PERK Regulates the Proliferation and Development of Insulin-Secreting Beta-Cell Tumors in the Endocrine Pancreas of Mice

PERK eIF2alpha kinase is required for the proliferation of the insulin-secreting beta- cells as well as insulin synthesis and secretion. In addition, PERK signaling has been found to be an important factor in determining growth and angiogenesis of specific types of tumors, and was attributed to PERK-dependent regulation of the hypoxic stress response. In this report we examine the role of PERK in regulating proliferation and angiogenesis of transformed beta-cells in the development of insulinomas.The SV40 Large T-antigen (Tag) was genetically introduced into the insulin secreting beta-cells of Perk KO mice under the control of an inducible promoter. Tumor growth and the related parameters of cell proliferation were measured. In late stage insulinomas the degree of vascularity was determined.The formation and growth of insulinomas in Perk-deficient mice was dramatically ablated with much fewer tumors, which averaged 38-fold smaller than seen in wild-type control mice. Beta-cell proliferation was ablated in Perk-deficient mice associated with reduced tumor growth. In the small number of large encapsulated insulinomas that developed in Perk-deficient mice, we found a dramatic reduction in tumor vascularity compared to similar sized insulinomas in wild-type mice. Although insulinoma growth in Perk-deficient mice was largely impaired, beta-cell mass was increased sufficiently by T-antigen induction to rescue the hypoinsulinemia and diabetes in these mice.We conclude that PERK has two roles in the development of beta-cell insulinomas, first to support rapid cell proliferation during the initial transition to islet hyperplasia and later to promote angiogenesis during the progression to late-stage encapsulated tumors

Acute ablation of PERK results in ER dysfunctions followed by reduced insulin secretion and cell proliferation

<p>Abstract</p> <p>Background</p> <p>A deficiency in <it>Perk </it>(EIF2AK3) causes multiple neonatal defects in humans known as the Wolcott Rallison syndrome. <it>Perk KO </it>mice exhibit the same array of defects including permanent neonatal diabetes (PND). PND in mice was previously shown by us to be due to a decrease in beta cell proliferation and insulin secretion. The aim of this study was to determine if acute ablation of PERK in the 832/13 beta cells recapitulates these defects and to identify the primary molecular basis for beta cell dysfunction.</p> <p>Results</p> <p>The INS1 832/13 transformed rat beta cell line was transduced with a dominant-negative <it>Perk </it>transgene via an adenoviral vector. <it>AdDNPerk</it>-832/13 beta cells exhibited reduced expression of <it>insulin </it>and <it>MafA </it>mRNAs, reduced insulin secretion, and reduced cell proliferation. Although proinsulin content was reduced in <it>AdDNPerk</it>-832/13 beta cells, proinsulin was abnormally retained in the endoplasmic reticulum. A temporal study of the acute ablation of <it>Perk </it>revealed that the earliest defect seen was induced expression of two ER chaperone proteins, GRP78/BiP and ERp72. The oxidized states of ERp72 and ERp57 were also increased suggesting an imbalance in the redox state of the ER.</p> <p>Conclusion</p> <p>Acute ablation of Perk in INS 832/13 beta cells exhibited all of the major defects seen in <it>Perk KO </it>mice and revealed abnormal expression and redox state of key ER chaperone proteins. Dysregulation of ER chaperone/folding enzymes ERp72 and GRP78/BiP occurred early after ablation of PERK function suggesting that changes in ER secretory functions may give rise to the other defects including reduced insulin gene expression, secretion, and cell proliferation.</p

Hyperthermia Induces the ER Stress Pathway

The ER chaperone GRP78/BiP is a homolog of the Hsp70 family of heat shock proteins, yet GRP78/BiP is not induced by heat shock but instead by ER stress. However, previous studies had not considered more physiologically relevant temperature elevation associated with febrile hyperthermia. In this report we examine the response of GRP78/BiP and other components of the ER stress pathway in cells exposed to 40°C.AD293 cells were exposed to 43°C heat shock to confirm inhibition of the ER stress response genes. Five mammalian cell types, including AD293 cells, were then exposed to 40°C hyperthermia for various time periods and induction of the ER stress pathway was assessed.The inhibition of the ER stress pathway by heat shock (43°C) was confirmed. In contrast cells subjected to more mild temperature elevation (40°C) showed either a partial or full ER stress pathway induction as determined by downstream targets of the three arms of the ER stress pathway as well as a heat shock response. Cells deficient for Perk or Gcn2 exhibit great sensitivity to ER stress induction by hyperthermia.The ER stress pathway is induced partially or fully as a consequence of hyperthermia in parallel with induction of Hsp70. These findings suggest that the ER and cytoplasm of cells contain parallel pathways to coordinately regulate adaptation to febrile hyperthermia associated with disease or infection

Beta-cell hyperplasia caused by T-antigen transformation reverses diabetes in <i>Perk KO</i> mice.

<p>Serum glucose levels (mg/dl) in wildtype (<i>WT</i>, closed squares and solid line), <i>wildtype-βTag</i> (<i>WT-βTag</i>, open squares and dashed line), <i>Perk KO</i> (<i>PKO</i>, closed triangles and solid line) and <i>Perk KO-βTag</i> (<i>PKO-βTag</i>, open triangles and dashed line) littermates during postnatal progression of insulinoma development. Each data point is the mean of 4 to 13 mice. Serum glucose in <i>PKO</i> mice often exceeded the detection capability of the blood glucose monitor (600 mg/dl) and these mice didn't survive beyond p45. In this figure and all others, error bars = SEM.</p

Beta-cell proliferation is impaired in <i>Perk</i>-deficient, T-antigen transformed islets.

<p>Beta-cell proliferation rates were assessed by BrdU incorporation in age and sex matched <i>PKO-βTag</i> mice and <i>WT-βTag</i> littermates at postnatal ages p50, p100 and p120. Cells = insulin-positive beta-cells.</p