Diabetes. 2014 Oct;63(10):3545-56. doi: 10.2337/db13-1562.

Blockade of Na+ channels in pancreatic α-cells has antidiabetic effects.

Dhalla AK1, Yang M2, Ning Y2, Kahlig KM2, Krause M2, Rajamani S2, Belardinelli L2.
  • 1Department of Biology, Cardiovascular Therapeutic Area, Gilead Sciences, Inc., Fremont, CA arvinder.dhalla@gilead.com.
  • 2Department of Biology, Cardiovascular Therapeutic Area, Gilead Sciences, Inc., Fremont, CA.

 

Abstract

Pancreatic α-cells express voltage-gated Na(+) channels (NaChs), which support the generation of electrical activity leading to an increase in intracellular calcium, and cause exocytosis of glucagon. Ranolazine, a NaCh blocker, is approved for treatment of angina. In addition to its antianginal effects, ranolazine has been shown to reduce HbA1c levels in patients with type 2 diabetes mellitus and coronary artery disease; however, the mechanism behind its antidiabetic effect has been unclear. We tested the hypothesis that ranolazine exerts its antidiabetic effects by inhibiting glucagon release via blockade of NaChs in the pancreatic α-cells. Our data show that ranolazine, via blockade of NaChs in pancreatic α-cells, inhibits their electrical activity and reduces glucagon release. We found that glucagon release in human pancreatic islets is mediated by the Nav1.3 isoform. In animal models of diabetes, ranolazine and a more selective NaCh blocker (GS-458967) lowered postprandial and basal glucagon levels, which were associated with a reduction in hyperglycemia, confirming that glucose-lowering effects of ranolazine are due to the blockade of NaChs. This mechanism of action is unique in that no other approved antidiabetic drugs act via this mechanism, and raises the prospect that selective Nav1.3 blockers may constitute a novel approach for the treatment of diabetes.

PMID: 24812428

 

Supplement

Glucose homeostasis is regulated primarily by the opposing actions of insulin and glucagon. Glucagon secreted by pancreatic α-cells regulates glucose levels by its effects on hepatic glucose production. Plasma glucagon levels are increased under both fasting and postprandial states in Type 2 diabetes and significantly contribute to hyperglycemia. Although this has been known for some time, the mechanisms of hyperglucagonemia in diabetes are still poorly understood. However, it has been shown that lowering of glucagon levels or antagonizing its actions via blockade of glucagon receptors can significantly reduce hyperglycemia (1, 2) and therefore can be potential strategies for treatment of patients with type 2 diabetes. Our current study has identified a novel mechanism to directly inhibit glucagon secretion from pancreatic α-cells.

It is known that pancreatic α-cells express tetrodotoxin (TTX)-sensitive sodium (Na+) channel isoforms and Na+ channel blockers like TTX inhibit glucagon secretion (3). Ranolazine, an anti-anginal drug, is a Na+ channel blocker with IC50 of 12 µM for Nav1.3 Na+ current (INa). In clinical and non-clinical studies, ranolazine has been shown to lower HbA1c, glucose and glucagon levels (4, 5). We hypothesized that inhibition of glucagon secretion from pancreatic islets via blockade of Na+ channels in α-cells can have anti-diabetic effects.

First we performed experiments to test the direct effects of various Na+ channel blockers (ranolazine, GS-458967 and TTX) on glucagon secretion in pancreatic islets in vitro. In human and rat pancreatic islets, treatment with ranolazine, selective Na+ channel blockers GS-458967 (IC50: 0.6 µM for Nav1.3 INa) or TTX significantly reduced glucagon secretion. In contrast, veratridine (a Na+ channel activator) significantly increased glucagon secretion. These data show that Na+ channels are functionally coupled to glucagon secretion. The veratridine-induced increase in glucagon secretion can be significantly reduced by ranolazine, GS-458967 and TTX in both human and rat islets, indicating that effect of ranolazine is mediated through blockade of Na+ channels in pancreatic α-cells. Indeed, ranolazine and GS-458967 inhibited INa and spontaneous electrical activity in rat pancreatic α-cells. Ranolazine and GS-458967 also inhibited veratridine-evoked electrical activity in rat pancreatic α-cells. The effects of ranolazine and Na+ channel blockers on glucagon secretion was further demonstrated in in vivo proof-of-concept studies in models of STZ-diabetic rats and ZDF rats. Treatment with ranolazine and GS-458967 significantly reduced postprandial hyperglucagonemia and glucose levels in STZ-diabetic rats in an oral glucose tolerance test. Long-term treatment with ranolazine and GS-458967 (over 4 weeks) also significantly reduced fasting plasma glucagon, fasting glucose and HbA1C in ZDF rats. In addition, long-term treatment with ranolazine and GS-458967 normalized the β to a cell ratio and improved pancreatic islet morphology compared to vehicle treatment (Figure 1).

 

AD fig1

Figure 1: Hematoxylin and Eosin (H/E) staining and fluorescent (Fluo) staining of pancreatic islets from lean and ZDF rats treated with ranolazine, GS-458967 or sitagliptin for 8 weeks. In H/E staining, treatment with ranolazine, GS-458967 or sitagliptin (a DPP-IV inhibitor) preserved islet structure compared to that treated with vehicle. In fluorescent staining, treatment with ranolazine or GS-458967 decreased the population of α-cells (green) and increased the population of β-cells (red) in islets compared to that treated with vehicle. This effect of Na+ channel blockers is comparable to that of sitagliptin.

 

Next we identified the Na+ channel isoforms responsible for glucagon secretion in pancreatic α-cells. Results of gene expression profiling of Na+ channel isoforms showed that Nav1.3 is the only isoform expressed in rat islets, suggesting that Nav1.3 is the isoform responsible for Na+ channel-mediated glucagon secretion in rat islets. In human islets, the major isoforms expressed are Nav1.3 and Nav1.7. To determine which isoform mediates glucagon secretion, we used gene silencing and loss-of-function approaches. Specific knockdown of Nav1.7 reduced gene expression by 55%, but had no impact on veratridine-induced glucagon secretion. In contrast, specific knockdown of Nav1.3 reduced gene expression by 65%, and glucagon secretion was reduced by more than 60% in the presence of different concentrations of veratridine. These data suggest that Nav1.3 is the isoform responsible for Na+ channel-mediated glucagon secretion in human islets. The possible mechanism of Na+ channel-mediated glucagon secretion in pancreatic α-cells is illustrated in a diagram in Figure 2.

 

AD fig2

Figure 2: Potential mechanism of Nav1.3-mediated glucagon secretion from pancreatic α-cells. The influx of Na+ through NaV1.3 depolarizes the plasma membrane to activate vesicular release of glucagon. Vm: Membrane potential, CaT: T-type calcium channel CaL: L-type calcium channel, KATP: ATP sensitive potassium channel, KDR: Delayed rectifier potassium channel, KCa: Calcium activated potassium channel.

 

Collectively, our results demonstrate that the anti-diabetic effect of ranolazine is likely mediated through inhibition of glucagon secretion by blocking Nav1.3 in pancreatic α-cells. Blocking Nav1.3 in pancreatic α-cells may represent a novel therapeutic approach for diabetes.

 

References

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