Progress 10/01/02 to 09/30/08
Outputs
OUTPUTS: This study examined factors that affect insulin sensitivity or glucose homeostasis as related to diabetes. The factors examined were phospholipase A2, fetuin-A, and leptin. (Phospholipase A2) Studies were conducted to investigate the role for phospholipase A2 (PLA2) in regulating insulin sensitivity through alterations in insulin-stimulated glucose uptake. We utilized skeletal muscle and adipocyte cell lines to perform experiments related to the role of group 1B PLA2 (PLA2-1B), cytosolic PLA2 (cPLA2), and calcium-independent PLA2 (iPLA2) in insulin-stimulated glucose uptake. These studies included the use of chemical inhibitors and gene knockdown techniques. In addition, dietary regulation in mice was determined for cPLA2 and iPLA2 in skeletal muscle and adipose tissue. (Fetuin-A) Alpha2-HS glycoprotein (AHSG), also known as fetuin-A, is a blood protein that inhibits insulin action at the level of the insulin receptor. Circulating levels of AHSG are shown to be increased in insulin resistant conditions such as obesity, metabolic syndrome, and type 2 diabetes. Our research has focused on characterizing the mechanisms mediating the inhibitory activity of AHSG. Specifically, we have analyzed insulin signal transduction in skeletal muscle cells leading to glucose uptake and glycogen synthesis. AHSG is a phosphorylated protein, and phosphorylation status has been shown to be critical for its activity. Therefore, we analyzed phosphorylation status of AHSG in several animal models of obesity and diabetes. Research findings of this study were presented at the 66th Scientific Sessions of the American Diabetes Association 2006, the 2007 Keystone Symposia on Diabetes, and the 2007 Alabama Diabetes and Obesity Conference organized by Alabama Cooperative Extension System, Montgomery, AL. These studies have led to the mentoring of 3 undergraduate students and one Master's student thesis. One patent application was filed on the basis of this study. (Leptin) Chronic administration of leptin into the ventricle of the brain has been found to normalize blood glucose concentrations in streptozoctoncin-induced diabetic rats. We performed a series of studies designed to determine how the leptin signal was communicated from the brain to the periphery and what peripheral factors were affected to cause the normalization of blood glucose concentrations. These findings were presented at the annual Keystone Symposia on Diabetes in the form of published abstracts and poster presentations, journal articles, and doctoral dissertations and master's theses. Two doctoral students, two masters students, and four undergraduate students were training in several techniques of laboratory research. The intended audience for this research is faculty and students engaged in diabetes research. PARTICIPANTS: (Phospholipase A2 studies): Kevin Huggins (Co-PI) - Wrote grant, designed experiments; Juan Yang (graduate student) - performed experiments, collected data, presented results at national meetings. (Fetuin-A studies): Suresh T. Mathews (Co-PI) - Wrote grant, designed experiments; Teayoun Kim (postdoctoral fellow) - supervised and performed data collection and laboratory assays; James Papizan (graduate student) - performed data collection and laboratory assays. Partner organizations: Suresh Mathews engaged in an active collaboration with Dr. Amira Klip, Professor of Physiology, Biochemistry, and Pediatrics at the University of Toronto, and Senior Scientist at the Hospital for Sick Children, Toronto. Dr. Klip also serves as the Chief Editor of the American Journal of Physiology - Endocrinology and Metabolism. Training: Dr. Teayoun Kim, a postdoctoral fellow was trained under the supervision of Dr. Mathews. (Leptin studies) B. Douglas White (Co-PI) wrote grant, designed experiments, performed ICV cannulations, and statistical analyses. Dr. Catherine Wernette (Research Associate) - supervised data collection and laboratory assays. Jinpin Wang, Yuan Kang (doctoral students) and MaryAnne Gragg, Jason Patten (Masters Students) performed animal care, data collection and laboratory assays. TARGET AUDIENCES: The primary target audiences were scientists and researchers. Experimental results were primarily disseminated through poster presentations at national meetings and published abstracts and journal articles. Dr. Mathews was an invited speaker at the Alabama Diabetes and Obesity Conference organized by the Alabama Cooperative Extension System, Montgomery, AL April 5, 2007, where data from the studies with fetuin-A were presented to the conference attendees that included nearly 200 health care workers, dietitians, nurses, and county agents. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(Phospholipase A2) The goal of these studies was to determine a role for phospholipase A2 (PLA2) in regulating insulin sensitivity through alterations in insulin-stimulated glucose uptake. Our approach was to focus on 3 main types of PLA2 expressed in tissues, group 1B PLA2 (PLA2-1B), cytosolic PLA2 (cPLA2), a calcium-independent PLA2 (iPLA2). Experiments were conducted in various cell types. Results from our studies indicate that PLA2-1B and cPLA2 do not play a direct role in regulating insulin-stimulated glucose uptake in skeletal muscle cells. Gene regulation of cPLA2 was not altered in skeletal muscle high fat-fed mice. However, we found that iPLA2 is required for insulin-stimulated glucose uptake in adipocytes. iPLA2 gene regulation was up-regulated in mature adipocytes compared to immature adipocytes and fibroblasts. Dietary fat appeared to have no effect on iPLA2 gene regulation. This work has established a role for iPLA2 in regulating insulin-stimulated glucose uptake in adipocytes and may represent a novel therapeutic target for the treatment of insulin resistance and diabetes. Future studies are designed to address the exact molecular mechanisms through which iPLA2 regulates insulin-stimulated glucose uptake in adipocytes and other cell types. (Fetuin-A) Our studies have significantly contributed to the current understanding of the mechanism of AHSG action in the regulation of insulin action. We have learned that AHSG inhibits phosphorylation of Akt and GSK3, thereby inhibiting glucose uptake in skeletal muscle cells. We have observed that AHSG phosphorylation status is significantly increased in Zucker Diabetic Fatty (ZDF) rats, consistent with the extreme insulin resistance observed in this animal model. Similarly, AHSG phosphorylation status was also increased in the obese, insulin resistant ob/ob mouse model. Additional studies carried out in intact cells and streptozotocin-diabetic rats indicate that both insulin and leptin can downregulate AHSG phosphorylation. This study has broadened our understanding of the mechanisms of AHSG action. Other scientists will be able to directly apply the new principles learned from this research to understand the molecular mechanisms of insulin resistance, as seen in obesity, metabolic syndrome, and type 2 diabetes. (Leptin) This research has as increased our understanding of how central leptin administration affects blood glucose concentrations during insulin-dependent diabetes. It appears that neither the sympathetic nervous system, nor the hepatic vagus nerve is required for central leptin to communicate with the periphery. Central leptin does not appear to enhance insulin sensitivity, but rather is related to an insulin-independent reduction in liver glycogen stores and decrease in hepatic gluconeogenesis. This suggests that leptin and its subsequent actions could point to novel targets for lowering blood glucose concentrations in diabetes independent of insulin. This is a change in our fundamental knowledge of leptin's actions, which may ultimately lead to a change in the quality of life for people with diabetes.
Publications
- Mathews, S.T. and Kim, T. (2009). Fetuin-A, a novel regulator of insulin action: Role in insulin resistance. In: Yao, EB (Ed.): Insulin Resistance: Recent Advances, Nova Science Publishers, Inc., (In Press).
- Wang, J., C.M. Wernette, R.L. Judd, K.W. Huggins, and B.D. White. Guanethidine treatment does not block the ability of central leptin administration to decrease blood glucose concentrations in streptozotocin-induced diabetic rats. Journal of Endocrinology 198(3):541-548, 2008.
- White, B.D., C.M. Wernette, J.R. Patten, and Y. Kang. Assessment of the gluconeogenic capabilities of leptin-treated diabetic rats by feeding albumin and fructose diets. Keystone Symposia, Diabetes Mellitus, Insulin Action and Resistance, Keystone, CO #347, p.105, 2008
- White, B.D., C.M. Wernette, M. Gragg, Y. Kang, R.L. Judd. Normalization of blood glucose concentrations in leptin-treated STZ-induced diabetic rats may not be due to enhanced insulin sensitivity. Keystone Symposia, Diabetes: Molecular Genetics, Signaling Pathways and Integrated Physiology, Keystone, CO #338, p.90, 2007
- Patten, J. Assessment of the gluconeogenic capabilities of leptin-treated diabetic rats by feeding albumin and fructose diets. Masters Thesis. Auburn University, (B.D. White, Major Professor) 2007.
- Wang, J. Central leptin, but not central insulin, attenuates the decrease of adiponectin concentrations and increases insulin sensitivity in streptozotocin (STZ)-induced diabetic rats. Doctoral Dissertation. Auburn University. (B.D. White, Major Professor) 2005.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This study examines factors that affect insulin sensitivity or glucose homeostasis as related to diabetes. The factors examined are phospholipase A2, fetuin-A, and leptin. (Phospholipase A2): Previous studies in this project were aimed at determining a role for cytosolic phospholipase A2 (cPLA2) in insulin-stimulated glucose uptake. Results from these experiments showed that cPLA2 did not play a role in insulin-stimulated glucose uptake. Experiments for this year of the project were aimed at investigating whether another intracellular class of phospholipase A2, calcium-independent phospholipase A2 (iPLA2), is required for insulin-stimulated glucose uptake. To accomplish this, we utilized a mouse adipocyte cell line (3T3-L1 cells) that is commonly used to investigate insulin-stimulated glucose uptake and has high expression of iPLA2. iPLA2 was inhibited via a known inhibitor of iPLA2 activity, bromoenol lactone (BEL). Glucose uptake assays were performed with and without
insulin in the presence of BEL to determine the role of iPLA2 in insulin-stimulated glucose uptake. In addition, the effects of iPLA2 inhibition were also assessed on insulin signaling pathways and plasma membrane incorporation of glucose transporter 4. (Fetuin A): Fetuin-A is a phosphorylated glycoprotein secreted by the liver into circulation. Previous studies have shown that phosphorylation status is critical for its activity as an inhibitor of insulin action. Thus, it was of significant interest to assess phosphorylation status of fetuin-A in animal models of diabetes. Accordingly, we analyzed plasma fetuin-A levels and its phosphorylation status in Zucker diabetic fatty rats, a model of impaired leptin signaling and insulin resistance, progressive beta-cell failure and type 2 diabetes, due to a mutation of the long form of the leptin receptor. We also analyzed fetuin-A levels and phosphorylation status in the ob/ob leptin-deficient, insulin resistant mouse model and in
streptozotocin-induced diabetes, a rat model of type 1 diabetes. (Leptin): Daily injections of leptin into the lateral ventricle of the brains of diabetic rats normalize blood glucose concentrations after 3-4 days. Furthermore, daily injections of leptin greatly decrease blood glucose concentrations in response to a short-term (hours) fast in both diabetic and nondiabetic animals. We had previously hypothesized that both of these effects were due to a leptin-induced increase in insulin sensitivity. However, since insulin sensitivity was determined by an insulin tolerance test, which by necessity must be performed in fasted animals, the possibility existed that what we interpreted as enhanced insulin sensitivity might actually be a similar response to insulin superimposed on a leptin-induced decrease in blood glucose due to fasting. To test this hypothesis we compared the glucose response of insulin tolerance tests with that of control injections of saline in diabetic and nondiabetic
rats treated daily with leptin or vehicle. This work and the work with fetuin-A were the basis of two masters student theses.
PARTICIPANTS: (Phospolipase A2 study): Kevin Huggins (Co-PI) - wrote grant, designed experiments Juan Yang (graduate student) - performed experiments, collected data, presented results at national meeting. (Fetuin-A study): Suresh T. Mathews (Co-PI); Teayoun Kim (Postdoctoral Fellow), James Papizan (graduate student). Partner organizations: The PI/PD engaged in active collaboration with Dr. Amira Klip, Professor of Physiology, Biochemistry and Pediatrics at the University of Toronto, and Senior Scientist at The Hospital for Sick Children, Toronto. Dr. Klip also serves as the Chief Editor of American Journal of Physiology - Endocrinology and Metabolism. Training: Dr. Teayoun Kim, a postdoctoral fellow was trained under the supervision of the Dr. Kim, the lead researcher on this project is a co-inventor on a patent application filed with the USPTO. Additionally, this project has afforded an opportunity to mentor Mr. James Papizan, a Master's student. Mr. Papizan presented his research
findings at the Keystone Diabetes Symposia and at several research forums at Auburn University. He was awarded the Malone-Zallen Graduate Research Fellowship from the Dept. of Nutrition and Food Sciences. He is a co-inventor on a patent application filed with the USPTO. (Leptin study): Doug White (Co-PI) - wrote grant, designed experiments, performed ICV cannulations, statistical analyses. Catherine Wernette (Research Associate) - supervised data collections and laboratory assays, MaryAnne Gragg (graduate student) - collected data and performed laboratory assays.
TARGET AUDIENCES: Primary target audiences were scientists and researchers. Data was primarily disseminated through a poster presentation at the Keystone Symposia on Diabetes. Additionally, Dr. Mathews was an invited speaker at the Alabama Diabetes and Obesity Conference organized by Alabama Cooperative Extension System, Montgomery, AL, Apr. 5, 2007 where data from the studies with fetuin-A were presented to the conference attendees that included nearly 200 health care workers, dietitians, nurses, and social workers, and county agents.
Impacts
(Phospholipase A2): The results from the experiments with phospholipase A2 demonstrate a role for iPLA2 in insulin-stimulated glucose uptake. We found that insulin-stimulated glucose uptake was inhibited approximately 40% with iPLA2 inhibition. This effect was not due to alterations in insulin signaling pathways, but due to decreased transport of GLUT4 to the adipocyte plasma membrane. These results demonstrate a novel role for iPLA2 in cellular glucose metabolism and may represent a novel therapeutic target for the treatment of diabetes and insulin resistance. Based on this result, future studies will assess the role of iPLA2 in obese and/or diabetic animal models. These studies will include gene expression and enzyme activity assays. Also, the role of iPLA2 in other aspects of glucose metabolism will be assessed. (Fetuin-A): For the fetuin-A study, use of a phospho-specific (312Ser) anti-fetuin-A antibody, demonstrated significantly elevated fetuin-A levels and
phosphorylation status in the insulin resistant Zucker diabetic fatty rats compared to lean controls (at 6 weeks of age). However, by 14 weeks, with beta-cell failure and full-blown diabetes, no significant differences were observed in fetuin-A levels and phosphorylation status. In ob/ob mice that have a mutation in the leptin gene, fetuin-A phosphorylation status is significantly elevated correlating with insulin resistance. In STZ-induced diabetes, fetuin-A levels were significantly elevated. Central administration of leptin into STZ-rats normalized fetuin-A levels and significantly decreased phosphorylation status of fetuin-A. Taken together, our findings indicate that fetuin-A levels and phosphorylation status are associated with insulin resistance. Additionally, these studies suggest a role for insulin and leptin in the regulation of fetuin-A phosphorylation status. (Leptin): Results of the study with leptin showed that both the diabetic and nondiabetic rats behaved similarly in
response to central leptin treatment. Leptin-treated animals showed an enhanced response, both to the injection of insulin and the injection of saline. This suggests that the additional leptin-induced decrease in blood glucose concentration was not due to enhance insulin sensitivity, but rather due to a leptin-induced decrease in glucose due to fasting. This study shows that the effect of leptin to decrease blood glucose concentration is independent of the effect insulin. Type 2 diabetes is characterized by insulin resistance. The ability of leptin to act through an insulin independent mechanism to decrease blood glucose concentrations could point to an important new treatment of type 2 diabetes.
Publications
- Yang, J. and Huggins, K.W. (2008) Inhibition of Calcium-Independent Phospholipase A2 Results in Decreased Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes. Keystone Symposia. Diabetes Mellitus, Insulin Action and Resistance, Breckenridge, CO. (p. 106).
- Papizan J.B., Kim T., Wernette C., White B.D., Mathews S.T. (2007) A novel role for leptin in regulating phosphorylation status of alpha2-HS glycoprotein, a physiological inhibitor of insulin action. Keystone Symposia, Diabetes: Molecular Genetics, Signaling Pathways and Integrated Physiology, Keystone, CO, Jan.14-19, 2007, #302/p.81.
- Mathews S.T., Kim T., Papizan J.B. (2007) Methods and compositions for treating obesity and diabetes. Patent pending, USPTO 07-011.
- Papizan, J.B. (2007) Phosphorylation of fetuin-A, a physiological inhibitor of insulin action, regulated by insulin and leptin. Masters Thesis, Auburn University.
- Mathews S.T, Rakhade S., Zhou X., Parker G., Coscina D.V., Grunberger G. (2006) Fetuin-null mice are protected against obesity and insulin resistance associated with aging. Biochemical and Biophysical Research Communications 350: 437-443.
- Gragg, M.G. (2007) The apparent increase in insulin sensitivity of leptin-treated rats appears to be due to a decrease in blood glucose concentrations due to fasting. Masters Thesis, Auburn University.
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Progress 01/01/06 to 12/31/06
Outputs
Phosphorylation of alpha 2-HS glycoprotein (AHSG) has been shown to be critical for its activity as an inhibitor of insulin receptor activation. Therefore, we assayed AHSG phosphorylation status in animal models of insulin resistance, diabetes and obesity. We demonstrate that models of extreme insulin resistance such as Zucker diabetic fatty rats and ob/ob mice, AHSG phosphorylation status is increased. In a high-fat fed rat model of mild insulin resistance, AHSG phosphorylation is decreased, indicative of counter-regulation. These studies suggest a role for insulin and leptin in the regulation of AHSG phosphorylation status. Consistent with this hypothesis, we observe that in vitro treatment of human hepatoma cells with insulin or leptin decreases AHSG phosphorylation. Further, central administration of leptin to STZ-treated type 1 diabetic rats, markedly decreases AHSG phosphorylation. Taken together, these studies implicate a role for AHSG in the pathogenesis of
insulin resistance and diabetes. Previous data showed that mice lacking group 1B phospholipase A2 (PLA21B) have improved insulin sensitivity and are resistant to diet-induced obesity. We hypothesized that PLA21B is inhibiting insulin signaling. We incubated L6 muscle cells with PLA21B (24 h) and measured glucose uptake in response to insulin. PLA21B (at high doses) inhibited insulin-stimulated glucose uptake approximately 50%. However, lower doses at more physiological concentrations did not inhibit insulin-stimulated glucose uptake. Also, when carried out for shorter treatment times (30 min - 2 h), there was no apparent decrease in insulin-stimulated glucose transport. Further studies revealed that incubation of high doses of PLA21B for 24 hours resulted in increased cell death. We conclude that PLA21B inhibition of insulin-stimulated glucose uptake was due to increased cell death presumably by stimulation of apoptotic pathways. These results suggest that physiological doses of
PLA21B do not play a significant role in inhibiting insulin-stimulated glucose uptake in L6 muscle cells. Chronic leptin administration normalizes blood glucose concentrations in STZ-induced diabetic rats. We found that when leptin-treated rats (both diabetic and nondiabetic) are fasted, their blood glucose concentrations decrease to very low levels in a few hours. Therefore, it appears that rats are feeding to help normalize their blood glucose concentrations. We hypothesize that chronic leptin administration decreases hepatic glucose production. In fed rats, liver glycogen content was lower in leptin-treated animals and was decreased further during a short-term (3-hour) fast. During this fasting period, liver glycogen content was not altered in non-leptin-treated rats. What we once thought was a leptin-induced increase in insulin sensitivity, appeared to be due to an additive effect of fasting, which suggests leptin may be acting independent of insulin. Chronic central leptin
decreased PEPCK gene expression in fed nondiabetic rats and decreased glucose 6-phosphatase activity in diabetic rats. These are constant with leptin causing a decrease in hepatic glucose production.
Impacts
Diabetes is epidemic in the United States. As a result of the consequences of diabetes and its prevalence, diabetes is the sixth leading cause of death in the United States. Currently, 20.8 million Americans have diabetes. This represents 7% of the population. It is estimated that if current tends continue one out of every three people born after the year 2000 will develop diabetes sometime in their life. Type 2 diabetes accounts for 90-95% of all cases of the disease, and it is an increase in this form of the disease that accounts for the increased incidence. Type 2 diabetes is characterized by insulin resistance with relative insulin insufficiency. Our group has examined various agents that affect insulin sensitivity or glucose homeostasis. Two of the agents, AHSG and PLA21B, are fairly novel inhibitors of insulin action. One agent, leptin, is a hormone secreted from fat that when administered chronically decreases blood glucose concentrations by an
insulin-independent mechanism. Understanding how these agents affect insulin sensitivity and glucose homeostasis will increase our understanding of the nature of insulin resistance, and hopefully will help to provide the scientific framework to aid in the prevention and treatment of type 2 diabetes.
Publications
- White, B.D., C.M. Wernette, S. Mathews, T. Kim, and R.L. Judd. Leptin-treated diabetic rats become hypoglycemic during a short-term fast and cannot derive glucose from lactate or alanine. Keystone Symposia, 2006.
- Mathews ST, Rakhade S, Zhou X, Parker G, Coscina DV, Grunberger G. Fetuin-null mice are protected against obesity and insulin resistance associated with aging. Biochemical and Biophysical Research Communications 350: 437-443, 2006
- Mathews ST, Kim T, Papizan JB. Methods and compositions for treating obesity and diabetes (USPTO 07-011, 2006, patent application submitted)
- Kim T, Price JC, Sunkara R, Mathews ST: Fetuin inhibits insulin-stimulated glucose uptake and glycogen synthesis in L6 GLUT4myc skeletal muscle cells. 66th Scientific Sessions, American Diabetes Association, Washington D.C., June 9-13, 2006. Diabetes 55 (Suppl.1): 1211P/A285, 2006
- Labonte, E.D., R. J. Kirby, N.M. Schildmeyer, A.M. Cannon, K.W. Huggins, and D.Y. Hui. Group1B Phospholipase A2-mediated lysophospholipid absorption directly contributes to postprandial hyperglycemia. Diabetes 55: 935-941, 2006
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Progress 01/01/05 to 12/31/05
Outputs
We have been interested in regulators of insulin sensitivity. Research from our group has concentrated on two inhibitors of insulin sensitivity, (alpha 2-HS glycoprotein (AHSG) and group 1B phospholipase A2 (PLA2-1B)) and one promoter of insulin sensitivity, (leptin in the brain). Alpha2-HS glycoprotein (AHSG), a liver-secreted phospho-glycoprotein, has been identified as an inhibitor of insulin action. During this study period, we directly tested the hypothesis that AHSG regulates glucose metabolism in skeletal muscle cells. Our studies demonstrate that physiological concentrations of AHSG inhibit insulin-stimulated glucose uptake and glycogen synthesis in rat and mouse skeletal muscle cells. To further delineate the signal transduction leading to glucose uptake and glycogen synthesis, phosphorylation status of Akt and GSK-3, and insulin-stimulated GLUT4 translocation were assayed. We demonstrated that AHSG blunted the insulin-stimulated translocation of GLUT4
vesicles from an intracellular compartment to the plasma membrane. This impairment was mediated by decreased phosphorylation status of Akt and GSK-3. These studies establish a novel role for AHSG in regulating insulin's metabolic action and lend credence to previous findings for AHSG as a physiological regulator of insulin action. We are also focused on elucidating a role for Group 1B phospholipase A2 (PLA2-1B) in the regulation of glucose metabolism. Specifically, we hypothesize that PLA2-1B functions as a negative regulator of insulin-stimulated glucose transport through it actions on the PLA2 receptor and not through its phospholipase hydrolytic activity. We used an in vitro cell culture system to address these studies. L6 myocytes are a rat skeletal muscle cell line that when differentiated into myotubes are the preferred cell system to study insulin-stimulated glucose uptake and have endogenous expression of the PLA2 receptor. When the L6 myotubes were pretreated with PLA2-1B for
24 hours, there was a significant reduction (p < 0.05) in insulin-stimulated glucose uptake compared to cells that had not been pretreated with PLA2-1B. Basal glucose uptake (absence of insulin) was not affected by pretreatment with PLA2-1B for 24 hours. Pretreatment with PLA2-1B for 1 hour or short time periods had no effect on glucose uptake. These results support our overall hypothesis that PLA2-1B is acting as a negative regulator of insulin-stimulated glucose uptake. We also examined the effect of central leptin to affect blood glucose concentrations in diabetic rats after an oral gavage of lactate (a gluconeogenic precursor). A previous study had demonstrated that central leptin negated an increase in hepatic PEPCK mRNA levels in diabetic rats, suggesting that gluconeogenesis was decreased. Leptin-treated diabetic rats had a severely blunted glucose response to the oral gavage of lactate, supporting the idea that central leptin inhibits gluconeogenesis in diabetic rats,
contributing to the decreased blood glucose concentrations found in leptin-treated diabetic rats.
Impacts
Type 2 diabetes has become epidemic in the United States. The State of Alabama leads the nation in the number of diagnosed cases per capita. If current trends continue, it is estimated that 1 out of every 3 children born after the year 2000 will become diabetic sometime in their lifetime. Prior to the development of full-blown type 2 diabetes, people develop a period of insulin resistance. Though their blood glucose concentrations are normal, they have to secrete higher the normal concentrations of insulin to overcome the insulin resistance. Work by our group is looking at various factors that can contribute to the development of insulin resistance. By understand the factors that can contribute to insulin resistance, we will be better prepared to prevent or treat the condition.
Publications
- Labonte, E.D., Kirby, R.J., Schildmeyer, N.M., Cannon, A.M., Huggins, K.W., Hui, D.Y. Group 1B-Phospholipase A2-mediated lysophospholipid absorption directly contributes to postprandial hyperglycemia. Diabetes, in press, 2006
- Kim, T., Price, J.C., Sunkara, R., Mathews, S.T. (2006) Fetuin inhibits insulin-stimulated glucose uptake and glycogen synthesis in L6-GLUT4myc skeletal muscle cells. 66th Scientific Sessions, American Diabetes Association, Washington D.C., June 9-13, 2006 (submitted)
- White, B.D., C.M. Wernette, S. Mathews, T. Kim, and R.L. Judd. Leptin-treated diabetic rats become hypoglycemic during a short-term fast and cannot derive glucose from lactate or alanine. Keystone Symposia. Diabetes Mellitus and the Control of Cellular Energy Metabolism. 2006
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Progress 01/01/04 to 12/31/04
Outputs
We have previously shown that leptin injected into the ventricle of the brain will normalize blood glucose concentrations in diabetic rats. The normalization of blood glucose concentrations appears to be due to a large increase in insulin sensitivity in the body. Interestingly, the leptin-induced increase in peripheral insulin sensitivity also occurs in non-diabetic animals, despite the fact that leptin treatment does not alter blood glucose concentrations in these animals. The reason leptin does not decrease blood glucose concentrations in non-diabetic animals is that leptin also causes a dramatic decrease in the concentration of circulating insulin, which appears to balance the increase in insulin sensitivity. Therefore, a consistent result of leptin administration into the brain of both diabetic and non-diabetic animals is a large increase in insulin sensitivity. How does leptin acting in the brain affect insulin sensitivity in the body? We hypothesized that leptin
stimulates the activity of the sympathetic nervous system, which in turn mediates metabolic changes in the periphery that results in the enhancement of insulin sensitivity. To test this hypothesis we pretreated rats with guanethidine, a blocker of the sympathetic nervous system, to determine whether this would negate or attenuate the leptin-mediated normalization of blood glucose concentration in diabetic rats. We found that guanethidine treatment (100mg/kg SQ every other day for 2 weeks) did not affect the ability of leptin to reduce blood glucose concentrations. We now report tissue norepinephrine levels from various tissues in these animals. This indicates the effectiveness of the guanethidine treatment at inhibiting the sympathetic activity. In spleen, norepinephrine levels were significantly (p<0.0001) reduced in guanethidine-treated animals to 34% of that of control animals. A similar reduction in norepinephrine levels was found in retroperitoneal fat (29% of control, p<0.0001).
Guanethidine had a significant, but less dramatic effect on norepinephrine levels in brown adipose tissue (53% of control, p<0.001). Interestingly, in liver, guanethidine treatment resulted in a 22% increase in norepinephrine levels (p=0.03). Additionally, leptin treatment increased norepinephrine levels in the liver of guanethidine-treated diabetic rats. Leptin did not have an effect on norepinephrine levels in any other tissue examined. These results suggest that guanethidine treatment did decrease sympathetic activity (with the exception of the liver); however, this decrease was not 100% efficient. We conclude that an approximate 70% reduction in sympathetic activity to some tissues does not attenuate the ability of leptin to increase insulin sensitivity in diabetic rats. This may mean that the sympathetic nervous is not involved in mediating the effect of central leptin. However, it could mean that only a small fraction of remaining sympathetic activity is necessary to mediate the
effect of leptin or alternatively, that sympathetic activity to a tissue whose sympathetic activity is not decreased by guanethidine treatment (like the liver) may mediate the effects of leptin.
Impacts
In Western society in general and in the State of Alabama in particular, there is an epidemic of obesity and type 2 diabetes. This lowers the quality of life of the population and places a larger burden on the health care system. A lack of leptin signaling in the brain has been suggested to play a role in both obesity and in the development of insulin resistance, a hallmark of type 2 diabetes. We feel that improving our understanding of the actions of brain leptin as it relates to insulin sensitivity will provide greater insight into the connection between obesity and type 2 diabetes. This may eventually lead to better methods of prevention and treatment of both obesity and type 2 diabetes.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We have previously demonstrated that serum leptin concentrations are greatly decreased in diabetic animals. Furthermore, diabetic animals develop resistance to peripherally administered insulin. This peripheral resistance to insulin can be reversed by central administration of small amounts of leptin into the brain. This greatly enhances peripheral insulin sensitivity and normalizes blood glucose concentrations in diabetic rats. This suggests that a lack of leptin signaling in the brain may play a role in the development of peripheral insulin resistance. Since obesity is also thought to be due to a relative lack of leptin signaling in the brain, this suggests a potential connection between obesity and the development of insulin resistance, (a characteristic of type 2 diabetes). Two experiments were performed to examine the role of central leptin on peripheral insulin sensitivity in diabetic rats. In the first experiment, we compared the ability of centrally
administered leptin to normalize blood glucose concentrations in diabetic rats with that of centrally administered insulin. As we had seen previously, leptin administered directly into the brain normalized blood glucose concentration in diabetic rats. This was accompanied by an increase in peripheral insulin sensitivity. The administration of insulin directly into the brain had no effect on the concentration of blood glucose or the sensitivity to peripheral insulin. We also assessed plasma adiponectin concentrations. Adiponectin has been suggested to increase peripheral insulin sensitivity. We found that the induction of diabetes decreased the plasma concentration of adiponectin and that this decrease was attenuated by central leptin administration. This finding is consistent will a role for adiponectin in the regulation of glucose homeostasis by central leptin. The second experiment attempted to determine the role of the sympathetic nervous system in mediating the ability of central
leptin to enhance insulin sensitivity. Rats were treated with a subcutaneous injection of guanethidine (100 mg/kg) every other day for 2 weeks. In a separate study, we had shown this paradigm of guanethidine treatment to decrease tissue norepinephrine levels to 30% of that of controls. Under the conditions of this study, guanethidine pretreatment did not alter the ability of central leptin to enhance insulin sensitive and normalize blood glucose concentrations in diabetic rats. While this finding suggests that the sympathetic nervous is not involved in mediate the effect of central leptin a glucose homeostasis, care should be taken in making this conclusion. It is possible that only a small fraction of remaining sympathetic activity is necessary to mediate the effect of central leptin. Therefore, in the future we will also pursue other approaches to examine the potential role of the sympathetic nervous system. Currently, we are examining AMP kinase activity in the two experiments
mentioned above to determine if alterations in AMP kinase activity could mediate the effect of central leptin on glucose homeostasis in diabetes.
Impacts
This work is examining the relationship between the effects of leptin in brain and the development of insulin resistance. Leptin, acting in the brain, appears to affect both the regulation of body fat (obesity) and the regulation of insulin sensitivity. Therefore, by improving our understanding of the actions of brain leptin, we will gain greater insight into connection between obesity and type 2 diabetes.
Publications
- White, B.D., J. Wang, D. Bedi, K. Clarke, B. Brunson, M. Ding, R.L. Judd. Central leptin and insulin administration on peripheral insulin sensitivity and plasma adiponectin concentrations. FASEB J. (submitted abstract) 2004
- White, B.D., F. Du , and D.A. Higginbotham. Low Dietary Protein is Associated with an Increase in Food Intake and a Decrease in the In Vitro Release of Radiolabeled Glutamate and GABA from the Lateral Hypothalamus. Nutritional Neuroscience 6:361-367, 2003
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