Role of vesicular monoamine transporter type 2 in rodent insulin secretion and glucose metabolism revealed by its specific antagonist tetrabenazine

Abstract

Despite different embryological origins, islet beta-cells and neurons share the expression of many genes and display multiple functional similarities. One shared gene product, vesicular monoamine transporter type 2 (VMAT2, also known as SLC18A2), is highly expressed in human beta-cells relative to other cells in the endocrine and exocrine pancreas. Recent reports suggest that the monoamine dopamine is an important paracrine and/or autocrine regulator of insulin release by beta-cells. Given the important role of VMAT2 in the economy of monoamines such as dopamine, we investigated the possible role of VMAT2 in insulin secretion and glucose metabolism. Using a VMAT2-specific antagonist, tetrabenazine (TBZ), we studied glucose homeostasis, insulin secretion both in vivo and ex vivo in cultures of purified rodent islets. During intraperitoneal glucose tolerance tests, control rats showed increased serum insulin concentrations and smaller glucose excursions relative to controls after a single intravenous dose of TBZ. One hour following TBZ administration we observed a significant depletion of total pancreas dopamine. Correspondingly, exogenous L-3,4-dihydroxyphenylalanine reversed the effects of TBZ on glucose clearance in vivo. In in vitro studies of rat islets, a significantly enhanced glucose-dependent insulin secretion was observed in the presence of dihydrotetrabenazine, the active metabolite of TBZ. Together, these data suggest that VMAT2 regulates in vivo glucose homeostasis and insulin production, most likely via its role in vesicular transport and storage of monoamines in beta-cells.

Figure 1. Tetrabenazine reduces the blood glucose excursion during IPGTT

Top Panel. Blood glucose values during IPGTT of Lewis rats treated with vehicle alone (open symbol) or with tetrabenazine at the indicated doses Error bars indicate the SEM (n=25) at the indicated dose (closed symbols). Bottom panel. Results presented as AUC IPGTT. The asterisk indicates AUC IPGTT for vehicle controls (n=25) was significantly higher than the AUC IPGTT of the same TBZ treated animals (n=25) (p < 0.05). The double asterisk indicates that the AUC IPGTT following treatment with TBZ and L-DOPA was significantly different than that of TBZ alone (n=6) Error bars represent S.E.M‥

Figure 2. TBZ reduces the dopamine content of brain and pancreas tissue

Tetrabenazine at 1.5 mg / Kg body weight was administered i.v. to Lewis rats. One hour later, the animals were euthanized and the brains and pancreata harvested and extracted in buffer. The dopamine concentration in the extract was determined by ELISA and normalized to the total protein content. The error bars represent the S.E.M. from measurements of three TBZ treated and three control Lewis rats. An asterisk represents a significant difference ( p < 0.05) from control‥

Figure 3. Tetrabenazine alters glucose stimulated insulin and glucagon secretion in vivo

Plasma insulin (panel A and C) and glucagon concentrations (panel B and D) were measured during IPGTT of Lewis rats (>11 weeks old) (n=6) treated with vehicle alone (open columns and circles). One week later, a second IPGTT was performed with TBZ (2.25 mg/Kg body weight)(filled columns and circles), TBZ (2.25 mg/Kg body weight) plus dopamine (6.0 mg/Kg), or following five daily injections TBZ (0.3 mg/Kg body weight). An asterisk represents a significant difference (p < 0.05) from control.

Figure 4. DTBZ enhances Glucose Stimulated Insulin secretion in rat islets ex vivo

Hand picked purified islets were cultured in high or low glucose containing media with and without DTBZ. Serial Insulin concentration measurements in the supernatant were performed and the means and S.E.M. calculated. An asterisk represents a significant difference ( p < 0.05) from control.

Figure 5. VMAT2 mRNA and protein in islets and pancreas of control and streptozotocin treated Lewis rats

Panel A, products of the qRT-PCR assay on total RNA from: brain, purified islets and total pancreas using VMAT2 primers that specifically amplify a 175 bp fragment. Untranscribed RNA from purified islets is used as a control.and GelPilot 200 bp ladder, as the molecular weight standard. Panel B, Relative accumulation of VMAT2 mRNA in pancreata of control and streptozotocin-induced diabetic Lewis rats. The average accumulation of VMAT2 mRNA in streptozotocin-treated rodents was approximately eight-fold lower than the average accumulation in untreated pancreata (p<0.005) Panel C, Western blot analysis of VMAT2 expression in protein lysates prepared from control and streptozotocin-induced diabetic Lewis rats.