01 <i>vs</i> WT.</p

Insulin secretion from islets of 8-week-old MT-tg and WT mice.

<p><b>(</b>A) MT-tg (n = 3) and WT (n = 3) male mice; (B) MT-tg (n = 5) and WT (n = 5) female mice. 2.8 mM = 2.8 mmol/l, 16.7 mM = 16.7 mmol/l glucose concentration in KRB, respectively; Arg = 10 mmol/l L-arginine; * p<0.05 <i>vs</i> WT.</p

Additional file 1 of A rare case of anti-DPPX encephalitis combined with neuroleptospirosis

Supplementary Material