F. Charles Brunicardi, MD: The Delta-to-Beta Cell Endocrine Axis

As academicians, we have three missions. One is to provide excellent clinical care. The next is to provide new treatments for disease through research. And our third mission is to train the next generation. I look at the Division of General Surgery laboratory as a training ground for those residents who are interested in going into academics. So I thought that I would present a project that was run entirely by surgical residents.

[ Slide 01 ]   The Endocrine Pancreas

The endocrine pancreas consists of 1 million islets that are hanging off the arterial system in parallel. When you see a change in glucose all 1 million islets respond almost simultaneously along with the brain. The brain then sends a neural signal that opens up the gates and you get a shunting of blood to the endocrine pancreas. There are also internal gates within the capillaries that open up and shunt blood to the center of the islet where the beta cells are that secrete insulin. I became very interested in cell-cell communication within the endocrine pancreas.

[ Slide 02 ]   Introduction

In particular, the current paradigm was that the somatostatin cell had no role within the islet and that made no sense because everywhere else that somatostatin is secreted, it has a paracine effect on inhibiting hormone secretion locally. It seems logical that somatostatin should inhibit insulin secretion within the islet.

[ Slide 03 ]   Isolated Perfuse Human Pancreas

To study the effect of intraislet somatostatin, the residents used a model called the isolated perfused human pancreas. This represents a human pancreas; the body and tail that has been harvested from a cadaver donor and brought back to the laboratory. The splenic artery is cannulated and perfused with a modified Kreb's buffer. The splenic vein is cannulated to collect the perfusate. The cut Surfaces are oversewn to prevent leakage and the pancreas is kept warm at 37°c

This is a basic standard physiologic prep, but in the human pancreas.

[ Slide 04 ]   Insulin and C-Peptide Secretion

We gave a very powerful monoclonal antibody to somatostatin. Essentially we removed somatostatin from the circulation. In response to the somatostatin monoclonal antibody, insulin C-peptide secretion went up. This was a very highly significant finding. We repeated this experiment seven different ways.

[ Slide 05 ]   Delta-to-Beta Cell Endocrine Axis

and we came up with a physiologic concept that there's a delta to beta cell endocrine axis within the human pancreas. It appears that the delta cell secretes somatostatin into the microcirculation. It then goes downstream and affects a receptor which then affects the beta cell secretion of insulin.

[ Slide 06 ]   Which Receptors Responsible?

We then wanted to examine which receptors were responsible for this effect. Shawn Fagan isolated mouse islets and exposed them to different antagonists

[ Slide 07 ]   Mean Percent Change in Insulin Secretion

and found that the agonist for the type 5 somatostatin receptor significantly inhibited insulin secretion. In the mouse model, it was a type 5 receptor that was responsible for mediating the delta to beta cell endocrine axis.

[ Slide 08 ]   RT-PCR of Mouse Pancreas

Shawn also did RtPCR of the mouse pancreas and found the presence of the SSTR 5 receptor, confirming our hypothesis.

[ Slide 09 ]   Somatostatin

In the mouse pancreas somatostatin was going downstream and affecting the type 5 receptor, causing inhibition of insulin secretion. In collaboration with Franco DeMayo, we decided to knock out this receptor. This work was done from by Dr. Stefan Moldovan, one of the residents in our program. The first step was to actually clone the gene to know it's exact sequence.

[ Slide 10 ]   Gene Ablation Steps: Gene Cloning

To to do the knockout, or gene ablation work, there are these five steps. First, you have to clone the gene so you know its exact sequence. You have to create a DNA targeting construct. You put that targeting construct into an embryonic stem cell and inject the embryonic stem cells into a blastocyst. The blastocyst is implanted into the uterus of a pseudo pregnant mouse to create chimeric mice that have the targeting construct. You breed those mice to get the gene ablated animals.

[ Slide 11 ]   Gene Sequencing

This was a three-year project that was done by Dr. Moldovan. His first step was to clone a gene and this process took one year of incredibly hard work. Once he had had the gene isolated, he then sequenced it. He did this by hand. Can you imagine 5,000 kilobases sequenced by hand? He found that the coding sequence was about 1,000 base pairs, the 5' flanking sequence was 3,000 and the 3' flanking sequence was 1,000 base pairs. The translated protein was 362 amino acids. The promoter was then cloned. We're doing some exciting work with the promoter of the SSTR5 gene.

[ Slide 12 ]   Sequence Homology

Dr. Moldovan then did a blast in which you compare the sequence of the gene to other known genes, and found there was an 80 percent homology to the human SSTR5 and a 92 percent homology to the rat SSTR5. The amino acid sequence had even a higher homology, therefore we concluded that we truly did have the mouse SSTR5.

[ Slide 13 ]   Gene Ablation Steps: DNA Targeting Construction

The next step was creating a DNA targeting construct.

[ Slide 14 ]   Targeting Construction

Although it's a very complex concept, I'll go through it as simply as I can. What you do is you take the SSTR5 gene and the 5' flanking sequences and replace it with the dummy gene. The body thinks that this is the whole SSTR5 gene, but the essential middle part of the gene has been knocked out of the construct.

[ Slide 15 ]   Gene Ablation Steps: Embryonic Stem Cells

You then take that construct and electroporate it into an embryonic stem cell.

[ Slide 16 ]   Embryonic Stem Cell Electroporated with New DNA

This is an embryonic stem cell that has been electroporated with the DNA construct. You grow the cells which is, once again, a very labor-intensive process. This whole process took Dr. Moldovan another year of hard work. These are called feeder cells in which the embryonic stem cell sits in culture on these feeders cells and actually uses them for nutrients.

[ Slide 17 ]   Gene Ablation Steps: Chimeras

Once you've created the embryonic stem cell, you then have to microinject it.

[ Slide 18 ]   Injection of Embryonic Stem Cells into Blastocyst - 1

And this is a spectacular set of slides. This shows you a blastocyst of a C57 mouse, which is held in place by a suction pipette. You take the embryonic stem cells and through a glass rod, you microinject them into the blastocyst. You start seeing the embryonic stem cells start filling the blastocyst cavity.

[ Slide 19 ]   Injection of Embryonic Stem Cells into Blastocyst - 2

This shows you the embryonic stem cells filling the blastocyst.

The blastocyst is then taken and implanted into a pseudo-pregnant mouse. This is done by creating a tract into the uterus using a larger needle

The blastocyst is implanted into the needle track of the uterus of the pseudo-pregnant mouse.

[ Slide 20 ]   Gene Ablated Mice

Once the mouse delivers, pups, they are screened for chimeras that are positive for the mutation. This is a chimera that has 1 gene which is missing the SSTR5 gene. You breed them with C57s and get a generation of heterozygotes. You take these mice and breed them to get mice that are totally missing the SSTR5. You have to take care and track the mouse colonies and it takes months to do these breedings.

[ Slide 21 ]   Mouse Screening

Once you think you have the mouse, you do PCR screening on the mice to find out which mice are missing the SSTR5. This 20 kilobase pair is the SSTR5 gene and this shows you the wild type which has the gene, the chimeras which also have it, and the knockouts which are missing the SSTR5 gene.

[ Slide 23 ]   Conclusion

We conclude that we developed SSTR5 ablated mice. This is a global knockout, model and the mice are missing the SSTR5 gene in their entire body. There are a lot of SSTR5 sites, including the brain, intestine, lung, and pancreas. We're currently studying these mice to determine whether there are alterations in phenotype.

[ Slide 22 ]   Delta to Beta Cell Endocrine Axis

We now have a mouse that is missing this receptor. One of the other residents in the lab, Dr. Tom Tirone, is setting up the perfused mouse pancreas, which is a difficult prep. He will perfuse the mouse pancreas the same way that we did the human pancreas to determine whether the delta to beta cell endocrine axis is altered when they're missing this receptor.

And I'd like to thank all the collaborators at the Baylor College of Medicine, especially Dr. Franco DeMayo, for his help with developing this project. But mostly I'd like to thank the surgical residents who really ran these projects and without their help, this work would not have been possible.

Thank you.