Tausif Alam

Chapter 8 Use of Recombinant Adenovirus for Metabolic Engineering of Mammalian Cells

Publisher Summary This chapter describes the utility of Adenovirus for transfer of genes involved in metabolic regulation into mammalian cells, with particular emphasis to primary cell types with low replicative activity, such as hepatocytes and cells of the islets of Langerhans. The chapter provides methods and procedures for constructing and propagating new recombinant virions. Recombinant adenoviruses have been useful for delivering genes to whole animals. Viruses have been administered as single injections via accessible blood vessels such as the external jugular vein. Adenovirus in its current form is unlikely to represent the ultimate transfer vector for human gene therapy, given problems such as the lack of integration of the viral genome and the potential for immunological response to injected virus. It is therefore likely that new research initiatives will focus on attempting to engineer “second generation” virions that combine functional features of different viruses—that is, the site-specific integration function of adeno-associated virus (AAV) with the growth and infectivity characteristics of adenovirus.

Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes.

The role of defective glucose transport in the pathogenesis of noninsulin-dependent diabetes (NIDDM) was examined in Zucker diabetic fatty rats, a model of NIDDM. As in human NIDDM, insulin secretion was unresponsive to 20 mM glucose. Uptake of 3-O-methylglucose by islet cells was less than 19% of controls. The beta cell glucose transporter (GLUT-2) immunoreactivity and amount of GLUT-2 messenger RNA were profoundly reduced. Whenever fewer than 60% of beta cells were GLUT-2-positive, the response to glucose was absent and hyperglycemia exceeded 11 mM plasma glucose. We conclude that in NIDDM underexpression of GLUT-2 messenger RNA lowers high Km glucose transport in beta cells, and thereby impairs glucose-stimulated insulin secretion and prevents correction of hyperglycemia.

Roles of insulin resistance and beta-cell dysfunction in dexamethasone-induced diabetes.

The roles of insulin resistance and beta-cell dysfunction in glucocorticoid-induced diabetes were determined in Wistar and Zucker (fa/fa) rats. All Wistar rats treated with 5 mg/kg per d of dexamethasone for 24 d exhibited increased beta-cell mass and basal and arginine-stimulated insulin secretion, indicating insulin resistance, but only 16% became diabetic. The insulin response to 20 mM glucose was normal in the perfused pancreas of all normoglycemic dexamethasone-treated rats but absent in every diabetic rat. Immunostainable high Km beta-cell transporter, GLUT-2, was present in approximately 100% of beta-cells of normoglycemic rats, but in only 25% of beta cells of diabetic rats. GLUT-2 mRNA was not reduced. All Zucker (fa/fa) rats treated with 0.2-0.4 mg/kg per d of dexamethasone for 24 d became diabetic and glucose-stimulated insulin secretion was absent in all. High Km glucose transport in islets was 50% below nondiabetic controls. Only 25% of beta cells of diabetic rats were GLUT-2-positive compared with approximately 100% in controls. Total pancreatic GLUT-2 mRNA was increased twofold suggesting a posttranscriptional abnormality. We conclude that dexamethasone induces insulin resistance, whether or not it induces hyperglycemia. Whenever hyperglycemia is present, GLUT-2-positive beta cells are reduced, high Km glucose transport into beta cells is attenuated and the insulin response to glucose is absent.

GLUT2 Expression and Function in β-cells of GK rats with NIDDM: Dissociation Between Reductions in Glucose Transport and Glucose-Stimulated Insulin Secretion

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

Effects of hypoglycemia and prolonged fasting on insulin and glucagon gene expression. Studies with in situ hybridization

In situ hybridization of proinsulin and proglucagon mRNA was performed in rat pancreas to assess prohormone gene expression during various glucopenic conditions. During a 4-d fast mean blood glucose declined by 48 mg/dl; proinsulin mRNA signal density remained normal while proglucagon mRNA signal density more than doubled. At the end of a continuous 12-d insulin infusion blood glucose averaged 53 +/- 12 mg/dl; proinsulin mRNA signal density declined to 30% of controls while proglucagon mRNA signal density more than doubled. In insulinoma-bearing NEDH rats blood glucose averaged 34 +/- 3.5 mg/dl; the proinsulin mRNA signal was virtually undetectable and proglucagon mRNA signal density was more than twice the controls. There was no detectable change in either beta-cell area or islet number in rats subjected to fasting or insulin infusion, but in insulinoma-bearing rats beta cell area was markedly reduced. Thus compensation during 4 d of starvation involves an increase in glucagon gene expression without change in insulin gene expression or beta cell mass. In moderate insulin-induced hypoglycemia glucagon gene expression is increased and insulin gene expression decreased. In more profound insulinoma-induced hypoglycemia, in addition to the foregoing changes in hormone gene expression, there is a profound reduction in the number of insulin-expressing cells.

Coordinate regulation of amylin and insulin expression in response to hypoglycemia and fasting

Amylin is a 37–amino acid peptide synthesized in the pancreatic β-cell and cosecreted with insulin. In situ hybridization of nondiabetic rat pancreas shows that insulin and amylin RNA are both localized within the islet of Langerhans in a similar distribution. After 12 days of insulin-induced hypoglycemia (mean blood glucose 3.0 ± 0.4 mM [54 ± 8 mg/dl]), both insulin and amylin RNA fell > 95%. However, maintenance of euglycemia by simultaneous infusion of glucose with insulin did not suppress insulin or amylin RNA. Fasting suppressed amylin and insulin secretion from the isolated, perfused pancreas 70 and 58%, respectively, and with refeeding, secretion rates recovered to fed levels. Despite these changes in the rates of secretion, the relative ratio of amylin to insulin was not significantly different in fed, fasted, or refed rats. The molar ratio of insulin to amylin was estimated to be 100:2.3–2.6. Both insulin and amylin RNA was suppressed ∼ 50% in response to fasting. Thus, although the absolute amounts of insulin and amylin change substantially under the conditions tested, the relative amounts of these peptides do not change.

Factors regulating islet regeneration in the post-insulinoma NEDH rat

Increases in islet β-cell mass can be induced in a variety of experimental models, and can also occur in the face of metabolic stress, such as obesity, or in certain genetic backgrounds such as the ob/ob mouse. The factors responsible for compensatory proliferation of islet β-cells are not understood, but as this volume attests, investigation in the area is intensifying. Our own work has heretofore utilized the insulinoma-bearing New England Deaconess Hospital (NEDH) rat1 as a model for studying islet regeneration.2,3 Implantation of a solid insulinoma tumor into NEDH rats causes dramatic suppression of the mass and function of their islet β-cells; this suppression is reversed rapidly by surgical removal of the tumor.2,3 We have used this model system to address two specific issues. First, we have investigated the regulation and site of expression of the reg gene, which was cloned by Okamoto and coworkers in 19881 by virtue of its preferential expression in a cDNA library prepared from isolated islets taken from 90% pancreatectomized, nicotinamide-injected rats, an alternate model of β-cell regeneration.4,5 The primary sequence of reg was subsequently shown to be identical to that of the pancreatic stone protein (PSP).6-8 an exocrine gene product whose only known function is to inhibit CaC03 crystal growth. thus helping to prevent chronic calcifying pancreatitis.9-13 Second, we have begun to develop differential screening strategies designed at identifying other genes that might be involved in the expansion of β-cell mass. These initiatives are reviewed herein, and new data on the site of expression of reg/PSP is also provided.