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Islet Cell Biology Core
The Core is currently located on the fifth floor of Stemmler Hall in Diabetes Center space and is organized to provide routine services and access to diverse more sophisticated approaches and equipment that promise to advance the research capabilities and productivity of about 2 dozen investigators in the pancreatic islet cell field.
Background
Islet research at Penn has greatly expanded since the inception of the DERC in 1977 and has had a significant impact on the field during the last 25 years. The Diabetes Center has always been the nucleus of this program following the establishment of the laboratories of Drs. Clyde Barker and Franz Matschinsky in the late 1970s. Following the inception of the DERC in 1977 several investigators have been trained here and have since established islet cell research 1aboratories at other academic institutions and in industry (K. Braymen, B.Corkey, D. Dafoe, P. Drain, Y. Liang, M. Meglasson, J. Parker, M. Prentki, P. Ronner and W. Zawalich). Others have continued their career here or have been attracted to the faculty because of unique research opportunities (e.g. R. Ahima, M. Birnbaum, C. Barker, N. Doliba, C. Deng, K. Gooch, Y. Imai, K. Kaestner, L. Kubin, J. Markmann, F. Matschinsky, A. Naji, R. Simmons, D. Stoffers, C. Stanley, K. Teff, B Wolf ). A subgroup of these (Barker, Deng, Markmann, Matschinsky, Naji, Wolf) has established an NIDDK supported pancreatic islet transplant program under the leadership of Dr. Ali Naji. At the present time, the Islet Cell Biology Core of the Penn Diabetes Center is designed to play a critical role in the scientific life of a multifaceted group of individuals studying islet cells in health and disease.
Scientific Purpose:
It is the purpose of this core to assist investigators who currently study or have plans to study independently or collaboratively various aspects of pancreatic islet cell biology. In order to accomplish this goal the Core provides four services:
¾ isolate, culture, and functionally assess pancreatic islets of rat and mouse including batch
incubations, perifusions, respirometry, measurements of Ca (^) i++, the P-potential (ATP, ADP, AMP and Pi ) and other metabolites, hormone contents and release.
¾ Will perform extra corporal phenotyping of the endocrine pancreas of mouse and rat using
the intact isolated perfused or minced perifused pancreas (both of these techniques to be fully established).
¾ maintains a broad and well characterized stock of transformed islet cells, grows large batches
of such cells or generates pseudo islets by embedding such cells into agarose beads for dynamic studies of metabolism and hormone release.
¾ provides in depth consultation and helps develop strategies how to use the services of the
core optimally or will attempt to modify available technologies to solve particular problems.
Personnel of the Islet Cell Biology Core
Dr. Franz Matschinsky serves as core director and he is involved in all aspects of the operation of the core in accordance with the general principles of operating DERC core facilities. He sets priorities and makes job assignments. He closely interacts with users to achieve the highest benefits for them and the highest scientific standards of the work to be conducted.
Dr. Nicolai Doliba serves as the technical director. He is responsible for operating the Ca 2+, NAD(P)H imaging setup and maintain and operate the system for optical analysis of O 2 consumption, participating also in the development of a more sensitive instrument. He participates in the planning of all islet studies that require the above approaches and supervises or performs many of the actual measurements. He also does cell work involving NMR technology. He is aided by Dr. C. Li (fully supported by Dr. Charles Stanley from Children’s Hospital). Drs. Doliba and Li are going to establish the isolated rat and mouse pancreas, methods previously operative in Dr. Matschinsky’s laboratory as documented by many publications. This team also does the 13 C-and 31 P-NMR studies using islet cell lines.
Carol Buettger: Ms. Buettger is a Senior Research Specialist with more than 40 years of experience who masters all technical aspects of tissue culture, in this particular case of culturing and maintaining stock of an extensive collection islet cell lines, of culturing islets, of performing hormone release assays with a multi well plate test with alpha- and beta-cell lines and GLUTag cells releasing GLP-1. She knows how to
¾ Islet Isolation, Organ Culture and Delayed in vitro Functional Testing: There are the facilities
and the personnel for isolating about 5,000 to 10,000 rat islets or 1000 to 1500 mouse islets per day using classical collagenase digestion combined with Ficoll gradient centrifugation with modification by the procedure of M.L. McDaniel et al (Meth. in Enzymol. 98:182-200, 1983). The Core includes a dedicated tissue culture room with two hoods, four incubators, table top centrifuges and stereo as well as inverted microscopes. Rat and mouse islets may be cultured for as long as 14 days using a variety of culture conditions with virtually no endocrine cell losses but variable secretory function (depending on conditions).
¾ Maintenance of Stock. Culturing and Characterization of Transformed Islet Cells: The
use of islet cell lines derived from spontaneous insulinomas and from insulinomas of transgenic mice which carry transgenes constructed of the SV40 T-antigen and promoters of the major islet cell hormones insulin, glucagon, somatostatin continues to play an important role in islet cell research (S. Efrat, et al. Proc. Natl. Acad. Sci. USA .82,9037-9041,1980; S. Efrat, et al. Neuron 1,605-613, 1988; F. Radvanyi, et al. Molecular & Cell BioI. 13, 4223- 4232, 1993). The islet cell resource assists investigators of the Center by maintaining stock of many useful cell lines and providing well characterized cultured cells for various experimental purposes. The Core has stocked early passages of the RIN-m5F, HIT-T15, INS- 1, β-TC3, β-TC7, MIN-6 and has established a comprehensive collection of β -HC cells. It provides seed cultures and generates large batches of these cells for investigators (i.e. as many as 2 billion cells per harvest). Available technology of the core is the superfusion of cultured islet cells imbedded in agarose beads (modified from D.L. Foxall et al. Experim. Cell Res. 1.1:1:, 521-529, 1984). This paradigm allows dynamic studies with large amounts of 0.5 to 1.0 ml of packed islet cells for the application of NMR/or mass spectrometry and energy balance studies using A/V differences of O 2 , lactate/pyruvate and other parameters (e.g. determination of cytosolic pH) during fuel stimulation of hormone release. We have noted a declining demand for this resource, but predict that this approach will show an upsurge in the next grant period because metabolomic research will increase and make use of this resource.
Freshly isolated or cultured islets are functionally tested by several
approaches available through the core:
¾ measuring insulin, glucagon and cAMP release in perifusion systems (in connection with the
Radioimmunoassay Core)
¾ quantitating oxygen consumption in large batches of cells
¾ studying glucose metabolism using 15 N, 14 C or 3 H labeled
substrate (in collaboration with Dr. M. Yudkoff and Dr. I. Nissim; to be arranged case by case)
¾ investigating the behavior of free Ca2+^ as well as the redox state of NAD(P)H using state of
the art fluorescence imaging techniques
¾ Under development: quantitating oxygen consumption of small groups of freshly isolated and
cultured islet tissue using novel optical methods as developed by Dr. D. Wilson.
Facilities, Equipment and Collaborations:
The Islet Cell Biology Core is located on the Fifth floor of Stemmler HaIl in the Diabetes Center space. There is about 750 SF of dedicated laboratory space, equipped for islet isolation, perifusion and metabolic testing. It is also used for preparing pseudoislets from transformed cells and for testing cells functionally. The Core uses a 300 SF tissue culture room for culturing islets and transformed cells. The Core has also established a computerized image analysis system (the Zeiss Atto-Fluor System) for studying Ca 2+ transients and native NAD(P)H fluorescence. Apparatuses for isolated pancreas perfusions and optical microrespirometry are being reestablished or are available, respectively. The equipment includes in addition centrifuges, microscopes, perifusion pumps, oxygen electrodes, water baths, 2 computerized gradient mixers to generate any desired stimulus profile for islet and pseudoislet perifusion studies. We have access to state of the art NMR machines located at CHOP through Dr. Susan Wehrli, a long time collaborator. We have also established a close working relationship with Drs. M. Yudkoff and I. Nissim from Children’s Hospital of Philadelphia to perform heavy isotope studies of isolated islets using 15 N or 13 C labeled substrates.
Highlights of Core Activities:
In collaboration with Dr. David Wilson we were able to establish a prototype apparatus for measuring oxygen consumption of perifused pancreatic islets during insulin secretion stimulated by fuels, neuroendocrine modifiers and drugs. This optical method is based on the phosphorescence quenching by oxygen and is significantly more sensitive and maintains far more stable baselines than the common oxygen electrode based assays (see Figure 1). We have applied the method to two projects. Pancreatic islets from SUR1 -/-^ mice were investigated and it was observed that insulin release and energy metabolism do not change in parallel consistent with current views of exocytosis which has a low requirement for ATP. In another project it was observed that epigallocatechingallate (one of the active ingredients of green tea) inhibits glutamate dehydrogenase, glutamine induced insulin release and the associated enhanced respiration. These studies have been published in the JBC and AJP (references 52 and 54). These investigations justify our proposal to develop a next generation of respirometers allowing to study 50-100 islets rather than the currently needed 500-1000. The project illustrates how the Core interacts productively with scientists from other fields.
We were able to assemble a unique team of investigators under the umbrella of the ICBC (Drs. Kelley, Li, Matschinsky, Nissim, Stanley and Yudkoff) which resulted in the successful application of heavy isotope technology for the study of glutamine metabolism in a mouse model of GDH linked hypoglycemia. We discovered that GDH in pancreatic islet tissue operates in the oxidative direction only, providing an explanation for many observations in man and with animal or cell models. This work exemplifies what we consider as an ideal activity of this core.
Under Dr. Matschinsky's direction it was possible to show for the first time that a newly discovered glucokinase activator drug enhanced glucose induced insulin release in normal rodent and human islets and enhanced insulin secretion from defective human islets. It is anticipated that glucokinase activators will be developed to a useful drug therapy for type 2 diabetes. This high light shows how interaction between
Figure 2: Changes in intracellular Ca2+ concentration in islets and INS-1 cells. Panel A: Effect of IBMX and glucose on intracellular Ca2+^ concentration in control and SUR1-/-^ islets. Panel B: Effect of glyburide on intracellular Ca2+^ concentration in INS-1 cells treated with scrambled or SCHAD siRNA.
The changes in cytosolic Ca2+ concentration were recorded using Fura-2AM. The Ca 2+^ tracings are typical examples (n=3).
List of Publications of Work Supported by the ICBC (2002-2006):
- Sund NJ, Vatamaniuk MZ, Casey M, Ang SL, Magnuson MA, Stoffers, DA, Matschinsky FM, Kaestner KH. (2001). Tissue-Specific Deletion of Foxa2 in Pacreatic B Cells Results in Hyperinsulinemic Hypoglycemia. Genes and Development 15: 1706-
- Lee CS, Sund NJ, Vatamaniuk MZ, Matschinsky FM, Stoffers DA, Kaestner KH. Foxa Controls Pdx1 Gene Expression in Pancreatic ß-Cells In Vivo. Diabetes 51: 2546-2551,
- Gao Z, Young RA, Trucco MM, Greene SR, Hewlett EL, Matschinsky FM, Wolf BA. Protein kinase A translocation and insulin secretion in pancreatic beta-cells. Studies with adenylate cyclase toxin from Bordetella pertussis. Biochem J. 2002 Aug 15
- Kelly A, Li C, Gao Z, Stanley CA, Matschinsky FM. Glutaminolysis and Insulin Secretion From Bedside to Bench and Back. Diabetes 51: S421-S426, 2002.
- Boloker J, Gertz S, and Simmons RA: Offspring of Diabetic Rats Develop Obesity and Type II Diabetes in Adulthood: Diabetes: 51: 1499-1506, 2002.
- Scearce, L.M., Brestelli, J.E., McWeeney, S.K., Lee, C.S., Mazzarelli, J., Pinney, D.F., Pizarro, A., Stoeckert, C.J., Clifton, S., Permutt, M.A., Brown, J., Melton, D.A. and Kaestner, K.H.: Functional genomics of the endocrine pancreas: the pancreas clone set and PancChip, new resources for diabetes research. Diabetes Vol. 51: 1997-2004, July 2002.
- Lee, C.S., Sund, N.J., Vatamaniuk, M.Z., Matschinsky, F.M., Stoffers, D.A. and Kaestner, K.H.: Foxa2 controls Pdx1 gene expression in pancreatic beta cells in vivo. Diabetes Vol. 51: 2546-2551, August 2002.
- Liu, Y., Shen, W., Brubaker, P.L., Kaestner, K.H. and Drucker, D.J.: Foxa3 (HNF-3 gamma) binds to and activates the rat proglucagon gene promoter but is not essential for proglucagon gene expression. Biochemical J. Vol. 366: 633-641, September 2002.
- Lee CS, Sund NJ, Vatamaniuk MZ, Matschinsky FM, Stoffers DA, Kaestner KH. (2002). Foxa controls Pdx1 gene expression in pancreatic beta-cells in vivo. Diabetes 51: 2546-2551.
- Gao ZY, Xu G, Young RA, Hewlett EL, Matschinsky FM, Wolf BA (2002) Different Protein Kinase A Isoforms mediate Insulin Secretion and Insulin Promoter Activity of Pancreatic b-Cells. Studies with adenylate cyclase toxin from Bordetella pertussis. Biochemical Journal 368:397-404.
- Li C, Najafi H, Daikhin Y, Nissim IB, Collins HW, Yudkoff M, Matschinsky FM , Stanley CA. Regulation of Leucine-stimulated Insulin Secretion and Glutamine Metabolism in Isolated Rat Islets. J. Biol. Chem. , 278: 2853 - 2858. Jan 2003.
- Doliba NM, Vatamaniuk MZ, Buettger CW, Qin W, Collins HW, Wehrli SL, Carr RD, Matschinsky FM. Differential Effects of Glucose and Glyburide on Energetics and Na +^ Levels of βHC9 Cells: NMR Spectroscopy and Respirometry Studies. Diabetes 52: 394-402, Feb 2003.
- Deng S, Vatamaniuk M, Lian MM, Doliba N, Wang J, Bell E, Wolf B, Raper S, Matschinsky FM, Markmann JF. Insulin gene transfer enhances the function of human islet grafts. Diabetologia. 2003 Mar;46(3):386-93.
- Gao Z, Young RA, Li G, Najafi H, Buettger C, Sukumvanich SS, Wong RK, Wolf BA, Matschinsky FM. Distinguishing features of leucine and alpha-ketoisocaproate sensing in pancreatic beta-cells. Endocrinology. 2003 May;144(5):1949-57.
- Grimsby J, Sarabu R, Corbett WL, Haynes N-E, Bizzarro FT, Coffey JW, Guertin KR, Hilliard DW, Kester RF, Mahaney PE, Marcus L, Qi L, Spence CL, Tengi J, Magnuson MA, Chu CA, Dvorozniak MT, Matschinsky FM, Grippo JF. Allosteric Activators of Glucokinase: Potential Role in Diabetes Therapy. Science 2003 July 18; 301: 370-373. (in Reports)
- Grimsby J., Matschinsky F.M., Grippo J.F.: Discovery and Actions of Glucokinase Activators. In: Glucokinase and Glycemic Disease: From Basics to Novel Therapeutics, Matschinsky F.M. and Magnuson M.A. (eds.) Basel, Karger, 2004, Vol. 16, pp 360-378.
- Li C, Buettger C, Kwagh J, Matter A, Daikhin Y, Nissim IB, Collins HW, Yudkoff M, Stanley CA, Matschinsky FM. A signaling role of glutamine in insulin secretion. J Biol Chem. 2004 Apr 2;279(14):13393-401. Epub 2004 Jan 20.
- Doliba NM, Qin W, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, Matschinsky FM. Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine. Am J Physiol Endocrinol Metab. 2004 May;286(5):E834-43. Epub 2004 Jan 21.
- Kobinger GP, Deng S, Louboutin JP, Vatamaniuk M, Matschinsky F, Markmann JF, Raper SE, Wilson JM. Related Articles, Transduction of human islets with pseudotyped lentiviral vectors. Hum Gene Ther. 2004 Feb;15(2):211-9.
- Shaoping Deng, Marko Vatamaniuk, Xiaolun Huang, Nicolai Doliba, Moh-Moh Lian, Adam Frank, Ergun Velidedeoglu, Niraj M. Desai, Brigitte Koeberlein, Bryan Wolf, Clyde F. Barker, Ali Naji, Franz M. Matschinsky, and James F. Markmann Structural and Functional Abnormalities in the Islets Isolated From Type 2 Diabetic Subjects. Diabetes 53: 624-632, March 2004.
- Schmitz A, Shiue CY, Feng Q, Shiue GG, Deng S, Pourdehnad MT, Schirrmacher R, Vatamaniuk M, Doliba N, Matschinsky F, Wolf B, Rosch F, Naji A, Alavi AA. Synthesis and evaluation of fluorine-18 labeled glyburide analogs as beta-cell imaging agents. Nucl Med Biol. 2004 May;31(4):483-91.
- Kristen A. Lantz, Marko Z. Vatamaniuk, John E. Brestelli, Joshua R. Friedman, Franz M. Matschinsky, and Klaus H. Kaestner. Foxa2 regulates multiple pathways of insulin secretion. J. Clin. Invest., Aug 2004; 114: 512 - 520.
- A Mancuso, NJ Beardsley, S Wehrli, S Pickup, FM Matschinsky, and JD Glickson. Real-time detection of 13C NMR labeling kinetics in perfused EMT6 mouse mammary tumor cells and betaHC9 mouse insulinomas. Biotechnol Bioeng, Sep 2004; 87(7): 835-48.
- Vuguin P, Raab E, Liu B, Barzilai N, Simmons RA: Hepatic Insulin Resistance Precedes the Development of Diabetes in a Model of Intrauterine Growth Retardation: Diabetes 53: 2617-2622,
- Esni F, Stoffers DA, Takeuchi T and Leach SD. (2004). Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas. Mechanisms of Development 121: 15-25.
- Schreiber FS, Deramaudt TB, Brunner TB, Boretti MI, Gooch KJ, Stoffers DA, Bernhard EJ, Rustgi AK. (2004). Successful growth and characterization of mouse pancreatic ductal cells: Functional properties of the Ki-RAS oncogene. Gastroenterology 127(1): 250-260.
- Stoffers DA: The development of beta cell mass: recent progress and potential role of GLP-1. Hormone & Metabolic Research Vol. 36: 811-821, 2004.
- Cao X, Yang J, Burkhardt BR, Gao ZY, Wong R, Greene S, Wu J, Wolf BA (2005) Effects of over- expression of Pancreatic-Derived Factor (FAM3B) in isolated mouse islets and bTC3 cells. American Journal of Physiology 289:E543-E
- Yang J, Wang X, Moibi J, Hessner MJ, Greene S, Wu J, Wong RK, Sukumvanich S, Wolf BA and Gao Z (2004) Leucine culture reveals that ATP synthase functions as a fuel sensor in pancreatic b cell. Journal of Biological Chemistry 279:53915-
- GP Kobinger, S Deng, JP Louboutin, M Vatamaniuk, VM Rivera, MM Lian, JF Markmann, T Clackson, SE Raper, F Matschinsky, and JM Wilson. Pharmacologically regulated regeneration of functional human pancreatic islets. Mol Ther, Jan 2005; 11(1): 105-11.
- Rana K. Gupta, Marko Z. Vatamaniuk, Catherine S. Lee, Reed C. Flaschen, James T. Fulmer, Franz M. Matschinsky, Stephen A. Duncan, and Klaus H. Kaestner. The MODY1 gene HNF-4 regulates selected genes involved in insulin secretion. J. Clin. Invest., Vol. 115: 1006-1015, April 2005.
- Rickels MR, Schutta MH, Markmann JF, Barker CF, Naji A, and Teff KL. ï ¢-Cell Function Following Human Islet Transplantation for Type 1 Diabetes. Diabetes 54: 100-106, 2005.
- Rickels MR, Schutta MH, Mueller R, Markmann JF, Barker CF, Naji A, and Teff KL. Islet Cell Hormonal Responses to Hypoglycemia After Human Islet Tranpslantation for Type 1 Diabetes. Diabetes 54: 3205-3211, 2005.
- Selak MA, Suponitsky-Kroyter I, Simmons RA: Progressive accumulation of MtDNA mutations and decline of mitochondrial function lead to ß-cell failure. J Biol Chem 280 28785-28791, 2005.
- Lee, C.S., Sund, N.J., Behr, R. and Kaestner, K.H.: Foxa2 is required for the differentiation of pancreatic alpha cells. Developmental Biology 278(2): 484-495, February 2005.
- De Leon DD, Crutchlow MF, Ham JN, Stoffers DA: Role of Glucagon-like Peptide-1 in the Pathogenesis and Treatment of Diabetes Mellitus. International Journal of Biochemistry and Cell Biology under revision 2005.
- Yang J, Gao Z, Robert CE, Burkhardt BR, Gaweska H, Wagner A, Wu J, Greene SR, Young RA and Wolf BA (2005) Structure-function studies of PANDER, an islet specific cytokine inducing apoptosis of insulin-secreting beta cells. Biochemistry 44:11342-
- Burkhardt BR, Yang MC, Robert CE, Yang J, Greene SR, McFadden KK, Wu J, Gao Z, and Wolf BA (2005) Tissue-Specific and Glucose-Responsive Expression of the Pancreatic Derived Factor (pander) Promoter. Biochimica Biophysica Acta 1730:215-
- Xu W, Gao Z, Wu J, Wolf BA (2005) Interferon-g-induced Regulation of the Pancreatic Derived Cytokine FAM3B in Islets and Insulin-secreting bTC3 Cells. Molecular Cellular Endocrinology 240:74-
Governance and Fees
We ask users, who require repeated services, to pay for animal purchase cost and per diems and contribute to cost of collagenase, Ficoll, tissue culture reagents and other materials.
¾ It costs the Core $400 to culture 2 billion cells when production is geared up (excluding
personnel).
¾ An NMR study using 13 C or 13 P containing metabolites to study the energetics of β-cells and
involving a reasonable (n) of 20 experiments would cost the investigator or a group of investigators $8000-9000, which is considered much less than required if an independent laboratory would generate the material not including training and startup cost.
¾ The actual cost to the Core of preparing isolated islets (fresh or cultured) is the result of prices
for animals, collagenase, Ficoll, plastic ware, media and fetal calf serum and amounts to $250/750-1000 cultured and about 2/3 that for freshly isolated islets. This does not include personnel costs.
¾ Responding to a recommendation of the External Advising Committeethe core offer the option
of services for fixed fees for two services of the core rather than ask for provision of reagents: islet isolation and cell expansion. We will charge $75 for isolation of islets from 1 adult rat or from 3-5 adult mice treated as one batch. Culturing the islets will add $30/2 days of culturing each islets harvest.
¾ We will continue to provide services of "side order" size without charge (i.e ≤100 islets). We
will also continue to offer initial services free of charge e.g. ≤500 islets 2-3 times, or batches of transformed cells ≤ 1 billion 2-3 times for the purpose of pilot studies to facilitate islet research.
¾ Occasional microscopic imaging and metabolic rate measurements (O 2 -consumption) are
performed free of charge as are pilot perifusion studies designed to test the viability of islets that have been prepared by users. Extensive projects involving the latter procedures will require special arrangements because of limited capacity and associated cost.
