Cystic Fibrosis Transmembrane Conductance Regulator Deficiency Exacerbates Islet Cell Dysfunction After -Cell Injury
http://www.100md.com
糖尿病学杂志 2006年第7期
1 Department of Pediatrics, University of Florida, College of Medicine, Gainesville, Florida
2 Department of Pathology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, Florida
3 General Clinical Research Center, University of Florida, College of Medicine, Gainesville, Florida
CFRD, cystic fibrosiseCrelated diabetes; CFTR, cystic fibrosis transmembrane conductance regulator; FABP, fatty acideCbinding protein; hCFTR, human CFTR; IPGTT, intraperitoneal glucose tolerance test; STZ, streptozotocin
ABSTRACT
The cause of cystic fibrosiseCrelated diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTReC/eC), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced -cell injury. Fasting blood glucose levels were similar among age-matched noneCSTZ-administered animals, but they were significantly higher in CFTReC/eC mice 4 weeks after STZ administration (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTReC/eC, C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTReC/eC mice. STZ reduced islets among all strains; however, only CFTReC/eC mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatuseCsensitized CFTReC/eC mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTReC/eC state.
Cystic fibrosis is a systemic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Some of the manifestations of this disease are caused by defective CFTR chloride channel function, but often they are related to cellular responses to mutant CFTR (1,2). In Caucasians, it is the leading life-limiting recessive genetic disorder (3). In 1938, the mean life expectancy for children with cystic fibrosis was <1 year (4). Over the past 30 years, the life expectancy of children with cystic fibrosis in the U.S. has increased from 16 to 32 years of age (4,5). The onset of diabetes heralds a great impediment to quality of life and is the leading comorbidity noted among the 23,000 patients with cystic fibrosis listed in the Cystic Fibrosis Foundation Patient Registry (5,6). The overall prevalence of cystic fibrosiseCrelated diabetes (CFRD) has been estimated from 4.9 to 12% (5,6,7). In the cystic fibrosis population, the prevalence of CFRD appears to be increasing at a rate of 5% per year. By the age of 30 years, one study indicated 50% of cystic fibrosis patients had clinical diabetes (8). In addition, Hardin and Moran (6) report that the incidence of glucose abnormalities in adults with cystic fibrosis is 75%.
The prevailing mechanistic belief is that CFRD results from a combination of chronic pancreatitis and eventual loss of the islet cells, resulting in less insulin reserve and production, together with a variable state of insulin resistance (6,9eC11). Clinical disease is associated with worsened glucose metabolism during times of lung exacerbation. Autopsy findings of individuals with cystic fibrosis have revealed minimal islet disease before the 2nd decade but extensive replacement of pancreatic acinar tissue with fibrous and fatty tissue. As patients age, autopsy data demonstrate the eventual loss of islet tissue and decreased -cell numbers (12,13). Other findings include deposition of amyloid in the islets, a chronic architectural change, found in cystic fibrosis patients who also have CFRD (6).
As an alternative hypothesis, we question whether the origin of the glucose abnormality observed in CFRD is directly associated with CFTR mutation. The most common CFTR mutation (F508) is a missense mutation that produces a misfolded version of CFTR; this mutation has been shown to trigger an endoplasmic reticulum overload response within cells (14), resulting in widespread changes in gene expression (1,15eC17). Among the most prominent of these changes is an upregulation of proinflammatory cytokines associated with innate immunity (1). This may correlate with the observation that cystic fibrosis patients (18) and CFTR knockout mice (CFTReC/eC) (19) show an exaggerated proinflammatory cytokine response to bacterial challenge compared with normal individuals. To address important questions relating to the formation of diabetes in cystic fibrosis, we developed a novel model involving administration of CFTReC/eC mice with streptozotocin (STZ), an agent capable of inducing -cell injury. These studies suggest that the CFTR mutation exacerbates islet cell dysfunction in CFRD after -cell injury.
RESEARCH DESIGN AND METHODS
We used mice with the CFTR S489XeC/eC neo insertion, developed initially at the University of North Carolina (20). These mice were also modified with transgenic overexpression of gut-specific human CFTR, from the fatty acid bindingeCprotein (FABP) promoter, to prevent intestinal obstruction and improve viability (21). These animals were developed on a background of C57BL/6J and FVB/NJ. Hence, age-matched C57BL/6J and FVB/NJ mice were used as control animals for all experiments. CFTR S489XeC/eC FABP-hCFTR+/+ (hereafter designated CFTReC/eC), C57BL/6J, and FVB/NJ mice were housed under specific pathogen-free conditions at the University of Florida Animal Care Services according to National Institutes of Health guidelines and allowed food and water ad libitum. All experimental procedures were approved by the institutional animal care and use committee of the University of Florida.
Eight CFTReC/eC, seven C57BL/6J, and six FVB/NJ mice received lactated ringers as a placebo via intraperitoneal injection at 7eC9 weeks of age. Blood glucose levels were measured in CFTReC/eC, C57BL/6J, and FVB/NJ mice at baseline. Random or fasting blood glucose values were checked weekly from the point of initial injection with lactated ringers until 4 weeks postinjection. In separate experiments, -cell injury was induced according to the protocol by Ito et al. (22). Low-dose STZ (100 mg/kg; Sigma, St. Louis, MO) was given as a single intraperitoneal injection. CFTReC/eC (n = 10), C57BL/6J (n = 11), and FVB/NJ (n = 6) mice received STZ at 7eC9 weeks of age. Mice were monitored with blood glucose values determined weekly from the point of initial STZ injection until 4 weeks postinjection. STZ- and lactated ringereCadministered mice underwent an intraperitoneal glucose tolerance test (IPGTT) 4 weeks after injection. Mice with random blood glucose levels >350 mg/dl at both week 1 and 2 after STZ administration were designated as early-onset diabetes. These mice were discontinued from the protocol, secondary to the health of the mouse, but also because of the inability to test blood glucose in excess of 600 mg/dl at time of IPGTT. Mice with fasting blood glucose levels >240 mg/dl at the end of 4 weeks posteCSTZ administration were classified as late-onset diabetes and were included in the IPGTT.
To assess the impact of lung injury on glycemia, we used a protocol by Muller et al. (23) previously demonstrating an increased inflammatory response. CFTR S489XeC/eC animals (4eC6 weeks old) were sensitized to Aspergillus fumigatus crude protein extract (Greer Laboratories, Lenoir, NC). Briefly, animals were administered intraperitoneal injections of 200 e蘥 A. fumigatus dissolved in 100 e蘬 PBS on days 0 and 14 (n = 4). Aerosol challenge was performed with 0.25% A. fumigatus for 20 min in a 30 x 30x 20 cm acrylic chamber, using a jet nebulizer (model LC-D; PARI Respiratory Equipment, Midlothian, VA) with an air (room air) flow of 6 l/min on days 28, 29, and 30 after the sensitization. Nonsensitized control mice (n = 5) received intraperitoneal injections with PBS alone and were challenged with A. fumigatus along with sensitized mice. At 48 h after lung challenge, IPGTTs were performed. Age-matched nonsensitized and unchallenged control animals (n = 6) were also compared with mice receiving inhalation challenge.
IPGTTs.
Mice were fasted for 4 h before receiving 2 g/kg dextrose (50% dextrose solution) as an intraperitoneal injection. Glucose tolerance was monitored via tail vein sampling at time 0 (just before 50% dextrose solution injection), 30, 60, 120, and 180 min. Glucose was measured with a LifeScan OneTouch Ultra glucometer (reported in milligrams per deciliter).
Histology and immunohistochemistry.
Pancreas, lung, duodenum, and liver specimens were obtained immediately after the mice were killed at the end of the IPGTT and fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA). Tissues were processed into paraffin blocks and sections stained with hematoxylin and eosin for morphologic evaluation. The entire pancreatic specimen was visualized under 10x magnification, and all islets were counted and inspected for insulitis. Any evidence of insulitis was scored (24). Insulin immunohistochemistry was performed as previously described, using guinea pig anti-insulin antibodies (1:2000; Dako, Carpinteria, CA) and routine avidin-biotin-peroxidase detection (Vectastain ABC kit; Vector Labs, Burlingame, CA) with 3,3'-diaminobenzidine (DAB staining kit; Vector Labs) as chromagen (24). Amyloid deposition was evaluated by staining with alkaline Congo Red (PolyScientific, Bay Shore, NY).
Human CFTR detection in tissue.
To ensure the absence of human CFTR (hCFTR) expression by the pancreas, after the CFTReC/eC mice were killed, pancreas, lung, and small intestine were collected. Using surgical tools sterilized and sprayed with RNase Away (Molecular BioProducts, San Diego, CA), tissue was removed and immediately frozen in liquid nitrogen. After homogenizing 100 mg of tissue, mRNA was extracted, using Oligotex Direct mRNA columns (Qiagen, Valencia, CA). For RT-PCR, a Qiagen One-Step RT-PCR kit was used. Briefly, 14 ng of mRNA were incubated at 50°C with reverse transcriptase for 30 min with gene-specific primers for hCFTR (hCFTR forward: 5'-aaacttctaatggtgatgaccag; reverse: 5'-agaaattcttgctcgttgac) or B-actin (B-actin1: 5'-agctgagagggaaatcgtgc; B-actin2: 5'-accagacagcactgtgttgg). This was followed by incubating the samples at 95°C for 15 min to inactivate the reverse transcriptase. The cDNA was then amplified with the same primers for 30 cycles by PCR. The noeCreverse transcriptase controls were subjected to the same PCR conditions and primers but in the absence of reverse transcriptase.
Statistical analysis.
Data were analyzed via one-way ANOVA, using Bonferroni corrections for multiple comparisons to evaluate individual differences in strain and treatments. Glucose tolerance was calculated, using the percent change from the baseline fasting blood glucose value, and compared as the total area under the curve (trapezoidal rule) followed by one-way ANOVA calculations and Bonferroni corrections as described above. Differences between groups were considered significant if the Bonferroni-corrected two-sided P value was <0.05. Correlations of the number of pancreatic islets with blood glucose values were calculated with a Pearson calculation. All statistical analyses were conducted using SAS 9.1.2 software (Cary, NC).
RESULTS
Detection of hCFTR expression.
Tissue obtained from the small intestine of the CFTReC/eC mice demonstrated hCFTR mRNA, whereas the lung and pancreas sections did not (Fig. 1).
Glucose challenge in mice receiving lactated ringers.
No significant differences were observed in the fasting glucose levels of lactated ringereCadministered CFTReC/eC mice in comparison to the C57BL/6J and FVB/NJ background strains (time 0, P = NS) (Fig. 2A). In addition, responses to glucose stimulation were similar among these three strains after intraperitoneal injection of 50% dextrose solution (P = NS) (Fig. 2A). These studies did not suggest intrinsic differences in glucose regulation by the CFTReC/eC mice.
Glucose challenge in STZ-administered mice.
Fasting blood glucose levels were higher in STZ-administered CFTReC/eC mice compared with C57BL/6J and FVB/NJ background strains subjected to identical treatment (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTReC/eC, C57BL/6J, and FVB/NJ, respectively; P < 0.05) (Fig. 2B). These glucose values remained higher throughout the entire 3-h time period after glucose infusion, but the percent increase did not achieve significance in comparison to the other STZ-administered strains (Fig. 2B). These results suggest that in the face of minimally compromised -cell function (in this situation, due to chemically induced injury), there is indeed an underlying defect in glucose metabolism associated with deficiencies in CFTR. A small subset of both CFTReC/eC (1 of 10) and C57BL/6J (3 of 11) mice developed diabetes early after STZ injection and were classified as early-onset diabetes, and they were thus removed from the animal protocol (Table 1). All early-onset diabetic mice were male (weight 25eC30 g) and had received more total STZ dosage (100 mg/kg) than the female counterparts (weight 20 g).
Glucose challenge in mice with lung inflammation.
Interestingly, no changes in glucose, either fasting or stimulated, were observed in A. fumigatuseCadministered mice in comparison to age-matched PBS-treated or untreated controls (Fig. 3). This suggests that acute lung inflammation does not play a dominant role in the predilection to diabetes in cystic fibrosis.
Pancreatic acinar and islet histology.
Both lactated ringereCand STZ-administered CFTReC/eC mice demonstrated focal mild acinar degeneration with fibrosis and inflammatory infiltrates consisting primarily of mononuclear cells (Fig. 4). Saponification of pancreatic fat was also observed in one animal (Fig. 4A). Though not subject to direct quantitation, intensive histological examination suggested that these local changes in pathology were similar among both lactated ringereCand STZ-administered CFTReC/eC mice. No C57BL/6J or FVB/NJ mice demonstrated evidence of pancreatitis.
Next, we compared the pancreatic islets to identify wherein variances in islet inflammation, insulin content, or number might provide partial explanation for the observed differences in glucose regulation between these strains. Insulitis was not observed in any strain, in either the absence or presence of STZ administration. To the question of insulin content, no qualitative differences were observed, as determined by immunohistochemical evaluation (data not shown). Likewise, no significant differences in islet number were observed in comparison to CFTReC/eC, C57BL/6J, and FVB/NJ strains (Fig. 5A), in either the absence or presence of STZ administration (all P = NS). Islet amyloid deposition was not observed in any strain either with or without STZ administration (data not shown).
However, a strong negative correlation between the number of islets and fasting blood glucose measurements were noted in STZ-administered CFTReC/eC mice (r = eC0.73, P = 0.02) (Fig. 5B). The absence of such a correlation in the other strains of mice suggests that the CFTR deficiency exacerbates glucose tolerance in injured islets and that glucose regulation in CFTReC/eC animals is dependent on the number of available islets.
DISCUSSION
The work presented here demonstrates differences in pancreatic inflammation between CFTReC/eC and control mice and an exacerbation of abnormal glycemic control in CFTReC/eC mice after a low-grade chemical injury with STZ. Without STZ, CFTReC/eC mice demonstrate focal inflammatory cell infiltrates and changes indicative of injury to the exocrine pancreas at baseline. This suggests the presence of intrinsic pancreatic disease leading to reduced effectiveness of -cell function in cystic fibrosis (Fig. 6) and a need for adequate -cell mass to maintain glycemic control.
The demonstration of higher fasting blood glucose levels and exaggerated IPGTT levels observed in STZ-administered CFTReC/eC mice mimic key features of diabetes in cystic fibrosis. The response to islet injury is also suggestive of an inherent difference in function of CFTR-deficient -cells. The absence of visible insulitis reinforces this conclusion. Although the background strains of the C57BL/6J and FVB/NJ mice maintained glucose control after STZ administration, despite a decrease in islets, the CFTReC/eC mice were unable to maintain glucose control, and the degree of hyperglycemia was reflected by the decrease in islet number. The lack of lung disease and other clinical manifestations allow for the assessment of isolated influences. Taken collectively, these studies support the notion that CFTReC/eC mice subjected to STZ administration provide an unequaled ability to recreate the CFRD clinical model.
Interestingly, the induction of airway inflammation in the CFTReC/eC mice did not result in a difference in glycemic control, even though proinflammatory cytokine and IgE responses are greater in these mice than in control mice when exposed to A. fumigatus (23). This suggests that pulmonary inflammation, and subsequent insulin resistance, is not necessary for the predisposition to diabetes in cystic fibrosis, once again pointing to the role of intrinsic, CFTR-dependent abnormalities in the pancreas. Further studies evaluating the effect of chronic (versus acute) inflammation are needed. The impact of diabetes on lung inflammation can be further studied in this model as well.
C57BL/6J CftreC/eC mice, a variation of CFTReC/eC mice, are the result of backcrossing CFTReC/eC mice into a C57BL/6J strain. This breeding effectively causes an inability to wean to a solid diet, which had been previously afforded by the gut expression of FABP-hCFTR. This variation has previously been described to have intestinal inflammatory changes, including focal pancreatic acinar damage (25eC27). In addition, these mice develop more of the cystic fibrosis phenotype, including lung pathology (27). Interestingly, the CFTR S489XeC/eC FABP-hCFTR+/+ model used in this experiment has not been previously reported to have pancreatic disease. However, in our series of experiments using this transgenic model, we found evidence of focal pancreatitis and saponification not previously described. This evidence of pancreatitis could impact -cell function. Similarly, the unique correlation of islet numbers to fasting blood glucose may imply a dysfunction of CFTReC/eC islets as well. Our conclusion is that CFTReC/eC islets maintain glucose control by quantity of islets. In humans, proliferation of existing islets can be seen during times of insulin resistance or increased need (i.e., obesity or pregnancy); however, preliminary data by our laboratory has not revealed increased insulin resistance in the baseline CFTReC/eC mouse as measured by fasting insulin levels (M.S.S., unpublished observations).
These findings provide the foundation for a large number of future studies aimed at addressing the pathogenesis of CFRD. Among them, our studies used mice 7eC9 weeks of age in an attempt to age-match additional studies of type 1 diabetes in the NOD mouse model. However, future studies are required to address the relationship of age to the degree of glucose metabolism in the noneCSTZ-administered CFTReC/eC model. This issue finds importance because age remains the most outstanding predictor of diabetes in the cystic fibrosis population.
As the population of individuals with CFRD grows, the need for animal models to study human disease becomes greater. The diagnosis of CFRD carries a poor prognosis, despite the lack of understanding regarding the influence on both lung and overall health. Through a better understanding of the etiology of CFRD, the full impact of disease can be seen, and strategies for a cure can be developed. The mouse model of this report provides one interesting opportunity to study the pathophysiology of CFRD and its systemic impact.
ACKNOWLEDGMENTS
These studies were supported in part by a Lawson Wilkins Pediatric Endocrinology Society Clinical fellowship, a Cystic Fibrosis Foundation clinical fellowship, and National Institutes of Health Grants M01RR00082 and P01HL51811.
We thank Robert F. Schwartz, Stacy Binns, and Todd Brusko for their technical support to these studies.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Virella-Lowell I, Herlihy JD, Liu B, Lopez C, Cruz P, Muller C, Baker HV, Flotte TR: Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell line. Mol Ther 10:562eC573, 2004
Sagel SD, Accurso FJ: Monitoring inflammation in CF: cytokines. Clin Rev Allergy Immunol 23:41eC57, 2002
Boat F: Cystic fibrosis. In Nelson Textbook of Pediatrics. 17th ed. Behrman RE, Kliegman RM, Jenson HB, Eds. Philadelphia, Saunders, 2004, p.1437eC1450
Orenstein DM, Winnie GB, Altman H: Cystic fibrosis: a 2002 update. J Pediatr 140:156eC164, 2002
Cystic Fibrosis Foundation Patient Registry 2003 Annual Report. Bethesda, MD, Cystic Fibrosis Foundation, 2004, p.1eC16
Hardin DS, Moran A: Diabetes mellitus in cystic fibrosis. Endocrinol Metab Clin North Am 28:787eC800, 1999
Rosenecker J, Eichler I, Kuhn L, Harms HK, von der Hardt H: Genetic determination of diabetes mellitus in patients with cystic fibrosis: Multicenter Cystic Fibrosis Study Group. J Pediatr 127:441eC443, 1995
Lanng S: Glucose intolerance in cystic fibrosis patients. Paediatr Respir Rev 2:253eC259, 2001
Marshall BC, Butler SM, Stoddard M, Moran AM, Liou TG, Morgan WJ: Epidemiology of cystic fibrosis-related diabetes. J Pediatr 146:681eC687, 2005
Moran A, Milla C: Abnormal glucose tolerance in cystic fibrosis: why should patients be screened J Pediatr 142:97eC99, 2003
Moran A, Pyzdrowski KL, Weinreb J, Kahn BB, Smith SA, Adams KS, Seaquist ER: Insulin sensitivity in cystic fibrosis. Diabetes 43:1020eC1026, 1994
Lohr M, Goertchen P, Nizze H, Gould NS, Gould VE, Oberholzer M, Heitz PU, Kloppel G: Cystic fibrosis associated islet changes may provide a basis for diabetes: an immunocytochemical and morphometrical study. Virchows Arch A Pathol Anat Histopathol 414:179eC185, 1989
Iannucci A, Mukai K, Johnson D, Burke B: Endocrine pancreas in cystic fibrosis: an immunohistochemical study. Hum Pathol 15:278eC284, 1984
Johnston JA, Ward CL, Kopito RR: Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883eC1898, 1998
Xu Y, Clark JC, Aronow BJ, Dey CR, Liu C, Wooldridge JL, Whitsett JA: Transcriptional adaptation to cystic fibrosis transmembrane conductance regulator deficiency. J Biol Chem 278:7674eC7682, 2003
Venkatakrishnan A, Stecenko AA, King G, Blackwell TR, Brigham KL, Christman JW, Blackwell TS: Exaggerated activation of nuclear factor-kappaB and altered IkappaB-beta processing in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol 23:396eC403, 2000
Lory S, Ichikawa JK: Pseudomonas-epithelial cell interactions dissected with DNA microarrays. Chest 121:36SeC39S, 2002
Kirchner KK, Wagener JS, Khan TZ, Copenhaver SC, Accurso FJ: Increased DNA levels in bronchoalveolar lavage fluid obtained from infants with cystic fibrosis. Am J Respir Crit Care Med 154:1426eC1429, 1996
Heeckeren A, Walenga R, Konstan MW, Bonfield T, Davis PB, Ferkol T: Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J Clin Invest 100:2810eC2815, 1997
Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, Koller BH: An animal model for cystic fibrosis made by gene targeting. Science 257:1083eC1088, 1992
Zhou L, Dey CR, Wert SE, DuVall MD, Frizzell RA, Whitsett JA: Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266:1705eC1708, 1994
Ito M, Kondo Y, Nakatani A, Hayashi K, Naruse A: Characterization of low dose streptozotocin-induced progressive diabetes in mice. Environ Toxicol Pharmacol 9:71eC78, 2001
Muller C, Braag SA, Herlihy JD, Wasserfall CH, Chesrown SE, Nick HS, Atkinson MA, Flotte TR: Enhanced IgE allergic response to Aspergillus fumigatus in CFTReC/eC mice. Lab Invest 86:130eC140, 2006
Goudy KS, Burkhardt BR, Wasserfall C, Song S, Campbell-Thompson ML, Brusko T, Powers MA, Clare-Salzler MJ, Sobel ES, Ellis TM, Flotte TR, Atkinson MA: Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion. J Immunol 171:2270eC2278, 2003
Norkina O, Kaur S, Ziemer D, De Lisle RC: Inflammation of the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 286:G1032eCG1041, 2004
De Lisle RC, Isom KS, Ziemer D, Cotton CU: Changes in the exocrine pancreas secondary to altered small intestinal function in the CF mouse. Am J Physiol Gastrointest Liver Physiol 281:G899eCG906, 2001
Durie PR, Kent G, Phillips MJ, Ackerley CA: Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model. Am J Pathol 164:1481eC1493, 2004(Michael S. Stalvey, Chris)
2 Department of Pathology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, Florida
3 General Clinical Research Center, University of Florida, College of Medicine, Gainesville, Florida
CFRD, cystic fibrosiseCrelated diabetes; CFTR, cystic fibrosis transmembrane conductance regulator; FABP, fatty acideCbinding protein; hCFTR, human CFTR; IPGTT, intraperitoneal glucose tolerance test; STZ, streptozotocin
ABSTRACT
The cause of cystic fibrosiseCrelated diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTReC/eC), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced -cell injury. Fasting blood glucose levels were similar among age-matched noneCSTZ-administered animals, but they were significantly higher in CFTReC/eC mice 4 weeks after STZ administration (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTReC/eC, C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTReC/eC mice. STZ reduced islets among all strains; however, only CFTReC/eC mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatuseCsensitized CFTReC/eC mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTReC/eC state.
Cystic fibrosis is a systemic disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Some of the manifestations of this disease are caused by defective CFTR chloride channel function, but often they are related to cellular responses to mutant CFTR (1,2). In Caucasians, it is the leading life-limiting recessive genetic disorder (3). In 1938, the mean life expectancy for children with cystic fibrosis was <1 year (4). Over the past 30 years, the life expectancy of children with cystic fibrosis in the U.S. has increased from 16 to 32 years of age (4,5). The onset of diabetes heralds a great impediment to quality of life and is the leading comorbidity noted among the 23,000 patients with cystic fibrosis listed in the Cystic Fibrosis Foundation Patient Registry (5,6). The overall prevalence of cystic fibrosiseCrelated diabetes (CFRD) has been estimated from 4.9 to 12% (5,6,7). In the cystic fibrosis population, the prevalence of CFRD appears to be increasing at a rate of 5% per year. By the age of 30 years, one study indicated 50% of cystic fibrosis patients had clinical diabetes (8). In addition, Hardin and Moran (6) report that the incidence of glucose abnormalities in adults with cystic fibrosis is 75%.
The prevailing mechanistic belief is that CFRD results from a combination of chronic pancreatitis and eventual loss of the islet cells, resulting in less insulin reserve and production, together with a variable state of insulin resistance (6,9eC11). Clinical disease is associated with worsened glucose metabolism during times of lung exacerbation. Autopsy findings of individuals with cystic fibrosis have revealed minimal islet disease before the 2nd decade but extensive replacement of pancreatic acinar tissue with fibrous and fatty tissue. As patients age, autopsy data demonstrate the eventual loss of islet tissue and decreased -cell numbers (12,13). Other findings include deposition of amyloid in the islets, a chronic architectural change, found in cystic fibrosis patients who also have CFRD (6).
As an alternative hypothesis, we question whether the origin of the glucose abnormality observed in CFRD is directly associated with CFTR mutation. The most common CFTR mutation (F508) is a missense mutation that produces a misfolded version of CFTR; this mutation has been shown to trigger an endoplasmic reticulum overload response within cells (14), resulting in widespread changes in gene expression (1,15eC17). Among the most prominent of these changes is an upregulation of proinflammatory cytokines associated with innate immunity (1). This may correlate with the observation that cystic fibrosis patients (18) and CFTR knockout mice (CFTReC/eC) (19) show an exaggerated proinflammatory cytokine response to bacterial challenge compared with normal individuals. To address important questions relating to the formation of diabetes in cystic fibrosis, we developed a novel model involving administration of CFTReC/eC mice with streptozotocin (STZ), an agent capable of inducing -cell injury. These studies suggest that the CFTR mutation exacerbates islet cell dysfunction in CFRD after -cell injury.
RESEARCH DESIGN AND METHODS
We used mice with the CFTR S489XeC/eC neo insertion, developed initially at the University of North Carolina (20). These mice were also modified with transgenic overexpression of gut-specific human CFTR, from the fatty acid bindingeCprotein (FABP) promoter, to prevent intestinal obstruction and improve viability (21). These animals were developed on a background of C57BL/6J and FVB/NJ. Hence, age-matched C57BL/6J and FVB/NJ mice were used as control animals for all experiments. CFTR S489XeC/eC FABP-hCFTR+/+ (hereafter designated CFTReC/eC), C57BL/6J, and FVB/NJ mice were housed under specific pathogen-free conditions at the University of Florida Animal Care Services according to National Institutes of Health guidelines and allowed food and water ad libitum. All experimental procedures were approved by the institutional animal care and use committee of the University of Florida.
Eight CFTReC/eC, seven C57BL/6J, and six FVB/NJ mice received lactated ringers as a placebo via intraperitoneal injection at 7eC9 weeks of age. Blood glucose levels were measured in CFTReC/eC, C57BL/6J, and FVB/NJ mice at baseline. Random or fasting blood glucose values were checked weekly from the point of initial injection with lactated ringers until 4 weeks postinjection. In separate experiments, -cell injury was induced according to the protocol by Ito et al. (22). Low-dose STZ (100 mg/kg; Sigma, St. Louis, MO) was given as a single intraperitoneal injection. CFTReC/eC (n = 10), C57BL/6J (n = 11), and FVB/NJ (n = 6) mice received STZ at 7eC9 weeks of age. Mice were monitored with blood glucose values determined weekly from the point of initial STZ injection until 4 weeks postinjection. STZ- and lactated ringereCadministered mice underwent an intraperitoneal glucose tolerance test (IPGTT) 4 weeks after injection. Mice with random blood glucose levels >350 mg/dl at both week 1 and 2 after STZ administration were designated as early-onset diabetes. These mice were discontinued from the protocol, secondary to the health of the mouse, but also because of the inability to test blood glucose in excess of 600 mg/dl at time of IPGTT. Mice with fasting blood glucose levels >240 mg/dl at the end of 4 weeks posteCSTZ administration were classified as late-onset diabetes and were included in the IPGTT.
To assess the impact of lung injury on glycemia, we used a protocol by Muller et al. (23) previously demonstrating an increased inflammatory response. CFTR S489XeC/eC animals (4eC6 weeks old) were sensitized to Aspergillus fumigatus crude protein extract (Greer Laboratories, Lenoir, NC). Briefly, animals were administered intraperitoneal injections of 200 e蘥 A. fumigatus dissolved in 100 e蘬 PBS on days 0 and 14 (n = 4). Aerosol challenge was performed with 0.25% A. fumigatus for 20 min in a 30 x 30x 20 cm acrylic chamber, using a jet nebulizer (model LC-D; PARI Respiratory Equipment, Midlothian, VA) with an air (room air) flow of 6 l/min on days 28, 29, and 30 after the sensitization. Nonsensitized control mice (n = 5) received intraperitoneal injections with PBS alone and were challenged with A. fumigatus along with sensitized mice. At 48 h after lung challenge, IPGTTs were performed. Age-matched nonsensitized and unchallenged control animals (n = 6) were also compared with mice receiving inhalation challenge.
IPGTTs.
Mice were fasted for 4 h before receiving 2 g/kg dextrose (50% dextrose solution) as an intraperitoneal injection. Glucose tolerance was monitored via tail vein sampling at time 0 (just before 50% dextrose solution injection), 30, 60, 120, and 180 min. Glucose was measured with a LifeScan OneTouch Ultra glucometer (reported in milligrams per deciliter).
Histology and immunohistochemistry.
Pancreas, lung, duodenum, and liver specimens were obtained immediately after the mice were killed at the end of the IPGTT and fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA). Tissues were processed into paraffin blocks and sections stained with hematoxylin and eosin for morphologic evaluation. The entire pancreatic specimen was visualized under 10x magnification, and all islets were counted and inspected for insulitis. Any evidence of insulitis was scored (24). Insulin immunohistochemistry was performed as previously described, using guinea pig anti-insulin antibodies (1:2000; Dako, Carpinteria, CA) and routine avidin-biotin-peroxidase detection (Vectastain ABC kit; Vector Labs, Burlingame, CA) with 3,3'-diaminobenzidine (DAB staining kit; Vector Labs) as chromagen (24). Amyloid deposition was evaluated by staining with alkaline Congo Red (PolyScientific, Bay Shore, NY).
Human CFTR detection in tissue.
To ensure the absence of human CFTR (hCFTR) expression by the pancreas, after the CFTReC/eC mice were killed, pancreas, lung, and small intestine were collected. Using surgical tools sterilized and sprayed with RNase Away (Molecular BioProducts, San Diego, CA), tissue was removed and immediately frozen in liquid nitrogen. After homogenizing 100 mg of tissue, mRNA was extracted, using Oligotex Direct mRNA columns (Qiagen, Valencia, CA). For RT-PCR, a Qiagen One-Step RT-PCR kit was used. Briefly, 14 ng of mRNA were incubated at 50°C with reverse transcriptase for 30 min with gene-specific primers for hCFTR (hCFTR forward: 5'-aaacttctaatggtgatgaccag; reverse: 5'-agaaattcttgctcgttgac) or B-actin (B-actin1: 5'-agctgagagggaaatcgtgc; B-actin2: 5'-accagacagcactgtgttgg). This was followed by incubating the samples at 95°C for 15 min to inactivate the reverse transcriptase. The cDNA was then amplified with the same primers for 30 cycles by PCR. The noeCreverse transcriptase controls were subjected to the same PCR conditions and primers but in the absence of reverse transcriptase.
Statistical analysis.
Data were analyzed via one-way ANOVA, using Bonferroni corrections for multiple comparisons to evaluate individual differences in strain and treatments. Glucose tolerance was calculated, using the percent change from the baseline fasting blood glucose value, and compared as the total area under the curve (trapezoidal rule) followed by one-way ANOVA calculations and Bonferroni corrections as described above. Differences between groups were considered significant if the Bonferroni-corrected two-sided P value was <0.05. Correlations of the number of pancreatic islets with blood glucose values were calculated with a Pearson calculation. All statistical analyses were conducted using SAS 9.1.2 software (Cary, NC).
RESULTS
Detection of hCFTR expression.
Tissue obtained from the small intestine of the CFTReC/eC mice demonstrated hCFTR mRNA, whereas the lung and pancreas sections did not (Fig. 1).
Glucose challenge in mice receiving lactated ringers.
No significant differences were observed in the fasting glucose levels of lactated ringereCadministered CFTReC/eC mice in comparison to the C57BL/6J and FVB/NJ background strains (time 0, P = NS) (Fig. 2A). In addition, responses to glucose stimulation were similar among these three strains after intraperitoneal injection of 50% dextrose solution (P = NS) (Fig. 2A). These studies did not suggest intrinsic differences in glucose regulation by the CFTReC/eC mice.
Glucose challenge in STZ-administered mice.
Fasting blood glucose levels were higher in STZ-administered CFTReC/eC mice compared with C57BL/6J and FVB/NJ background strains subjected to identical treatment (288.4 ± 97.4, 168.4 ± 35.9, and 188.0 ± 42.3 mg/dl for CFTReC/eC, C57BL/6J, and FVB/NJ, respectively; P < 0.05) (Fig. 2B). These glucose values remained higher throughout the entire 3-h time period after glucose infusion, but the percent increase did not achieve significance in comparison to the other STZ-administered strains (Fig. 2B). These results suggest that in the face of minimally compromised -cell function (in this situation, due to chemically induced injury), there is indeed an underlying defect in glucose metabolism associated with deficiencies in CFTR. A small subset of both CFTReC/eC (1 of 10) and C57BL/6J (3 of 11) mice developed diabetes early after STZ injection and were classified as early-onset diabetes, and they were thus removed from the animal protocol (Table 1). All early-onset diabetic mice were male (weight 25eC30 g) and had received more total STZ dosage (100 mg/kg) than the female counterparts (weight 20 g).
Glucose challenge in mice with lung inflammation.
Interestingly, no changes in glucose, either fasting or stimulated, were observed in A. fumigatuseCadministered mice in comparison to age-matched PBS-treated or untreated controls (Fig. 3). This suggests that acute lung inflammation does not play a dominant role in the predilection to diabetes in cystic fibrosis.
Pancreatic acinar and islet histology.
Both lactated ringereCand STZ-administered CFTReC/eC mice demonstrated focal mild acinar degeneration with fibrosis and inflammatory infiltrates consisting primarily of mononuclear cells (Fig. 4). Saponification of pancreatic fat was also observed in one animal (Fig. 4A). Though not subject to direct quantitation, intensive histological examination suggested that these local changes in pathology were similar among both lactated ringereCand STZ-administered CFTReC/eC mice. No C57BL/6J or FVB/NJ mice demonstrated evidence of pancreatitis.
Next, we compared the pancreatic islets to identify wherein variances in islet inflammation, insulin content, or number might provide partial explanation for the observed differences in glucose regulation between these strains. Insulitis was not observed in any strain, in either the absence or presence of STZ administration. To the question of insulin content, no qualitative differences were observed, as determined by immunohistochemical evaluation (data not shown). Likewise, no significant differences in islet number were observed in comparison to CFTReC/eC, C57BL/6J, and FVB/NJ strains (Fig. 5A), in either the absence or presence of STZ administration (all P = NS). Islet amyloid deposition was not observed in any strain either with or without STZ administration (data not shown).
However, a strong negative correlation between the number of islets and fasting blood glucose measurements were noted in STZ-administered CFTReC/eC mice (r = eC0.73, P = 0.02) (Fig. 5B). The absence of such a correlation in the other strains of mice suggests that the CFTR deficiency exacerbates glucose tolerance in injured islets and that glucose regulation in CFTReC/eC animals is dependent on the number of available islets.
DISCUSSION
The work presented here demonstrates differences in pancreatic inflammation between CFTReC/eC and control mice and an exacerbation of abnormal glycemic control in CFTReC/eC mice after a low-grade chemical injury with STZ. Without STZ, CFTReC/eC mice demonstrate focal inflammatory cell infiltrates and changes indicative of injury to the exocrine pancreas at baseline. This suggests the presence of intrinsic pancreatic disease leading to reduced effectiveness of -cell function in cystic fibrosis (Fig. 6) and a need for adequate -cell mass to maintain glycemic control.
The demonstration of higher fasting blood glucose levels and exaggerated IPGTT levels observed in STZ-administered CFTReC/eC mice mimic key features of diabetes in cystic fibrosis. The response to islet injury is also suggestive of an inherent difference in function of CFTR-deficient -cells. The absence of visible insulitis reinforces this conclusion. Although the background strains of the C57BL/6J and FVB/NJ mice maintained glucose control after STZ administration, despite a decrease in islets, the CFTReC/eC mice were unable to maintain glucose control, and the degree of hyperglycemia was reflected by the decrease in islet number. The lack of lung disease and other clinical manifestations allow for the assessment of isolated influences. Taken collectively, these studies support the notion that CFTReC/eC mice subjected to STZ administration provide an unequaled ability to recreate the CFRD clinical model.
Interestingly, the induction of airway inflammation in the CFTReC/eC mice did not result in a difference in glycemic control, even though proinflammatory cytokine and IgE responses are greater in these mice than in control mice when exposed to A. fumigatus (23). This suggests that pulmonary inflammation, and subsequent insulin resistance, is not necessary for the predisposition to diabetes in cystic fibrosis, once again pointing to the role of intrinsic, CFTR-dependent abnormalities in the pancreas. Further studies evaluating the effect of chronic (versus acute) inflammation are needed. The impact of diabetes on lung inflammation can be further studied in this model as well.
C57BL/6J CftreC/eC mice, a variation of CFTReC/eC mice, are the result of backcrossing CFTReC/eC mice into a C57BL/6J strain. This breeding effectively causes an inability to wean to a solid diet, which had been previously afforded by the gut expression of FABP-hCFTR. This variation has previously been described to have intestinal inflammatory changes, including focal pancreatic acinar damage (25eC27). In addition, these mice develop more of the cystic fibrosis phenotype, including lung pathology (27). Interestingly, the CFTR S489XeC/eC FABP-hCFTR+/+ model used in this experiment has not been previously reported to have pancreatic disease. However, in our series of experiments using this transgenic model, we found evidence of focal pancreatitis and saponification not previously described. This evidence of pancreatitis could impact -cell function. Similarly, the unique correlation of islet numbers to fasting blood glucose may imply a dysfunction of CFTReC/eC islets as well. Our conclusion is that CFTReC/eC islets maintain glucose control by quantity of islets. In humans, proliferation of existing islets can be seen during times of insulin resistance or increased need (i.e., obesity or pregnancy); however, preliminary data by our laboratory has not revealed increased insulin resistance in the baseline CFTReC/eC mouse as measured by fasting insulin levels (M.S.S., unpublished observations).
These findings provide the foundation for a large number of future studies aimed at addressing the pathogenesis of CFRD. Among them, our studies used mice 7eC9 weeks of age in an attempt to age-match additional studies of type 1 diabetes in the NOD mouse model. However, future studies are required to address the relationship of age to the degree of glucose metabolism in the noneCSTZ-administered CFTReC/eC model. This issue finds importance because age remains the most outstanding predictor of diabetes in the cystic fibrosis population.
As the population of individuals with CFRD grows, the need for animal models to study human disease becomes greater. The diagnosis of CFRD carries a poor prognosis, despite the lack of understanding regarding the influence on both lung and overall health. Through a better understanding of the etiology of CFRD, the full impact of disease can be seen, and strategies for a cure can be developed. The mouse model of this report provides one interesting opportunity to study the pathophysiology of CFRD and its systemic impact.
ACKNOWLEDGMENTS
These studies were supported in part by a Lawson Wilkins Pediatric Endocrinology Society Clinical fellowship, a Cystic Fibrosis Foundation clinical fellowship, and National Institutes of Health Grants M01RR00082 and P01HL51811.
We thank Robert F. Schwartz, Stacy Binns, and Todd Brusko for their technical support to these studies.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Virella-Lowell I, Herlihy JD, Liu B, Lopez C, Cruz P, Muller C, Baker HV, Flotte TR: Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell line. Mol Ther 10:562eC573, 2004
Sagel SD, Accurso FJ: Monitoring inflammation in CF: cytokines. Clin Rev Allergy Immunol 23:41eC57, 2002
Boat F: Cystic fibrosis. In Nelson Textbook of Pediatrics. 17th ed. Behrman RE, Kliegman RM, Jenson HB, Eds. Philadelphia, Saunders, 2004, p.1437eC1450
Orenstein DM, Winnie GB, Altman H: Cystic fibrosis: a 2002 update. J Pediatr 140:156eC164, 2002
Cystic Fibrosis Foundation Patient Registry 2003 Annual Report. Bethesda, MD, Cystic Fibrosis Foundation, 2004, p.1eC16
Hardin DS, Moran A: Diabetes mellitus in cystic fibrosis. Endocrinol Metab Clin North Am 28:787eC800, 1999
Rosenecker J, Eichler I, Kuhn L, Harms HK, von der Hardt H: Genetic determination of diabetes mellitus in patients with cystic fibrosis: Multicenter Cystic Fibrosis Study Group. J Pediatr 127:441eC443, 1995
Lanng S: Glucose intolerance in cystic fibrosis patients. Paediatr Respir Rev 2:253eC259, 2001
Marshall BC, Butler SM, Stoddard M, Moran AM, Liou TG, Morgan WJ: Epidemiology of cystic fibrosis-related diabetes. J Pediatr 146:681eC687, 2005
Moran A, Milla C: Abnormal glucose tolerance in cystic fibrosis: why should patients be screened J Pediatr 142:97eC99, 2003
Moran A, Pyzdrowski KL, Weinreb J, Kahn BB, Smith SA, Adams KS, Seaquist ER: Insulin sensitivity in cystic fibrosis. Diabetes 43:1020eC1026, 1994
Lohr M, Goertchen P, Nizze H, Gould NS, Gould VE, Oberholzer M, Heitz PU, Kloppel G: Cystic fibrosis associated islet changes may provide a basis for diabetes: an immunocytochemical and morphometrical study. Virchows Arch A Pathol Anat Histopathol 414:179eC185, 1989
Iannucci A, Mukai K, Johnson D, Burke B: Endocrine pancreas in cystic fibrosis: an immunohistochemical study. Hum Pathol 15:278eC284, 1984
Johnston JA, Ward CL, Kopito RR: Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883eC1898, 1998
Xu Y, Clark JC, Aronow BJ, Dey CR, Liu C, Wooldridge JL, Whitsett JA: Transcriptional adaptation to cystic fibrosis transmembrane conductance regulator deficiency. J Biol Chem 278:7674eC7682, 2003
Venkatakrishnan A, Stecenko AA, King G, Blackwell TR, Brigham KL, Christman JW, Blackwell TS: Exaggerated activation of nuclear factor-kappaB and altered IkappaB-beta processing in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol 23:396eC403, 2000
Lory S, Ichikawa JK: Pseudomonas-epithelial cell interactions dissected with DNA microarrays. Chest 121:36SeC39S, 2002
Kirchner KK, Wagener JS, Khan TZ, Copenhaver SC, Accurso FJ: Increased DNA levels in bronchoalveolar lavage fluid obtained from infants with cystic fibrosis. Am J Respir Crit Care Med 154:1426eC1429, 1996
Heeckeren A, Walenga R, Konstan MW, Bonfield T, Davis PB, Ferkol T: Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J Clin Invest 100:2810eC2815, 1997
Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, Koller BH: An animal model for cystic fibrosis made by gene targeting. Science 257:1083eC1088, 1992
Zhou L, Dey CR, Wert SE, DuVall MD, Frizzell RA, Whitsett JA: Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266:1705eC1708, 1994
Ito M, Kondo Y, Nakatani A, Hayashi K, Naruse A: Characterization of low dose streptozotocin-induced progressive diabetes in mice. Environ Toxicol Pharmacol 9:71eC78, 2001
Muller C, Braag SA, Herlihy JD, Wasserfall CH, Chesrown SE, Nick HS, Atkinson MA, Flotte TR: Enhanced IgE allergic response to Aspergillus fumigatus in CFTReC/eC mice. Lab Invest 86:130eC140, 2006
Goudy KS, Burkhardt BR, Wasserfall C, Song S, Campbell-Thompson ML, Brusko T, Powers MA, Clare-Salzler MJ, Sobel ES, Ellis TM, Flotte TR, Atkinson MA: Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion. J Immunol 171:2270eC2278, 2003
Norkina O, Kaur S, Ziemer D, De Lisle RC: Inflammation of the cystic fibrosis mouse small intestine. Am J Physiol Gastrointest Liver Physiol 286:G1032eCG1041, 2004
De Lisle RC, Isom KS, Ziemer D, Cotton CU: Changes in the exocrine pancreas secondary to altered small intestinal function in the CF mouse. Am J Physiol Gastrointest Liver Physiol 281:G899eCG906, 2001
Durie PR, Kent G, Phillips MJ, Ackerley CA: Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model. Am J Pathol 164:1481eC1493, 2004(Michael S. Stalvey, Chris)