Diabetes

OR30-1

Induction of Insulin Production from Non-Human Primate Embryonic Stem Cells.

Hung-Chih Kuo1, Brian Nauert2, Don Wolf1, Linda B Lester*2. 1Andrology and Embryology, Oregon Regional Primate Res Ctr, Beaverton, OR; 2Endocrinology, Diabetes and Nutrition, Oregon Hlth & Scis Univ, Portland, OR.

Embryonic stem cells (ES) hold great promise as a source of tissue for transplantation in the treatment of diabetes mellitus (DM). Recent studies have shown that mouse and human ES cells can be induced to produce insulin ex vivo. However, before these cells can be used for clinical transplantation many questions must be answered. Therefore, developing a pre-clinical model of ES cell transplantation for the treatment of DM using non-human primates is critical. As the first step in developing a non-human primate model of islet-cell transplantation we have induced non-human primate (Rhesus macaque) ES cells into insulin production. Small pieces of an ES cell colony were grown in tissue culture media in hanging drops for 1 week to develop embryoid bodies (EB). The EBs were isolated and grown for 7-10 days in serum-free media supplemented with insulin, transferin, selenium and fibronectin to expand the cell population (Stage 1). Cells at the end of stage 1 were trypsinized and plated on tissue culture dishes in 1:1 DMEM/F12, glucose (8 mM), with N-2 supplement (insulin, transferrin, progesterone, putrascine and selenite) with 10 ng/ml of bFGF (Stage 2). In stage 3, the bFGF was removed from the media and the cells grown without bFGF and with 10 mM nicotinamide for an additional 1-3 weeks (stage 3). Islet cell development was monitored by the identification of markers nestin and insulin by RT-PCR and immunohistochemistry. Nestin positive cells made up 80% of the cells at the end of stage 2 but nestin expression decrease to < 10% by the end of stage 3. Insulin expression was not detected by immunocytochemistry until stage 3. At the end of stage 3 50- 70% of cells were insulin positive. Insulin release was measured from cells from stage 2 and 3 in response to glucose and glucose + forskolin. Only cells at stage 3 showed secretagogue mediated insulin release confirming the ability to induce Rhesus ES cells into insulin producing cells. We plan to increase the number of ES cells undergoing differentiation for eventual transplantation into diabetic monkeys. This would provide a model system for ES transplantation allowing us to study the risks and benefits of ES transplantation in the treatment of DM.

Basic Oral: Diabetes: Islet Biology (1:00 PM - 2:30 PM)

Presentation Date: Friday, June 21, 2002; Time: 1:00:00 PM; Location: 302

Diabetes

OR30-1 News Summary

Transplanting embryonic stem cells may be future diabetes treatment

Embryonic stem cells can be considered as a future source for treating diabetes through tissue transplantation, according to new data from an animal study being presented on Friday, June 21, at The Endocrine Society’s 84th Annual Meeting in San Francisco.

Embryonic stem cells have the potential to grow into any cell type in the body, including the insulin-producing beta cell. Therefore, embryonic stem cells could be used to treat people with diabetes who have damaged beta cells. To understand how to promote the growth of beta cells from stem cells, researchers in Oregon are developing a monkey model of stem cell transplantation. Their first step was to make monkey embryonic stem cells grow into insulin- producing beta cells.

In this study, the researchers, led by Dr. Linda B. Lester, induced monkey stem cells to make and release insulin. Although these cells release less insulin than a normal monkey or human beta cells, their research confirms that stem cells may be a potential source of tissue for transplantation and the treatment of diabetes.

The scientists believe that they are the first laboratory to accomplish this research with monkey cells. In the future, they plan to transplant similar cells into diabetic animals, which they hope will treat the animals’ diabetes and allow them to determine the safety of transplanting beta cells derived from stem cells.

This study was supported by the National Institutes of Health primate research fund and the American Diabetes Association.

Diabetes

OR52-2

Reversal of Diabetes by Leptin in IRS-1, IRS-3 Double Knockout Mice.

Palle G Laustsen*1, M Dodson Michael1, Gustav E Lienhard2, C Ronald Kahn1. 1Res Div, Joslin Diabetes Ctr, Boston, MA; 2Dept of Biochemistry, Dartmouth Med Sch, Hanover, NH.

Both obesity and lipoatrophy are associated with insulin resistance and diabetes. We have produced a novel animal model of lipoatrophic diabetes by generating mice with a combined deficiency of insulin receptor substrate 1 (IRS-1) and IRS-3 (IRS-1, 3 -/-). These mice develop severe insulin resistance and glucose intolerance and display hyperglycemia and hyperinsulinemia in both fed and fasted state as early as five weeks after birth. Epididymal fat pad size is reduced by 80-90% compared with wild-type control mice and the fat pads are composed of small and PPARgamma-deficient cells, suggesting that the absence of IRS-1 and IRS-3 results in defective adipogenesis. Correlating with the marked reduction in white adipose tissue mass, plasma leptin levels in the fed state are significantly decreased in the IRS-1, 3 -/- mice compared to wild-type mice. Leptin deficiency has been suggested to account for insulin resistance in lipoatrophic syndromes. To investigate whether the insulin resistance and diabetes observed in IRS-1, 3 -/- mice can be explained by leptin deficiency, recombinant adenovirus carrying leptin cDNA was injected into IRS-1, 3 -/- mice as well as wild-type and leptin-deficient ob/ob control mice. As a further control, the effect of injecting leptin adenovirus was in each case compared with the effect of injecting adenovirus carrying the gene for beta-galactosidase (LacZ). Injection of leptin adenovirus or LacZ adenovirus had no significant effect on blood glucose and plasma insulin levels in wild-type mice, whereas hyperglycemic and hyperinsulinemic ob/ob mice responded to leptin adenovirus, but not to LacZ adenovirus, with a normalization of these parameters. In the IRS-1, 3 -/- mice, injection of leptin adenovirus resulted in normalization of blood glucose and plasma insulin levels, whereas injection of LacZ adenovirus had no effect on these parameters. Taken together, these data show that leptin can reverse hyperglycemia and hyperinsulinemia in lipoatrophic IRS-1, 3 -/- mice, suggesting that the diabetic phenotype of these animals is due to leptin deficiency. This observation lends further support to the view that leptin can act as a modulator of insulin action.

Basic Oral: Diabetes (1:30 PM - 3:00 PM)

Presentation Date: Saturday, June 22, 2002; Time: 1:45:00 PM; Location: 104

Diabetes

OR52-2 News Summary

A fat-related hormone may be a potential diabetes treatment

Mice that are deficient in fat tissue develop diabetes, but it can be reversed by increasing the amount of leptin, a fat cell-derived hormone in their bodies, according to a study being presented on Saturday, June 22, at The Endocrine Society’s 84th Annual Meeting in San Francisco. Based on these and other data, researchers believe that a leptin deficiency is a major contributor to the development of diabetes in these animals and possibly in patients, and that leptin-replacement therapy might be beneficial.

By disrupting two genes involved in the cellular response to the hormones insulin and insulin-like growth factor, researchers in Boston and Hanover, N.H., have created mice deficient in fat tissue. These mice have highly elevated blood sugar levels, the hallmark of diabetes, because they are insensitive to the blood sugar–lowering effect of insulin and show an impaired ability to dispose of sugar from the blood.

To test the hypothesis that leptin deficiency contributes to the development of diabetes in these animals, the researchers, led by Dr. Palle G. Laustsen, injected fat cell–deficient mice and normal control mice with a leptin-producing virus. As a further control, they also injected fat cell–deficient mice and normal control mice with a different virus, which did not produce leptin. Injection of either virus did not affect blood sugar levels in normal control mice. However, fat cell–deficient mice responded to the leptin-producing virus by lowering blood sugar levels to near normal. This effect was not seen in fat cell–deficient mice injected with the control virus.

Together with results from previous studies of fat cell–deficient mice, these findings may be of potential clinical importance for patients with metabolic disorders related to a deficiency in fat tissue. Collectively, the studies suggest that leptin deficiency is a major contributor to the development of diabetes in fat cell–deficient patients, independent of the underlying molecular defect. In a recent clinical study involving nine fat cell–deficient patients, leptin replacement therapy led to significant metabolic improvements.

This study was funded by a Juvenile Diabetes Research Foundation Postdoctoral Fellowship and grants from the National Institutes of Health.

Diabetes

P1-13

Treatment with LY307161 SR, a Sustained Release Formulation of a GLP-1 Analog, Induces Weight Loss in Patients with Type 2 Diabetes.

Michael E Trautmann*1, Christoph Kapitza2, Lutz Heinemann2, Tim Heise2, Kenneth F Mace3, Beverley Patterson4. 1Clinal Investigation, Lilly Res, Hamburg, Germany; 2Profil Res, Neuss, Germany; 3Drug Disposition, Lilly Res Labs, Indianapolis, IN; 4Global Pharmacokinetics, Lilly Res Ctr, Windlesham, United Kingdom.

The human incretin hormone GLP-1 has several antidiabetic actions which have been demonstrated in patients with type 2 diabetes: Strictly glucose-dependant stimulation of insulin secretion, suppression of glucagon levels, delay of gastric emptying, and weight loss. Since native GLP-1 is rapidly degraded by the peptidase DPP IV, a sustained release formulation of a DPP IV resistant analogue, LY307161SR, was developed for single daily administration. In this study, 24 patients with Type 2 diabetes (age: 58+/-1.5 y; weight: 90.9+/-2.1 kg; BMI: 30.2 +/-0.6 kg/m2; HbA1c: 7.3+/-0.1 %(mean+SEM); on diet (16), metformin (7), or metformin plus sulfonylurea ((1) SU washed-out prior to dosing)) participated in a single blind dose-escalation study. Patients received LY307161 SR treatment as a single injection prior to breakfast with either 2.5 mg (n=8) or 3.5 mg (n=7) for 6 days. In a third group a dose of 4.5 mg was given for 21 days (n=8). Treatment with LY307161 SR was well tolerated, 1 patient dropped out of the study because of hypokalemia before dosing. Once daily administration of LY307161 SR resulted in sustained drug concentrations for the duration of the 24 hour dosing interval. LY307161 plasma concentrations after once daily dosing for 6 days were approximately 3-fold higher than single dose values. Mean average LY307161 concentrations ranged from 370 to 620 pg/mL across the entire dose range. Fasting glucose was lowered by 19, 24, and 15% and 2 hour post prandial glucose peaks were reduced by 26, 37, and 27% in the 2.5, 3.5, and 4.5 mg dosing groups, respectively, compared to pre-treatment values. No hypoglycemia was observed. Body weight was lowered at all doses. Six day treatment with 2.5 and 3.5 mg/d resulted in 1.1+/-0.7kg (1.2%) and 1+/-0.7 kg (1%) weight loss respectively. Following 4.5 mg/d for 21 days patients had lost 2.1+/-0.5 kg (2.3%) body weight, indicating a continuous weight reducing effect. The long term extent and durability of this effect will be evaluated in future studies. LY307161 SR may offer a unique new treatment for patients with type 2 diabetes that combines the benefits of weight loss with improved glycemic control.

Basic Poster: Diabetes I (11:00 AM - 12:00 PM and 2:30 PM - 3:30 PM)

Presentation Date: Wednesday, June 19, 2002

Diabetes

P1-13 News Summary

New drug for diabetics treats disease and helps with weight loss

A new anti-diabetic medication, modeled after a naturally occuring hormone, appears to lower blood sugar and encourage weight loss through appetite suppression, according to results of a study being presented on Wednesday, June 19, at The Endocrine Society’s 84th Annual Meeting in San Francisco.

Glucagon-like peptide-1 (GLP-1) is a naturally occurring hormone that plays an important role in controlling blood glucose levels and, therefore, if given as treatment for type-2 diabetes, is expected to act as a potent anti-diabetic therapy. GLP-1 is produced in the intestine and released into the bloodstream during meals. GLP-1 stimulates insulin secretion, suppresses glucagon production, reduces appetite and delays absorption of food.

In contrast to other anti-diabetic medications that increase insulin levels, the mechanism of action for GLP-1 is strictly glucose dependent, as it exerts its anti-diabetic effects only when blood glucose levels are elevated. The glucose-dependent action of GLP-1 means that both post-meal and fasting blood sugar can be well controlled without the risk of hypoglycemia. In addition, GLP-1 administration is expected to result in a therapy without the burden of weight gain sometimes seen with anti-diabetic agents and may even result in weight loss. Unfortunately, the therapeutic value of the naturally occurring GLP-1 hormone is limited by a very rapid degradation, which occurs within the body.

Researchers at Eli Lilly and Company of Indianapolis have developed an analog of GLP-1, which is protected against the rapid destruction that would otherwise occur after dosing. A sustained release formulation, LY307161 SR, is now in early clinical trials—with reports of the blood levels required to lower glucose over 24 hours following a single daily injection.

The current study, led by Dr. Michael E. Trautmann, is a phase-1 study of 24 overweight patients with type-2 diabetes. Groups of approximately eight patients received treatment with a single daily injection of one of three doses of LY307161 SR for either six or 21 days. The treatment was found to be well tolerated. Blood glucose was lowered to near normal levels in all groups both in the fasting state and after meals. After six days of treatment, patients had lost approximately one kilogram (or 2.2 pounds) of body weight. In the group receiving treatment with LY307161 SR for 21 days, the weight loss continued and amounted to approximately two kilograms (or 4.4 pounds) at the end of the study. Future clinical trials will further evaluate the long-term effects of this compound on blood glucose and body weight.

This study was funded by Eli Lilly and Company.

Diabetes

P1-32

Effects of Marginal Vitamin A Deficiency on Replication of Fetal b-Cells and the Development of Glucose Intolerance in the Adult Rat.

Kimberly A Matthews*1,2, Raghda Salhoul1,2, Henry K Driscoll1,2, Bruce S Chertow1,2. 1Med, Joan C. Edwards Sch of Med at Marshall Univ, Huntington, WV; 2Res and Med Services, VA Med Ctr, Huntington, WV.

Retinoids and their nuclear transcription factors are important mediators in the growth and differentiation of epithelial-derived structures including embryonic foregut derivatives. Given the importance of the intrauterine environment on development and the requirement for retinoids for insulin secretion, we hypothesized that vitamin A deficiency may cause defects in pancreatic and islet neogenesis and differentiation, leading to impaired glucose tolerance later in life. Previously, we have demonstrated that b-cell area was reduced by 50% in fetuses of marginally vitamin A-deficient mothers compared to those of mothers fed a diet sufficient in vitamin A. To examine the role of retinoids in islet development, replication of fetal b-cells and adult glucose tolerance were examined in Sprague-Dawley rats using a model of marginal vitamin A deficiency. Female rats prior to and during pregnancy and their offspring postweaning were fed a diet containing retinol as retinyl palmitate at a low marginal (LM, 0.25 mg/kg diet) or a sufficient (SUFF, 4.0 mg/kg diet) level. On day 20 of pregnancy, female rats were injected IP with 5-bromo-2’deoxyuridine (BrdU), 100mg/kg body weight to label newly replicated cells. Six hours later, the fetuses were removed and the pancreases were collected. Five-micron sections of paraffin embedded pancreas were stained immunohistochemically for BrdU as well as glucagon to distinguish non b-cell from b-cell area. The number of new b-cells and the total number of b-cells per islet were counted. The percent of newly replicated cells/islet in the LM fetal pancreas was 42% less than that in the SUFF offspring (p<0.05; LM = 2.07 ± .26, SUFF = 3.55 ± .65). One minute following an intracardiac injection of glucose, 1g/kg, 65 day old LM rats had a 55% lower plasma insulin level than SUFF rats (p<0.001; LM = 1.34 ± .27, SUFF = 2.99 ± .19 ng/ml). At fifteen minutes following glucose injection, LM rats had 76% higher serum glucose (p<0.01; LM = 532.1 ± 114.7, SUFF = 301.6 ± 26.0 mg/dl). These findings show that the reduced b-cell mass found in marginally vitamin A-deficient fetuses can be attributed to a reduced rate of replication in the islet, and that this may contribute to impaired glucose tolerance later in adult life.

Basic Poster: Diabetes I (11:00 AM - 12:00 PM and 2:30 PM - 3:30 PM)

Presentation Date: Wednesday, June 19, 2002

Diabetes

P1-32 News Summary

Vitamin A deficiency in pregnancy can lead to diabetes for offspring later in life

A marginal deficiency of vitamin A in the womb of a rat affected the development of the pancreas, causing symptoms of diabetes later in life, according to new data being presented on Wednesday, June 19, at The Endocrine Society’s 84th Annual Meeting in San Francisco. Investigators believe that this research may influence nutritional requirements and guidelines for pregnant mothers to prevent diabetes from occurring in later life.

The importance of the environment inside the uterus on fetal development is becoming increasingly clear. Vitamin A is an essential dietary nutrient and is required for normal growth, reproduction and vision, with a deficiency causing developmental defects and a reduction in birth weight. Vitamin A deficiency in third-world countries is a world health problem. It is estimated that almost 200 million people are at risk for and affected by vitamin A deficiency. High-risk populations include socioeconomically disadvantaged pregnant women in the United States.

Researchers from Huntington, W.V., led by Dr. Kimberly A. Matthews, hypothesized that a vitamin A deficiency may cause defects in the development of the pancreas and islets, or islands of cells composing the endocrine portion of the pancreas—the source of the hormones insulin and glucagon. From previous work with rats, they had found that maternal vitamin A deficiency leads to a 50 percent reduction in the mass of the fetal β-cell, insulin-secreting cells in the islet.

To examine the role of vitamin A on islet development, replication of fetal insulin-secreting β-cells and adult glucose tolerance were examined in rats using a model of marginal vitamin A deficiency. Female rats before and during pregnancy and their offspring post-weaning were fed a diet containing vitamin A at a low marginal or a sufficient level. On day 20 of pregnancy, female rats were injected with a chemical that substitutes for thymidine in the formation of new DNA as the cell replicates. Six hours later, the fetuses were removed and the pancreases were collected. Thin sections of pancreas were stained with special antibodies to identify the various cells. The percent of newly replicated cells/islet in the low marginal vitamin A–deficient fetal pancreas was 42 percent less than that in the sufficient offspring, showing that marginally vitamin A–deficient fetuses produce fewer insulin-secreting β-cells than vitamin A–sufficient fetuses.

A glucose tolerance was then given. One minute following an injection of glucose into the heart, 65-day-old, low-marginal vitamin A–deficient rats had a 55-percent lower plasma insulin level than sufficient rats. At 15 minutes following glucose injection, low-marginal rats had a 76 percent higher serum glucose. In response to a glucose challenge, the low-marginal adult rats secreted less insulin and demonstrated impaired glucose tolerance. This impairment of islet function in later life may be attributed to the reduced fetal β-cell development during fetal life.

This study was funded by a Public Health Service grant from the U.S . Department of Health and Human Services.

Diabetes

P1-43

Oxidative Stress Mediates the Onset of Peripheral Neuronal Dysfunction and Degeneration in Diabetes.

Andrea Vincent*1, James Olzmann1, James Russell1, Eva Feldman1, JDRF Center1. 1Neurology, Univ of Michigan, Ann Arbor, MI.

Oxidative stress is emerging as a unifying feature of disease progression in diabetic complications-prone tissues. In peripheral neuropathy, however, the most common complication of diabetes, the presence and involvement of oxidative stress have not been characterized. This study takes a multifaceted approach to defining the development of oxidative stress in animal and cell culture models of diabetic neuropathy. Nerve conduction studies in diabetic streptozotocin-treated (STZ) rats demonstrate loss of peripheral neuron function that is initially reversible at 4 and 12 weeks of diabetes. Cultured dorsal root ganglia (DRG) neurons provide a model of acute hyperglycemia in the presence of 20mM extra glucose that induces neuronal programmed cell death through a direct glucose-mediated mechanism. Biochemical, immunocytochemical, immunoblotting, and fluorimetric analyses were used to define the development of oxidative stress in both the animal and cell culture models of diabetic neuropathy. End-products of oxidative stress were significantly increased in both cultured DRG exposed to hyperglycemia and in STZ rats. Malondialdehyde, 8-isoprostane F2a, 4-hydroxynonenyl, and nitrotyrosine all significantly increased by 4 weeks of diabetes compared with age and weight-matched control animals (p < 0.05) but did not increase further between 4 and 12 weeks. Fluorimetric real-time analysis of reactive oxygen species (ROS) in cultured DRG demonstrated that general ROS (using DCFDA), superoxide (using dihydroethidium) and nitric oxide (using DAF-2DA) all rapidly increased by 100% approximately 5 h following the application of 20 mM glucose. Cellular antioxidant systems such as reduced glutathione and superoxide dismutase initially increased in cultured DRG within 3 h but longer-term studies demonstrated a significant decrease in these molecules that can avert oxidative stress. In a similar manner, cultured DRG initially increased energy levels through increased ATP at 3 h, but over the subsequent 3 h, ATP levels decreased to 25% of basal levels and did not recover. Taken together the studies provide strong evidence that oxidative stress is an early and robust response to hyperglycemia in peripheral neurons. Similar to other complications-prone tissues, therapies to promote the cellular antioxidant status may provide relief from disease progression.

Supported by NIH RO1 NS38849, KO2 NS42056 and the Juvenile Diabetes Research Foundation.

Basic Poster: Diabetes I (11:00 AM - 12:00 PM and 2:30 PM - 3:30 PM)

Presentation Date: Wednesday, June 19, 2002

Diabetes

P1-43 News Summary

Damage by “radical” molecules to healthy cells is linked to diabetic complications

A study of healthy cells and how they are damaged by the body’s own attack mechanism links the attack process—called oxidative stress—to nerve-cell injury in diabetes, according to a study being presented on Wednesday, June 19, at The Endocrine Society’s 84th Annual Meeting in San Francisco.  Researchers report that these findings are influencing the design of a clinical trial that will test the ability of a potent antioxidant to decrease diabetic nerve damage.

Peaks of increased blood glucose occur in even the best controlled diabetes and cause the development of complications over time. The most common complication of diabetes is peripheral neuropathy. In this condition, patients experience a progressive loss of nerve function that can cause pain and/or loss of sensation. Approximately 20 percent of patients who have diabetes for 20 years or more will require lower limb amputations.

Examination of the normal pathway of glucose metabolism has suggested that overloading the system could lead to the formation of oxygen free radicals. Free radicals damage cells by reacting with lipids and proteins and inhibiting their activities. The uncontrolled production of free radicals is known as oxidative stress. Healthy cells defend themselves against oxidative stress by containing high levels of antioxidants like glutathione and also by making enzymes that quickly convert free radicals to non-destructive molecules like oxygen and water.

In this study, led by Dr. Andrea Vincent of the University of Michigan, Ann Arbor, the profiles of antioxidants, antioxidant enzymes, free radicals and free-radical damaged proteins and lipids were analyzed in both diabetic rats and in cultured neuronal cells acutely exposed to increased glucose. Neurons initially increased the levels of antioxidants and the activities of antioxidant enzymes also increased. However, this defense is only sustained for up to three hours while peaks of increased glucose can be longer. The result of the loss of antioxidant defense is seen by increased free-radical derivatives of proteins and lipids in the neurons. Also, the production of the essential energy-storing molecule ATP was decreased after three hours of glucose exposure and, after this point, it did not recover.

This research was funded by the Juvenile Diabetes Research Foundation and by the National Institute of Neurological Disorders and Stroke.

Diabetes

P3-387

Oral Insulin Spray as an Add-On Therapy in Combination with Failing Oral Agents (OHAs) for Treatment of Type-2 Diabetes.

Jaime Guevara-Aguirre*1, Marco Guevara1, Jeannette Saavedra1, Gladys Baldeon1, Patricia Moncayo1, Ligia Llore1, Andres Cando1, Pankaj Modi2. 1Inst of Endocrinology IEMIR, Quito, Ecuador; 2Res & Devel, Generex Biotechnology Corp., Toronto, Ontario, Canada.

Hyperglycemia and subsequent complications are the most important health concerns in Type-2 diabetics failing on OHAs. Insulin treatment in combination with failing OHAs can help overcome those outstanding issues. The introduction of an oral insulin spray (Oralin) as an add-on therapy in place of painful injections offers a needle free, pain free Diabetes Management System. In the present study we evaluated the efficacy of Oralin in combination with OHAs versus OHAs alone in controlling post-prandial hyperglycemia in Type-2 diabetics after a standard meal challenge. In a single blind, randomized, crossover study, 13, Type-2 diabetic subjects received Oralin (15 puffs) in combination with OHAs or OHAs alone, followed by a 360 calories Ensure meal 15 minutes after the dose. The table below shows serum glucose, insulin and C-peptide changes from the baseline. 

We conclude that oral insulin (Oralin) was rapidly absorbed and increased serum insulin by 233% (p<0.0003) in comparison to s.c. insulin. The rise in glucose and C-peptide were much lower (30% and 87%) after the standard meal challenge. Oralin can be used safely in combination with OHAs to control post-prandial glucose levels.

Basic Poster: Diabetes II (11:00 AM - 12:00 PM and 2:30 PM - 3:30 PM)

Presentation Date: Friday, June 21, 2002

Diabetes

P3-387 News Summary

New oral insulin spray and oral glucose show needle-free, promising results for diabetics

A combination of a new oral insulin spray and oral glucose is more effective than glucose alone in controlling glucose levels after meals in type-2 diabetics, reports data from a study being presented on Friday, June 21, at The Endocrine Society’s 84th Annual Meeting in San Francisco. Researchers believe that this strategy could represent an improvement in therapy for the most common type of diabetes.

Researchers have been searching for an alternative to injected insulin since its discovery in the early 1920s. Virtually every membrane in the body has been explored, including the rectum, vagina, skin, nose, intestines, lungs and the buccal mucosa. While many of these routes have been abandoned for a variety of reasons, there is renewed interest for insulin delivery in the buccal mucosa, or inside of the mouth. Insulin is administered in the form of an aqueous spray that gets absorbed rapidly through the cheeks and back of the throat. To date studies have shown the insulin administration method to be safe and effective.

Type 2 diabetic (non-insulin dependent) patients are often not open to or compliant with insulin therapy due to its invasive nature. As a result, physicians can be reluctant to prescribe insulin to type-2 diabetic patients early in treatment. Type-2 diabetes patients that are able to make some insulin are often treated with oral agents (OHAs) that can help to control blood glucose. While this treatment may work for a period of time, often plasma glucose is not adequately controlled. Poor control of blood glucose can lead to costly, debilitating and, eventually even fatal complications.

A combination therapy of insulin and OHAs may be considered, which urged researchers from Ecuador and Generex Biotechnology Corporation, led by Dr. Jaime Guevara-Aguirre of Ecuador, to test the company’s oral insulin spray, Oralin™, as an option for needle-free treatment. In the study, 13 type-2 diabetic subjects received Oralin in combination with OHAs or OHAs alone, followed by a 360-calorie meal 15 minutes after the dose.

The study showed a significant increase in insulin when the oral insulin spray was used, while the increase in glucose and c-peptide was much lower versus the OHAs alone.

This study was funded by Generex Biotechnology Corporation.

Diabetes-Lipids-Metabolism

OR11-4

Rosiglitazone-Mediated Improvement of Insulin Sensitivity Is Highly Correlated with Increases in Circulating Acrp30/Adiponectin in HIV-Associated Lipodystrophy.

Dennis C Mynarcik*1, Terry P Combs2, Margaret A McNurlan3, Philipp E Scherer2, Marie C Gelato1. 1Med / Endocrinology, SUNY at Stony Brook, Stony Brook, NY; 2Cell Biology, Albert Einstein Coll of Med, Bronx, NY; 3Surgery, SUNY at Stony Brook, Stony Brook, NY.

Loss of subcutaneous fat depots in HIV disease (HIV-LD) is associated with hyperlipidemia and severe peripheral insulin resistance, predisposing this patient population to significantly increased risk for the development of vascular disease. Gaining insight into the mechanism(s) underlying the pathophysiology of this syndrome can facilitate the design of effective therapeutic regimens. Lipodystrophy syndromes have consistently been associated with insulin resistance. This reduced insulin sensitivity has been linked to the inadequate production of an adipose tissue-derived secreted protein complex, ACRP30 or Adiponectin. But in HIV-LD visceral fat accumulates in the abdomen, presumably in compensation for the loss of the peripheral adipose tissue. A study, approved by the Institutional Review Board at SUNY at Stony Brook, was designed to determine the effect of the thiazolidinedione Rosiglitazone on Acrp30 levels and insulin resistance in HIV-LD. Seven individuals with HIV-LD were assessed for insulin sensitivity (Rd, hyperinsulinemic-euglycemic clamp), subcutaneous adipose tissue (SAT from CT scan planimetry), and serum ACRP30 levels (scanning western blots) prior to and following 12 weeks treatment with Rosiglitazone, Avandia (8mg/d). Individuals treated with Avandia experienced significantly increased SAT (130±54 cm2, P=0.046), Rd (2.18±0.71 mg glucose/kg lean body mass/min, P=0.022), and Acrp30 (24.29±8.29, P=0.022). The changes in Rd and Acrp30 were highly correlated (R=0.757, P=0.049), suggesting that the improvement in Rd, in HIV-LD, is a consequence of the action of Acrp30 on skeletal muscle, the primary site of peripheral glucose disposal. The results from the present study support findings from animal and tissue culture experiments indicating that thiazolidinediones may not regulate insulin sensitivity directly in the skeletal muscle, but rather enhance skeletal muscle physiology secondary to their action in adipose tissue through the production of Acrp30. Furthermore, the results suggest that the visceral adipose tissue production of Acrp30 does not adequately compensate for the lost production from the SAT.

Clinical Oral: Diabetes & Lipids (1:00 PM - 2:30 PM)

Presentation Date: Wednesday, June 19, 2002; Time: 1:45:00 PM; Location: 130

Diabetes-Lipids-Metabolism

OR11-4 News Summary

Diabetic drug can help HIV patients with glucose control and abnormal body fat distribution

Researchers found a potential solution for human immunodeficiency virus (HIV) patients who have risk factors leading to diabetes and heart disease, according to a study being presented on Wednesday, June 19, at The Endocrine Society’s 84th Annual Meeting in San Francisco. Impaired glucose control and an abnormal distribution of body fat in HIV patients respond well to insulin sensitizers because the drugs stimulate the action of a hormone that counteracts these effects.

Many people with HIV disease lose fat from their arms, legs and face and gain it in their abdomen. In addition, they have reduced glucose control, which can lead to type-2 diabetes, and they also have very high levels of fat in their blood. All of these are important risk factors for developing heart disease. To help restore their normal distribution of fat and improve their glucose control, researchers from New York state treated a group of these patients with a class of drugs called insulin sensitizers.

Insulin sensitizers improve glucose control in individuals with diabetes but the drugs can also cause the patients to gain weight. When these researchers, led by Dr. Dennis C. Mynarcik, treated a group of HIV-positive patients with an insulin sensitizer, the fat in their arms and legs was increased, the fat in their abdomens was reduced and their glucose control improved. In general, the increase in fat coupled with the improvement in glucose control, caused by the insulin sensitizers, has created confusion among physicians and scientists. Increases in fat usually contribute to the development of diabetes, not improve it.

The researchers found that the insulin sensitizers stimulate the fat to make a hormone called Acrp30 or Adiponectin. Acrp30 causes the improvement in glucose control. Fat cells normally make Acrp30 to properly regulate glucose control in the body. But HIV-positive individuals with altered fat distribution do not make adequate quantities of Acrp30 due to a disease- or drug-related defect in their fat cells.

This study demonstrates that the control of Acrp30 is defective in the fat cells in the HIV patients. It also provides strong evidence that the loss of fat is the primary cause of the loss of glucose control. More importantly, the study demonstrates that treatment can both restore normal fat distribution and improve glucose control, potentially reducing the risk of heart disease in these patients.

This study was funded by the National Institutes of Health.

Inhibins-Activins-TGF Betas

OR16-5

The Role of Activin Receptor/Smad2 Signaling in Pancreas Development.

Yutaka Goto*1,2, Masatoshi Nomura1,2, Kimitaka Tanaka1,2, Taijiro Okabe1,2, Kiminobu Goto1,2, Toshihiko Yanase1,2, Hajime Nawata1,2. 1Dept of Med and Bioregulatory Sci, Grad Sch of Med Scis, Kyushu Univ, Fukuoka, Fukuoka, Japan; 2CREST, JST(Japan Sci and Technology Corp), Kawaguchi, Saitama, Japan.

Activin, a member of the transforming growth factor-b (TGF-b) family, has been shown to regulate such a broad range of cellular responses as proliferation, apoptosis and cell fate specification, and also shown to play an important role in embryonic development. Activin signaling has been shown to be mediated by complex of type I and type II activin receptors (ActRI and ActRII) and intracellular Smad proteins. Recently, mutations of type II activin receptor genes ActRIIA and ActRIIB in mice have revealed their important roles in pancreas development. The mice carrying such the mutations as ActRIIA+/-IIB+/- and IIB-/- exhibit hypoplastic pancreas islet and impaired glucose tolerance. However, little is known about the signaling pathway in which Smad proteins are involved. In early embryogenesis, Smad2 and ActRIIB have been suggested to be in the same genetic pathway to regulate the left-right patterning of the body. It is therefore interesting to speculate that Smad2 acts downstream to ActRIIB in the pancreas development. As Smad2 functions in a dosage-sensitive manner, we predicted that a reduction of the gene dosage of Smad2 in ActRIIB homozygous would result in increase of severity of the pancreas defects. We therefore generated and analyzed the mice heterozygous for Smad2 and either heterozygous or homozygous for ActRIIB. To generate such the mice, the compound heterozygous mice for ActRIIB and Smad2 were crossed with the mice heterozygous for ActRIIB. Histological analyses of the pancreas from the embryos at 18.5 dpc revealed that hypoplastic islet was found in not only ActRIIB homozygous but also Smad2 heterozygous mice. Interestingly, severer phenotypes were found in the compound heterozygous. In addition, these mutant mice exhibited impaired glucose tolerance at an adult stage. These results suggest that Smad2 and ActRIIB function in the same signaling pathway to regulate the development of pancreas. In conclusion, we provide compelling genetic evidence that Smad2 interacts with ActRIIB in pancreas development and raise an interesting question whether Smad2 or ActRIIB is one of the causative genes for typeII diabetes mellitus.

Basic Oral: Inhibins-Activins-TGF Betas (1:00 PM - 2:30 PM)

Presentation Date: Thursday, June 20, 2002; Time: 2:00:00 PM; Location: 301

Inhibins-Activins-TGF Betas

OR16-5 News Summary

Cellular components provide clues about pancreas development, possible diabetes therapy

A study of mice and a specific cellular pathway have led Japanese researchers to better understand pancreas development and potentially uncover a new treatment for diabetes, according to new data being presented on Thursday, June 20, at The Endocrine Society’s 84th Annual Meeting in San Francisco.

Activin, one of the members of transforming growth factor-b family, has been shown to regulate a broad range of cellular responses such as growth, death and cell-fate specification and, thus, play an important role in organ development. To exert such an important function, activin signals must be mediated from the cell surface membrane to the nucleus. The basal components in the activin signaling pathway are type I and type II activin receptors and intracellular Smad proteins.

Recently, mutations of type II activin receptor genes in mice have revealed their important roles in pancreas development. The mutant mice exhibit hypoplastic pancreas islet, which occurs when the endocrine portion of the pancreas is underdeveloped, and impaired glucose tolerance. However, little is known about the downstream signaling pathway in which Smad proteins are involved. Signaling pathways carry out cell activities. In early embryogenesis, Smad2 and activin type II receptors have been suggested to be in the same genetic pathway to regulate gastrulation and left-right patterning of the body.

Therefore, Japanese researchers, led by Dr. Yutaka Goto, speculated that Smad2 proteins also function to regulate pancreas development as well as body planning. To address this possibility, they generated and analyzed the mice heterozygous for Smad2 and either heterozygous or homozygous for activin type II receptor gene, ActRIIB. Histological analyses of the embryos at 18.5 dpc revealed hypoplastic islet in not only ActRIIB homozygous but also Smad2 heterozygous mice. Interestingly, several phenotypes were found in the compound heterozygous. In addition, these mutant mice exhibited impaired glucose tolerance at an adult stage.

These results suggest that both Smad2 and ActRIIB function to regulate pancreas development, probably in the same signaling pathway. They propose that activin type II receptor/Smad2 mutants are some of the polygenic animal models of impaired glucose tolerance. It might be possible that Smad2 or ActRIIB is one of the susceptible genes for diabetes mellitus, which is known as a polygenetic disease. The researchers plan to investigate the role of activin receptor/Smad signaling in the pancreas as a contributor to cell-replacement/regeneration therapy for diabetes mellitus.

This study has been supported by CREST of Japan Science and Technology Corporation.

Intracellular Signaling

OR42-5

HIV-1 Accessory Protein Vpr Interacts with 14-3-3 Proteins Causing Cell Cycle Arrest at G2/M and Insulin Resistance: Potential Involvement of Cdc25C and Forkhead Transcription Factor (FKHR).

Tomoshige Kino*1, Alexander Gragerov2, George N Pavlakis2, George P Chrousos1. 1Pediatric and Reproductive Endocrinology Br, Natl Inst of Child Hlth and Human Devel, Natl Insts of Hlth, Bethesda, MD; 2Human Retrovirus Sect, Ctr for Cancer Reserach, Natl Cancer Inst-Frederick, Frederick, MD.

Human Immunodeficiency Syndrome type-1 (HIV-1) accessory protein Vpr is a small multifunctional protein that may cause a variety of clinical manifestations in patients with Acquired Immunodeficiency Syndrome (AIDS). We reported earlier that Vpr functions as a nuclear receptor coactivator through its LXXLL motif, increasing the sensitivity of cells to glucocorticoids. Vpr also arrests host cells at the G2/M transition and induces apoptosis by inhibiting mitochondrial function. To explore the cellular factor(s) that specifically support(s) the cell cycle-arresting activity of Vpr, we performed yeast two-hybrid screening using one of its mutants, VprL64A, which causes cell cycle arrest but has no glucocorticoid receptor coactivator activity. We found that Vpr physically interacts with 14-3-3 proteins, a family of intracellular polypeptides that bind to phosphorylated serine/threonine residues of several “partner” molecules, modulating their activities by changing their subcellular localization or protecting them from proteolysis. Vpr bound to 14-3-3 at its C-terminal domain, sparing the binding site for the phosphorylation sequences of partner proteins. One of these proteins, Cdc25C, which promotes cell cycle transition at G2/M, also bound Vpr through its amino acids 217-253, determined in a GST pull-down assay. Thus, Vpr supports complex formation with 14-3-3 and Cdc25C, shown in both coimmunoprecipitation and GST pull-down assays. Furthermore, Vpr induced retention of Cdc25C in the cytoplasm, independently of the phosphorylation status of serine 216 of the latter, in an experiment using GFP-Cdc25C. Another 14-3-3 partner protein, forkhead transcription factor (FKHR), which functions as a negative transcription factor for the expression of insulin-dependent genes, was examined. In the presence of Vpr, formation of FKHR-14-3-3 complexes were disrupted in coimmunoprecipitation experiments. Furthermore, Vpr moderately suppressed insulin-induced cytoplasmic translocation of FKHR from the nucleus and inhibited insulin’s effect on the FKHR’s transactivation of its responsive promoter. Thus, it appears that Vpr modulates 14-3-3 functions, changing the binding specificity of these polypeptides to partner proteins. We suggest that Vpr induces cell cycle arrest by facilitating formation of 14-3-3/Cdc25C complexes, while it antagonizes the negative effect of insulin on FKHR-responsive promoters by inhibiting formation of 14-3-3/FKHR complexes.

Basic Oral: Intracellular Signaling I: Insulin Action (11:00 AM - 12:30 PM)

Presentation Date: Saturday, June 22, 2002; Time: 12:00:00 PM; Location: 132

Intracellular Signaling

OR42-5 News Summary

Scientists discover molecular steps HIV takes to prevent cells from dividing, may also explain diabetes in AIDS patients

Scientists at the National Institute of Child Health and Human Development (NICHD) have discovered the molecular steps that the human immunodeficiency virus type-1 (HIV-1) takes to prevent cells from dividing, according to a study being presented on Saturday, June 22, at the 84th Annual Meeting of the Endocrine Society. The researchers, led by Dr. Tomoshige Kino, also found that these are the molecular steps that the virus uses to cause diabetes mellitus in acquired immunodeficiency syndrome (AIDS) patients.

AIDS, caused by HIV-1, is a lethal disease that destroys a host defense mechanism leading to multiple fatal infections. Recently, physicians and scientists developed a strong therapeutic method, called highly active antiretroviral therapy, using any of three different types of anti-viral drugs. Once this therapeutic regimen started, the viruses in the patients disappeared at almost undetectable levels and patients’ condition was dramatically improved. However, at the same time, prolonged life with AIDS and the use of many therapeutic compounds has raised many complications. Two of such complication is AIDS-related diabetes and high cholesterol, which may increase problems in heart and vascular systems.

Once its hereditary material is incorporated into the cell, HIV-1 causes the cells to make a chemical known as VPR. Basic researchers have also known that once cells begin making VPR, the cells soon become incapable of dividing, but have not known why. Independently, physicians who treat AIDS patients have noticed that the patients frequently develop diabetes, but have not known why they do so.

The NICHD researchers have discovered that VPR binds to a cellular protein known as 14-3-3. In a normal situation without VPR, 14-3-3 binds to “partner proteins,” which help the cell carry out various biological processes. After it binds to a partner protein known as Cdc25C, 14-3-3 inactivates this compound and allows cells to begin the process of cell division. Similarly, after 14-3-3 binds to another partner protein, known as forkhead transcription factor, it inactivates this molecule and helps mediate insulin’s effect to the cells. However, in the presence of VPR, this viral peptide binds 14-3-3 and changes the binding activity of 14-3-3 to its partner proteins. VPR helps 14-3-3 bind Cdc25C while it prevents binding to forkhead transcription factor. Thus, VPR effectively prevents cell division and inhibits insulin action by binding with 14-3-3.

This study was funded by the National Institutes of Health Intramural AIDS Targeted Antiviral Program.