The Phelps lab at the University of Florida used the Eicom HTEC-500 HPLC-ECD to determine the time-resolved secretion of GABA, taurine, and other amino acids from pancreatic islet beta cells. This helped them show:

• GABA in human beta cells is mostly stored in the cytosol, not in vesicles.
• An ion channel (VRAC) handles GABA secretion from beta cells.
• Pulsatile release of GABA synchronizes and regulates the secretion of insulin from islets.
• The depletion of GABA reserves in beta cells correlates with both type 1 and type 2 diabetes in human patients.

Roles of GABA

GABA, short for gamma-aminobutyric acid, has many roles in biology. It is best known as an inhibitory neurotransmitter in the nervous system. In fact, impaired clearance of GABA in the amygdala may contribute to alcoholism. GABA also acts to inhibit immune responses and regulates the secretion of hormones in the pancreas.

How GABA, Insulin, and Glucagon are related to diabetes.

Insulin is secreted in response to high blood sugar levels. It directs cells throughout the body to take up glucose from the blood, thus lowering blood sugar levels. As insulin and blood sugar levels drop, glucagon is secreted. Glucagon directs the liver to convert glycogen into glucose and release it. Bringing blood sugar levels back up and balancing the action of insulin. Beta cells secrete both of these hormones in pancreatic islets. The secretion of both of these hormones is regulated in part by GABA, which is also secreted by beta cells. In type 1 diabetes an autoimmune response attacks and destroys beta cells. But this immune response can be damped by the GABA secreted by beta cells. Administering GABA as a drug not only protects beta cells, it can also cause other pancreatic islet cells (alpha cells) to convert into beta cells. Helping to restore the beta-cell population. Because of this protective role, GABA is already being studied as a treatment for diabetes by the Swedish biotech Diamyd. With clinical trials led by Prof. Kenneth McCormick at the University of Alabama.

How is GABA secreted in the pancreas?

In the brain, secretory vesicles are responsible for storing and then releasing GABA from neurons. This same mechanism was long thought to be at work in the pancreas as well. Yet, the GABA transporter proteins necessary for this mechanism remained elusive in beta cells. Leaving a gap in the understanding of how GABA release occurs. Prof. Edward Phelps previously observed that instead of being held in vesicles, the bulk of GABA is loose within the cytosol of beta cells. This meant that another mechanism could be in play. He and his student Walker Hagan hypothesized that an ion channel, specifically VRAC (Volume Regulatory Anion Channel), could be responsible.

“There is a channel between the interior of the beta cell and the extracellular space, which we thought was worth investigating,” Phelps said. “The volume regulatory anion channel (VRAC) is known for another purpose. It is used to help cells maintain their shape by keeping the osmotic pressure inside and outside the cell in equilibrium. When this balance is disturbed and the cell shape changes, small organic chemicals known as osmolytes are expelled from the cell via the VRAC channel to help the cell regain its shape. When we artificially opened this channel in beta cells using low saline, we found that this channel also transports GABA.”

Changes in cell volume had already been implicated as a trigger for insulin secretion. Further supporting the possibility that VRAC channels could be the principal mechanism for GABA secretion.

Measuring GABA secretion in the pancreas

The Phelps lab used their HTEC-500 HPLC-ECD system to measure GABA and other amino acids in three phases of this project.

First, they confirmed that human pancreatic islets from both type 1 and type 2 diabetic donors are deficient in GABA when compared to those of non-diabetics. Then they measured GABA in perifusate from islets cultured in a Biorep perifusion system. Perifusion washes media though a small group of islets and allows recovery of the perfusate as time-resolved fractions. This way conditions can be varied and resulting changes in the secreted amino acids and hormones can be monitored over time. By varying the media washing through the islets, The Phelps lab was able to confirm that hypotonic media triggers the release of GABA from beta cells. Consistent with VRAC being responsible for secretion. Perifusion also allowed them to study the time-resolved relationship between GABA release and insulin secretion.

Finally, they created a knockout strain of mice unable to express the VRAC channel in beta cells: (βc-LRRC8A−/−). The beta cells in these mice are missing one of the subunits (LRRC8A) of VRAC, and no longer secreted GABA in response to osmotic stress. This lent further support to the role of VRAC in GABA release.

Representative HPLC chromatograms from the supernatant of a culture of 200 pancreatic islets. Hypotonic media opens VRAC channels as the cell attempts to relieve osmotic stress. Allowing the release of both GABA and taurine from islets into the supernatant (blue). VRAC channels remain closed in Isotonic media (gray). Resulting in lower concentrations of GABA and taurine in the supernatant. Samples were injected onto an Eicom HTEC-500 equipped with an FA-3ODS column, used for separating GABA, Glu, and other amino acids. An AS-700 autosampler was used to automate both derivatization and subsequent injection of the samples. Data courtesy of the Phelps lab.

Levels of GABA and Taurine released into the supernatant of islets suspended in low saline (hypotonic) and isotonic (3G) media. Figure courtesy of the Phelps lab.

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The Phelps lab research is featured on the cover of the November 2019 issue of Nature Metabolism

Menegaz, D., Hagan, D.W., Almaça, J. et al. Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell. Nat Metab 1, 1110–1126 (2019) doi:10.1038/s42255-019-0135-7