Research initiatives for turning the tide on diabetes are directed by Jerry Nadler, MD and Aaron Vinik, MD, PhD and an expansive team of researchers at EVMS Strelitz Diabetes Center with worldwide collaborations.

A Marriage of Two Proteins: INGAP (Islet NeoGenesis Associated-Protein)­ and LSF (Lisofylline) as a Possible Cure for Diabetes

History of INGAP

A New Target for Preventing Beta Cell Destruction - 12-Lipoxygenase

Role of Obesity and Inflammation in Fat Tissue in Leading to Diabetes

Cardiovascular Disease Due to Diabetes

Reversing Complications of Diabetes

Stem Cell Research for Diabetes

Neuroendocrine Tumors

A Marriage of Two Proteins: INGAP (Islet NeoGenesis Associated-Protein)­ and LSF (Lisofylline) as a Possible Cure for Diabetes

The Strelitz Diabetes Center’s (SDC) announcement in April, 1997 of the discovery of the INGAP gene by Aaron I. Vinik, MD, PhD and his research team caused international excitement among scientists (INGAP is the acronym for Islet Neogenesis-Associated Protein). INGAP is able to regenerate insulin-producing cells but on the other hand, the autoimmune process which characterizes type 1 diabetes, continues on its destructive course. In July 2008, Jerry Nadler, MD brought his research program geared to stop autoimmune damage and inflammation of cells to Eastern Virginia Medical School (EVMS) and the Strelitz Diabetes Center’s Research Program. Dr. Nadler pioneered a drug called Lisofylline (LSF), which prevents destruction of insulin producing cells. What a great pairing of INGAP and Lisofylline in the search for the cure and turning the tide on diabetes!

The research team continues to investigate genes and protein products that will cause pancreatic islet cells to grow (neogenesis) and make insulin, as well as identify newer medications that prevent the destruction of islet cells.

History of INGAP

After cloning and sequencing INGAP, the product was first licensed to Eli Lilly. Not satisfied with the ability of INGAP to cause remission of artificially-induced diabetes in 40% of animals, Eli Lilly withdrew and EVMS licensed the peptide to Global Medical Products (GMP) Companies who had no reservation in aggressively pursuing clinical studies in both Type 1 and Type 2 diabetes. These multicenter studies were carried out by some of the most highly recognized investigators in the country; the results showed that even in Type 1 diabetes (totally depleted of C-peptide, a measure of islet function), an increase in C- Peptide could be induced after 3 months of treatment. In fact, the effect persisted after cessation of INGAP administration and there was a reduction of about 0.8 % in HbA1c. In Type 2 diabetes INGAP was able to stimulate an increase in C-Peptide and reduce HbA1c levels by about 1%. These findings led Dr. Robert Ratner, the Principal Investigator in the trials, to conclude, “With appropriate adjustment of dosage, site of administration and duration of therapy this approach holds promise to evolve into a new therapeutic approach to Type 1 and Type 2 diabetes.” The results of the study have been published in Diabetes Metabolism Research Reviews, 2009 Sep;25(6):558-65. Additionally, a second Phase 2 study in Type 2 diabetes has been initiated at Mayo Clinic.

Meanwhile, researchers at the SDC elected to pursue probing further into the induction of islet cell neogenesis, focusing on investigating growth factors that initiate growth, proliferation, and differentiation of islet cells using a molecular biology approach to investigate genes that are activated during the process of islet cell regeneration. These studies have been conducted by Gary Pittenger, PhD and David Taylor-Fishwick, PhD. The researchers also pursued a protein chemistry approach to investigate the growth factor proteins that stimulate the growth, proliferation, and differentiation of ductal epithelial cells. They were able to show that INGAP induces a protein (known as pancreatic transcription factor PDX) that binds to specific DNA sequences. PDX is necessary not only for islet development but also for beta cell function. Dr. Taylor-Fishwick discovered that PDX had a “recognition site” in the INGAP gene, which could also turn off the expression of INGAP. This demonstrated the presence of a short feedback inhibition loop that would protect unbridled growth of islets. The research also showed that INGAP was expressed during fetal development and that it joined the cluster of factors necessary for growth and reproduction of pancreatic beta cells. In addition, these molecular studies demonstrated that other small molecules like LIF (Leukemia Inhibitory Factor) bound to the INGAP gene promoter and was capable of inducing its expression, raising the hope that in the future small molecules could be used to stimulate INGAP production in diabetes.

A new model for INGAP was shaped when, in collaboration with Dr. David Harlan at the National Institutes of Health (NIH), the INGAP gene was inserted into the pancreas of several “transgenic” animals, created as carriers to transfer the genetic material. The insertion of INGAP was driven by “promoter” genes - the elastase promoter or the insulin promoter. Dr. Taylor-Fishwick demonstrated that the site of expression of INGAP is critical to its beneficial effects and that the most profound effect occurred when INGAP was driven by the elastase promoter and expressed in the acinar (cluster) cells in the pancreas. Not only could this reverse diabetes but it was shown to prevent chemically-induced diabetes, using Streptozotocin (STZ). This raised the possibility that INGAP may become a prevention agent for diabetes.

Additionally, Dr. Pittenger has been funded to determine if a combination of INGAP with a Glitazone or a DPP1V inhibitor will inhibit apoptosis and is a potential for preventing loss of the beta cell mass in type 2 diabetes. The animal model used is the transgenic mouse expressing INGAP in the acinar tissue and the fat fed mouse which resembles type 2 diabetes.

The exciting bonus to this work occurred when Dr. Nadler, while at University of Virginia, combined INGAP with the anti inflammatory agent Lisofylline (LSF) in a pretreatment paradigm and showed that diabetes could be reversed in approximately 70% of the time in the notoriously resistant NOD mouse which develops an aggressive form of autoimmune diabetes. The anti-inflammatory compound, LSF inhibits pathways associated with destructive autoimmunity. In combination, LSF and INGAP can reverse new onset Type 1 diabetes in NOD mice. The research team has also identified, in a synthesized chemical library, novel analogs of LSF that have enhanced efficacy and improved drug properties. LSF, in a new subcutaneous formulation, has now completed a phase I/II trial in healthy normal subjects and people with Type 1 diabetes. We are now poised for a clinical trial of INGAP + Lisofylline in Type 1 diabetes in humans! There are also plans are to initiate a trial of LSF alone to halt beta cell damage in newly diagnosed people with Type 1 diabetes.

As this work continues, the SDC’s research team has also formed research collaborations with scientists from around the world for the development of combination approaches to lead to safe islet cell regeneration as a means of treating diabetes. The research evaluating the combination of LSF and INGAP has been funded by the ADA and Iacocca Foundations.

A New Target for Preventing Beta Cell Destruction - 12-Lipoxygenase

The on-going autoimmunity (self–destruction of cells), which is typical of type 1 diabetes, prevents the body from effectively retaining newly-produced functional beta cells. There is an enzyme called 12/15-lipoxygenase (12/15-LO) which is present in beta cells and immune cells called macrophages. Fats produced by 12/15-LO are extremely high promoters of inflammation, linked to diseases and can lead to damage and death of pancreatic beta cells. When the gene that produces 12/15-LO is deleted (removed by a laboratory process), almost 100% protection from type 1 diabetes development in female non-obese diabetic (NOD) mice - a model for type 1 diabetes in humans - occurs. Therefore, targeting 12/15-LO expression at the appropriate time could offer a novel therapeutic approach to allow functional regeneration of beta cells in type 1 diabetes. This notion will be moved from concept to reality using a multidisciplinary approach with experienced beta cell biology investigators at the EVMS Strelitz Diabetes Center in collaboration with scientists in other academic centers. Margaret Morris, PhD a scientist with the SDC and Dr. Taylor-Fishwick are carrying out studies to identify the way that deletion of 12/15-LO gene protects beta cells.

There is also new evidence showing that human islets exposed to the same inflammatory lipids (fats) will trigger (up-regulate) an increase of 12/15-LO, inducing beta cell apoptosis (cell death) in type 1 diabetes. Development and use of drugs to reduce 12/15-LO activity could offer an innovative approach to allow functional regeneration and prevent progression to fully developed beta cell destruction. Kaiwen Ma, PhD is also collaborating with the excellent proteomics group at EVMS to study how 12/15-LO damages beta cells.

The hypothesis is that continued activation of 12/15-LO leads to progressive decline in beta cell function and mass. In addition, use of a 12/15-LO inhibitor will reduce beta cell apoptosis and protect beta cells from inflammatory damage. Currently there are no suitable 12/15-LO inhibitors available for pre-clinical or human testing. Jerry Nadler, MD, Swarup Chakrabarti, PhD and David Taylor-Fishwick, PhD are collaborating with a medicinal chemist at the University of California and the National Institutes of Health to develop several new 12/15-LO inhibitors to move forward in clinical testing.

To facilitate these investigations, Dr. Nadler has competitively applied for and received human pancreatic islet cells, as well as fixed and frozen human pancreatic and spleen tissue. These samples are provided by the Juvenile Diabetes Research Foundation (JDRF) and the Network for Pancreatic Organ Donors with Diabetes (nPOD), respectively. Dr. Nadler has recently been added as one of the 30 diabetes investigators in the world to be part of the nPOD JDRF program to obtain rare and difficult to obtain tissues to further his important research.

Yumi Imai, MD, has studies underway to elucidate additional targets for the prevention and treatment of type 2 diabetes, the most common form of diabetes. Dr. Imai is funded by National Institute of Diabetes, Digestive, and Kidney Diseases to clarify the contribution of neuropeptide Y in the development of Type 2 diabetes in obesity.

Role of Obesity and Inflammation in Fat Tissue in Leading to Diabetes

EVMS investigators are conducting studies focused on understanding the mechanism by which bariatric surgery improves diabetes and cardiovascular risk factors. They are also conducting the first study to determine the specific characteristics of the inflammatory cells in the fat tissue and in the circulation of obese individuals - with and without - Type 2 diabetes. Dr. Nadler is collaborating in this study with two EVMS investigators, Elena Galkina, PhD and Anca Dobrian, PhD , as well as a physician at the Strelitz Diabetes Center, David Lieb, MD.

The research aims address the key role of inflammation and immune cell activation in subjects undergoing both common forms of bariatric surgery, (Roux-en-Y and Gastric Banding). The overall hypothesis is that adipose tissue and immune cell-related inflammation are linked to the occurrence of Type 2 diabetes and cardiovascular disease (CVD) in obesity. Further, an important part of the mechanism by which specific gastric peptides induce remission of type 2 diabetes after the Roux-en-Y bariatric surgery, is “normalizing” the inflammatory state. We suggest that gastric bypass surgery will change those physical or biochemical characteristics (phenotype) in the diabetic patient by reducing the fat (adipose) cells under the skin and surrounding the internal organs that stimulate inflammation and activate the immune cells that lead to disease.

Recognizing and studying the mechanisms of the inflammatory and immune cells in fat tissue and in the circulation should help to identify specific factors that could be targeted for innovative therapies to treat diabetes and prevent cardiovascular risk and mortality.

Cardiovascular Disease Due to Diabetes

Almost 80% of people with diabetes die due to cardiovascular disease (CVD). Given the current epidemic of diabetes, the prevalence of CVD will become a greater problem. Dr. Nadler is tackling this problem in collaboration with SDC investigators, Dr. Chakrabarti and Banumathi Cole, PhD and other basic scientist at EVMS, Anca Dobrian, PhD and Elena Galkina, PhD. The research is funded by a major project in a Program Project grant funded by the National Heart Lund and Blood Institute, NIH. The role of obesity, inflammation and immune cell activation is being tested in new animal models that simulate human disease. New therapeutic targets have been identified and the goal will be to develop new treatment to prevent this major complication. Dr. Nadler and Joseph Aloi, MD, Clinical Director of the Strelitz Diabetes Center, are also closely collaborating with cardiologists at Sentara Heart Hospital to identify improved ways to treat people with diabetes who are at risk of CVD.

Reversing Complications of Diabetes

Neuropathy

Research on reversing complications of diabetes at the EVMS Strelitz Diabetes Research Center focuses on basic science in the laboratory, as well as, clinical neuropathy research projects; laboratory research investigates the causes of nerve cell death, while clinical research evaluates preventative measures and tests new treatments.

Dr. Aaron Vinik’s research team at the Strelitz Diabetes Research Center has been extensively funded by the National Institutes of Health (NIH), American Diabetes Association (ADA), Housing and Urban Development (HUD) as well as several industries interested in supporting investigational research into the complexity of neuropathy, and its many causal factors. Research studies are geared to finding out about those factors that contribute to the causes of neuropathy and to tailor therapies to the specific cause. The Strelitz Diabetes Center’s researchers are investigating neuropathy (nerve damage) in six initiatives:

• Preventing programmed nerve cell death - a process called apoptosis - in neuroblastoma cells (developing malignant nerve cells) by investigating the possible immunological factors that cause nerve cell death
• Reversing nerve damage through the use of drugs that reduce accumulation of sugar alcohols or drugs that supply needed fatty acids
• Regenerating nerves through the application of growth factors, such as Vascular Endothelial Growth Factor (VEGF), Nerve Growth Factor (NGF) and Neurotrophin 3Ascertaining the cause and effect of hyperglycemia and other elements of the metabolic syndrome, e.g. dyslipidemia
• Determining the role of the small blood vessel system (microvasculature) that supplies blood to the vas nervorum of the nerve fibers, in particular nitric oxide endothelial derived hyperpolarizing factor, prostaglandins and neuropeptide
• Investigating the ability to influence nerve function by different forms of exercise
• Testing new combination pain medications in alleviating chronic painful diabetic neuropathy

The unit has been remarkably successful in obtaining grants to carry out these studies. In collaboration with Dr. Elsa Strotmeyer at the University of Pittsburgh funded by NIH, studies have been undertaken to demonstrate that the increased predisposition to fractures in the elderly (15x the young) is not due to osteopenia but due to falling - a consequence of large fiber neuropathy. In addition, the presence of neuropathy was a contributing factor to the development of osteopenia, further compounding the problem. This raises interesting prospects for trying to prevent falls and fractures, which exert a huge impact on morbidity and mortality in the old.

In collaboration with Sheri Colberg, PhD at Old Dominion University, funded by the American Diabetes Association, researchers at the Strelitz Diabetes Center have shown that there is a profound defect in microvascular perfusion (ability for blood to circulate through the blood vessels) in people with diabetes, which may contribute to the 96,000 amputations each year in the USA. They showed that exercise improves circulation but does not cure the defect.

Also in collaboration with Steve Morrison, PhD of Old Dominion University, the Strelitz Diabetes Center clinical research group was funded by a developmental grant from Old Dominion University to implement a program of gait, strength and balance training to prevent falls in people with diabetes and the elderly, to reduce the risk of falls and fractures.

In collaboration with Kara Witzke, PhD, California State University, San Marcos funded by a Virginia Commonwealth grant, diabetes researchers were able to show that the normal defense mechanisms present in the circulation and capable of “mopping up” the end products of advanced diabetes that cause damage to nerves, are deficient in people with a type of neuropathy, called “Charcot” neuropathy. This is the first and most exciting development in this previously unknown condition, and suggests that with the administration of the missing defense mechanisms called sRAGE, fractures could be prevented and/or treated.

Henri Parson, PhD, Director of the Clinical Research Laboratory has received funding with Dr. Vinik as Principal Investigator, (PI) to study the factors that contribute to the disturbed vascular function in patients with neuropathy. Perhaps her most striking finding is the fact that there is a profound defect in skin (cutaneous) blood flow in diabetic neuropathies. This appears to be mediated by defective prostaglandin and endothelial-derived hyperpolarizing fact and not by the traditional nitric oxide, leading to the possibility of a new form of increasing blood flow by relaxing smooth muscle cells within the blood vessel (vasodilatation) to protect the feet of people with diabetic neuropathy.

Dr. Parson’s newest findings show that the African American population, which has the highest rate of amputations in the USA, also has compromised blood flow even without diabetes and that superimposition of diabetes in this environment has disastrous consequences. Funding has now been secured to determine the genetic susceptibility to compromised blood vessels and to explore pathways in the prostaglandin synthetic pathways, which so far has provided the greatest yield.

Funding has been received in a series of investigator-initiated studies, yielding reports on the effects of many candidate genes in the pathogenetic pathway but so far none have provided the ultimate solution to the treatment or prevention of neuropathy. However, our recognition that oxidative stress was a key player in the development of neuropathy led to the development of a mixture know as NutriNerve, licensed by Eastern Virginia Medical School to Neuroeffex, a company that is now selling a product capable of preventing the progression of neuropathy as well as in some cases achieving a reversal.

Perhaps one of the most valuable findings in the laboratory was the discovery that autoimmune mechanisms were able to damage nerves and that the metabolic syndrome causes damage to small intraepidermal nerve fibers before the advent of hyperglycemia. This has led to the use of Topiramate to reverse certain neuropathies, the use of intravenous immunoglobulin and Enbrel with somewhat astounding results, as well as a particularly salutary reward for the physician scientist in recognizing the possibility of a therapy and then applying it in the clinic - the most rapid form of translational research.

A recent finding on neuropathy studies funded by a grant from the American Diabetes Association in Dr. Parson’s and Dr. Pittenger’s laboratories is the very large increase in Langerhans cells in the skin of patients with diabetic neuropathy. These cells are similar to the macrophages referred to in the islet and adipose tissue in the studies by Dr. Nadler and his team. The Langerhans cells may have a significant impact on nerve damage and may be prove to be a means of abrogating the inflammation which may be a consequence of neuropathy.

In collaboration with Irina Obrosova, PhD at the Pennington Institute for a pending National Institutes of Health grant, the neuropathy team is studying the role of Nitrosative stress on nerve damage with the potential of developing new compounds to treat or prevent neuropathy. These studies illustrate that too much of a good thing, in this case Nitric Oxide (NO) can actually do harm. When the excess NO combines with reactive Oxygen species it leads to the formation of Peroxynitrite, which is highly damaging to nerves and blood vessels.

Dr. Nadler’s group is also collaborating with scientists in Dr. Vinik’s group and Dr. Obrosova on a National Institutes of Health grant to develop new ways to prevent inflammatory damage to nerve cells.

Last, but not least, is concern for the quality of life (QOL) of the individual with neuropathy. With the involvement of Etta Vinik,MA (Ed) the Associate Director of the Education Unit of the Strelitz Diabetes Center, a quality of life (QOL) questionnaire has been developed, sensitive to the specific nerve fiber deficits. This tool has been translated into seventeen different languages and licensed five times to different companies or institutions and is now “The Gold Standard” of QOL endpoints in diabetic neuropathy trials.

Neuroendocrine Tumors

In the Neuroendocrine Tumor (NET) world, in collaboration with Drs. Woltering, Go, and O’Dorisio, in medical centers around the country, a new finding has emerged that the breakdown in controlling the symptoms of patients with Carcinoid tumors is due to inadequate delivery of somatostatin into the body, using the long acting analog (LAR). This group of researchers has also pioneered the development of new assays for Chromogranin A, LAR, Pancreastatin, all of which have proved to be useful diagnostic criteria and are the most useful markers for prognosis and progression of the tumor. Dr. Vinik’s group has received funding to study the effects of tyrosine kinase inhibitors as well as different somatostatin analogs and the mTOR inhibitor drugs. Because of our concern for the quality of life (QOL) of patients with neuroendocrine tumors, as in the case of patients with neuropathy, a QOL questionnaire sensitive to the specific symptoms of the disease has been developed and validated with the involvement of Etta Vinik, MA (Ed). The article on the development of the questionnaire, designed to measure patient-reported QOL, has been published in the journal Pancreas.