Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Summary

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Abstract

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes ^1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.

Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.

By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass ^4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets ^4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function ^{5,6,8,9,16,17}.

Protocol

1. Adenoviral Transduction and Culturing of Rat Islets

1. Prepare a 6-well non-tissue culture coated plate by adding 2 ml of media (RPMI 1640 media containing 8 mM glucose, 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin) to the required number of wells. For example, a typical experiment may require three wells – one each for a no-virus control, a virus control (e.g., GFP-expressing adenovirus), and the experimental group.
2. Warm the plate to 37 °C by placing it into a tissue culture incubator for at least 30 min.
3. Immediately following rat islet isolation ^18,19, place 100-200 islets into in....

Discussion

Establishing pathways that can be modulated to stimulate the replication and enhance the function of beta cells are relevant to both major forms of diabetes. Because functional beta cell mass is dependent on the existence and function of insulin-secreting cells, assessing these determinants simultaneously has its advantages. This protocol describes a streamlined protocol for identifying whether overexpression or suppression of a protein leads to changes in functional beta cell mass in vitro, which can the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 media	Gibco	11879
Penicillin/streptomycin	Gibco	15140
6-well plate	BD-Falcon	35-1146	Non-TC treated
[methyl-3H]-thymidine	Perkin Elmer	NET027Z001MC	1 mCi/ml
Micro-centrifuge tubes	Denville	C2170	1.7 ml
NaCl	Sigma	59888
KCl	Acros	42409
KH2PO4	Acros	20592
MgSO4	Acros	41348
CaCl2	Acros	34961
HEPES	Sigma	H0887	1 M solution
35% BSA	Sigma	A7979
NaHCO3	Acros	42427
d-glucose	Sigma	G8769
TCA	Fisher Scientific	SA9410-1	10% w/v
NaOH	Acros	12426
Scintillation counting tube	Sarstedt	58.536	7 ml, PP
Scintillation counting tube cap	Sarstedt	65.816
Econo-Safe counting cocktail	RPI	111175
Insulin RIA	Siemens	TKIN2
BCA Assay Kit	Thermo Scientific	23250
Equipment
Centrifuge	Eppendorf	5415R
Scintillation counting tube rack	Sarstedt	93.1431.001
Liquid scintillation counter	Perkin Elmer	Tri-Carb 2910TR