mRNA destabilization improves glycemic responsiveness of transcriptionally regulated hepatic insulin gene therapy in vitro and in vivo.
BACKGROUND
Hepatic insulin gene therapy (HIGT) employing a glucose and insulin sensitive promoter to direct insulin transcription can lower blood sugars within 2 h of an intraperitoneal glucose challenge. However, post-challenge blood sugars frequently decline to below baseline. We hypothesize that this 'over-shoot' hypoglycemia results from sustained translation of long-lived transgene message, and that reducing pro-insulin message half-life will ameliorate post-challenge hypoglycemia.


METHODS
We compared pro-insulin message content and insulin secretion from primary rat hepatocytes expressing insulin from either a standard construct (2xfur), or a construct producing a destabilized pro-insulin message (InsTail), following exposure to stimulating or inhibitory conditions.


RESULTS
Hepatocytes transduced with a 2xfur construct accumulated pro-insulin message, and exhibited increased insulin secretion, under conditions that both inhibit or stimulate transcription. By contrast, pro-insulin message content remained stable in InsTail expressing cells, and insulin secretion increased less than 2xfur during prolonged stimulation. During transitions from stimulatory to inhibitory conditions, or vice versa, amounts of pro-insulin message changed more rapidly in InsTail expressing cells than 2xfur expressing cells. Importantly, insulin secretion increased during the transition from stimulation to inhibition in 2xfur expressing cells, although it remained unchanged in InsTail expressing cells. Use of the InsTail destabilized insulin message tended to more rapidly reduce glucose induced glycemic excursions, and limit post-load hypoglycemia in STZ-diabetic mice in vivo.


CONCLUSIONS
The data obtained in the present study suggest that combining transcriptional and post-transcriptional regulatory strategies may reduce undesirable glycemic excursion in models of HIGT.
Atlanta VA Medical Center, Division of Endocrinology, Diabetes, & Lipids, Emory University School of Medicine, Decatur, Georgia, USA.|Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.
Peter M
PM
Thulé
Atlanta VA Medical Center, Division of Endocrinology, Diabetes, & Lipids, Emory University School of Medicine, Decatur, Georgia, USA.
Yulin
Y
Lin
Atlanta VA Medical Center, Division of Endocrinology, Diabetes, & Lipids, Emory University School of Medicine, Decatur, Georgia, USA.
Dingwu
D
Jia
Atlanta VA Medical Center, Division of Endocrinology, Diabetes, & Lipids, Emory University School of Medicine, Decatur, Georgia, USA.
Darin E
DE
Olson
Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.|School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.|Department of Medical Science, National Tsing Hua University, Taiwan, USA.
Shiue-Cheng
SC
Tang
Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, USA.|School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.|Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
Athanassios
A
Sambanis
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001786:Blood Glucose / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental; D005786:Gene Expression Regulation; D023061:Gene Order; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D061251:Primary Cell Culture; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D001786:Blood Glucose; D007328:Insulin; D012333:RNA, Messenger
adenovirus; gene therapy; hypoglycemia; insulin; liver