Experience

Duke University

 

Supervisor: Dr. C. Newgard (Department of Pharmacology and Cancer Biology)

 

 

Do you want to learn more about Dr. Newgard  (click here) or (click here)

Do you want to learn more about the Sarah Stedman Nutrition and Metabolism Center or the Metabolomics platform (click here)

 

Introduction:  Metabolic regulation of insulin secretion in the pancreatic b-cell.

 

In order to gain insight into the projects that I am involved in at Dr. Newgard’s laboratory I will first give a brief introduction to insulin secretion and then talk about potential novel pathways for insulin secretion.  The mechanism of nutrient controlled insulin release from insulin granules is still not completely understood.  One key player in insulin secretion is the ability of glucose to stimulate an increase in mitochondrial ATP production.  Mitochondrial oxidative metabolism has been estimated to produce 98% of β-cell ATP.  The currently accepted pathway for insulin secretion involves glucose increasing cytosolic ATP/ADP ratio which in turn leads to the closure of the K_ATP channels on the plasma membrane.  K_ATP channels closure results in membrane depolarization and activation of voltage-dependent Ca²⁺ channels, increasing the concentration of cytosolic Ca²⁺.  Elevated cytosolic Ca²⁺ leads to stimulation of exocytosis of insulin.  However, the Ca²⁺ signal alone is not the only signal because under clamped cytosolic Ca²⁺ concentrations, glucose can still elicit further insulin secretion.  This suggests that a messenger must exist which is distinct from ATP and membrane depolarization that is involved in stimulation of insulin secretion.  Some of the suggested mitochondrial factors include glutamate, malonyl CoA, long-chain acyl CoAs (LC-CoA), and/or NADPH. 

 

The production of malonyl CoA, LC-CoA and NADPH depends on the export of mitochondrial metabolites.  In particular, mitochondrial malate and/or citrate transport appears to be important for cytosolic NADPH production via malic enzyme and cytosolic isocitrate dehydrogenase (cICD), whereas citrate alone appears to be critical for malonyl CoA and LC-CoA formation.  In addition, our lab has shown that pyruvate cycling strongly correlates with glucose-stimulated insulin secretion (GSIS).  Pyruvate cycling involves recycling of pyruvate across the mitochondrial inner membrane (see figure below).  There are several pathways for pyruvate recycling and its potential downstream product NADPH.  One key pathway is the pyruvate-malate shuttle system.  The first step of this pathway involves a TCA cycle anaplerotic step where pyruvate is converted to oxaloacetate via the pyruvate carboxylase (PC) reaction.  This anaplerotic step is a key step in β-cells since 40% of all pyruvate in mitochondria enters the PC reaction.  Oxaloacetate is part of the TCA cycle and has several potential fates in mitochondria.  Oxaloacetate can be converted to malate by the TCA cycle enzyme mitochondrial malate dehydrogenase (MDH_m).  Oxaloacetate after its conversion to malate, can participate in a number of pathways including the malate-aspartate shuttle (shuttles NADH into mitochondria) or the pyruvate-malate shuttle (shuttles NADPH out of mitochondria).  In the pyruvate-malate shuttle system malate is transported out of the mitochondria by the dicarboxylate carrier (DIC) in exchange for P_i.  In the cytosol, malate can then be converted to pyruvate via malic enzyme resulting in the generation of CO₂ and NADPH.  Pyruvate can then be transported back into the mitochondria to begin the cycle again.

 

In addition to the above pathway for pyruvate cycling, two other potential pathways for pyruvate cycling exist that involve citrate or isocitrate transport via the citrate carrier (CIC).  After conversion of pyruvate to oxaloacetate by PC, oxaloacetate then is converted to citrate or isocitrate, both of which can be transported out by CIC.  In one potential downstream pathway, citrate can be metabolized by citrate lyase to acetyl CoA and oxaloacetate; oxaloacetate can then be converted to malate.  Malate can then be metabolized to pyruvate, CO₂ and NADPH as described above by malic enzyme.  In a third pathway for NADPH production, citrate can also be converted to isocitrate by aconitase and then be utilized by cytosolic isocitrate dehydrogenase (cICD) to form a-ketoglutarate and NADPH.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

My goals in Dr. Chris Newgard’s laboratory is to investigate the roles of these pathways in collaboration with Mette Jensen (postdoctoral fellow) and Sarah Ronnebaum (PhD student). 

 

 

Project 1:  The role of the mitochondrial citrate carrier (CIC) in glucose-stimulated insulin secretion.

 

Abstract:  Cellular metabolic factors are required to dose-dependently regulate glucose-stimulated insulin secretion (GSIS) in the pancreatic b-cell.  While mitochondrial derived substrates such as ATP play a key role in GSIS evidence from our laboratory and others suggest that there are other key mitochondrial metabolic modulators of insulin secretion.  The mitochondrial citrate carrier (CIC) catalyzes an electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (i.e. citrate, isocitrate, and cis-aconitate) plus a proton, for either another tricarboxylate-H⁺, a dicarboxylate (i.e. malate or succinate), or phosphoenolpyruvate (see figure below).  The mitochondrial CIC occupies a critical position in intermediary metabolism since it is the only source of cytoplasmic citrate and because it serves as a key carbon source, which fuels both the fatty acid and the sterol biosynthetic pathways.  To date no paper has described the expression of CIC in pancreatic b-cells.  Inhibition of citrate transport by the specific substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in reduced glucose-stimulated citrate accumulation and inhibition of glucose-stimulated insulin secretion (GSIS) in 832/13 cells.  Perifusion of isolated rat islets with BTC dose-dependently inhibited both first- and second-phase insulin secretion.  BTC enhanced glucose utilization and decreased glucose oxidation, ATP/ADP ratio and glucose incorporation into fatty acids in 832/13 cells.  In order to further investigate the role of CIC we developed a siRNA adenovirus against CIC (AdsiCIC). AdsiCIC dose-dependently reduced CIC mRNA levels in 832/13 cells, and dose-dependently inhibited GSIS. AdsiCIC did not affect glucose utilization, glucose oxidation or ATP/ADP ratio however there was lower glucose-stimulated citrate accumulation and glucose incorporation into fatty acids in 832/13 cells.  AdsiCIC also reduced CIC expression by 55% and inhibited GSIS in isolated rat islets.  These studies suggest that CIC plays an important role in GSIS.

 

Notes:  This work was done in collaboration with Mette Jensen.  For the paper I will be listed as first author.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Project 2:  The role of the mitochondrial dicarboxylate carrier (DIC) in glucose-stimulated insulin secretion.

 

Abstract:  The dicarboxylate carrier (DIC) catalyses the electroneutral exchange of certain dicarboxylates (e.g. malate and succinate) in exchange for inorganic phosphate or inorganic sulphur-containing compounds (see figure above).  DIC is thought to be primarily involved in gluconeogenesis from pyruvate (and amino acids) and ureogenesis.  In liver cells pyruvate is converted in the matrix into oxaloacetate and then into malate; this dicarboxylate is exported by the DIC to the cytosol where it reduces NAD⁺ to NADH plus H⁺ and is converted into oxaloacetate and then into phosphoenolpyruvate, which is used by the gluconeogenetic pathway.  I have been able to show that DIC is highly expressed in clonal b-cells lines and in isolated rat islets.  In order to investigate the involvement of DIC and malate transport in glucose-stimulated insulin secretion I have transfected 832/13 cells (a clonal b-cells line) with a siRNA construct against DIC.  The siRNA duplex against DIC reduces DIC mRNA levels by 70-80% and reduced proteins levels by 60-70%.  The siRNA duplex did not affect glucose utilization, glucose oxidation or alter the ability of glucose to stimulate an increase in the ATP/ADP ratio.  The siRNA duplex however did inhibit glucose-stimulated insulin secretion suggesting an important role of this carrier in glucose-stimulated insulin secretion.  Work is currently ongoing to further investigate the role of this carrier in insulin secretion.

 

Notes:  For the paper I will be listed as first author.

 

Project 3:  A Pyruvate Cycling Pathway Involving Cytosolic NADP-dependent Isocitrate Dehydrogenase and NADPH Production Regulates Glucose-Stimulated Insulin Secretion.

 

Abstract:  Glucose-stimulated insulin secretion (GSIS) from pancreatic islet b-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-sensitive K⁺ channels, but multiple lines of evidence suggest participation of other, as yet unidentified signals. Here we show that cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) regulates NADPH levels and controls GSIS in b-cells.  Adenovirus-mediated delivery of a small interfering RNA (siRNA) specific for ICDc caused a large impairment in GSIS in two independent robustly glucose responsive rat insulinoma (INS-1-derived) cell lines, and in primary rat islets.  In contrast, siRNA-mediated suppression of citrate lyase, the first step in the cytosolic conversion of citrate to pyruvate, had no effect on GSIS.   Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity, as measured by ¹³C NMR, and in NADPH levels, a predicted byproduct of pyruvate cycling pathways. Mass spectrometry-based metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused a 2.5-fold increase in lactate levels, with no significant changes in other intermediates, consistent with the attenuation of pyruvate cycling. Based on these studies, we propose a new b-cell stimulus/secretion coupling pathway by which cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) regulates NADPH levels and controls GSIS in b-cells, involving generation of a-ketoglutarate in the cytosol and its subsequent recycling to pyruvate via mitochondrial enzymes. 

 

Notes:  This work was done in collaboration with Mette Jensen and Sarah Ronnebaum.  For the paper I am listed as second author.

 

 

Project 4:  de novo production of Free fatty acids is not critical for glucose-stimulated insulin secretion.

 

Abstract:  Free fatty acids have been proposed to be an important signaling molecule for insulin secretion.  Dr. M. Prentki and Dr. B. Corkey have proposed that a second messenger for insulin secretion outside of alterations in the ATP/ADP ratio may be long-chain acyl CoA (LC-CoA).  They propose that glucose stimulates the production of LC-CoA and that these LC-CoA are then able to modulate insulin secretion by a number of mechanism.  An important enzyme in the de novo production of LC-CoA from glucose is citrate lyase (CL).  The enzyme catalyzes the breakdown of citrate to oxaloacetate and acetyl CoA.  Acetyl CoA is then converted to malonyl CoA which is used by fatty acid synthase to generate palmitate.  There is controversy over the potential role CL plays in insulin secretion.  An inhibitor of CL, hydroxycitrate, appears to inhibit insulin secretion in some investigators hands whereas in others it does not.  I have investigated this controversy and have determined that the method of preparation of this reagent leads to a two fold increase in NaCl in the insulin secretion buffer and that this effect alone explains the ability of hydroxycitrate to inhibit insulin secretion since the removal of the excess NaCl prevents hydroxycitrates ability to inhibit insulin secretion.  In order to further investigate the role of CL in insulin secretion I have used a siRNA duplex against CL.  The siRNA duplex reduces CL expression by 90-95% and reduces CL protein expression by 85-95%.  The reduction in CL protein translates into a 90% reduction in CL activity.  The siRNA duplex against CL dose not affect glucose utilization, oxidation or glucose-stimulated increase in the ATP/ADP ratio.  The siRNA duplex also does not affect insulin secretion.  These pieces of data suggest that CL and de novo production of free fatty acids from glucose is not critical for insulin secretion.

 

Notes:  This work was done in collaboration with a summer student Jeff Muehlbauer.  For the paper I will be listed as first author.

 

 

Project 5:  The search for novel NADPH binding proteins that modulate insulin secretion.

 

Abstract:  Since our laboratory has provided a significant amount of data suggesting that NADPH plays a signaling role in insulin secretion, I have undertaken studies to determine what is the downstream effector of NADPH.  In particular, I am searching for novel signaling molecules that can be modulated by NADPH.  In collaboration with Tim Haystead we have developed a novel column that binds to NADPH binding proteins.  We are currently using this column to search for NADPH binding proteins in a b-cell line 832/13 cells.  After eluting proteins that bind to the column they are identified using peptide mass fingerprinting by MALDI-MS.  These identified proteins will then be tested for their ability to modulate insulin secretion.

 

Notes:  This work was done in collaboration with Tim Haystead.  For the paper I will be listed as first author.

 

 

Project 6:  Metabolic Regulation of Insulin Secretion: Importance of Pyruvate Carboxylase.

 

Abstract: Cellular metabolism of glucose is required for stimulation of insulin secretion from pancreatic b-cells, but the underlining biochemical pathways involved are incompletely understood.  Pyruvate carboxylase (PC) is highly active in b-cells, accounting for approximately 40-50% of pyruvate entry into mitochondrial metabolism at stimulatory glucose concentrations.  Furthermore, ¹³C-NMR isotopomer analysis demonstrates a linear correlation between glucose-stimulated insulin secretion (GSIS) and pyruvate cycling activity (substrate flux from pyruvate to oxaloacetate and back to pyruvate) in INS-1-derived cell lines.  Moreover, in lipid cultured cells, the normal glucose-induced increment in pyruvate cycling is eliminated in concert with a near complete inhibition of GSIS.  In order to further investigate the role of pyruvate cycling in regulation of insulin secretion, we used small interfering RNA (siRNA) technology to suppress PC mRNA levels by 70-80%, and PC protein by 60-70% in INS-1-derived 832/13 cells.  Interestingly, this manipulation increased insulin content by 2.3 ± 0.6 fold compared to control cells, resulting in 73 ± 28% and 30 ± 14% increases in basal (3 mM glucose) and stimulated (12 mM glucose) insulin secretion, respectively.  We speculate that the increment in content might be due to an accumulation of glycolytic intermediates, which in turn, by a yet unknown mechanism, stimulates insulin expression as part of b-cell glucose sensing.  When it comes to relative insulin secretion, diminution of PC levels translates to a net decrease in fold-response to stimulatory glucose from 7.0 ± 0.7 fold in control cells to 5.3 ± 0.5 fold in PC siRNA-treated cells, further demonstrating the importance of pyruvate cycling in insulin secretion. 

 

Notes:  This work was done in collaboration with Mette Jensen and Sarah Ronnebaum.  For the paper I am listed as second author.