May 19, 2000

Researchers track signals that lead to release of insulin

Featured Image

Dr. Harry Jacobson (left), Monroe Carell (fourth from left) and a host of children and families turned the dirt earlier this week to mark the groundbreaking for the new children’s hospital. (photo by Dana Johnson)

Researchers track signals that lead to release of insulin

Beta cells in the pancreas have a seemingly simple job — take in blood sugar and ship out insulin. Inside the cell, however, this apparently easy task involves a complicated series of steps to convert the captured sugar into the signal to release insulin.

Vanderbilt University Medical Center investigators have now used sophisticated imaging technology to follow glucose signaling inside beta cells. Their findings, published in the Proceedings of the National Academy of Sciences, lay the groundwork for understanding how beta cell signaling fails, leading to some forms of type II diabetes and its myriad complications.

"In terms of glucose (sugar)-induced insulin release, there's a big question about where the signal comes from," said David W. Piston, Ph.D., associate professor of Molecular Physiology and Biophysics and Physics. "It's really a big black box."

It is known that glucose enters beta cells and begins the cell's metabolic energy-generating pathways. The molecule ATP, the cell's energy currency, then plays a key role in the last steps before insulin is released.

But metabolic pathways that generate ATP exist in two places–free inside the cell cytoplasm and contained in the cell power plants called mitochondria, and it was unclear which pathways were important to glucose signaling.

Using a technique called "two-photon excitation microscopy," Piston and colleagues imaged the metabolism of glucose in living pancreatic beta cells and separated the energy generated in the cell cytoplasm from that produced by the mitochondria.

They found that the mitochondria dominate the total signal. And their data also suggest that the mitochondria require the smaller signal in the cytoplasm to be produced first.

"It looks like there is an extra regulatory step at the mitochondria," Piston said. "This could have a lot of impact, because people haven't been thinking about that possibility."

A regulatory step at the mitochondria might be impaired in the malfunctioning beta cells of type II diabetics and could possibly be a drug target, Piston said.

The signal that the researchers followed using microscopy was the fluorescence of NADH, one of the molecular byproducts of the metabolic reactions. Although NADH can be made to emit light that can be detected and measured, it requires the special technology afforded by two-photon excitation microscopy.

"Two-photon microscopy is fairly unique, and our instrument is specially built to image NADH," Piston said. "We're probably the only people who do a lot of NADH imaging, because it is difficult. NADH is desirable to follow, though, because it's a molecular reporter of the action. It's actually one of the players — you don't have to add anything foreign to the cell."

Because two-photon excitation microscopy uses infrared instead of ultraviolet lasers, it does less damage to cells or tissue than other forms of laser microscopy. This makes it advantageous for looking at fluorescence in living tissue, Piston said.

His group will now use the technique to examine glucose-induced responses in living pancreatic cells from diabetic mice. There are a number of different strains of knockout mice that develop diabetes, and the investigators will determine if any of them have an imbalance in mitochondrial versus cytoplasmic signaling in beta cells.

"We think that there will be disease pathologies that are associated with altered signaling," he said. "If we see a difference, we can start looking at how drugs might affect this imbalance.

"Our current findings really provide a new way to probe beta cell signaling."

Piston's colleagues include George H. Patterson and Susan M. Knobel in Molecular Physiology and Biophysics and Per Arkhammar and Ole Thastrup at BioImage in Denmark. The work was supported by the National Institutes of Health and the National Science Foundation.