MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia
Rahul Agrawal, Griffin Durupt, Dinesh Verma, Michael Montgomery, Adriana Vieira-de Abreu, Casey Taylor, Sankar Swaminathan, Simon J. Fisher
Rahul Agrawal, Griffin Durupt, Dinesh Verma, Michael Montgomery, Adriana Vieira-de Abreu, Casey Taylor, Sankar Swaminathan, Simon J. Fisher
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Research Article Endocrinology

MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia

Abstract

It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3′-UTR of GABAA receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.

Authors

Rahul Agrawal, Griffin Durupt, Dinesh Verma, Michael Montgomery, Adriana Vieira-de Abreu, Casey Taylor, Sankar Swaminathan, Simon J. Fisher

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Figure 3

Schematic timeline representing study design for lentiviral-mediated microRNA manipulation in the VMH and hypoglycemic clamps.

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Schematic timeline representing study design for lentiviral-mediated mic...
(A) Male Sprague-Dawley rats were subjected to either artificial cerebrospinal fluid (aCSF) or lentivirus (containing either control or targeted microRNA sequence) injection into the VMH by using a stereotaxic frame. Three weeks following intra-VMH injection, all rats subsequently had vascular catheters implanted under ketamine anesthesia. Four days following vascular surgery, rats that received aCSF into the VMH were subjected to either RS or RH for 3 days, while rats that received lentiviruses into the VMH were subjected to the RH only. Following overnight fasting (i.e., after 4 weeks of intra-VMH injection), all rats were subjected to the hyperinsulinemic (20 mU/kg/min) hypoglycemic (40–50 mg/dL for 90 minutes) clamps for assessing counterregulatory hormonal response in plasma. (B and C) Blood glucose levels (mg/dL) during the 3-day protocol in RS rats treated with intra-VMH aCSF-RS and RH rats treated with either aCSF-RH or control lentivirus (lenti-7a-cont-RH or lenti-665-cont-RH) or microRNA-encoding lentivirus (lenti-miR-7a-RH or lenti-miR-665-RH). Data are expressed as mean ± SEM (n = 6–7/group).