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Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome.

Bixler GV, Vanguilder HD, Brucklacher RM, Kimball SR, Bronson SK, Freeman WM - BMC Med Genomics (2011)

Bottom Line: A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes.Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.

ABSTRACT

Background: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.

Methods: The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.

Results: Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.

Conclusions: These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

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Transcript expression not rescued by insulin treatment. qPCR confirmation experiments also included 1 month control and diabetic groups to determine if retinal mRNA expression changes occurred before initiation of insulin treatment. Lad1, Litaf1, Mt1a, and Txnip all demonstrated a 'not rescued' molecular phenotype of increased expression in diabetic animals at 1 month of diabetes which remained elevated at 3 months in diabetic and insulin-treated diabetic groups. Similarly, Rgr expression was decreased at one month of diabetes and remained depressed at three months in diabetic animals with and without insulin treatment. One-way ANOVA, Student Newman Keuls pair-wise post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 for 3 month data; t-test #P < 0.05, ##P < 0.01, ###P < 0.001 for 1 month data; n = 7-11/group for both time points. ND - Non-Diabetic control, D - Diabetic, D+I - Insulin-treated Diabetic, 1M - 1 month, 3M - 3 Months.
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Figure 4: Transcript expression not rescued by insulin treatment. qPCR confirmation experiments also included 1 month control and diabetic groups to determine if retinal mRNA expression changes occurred before initiation of insulin treatment. Lad1, Litaf1, Mt1a, and Txnip all demonstrated a 'not rescued' molecular phenotype of increased expression in diabetic animals at 1 month of diabetes which remained elevated at 3 months in diabetic and insulin-treated diabetic groups. Similarly, Rgr expression was decreased at one month of diabetes and remained depressed at three months in diabetic animals with and without insulin treatment. One-way ANOVA, Student Newman Keuls pair-wise post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 for 3 month data; t-test #P < 0.05, ##P < 0.01, ###P < 0.001 for 1 month data; n = 7-11/group for both time points. ND - Non-Diabetic control, D - Diabetic, D+I - Insulin-treated Diabetic, 1M - 1 month, 3M - 3 Months.

Mentions: A total of 27 targets from the microarray analysis were chosen for confirmation analysis including 15 from the Not Normalized, 5 from the Partially Normalized and 7 from the Normalized groups. While not comprehensive of all the genes observed with differential expression in the microarray analysis, the extensive qPCR confirmation experiments validate the quantitative accuracy of the discovery experiments. Twenty six of the 27 (96%) genes examined in this analysis were confirmed as being differentially regulated with diabetes in a statistically significant manner at the three month time-point (ANOVA, SNK post-hoc, P < 0.05). Eight of the 26 genes tested by qPCR were found to be differentially regulated after one month of diabetes. Using the qPCR data, genes were separated into different expression categories. Five were Not Rescued (Figure 4) and three were Rescued (Figure 5), with the remainder of genes dividing into four Not Prevented (Figure 6), four Partially Prevented (Figure 7), and 10 Prevented (Figure 8).


Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome.

Bixler GV, Vanguilder HD, Brucklacher RM, Kimball SR, Bronson SK, Freeman WM - BMC Med Genomics (2011)

Transcript expression not rescued by insulin treatment. qPCR confirmation experiments also included 1 month control and diabetic groups to determine if retinal mRNA expression changes occurred before initiation of insulin treatment. Lad1, Litaf1, Mt1a, and Txnip all demonstrated a 'not rescued' molecular phenotype of increased expression in diabetic animals at 1 month of diabetes which remained elevated at 3 months in diabetic and insulin-treated diabetic groups. Similarly, Rgr expression was decreased at one month of diabetes and remained depressed at three months in diabetic animals with and without insulin treatment. One-way ANOVA, Student Newman Keuls pair-wise post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 for 3 month data; t-test #P < 0.05, ##P < 0.01, ###P < 0.001 for 1 month data; n = 7-11/group for both time points. ND - Non-Diabetic control, D - Diabetic, D+I - Insulin-treated Diabetic, 1M - 1 month, 3M - 3 Months.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113923&req=5

Figure 4: Transcript expression not rescued by insulin treatment. qPCR confirmation experiments also included 1 month control and diabetic groups to determine if retinal mRNA expression changes occurred before initiation of insulin treatment. Lad1, Litaf1, Mt1a, and Txnip all demonstrated a 'not rescued' molecular phenotype of increased expression in diabetic animals at 1 month of diabetes which remained elevated at 3 months in diabetic and insulin-treated diabetic groups. Similarly, Rgr expression was decreased at one month of diabetes and remained depressed at three months in diabetic animals with and without insulin treatment. One-way ANOVA, Student Newman Keuls pair-wise post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 for 3 month data; t-test #P < 0.05, ##P < 0.01, ###P < 0.001 for 1 month data; n = 7-11/group for both time points. ND - Non-Diabetic control, D - Diabetic, D+I - Insulin-treated Diabetic, 1M - 1 month, 3M - 3 Months.
Mentions: A total of 27 targets from the microarray analysis were chosen for confirmation analysis including 15 from the Not Normalized, 5 from the Partially Normalized and 7 from the Normalized groups. While not comprehensive of all the genes observed with differential expression in the microarray analysis, the extensive qPCR confirmation experiments validate the quantitative accuracy of the discovery experiments. Twenty six of the 27 (96%) genes examined in this analysis were confirmed as being differentially regulated with diabetes in a statistically significant manner at the three month time-point (ANOVA, SNK post-hoc, P < 0.05). Eight of the 26 genes tested by qPCR were found to be differentially regulated after one month of diabetes. Using the qPCR data, genes were separated into different expression categories. Five were Not Rescued (Figure 4) and three were Rescued (Figure 5), with the remainder of genes dividing into four Not Prevented (Figure 6), four Partially Prevented (Figure 7), and 10 Prevented (Figure 8).

Bottom Line: A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes.Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.

ABSTRACT

Background: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.

Methods: The retina transcriptome (22,523 genes and transcript variants) was examined after three months of streptozotocin-induced diabetes in male Sprague Dawley rats with and without insulin replacement for the later one and a half months of diabetes. Selected gene expression changes were confirmed by qPCR, and also examined in independent control and diabetic rats at a one month time-point.

Results: Transcriptomic alterations in response to diabetes (1376 probes) were clustered according to insulin responsiveness. More than half (57%) of diabetes-induced mRNA changes (789 probes) observed at three months were fully normalized to control levels with insulin therapy, while 37% of probes (514) were only partially normalized. A small set of genes (5%, 65 probes) was significantly dysregulated in the insulin-treated diabetic rats. qPCR confirmation of findings and examination of a one month time point allowed genes to be further categorized as prevented or rescued with insulin therapy. A subset of genes (Ccr5, Jak3, Litaf) was confirmed at the level of protein expression, with protein levels recapitulating changes in mRNA expression.

Conclusions: These results provide the first genome-wide examination of the effects of insulin therapy on retinal gene expression changes with diabetes. While insulin clearly normalizes the majority of genes dysregulated in response to diabetes, a number of genes related to inflammatory processes, microvascular integrity, and neuronal function are still altered in expression in euglycemic diabetic rats. Gene expression changes not rescued or prevented by insulin treatment may be critical to the pathogenesis of diabetic retinopathy, as it occurs in diabetic patients receiving insulin replacement, and are prototypical of metabolic memory.

Show MeSH
Related in: MedlinePlus