Jake Kushner Lab

Division of Endocrinology, Children's Hosptital of Philadelphia, University of Pennsylvania School of Medicine


all © 2008 Jake Kushner



Mission Statement
Overview
Beta Cell Progenitors  Beta Cell Turnover
Cell Cycle Regulation of Beta Cells Conclusions

Publications The P.I.
Group Members Contact Us
Directions Parent Organizations
Organizations We Support


Mission Statement:

Despite advances in basic science knowledge and clinical care, Type 1 and 2 diabetes are serious illnesses that are associated with profound individual and societal consequences.  At the heart of abnormal glucose homeostasis is an inability to make enough insulin to control blood glucose levels. We seek improved treatments for individuals affected with diabetes that will restore or preserve insulin secretion, in order to delay or prevent long term diabetes complications.


Overview

The Islets of Langerhans secrete insulin and other hormones to regulate glucose homeostasis. Even though insulin-secreting beta-cells replicate at very low rates, early in adulthood total islet mass can slowly expand and adapt to increased insulin requirements. In type 1 diabetes islet mass is greatly decreased by autoimmune attack, resulting in an absolute insulin deficiency. In type 2 diabetes islet growth adaptive mechanisms fail, resulting in insufficient insulin secretion. Thus, understanding the molecular mechanism of islet growth is central to the development of definitive cures for both type 1 and type 2 diabetes. However, very little is known about the molecular regulation of adult beta-cell growth and survival. Moreover, even the normal lifecycle of islets remains poorly understood.

How does beta-cell failure occur in type 2 diabetes?  Where do beta-cells come from?  Do all beta-cells replicate? What signaling pathways influence beta-cell replication?  How long to beta-cells survive?  These are the questions our laboratory hopes to answer using genetic manipulation in mice, along with a variety of in vitro techniques.

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Beta Cell Progenitors:

Specialized progenitors play essential roles throughout the body in tissue development, maintenance, and regeneration. For example, in gastrointestinal epithelia and skin specialized progenitors (stem cells) both self renew and give rise to rapidly dividing progeny (the transit amplifying cells), which migrate away from stem cell niches, terminally differentiate, and cease to replicate. Similarly, specialized progenitors have been speculated to occur in pancreatic ß-cell mass expansion, with a “proliferative compartment” of very rapidly proliferating ß-cells or proliferating progenitors (Bonner-Weir, 1992; Gershengorn et al., 2004; Swenne, 1983). However, no direct evidence exists to support or refute a proliferative compartment model within the ß-cell lineage. Do specialized progenitors (insulin positive or otherwise) have any contribution to the adult ß-cell lineage?

We decided to address this fundamental question by determining the cell division histories of ß-cells and their progenitors.  To carry out this work we adapted existing DNA precursor labeling techniques to reveal the cell division histories of mature tissues over long periods of time. Remarkably, we do not observe any contribution to adult ß-cell mass by specialized progenitors or stem cells. Instead, we find that adult ß-cells uniformly self-renew. We find that adult ß-cells exhibit equal proliferation potential, and expand from within a vast and uniform pool of mature ß-cells.




We adapted techniques to detect incorporation of the thymidine analogues 5-chloro-2-deoxyuridine (CldU) and 5-iodo-2-deoxyuridine (IdU) into tissues of mice that were labelled with the analogues over prolonged periods via the drinking water. This strategy employs two different forms of anti-BrdU antisera raised in different species, which bind to CldU and IdU with different affinities (a). As each analogue could theoretically detect a distinct round of cell division, we speculated that this technique could allow detection of more than one round of cell division in vivo. If specialized progenitors substantially contribute to a mature tissue, recently divided cells should have undergone multiple rounds of cell division and therefore be doubly labelled (b). Alternatively, a mature tissue could renew or expand by self-renewal, and recently divided cells would not be comprised of cells that had undergone multiple rounds of cell division (c).



In this series of pictures, mice were treated with CldU for 24 hours followed by Idu for 24 hours.  Consequently, dividing cells were first marked with CldU (viewed here as red), then with IdU (viewed here as green).  In the surface lining of the gastrointestinal tract (above), double colored cells are present deep in the folds of the crypts. This indicates that these cells divide multiple times from "specialized progenitors". 



Similarly, in the skin cells can be observed that undergo multiple rounds of cell division.  In the above picture, three  hair follicles are seen.  Notice the CldU (red) IdU (green) co-positive cells that sit in the outer root sheath of the hair follicle in the center.  These cells repeatedly divided in the presence of CldU and then IdU.




In comparison, pancreatic islets show only beta cells that are singly labeled by either CldU (red) or IdU (green). This result indicates that the insulin secreting beta cells are the products of cells that divide very infrequently, and are not the products of specialized progenitors.

Teta M, Rankin MM, Long SY, Stein GM, Kushner JA. (2007). Growth and Regeneration of Adult Beta Cells Does Not Involve Specialized Progenitors. Developmental Cell 12(5): 817-826.

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Beta Cell Turnover

Our lab is also interested in the normal life cycle of
β-cells. Surprisingly little is known about the normal lifecycle of β-cells or the signaling pathways required for β-cell survival. It has been known for some time that β-cell replication declines as rodents age, but widely assumed that proliferation rates eventually stabilized at a rate of 1-3% per day. As such, adult β-cells have been speculated to have a finite lifespan, with ongoing adult β-cell replication throughout life to replace lost cells. However, little solid evidence supports this idea. To more accurately measure adult β-cell turnover we performed continuous long-term labeling of proliferating cells with the DNA precursor analog 5-bromo-2-deoxyuridine (BrdU) in one-year-old mice.



A priori, we predicted at least 3 possible models of BrdU labeling of β-cells using continuous BrU (see figure above). If β-cells rapidly turn over, as predicted by several authors, near complete β-cell BrdU labeling would occur if the period of label exceeded typical β-cell lifespan. In contrast, if β-cells exhibit slow turn over, limited accrual of BrdU label would occur in β-cells. Finally, heterogeneity within islets might result in non-linear BrdU labeling: a limited population of rapidly dividing but short lived β-cells could co-exist with slowly proliferating long-lived β-cells.



Islet β-cell histology of pancreas sections immunostained with antibodies against insulin (green) and BrdU (red) and counterstained with DAPI (blue) and photographed with a 20x (left) or a 100x (right) objective.

Surprisingly, our results show that adult mouse β-cells acquire BrdU in a very slow manner and appear to be very long lived, with approximately 1 in 1400 mature β-cells turning over per day. In contrast to other cells contained within pancreatic preparations, insulin/BrdU co-positive cells were very rare and comprised only a small portion of total β-cells, even in mice exposed to BrdU for very long periods. β-cell proliferation rates averaged less than 0.1 % per day, far lower than expected given previously published data of 3% daily β-cell proliferation in rats.

Thus, our data suggests that β-cell replication is a rare event in aged adult mice, with minimal evidence of β-cell turnover. We conclude that aged murine β-cells are in a mostly quiescent G0 state. Although it has been assumed that signaling pathways that promote β-cell growth are also active in β-cell remodeling and adaptation, this has not been formally proven in most cases. Therefore, it is possible that some factors shown to regulate β-cell growth in adolescence might not be required for adult β-cell survival. Similarly, other β-cell mitogenic signals might only be involved in adult β-cell adaptation, allowing β-cells to emerge from the quiescent G0 state to enter the G1 state and divide. Moreover, human type 2 diabetes could conceivably occur from either scenario: inadequate β-cell growth, or insufficient β-cell adaptation. If true, drugs that promote β-cell adaptation might be especially effective diabetes treatments, even in the face of severe insulin resistance.

Teta M, Long SY, Wartschow LM, Rankin MM, Kushner JA (2005). Very Slow Turnover Of Beta Cells In Aged Adult Mice. Diabetes, 54(8).

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Cell Cycle Regulation of Beta Cells

As a result of these observations, we became interested in the molecular regulation of β-cell proliferation. In many mammalian cells growth factors stimulate D-type cyclin/cyclin dependent kinase (cdk) activity, which phosphorylates and inactivates retinoblastoma (Rb) tumor suppressor proteins, resulting in G1 cell cycle progression. Our recent work illustrates that cyclins D2 and D1 are essential for normal postnatal islet growth and function. Although neonatal islet development is normal in the absence of cyclins D2 and/or D1, cyclin D2-/- mice develop diabetes from deficient islet mass by one year, and cyclin D1+/- D2-/- have an even more severe islet phenotype, which typically results in death by 4 months of age.
Wild type mouse islet stained with insulin (green) and gucagon (red). 

cyclin D2-/- mouse islet stained with insulin (green) and gucagon (red).

cyclin D1+/- D2-/- mouse islet stained with insulin (green) and gucagon (red).
ß-cell proliferation measured by performing BrdU labeling at 16 days of life. Pancreas sections from wild-type and cyclin D2–/– mice displayed equal numbers of insulin and BrdU copositive cells. By contrast, ß-cell proliferation was barely detected in cyclin D1+/– D2–/– mice at 16 days of age; when D-type cyclin activity drops below a critical level ß-cell duplication is almost totally ablated.

By 3 months ß-cell proliferation was extremely low in cyclin D2–/– mice in comparison to wild-type mice or cyclin D1+/– mice and not detected at all in cyclin D1+/– D2–/– mice.
These results clearly show that cyclins D2 and D1 are essential for normal islet growth and maintenance. 

Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF (2005). Cyclins D2 And D1 Are Essential for Postnatal Pancreatic Beta-Cell Growth. Molecular and Cellular Biology, 25(9436): 3752-3762.

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Conclusions:
How is D-type cyclin activity regulated in β-cells?  What are the downstream targets of D-type cyclins in β-cells?  Can this pathway be manipulated to increase adult β-cell replication?

Based on this new understanding of the normal life cycle of β-cells, we feel that much remains to be learned in order to make adult β-cell replication therapies a reality.  Why do β-cells proliferate so infrequently? Which β-cells proliferate? To be honest, we have no idea. However, these questions suggest experimental avenues which we are actively pursuing with rigorous hypothesis-driven studies.


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Recent Publications :
Sherry NA, Chen W, Kushner JA, Glandt M, Tang Q, Tsai S, Santamaria P, Bluestone JA, Brillantes AB, Herold KA. (2007). Exendin-4 improves reversal of diabetes in NOD mice treated with anti-CD3 mAb by enhancing recovery of  beta-cells. Endocrinology 148(11):5136-44. Link

Podcheko A, Northcott P, Bikopoulos G, Lee A, Bommareddi SR, Kushner JA, Farhang-Fallah J, Rozakis-Adcock M. (2007). Identification of a SD-40 repeat-containing isoform of PHIP as a novel regulator of beta-cell growth and survival Molecular and Cellular Biology 27(18): 6484-96. Link

Kiel MJ, He S, Ashkenazi R, Gentry SN, Teta M, Kushner JA, Jackson TL, Morrison SJ. (2007). Hematopoietic stem cells do not asymmetrically segregate chromosomes or retain bromodeoxyuridine. Nature 449(7159): 238-42.
Link

Teta M, Rankin MM, Long SY, Stein GM, Kushner JA. (2007). Growth and Regeneration of Adult Beta Cells Does Not Involve Specialized Progenitors. Developmental Cell 12(5): 817-826.
Link

Ablamunits V, Sherry NA, Kushner JA, Herold KC. Autoimmunity and beta cell regeneration in mouse and human type 1 diabetes: the peace is not enough.
Ann N Y Acad Sci. 2007 Apr;1103:19-32.
Link

Sherry NA, Kushner JA, Glandt M, Kitamura T, Brillantes AM, Herold KC
(2006). Effects of autoimmunity and immune therapy on beta-cell turnover in type 1
diabetes.
Diabetes. 55(12):3238-45. Link

Kushner JA (2006). Beta-Cell Growth: An Unusual Paradigm of Organogenesis That is Cyclin D2/Cdk4 Dependent. Cell Cycle. (3):234-7
Link

Kushner JA, Simpson L, Wartschow LM, Guo S, Rankin MM, Parsons R, and WhiteMF (2005).
Phosphatase and tensin homolog regulation of islet growth and glucose homeostasis. Journal of Biological Chemistry, 280(47): 39388-93. Link

Teta M, Long SY, Wartschow LM, Rankin MM, Kushner JA (2005). Very Slow Turnover Of Beta Cells In Aged Adult Mice. Diabetes, 54(8).
Link

Kushner JA, Ciemerych MA, Sicinska E, Wartschow LM, Teta M, Long SY, Sicinski P, White MF (2005). Cyclins D2 And D1 Are Essential for Postnatal Pancreatic Beta-Cell Growth. Molecular and Cellular Biology, 25(9436): 3752-3762.
Link

Yi X, Schubert M, Peachey NS, Suzuma K, Burks DS, Kushner JA, Suzuma I, Cahill C, Flint CL, Dow MA, Leshan RL, King GL, White MF. (2005). Insulin Receptor Substrate 2 is Essential for Maturation and Survival of Photoreceptor Cells. The Journal of Neuroscience 25(7). Link

Lin X, Taguchi A, Park S, Kushner JA, Li F, Li Y, White MF (2004). Dysregulation of insulin receptor substrate 2 in ß cells and brain causes obesity and diabetes. The Journal of Clinical Investigation 114(7): 908-916. Link

Pawlak DB, Kushner JA, Ludwig DS. (2004). Effects of dietary glycemic index on adiposity, glucose homeostasis and plasma lipids in an animal model. The Lancet 364(9436): 778-85.
Link

Kushner JA, Haj HG, Klaman LD, Dow MA, Kahn BB,Neel BG, and White MF. (2004). Islet-Sparing Effects of Protein Tyrosine Phosphatase-1b Deficiency Delays Onset of Diabetes in IRS2 Knockout Mice. Diabetes 53(1): 61-66. Link

Kushner JA, Ye J, Schubert M, Burks DJ, Dow MA, Flint CL, Dutta S, Wright CVE, Montminy MR, White MF. (2002). Pdx1 restores ß cell function in Irs2 knockout mice. The Journal of Clinical Investigation 109(9436): 1193-1200. Link


Medline Search for Jake Kushner


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The Principal Investigator :
Jake Kushner

 




Education:
1987   B.A.    University of California at Berkeley (Biochemistry)
1994   M.D.    Albany Medical College

Postgraduate Training and Fellowship Appointments:
1994-1997    Pediatrics Resident, Rhode Island Hospital, Brown University
1997-2000    Clinical Fellow in Pediatric Endocrinology, Children's Hospital, Boston
1999-2003     Postdoctoral Research Fellow, Laboratory of Morris White, Howard Hughes Medical Institute, Joslin Diabetes Center, Harvard Medical School

Faculty Appointments:
2000-2003    Instructor in Medicine, Department of Pediatrics, Harvard Medical School
2004-present    Assistant Professor of Pediatrics, University of Pennsylvania School of Medicine
2004-present    Faculty, Developmental Biology Graduate Group, Cell Biology and Physiology Program, University of Pennsylvania School of Medicine


Awards and Honors:
1999-2000    Lawson Wilkins Pediatric Endocrinology Society, Eli Lilly Fellowship
2001-2003    Juvenile Diabetes Research Foundation International Postdoctoral Research Fellowship
2003-2005    Charles H. Hood Foundation Child Health Research Grant
2003-2005    Lawson Wilkins Pediatric Endocrinology Society Clinical Scholar Award
2003-2008    NIH Mentored Clinical Scientist Career Development Award
2003-2009    NIH Pediatric Research Loan Repayment Program





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Lab Members :

Matthew Rankin, BA Kim Rak, BA

Research Technician  Research Technician



Alex Tuttle, BA

Jennifer Heim, BA


Research Technician
Research Technician



Dan Sartori, BA

Chris Wilbur, BA


Research Technician Research Technician


Anne Granger PhD
Alisa Schiffman DO


Postdoctoral Research Fellow Pediatric Endocrinology Fellow


Di Zhou Gina Kim
Undergraduate Research Fellow (U Penn) Undergraduate Research Fellow  (U Penn)


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Directions :

The Children's Hospital of Philadelphia
 The Joseph Stokes Jr. Research Institute · Leonard and Madlyn Abramson Pediatric Research Center
  3615 Civic Center Blvd.
Philadelphia, PA 19104
 
Click on this link for a map of the Abramson Building (Stokes Institute) at CHOP
NOTE: Our building is at the intersection of Osler Circle and Civic Center Blvd, and is not shown on the linked map. To find us, check in with security and take the elevators to the 8th floor.  Turn right.
Office: ARC 802c     Lab: ARC 804


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Contact Us:
Children's Hospital of Philadelphia
Division of Endocrinology, ARC 802c
3615 Civic Center Blvd.
Philadelphia, PA 19104 USA

email: last name immediately followed by first initial (and all one word) at mail dot med dot upenn dot edu

example for a fictional person whose first name is "First" and last name is "Last": lastf@mail.med.upenn.edu


Tel : 1 267 426 5717
Fax: 1 215 590 1605
Lab Tel: 215-590-4572
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Parent Organizations:

CHOP Division of Endocrinology Joseph R Stokes Research Institute
U Penn Cellular and Molecular Biology Graduate Group Children's Hospital of Philadelphia
UPenn Division of Endocrinology
The Institute for Diabetes, Obesity, and Metabolism (IDOM) at U Penn

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Organizations We Support:

Juvenile Diabetes Research Foundation
American Diabetes Association
Lawson Wilkins Pediatric Endocrine Society
Society for Pediatric Research

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