1Institute of Biology and 2School of Medical Sciences, State University of Campinas, Campinas; and 3School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Sao Paulo, Brazil
Submitted 5 October 2004 ; accepted in final form 20 June 2005
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ABSTRACT |
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glucose transporter; tear film; ocular surface
Insulin mediates nutrient influx, energy storage, gene expression, and DNA and protein synthesis for various tissues by a process based on binding to its tetrameric transmembrane-specific receptor (25). Insulin has been detected in other exocrine gland secretions like saliva and milk (7, 45), and its pleiotropic action has been identified in the lachrymal tissue and on the ocular surface as well (13, 42, 45). In addition, topical insulin therapy has been used to promote corneal wound healing and to treat diabetes mellitus (DM) (15, 28, 34, 47).
Recent studies have detected unique insulin activities related to the eye and visual pathways, such as orientation of axonal linkage between retina and visual cortex in the embryonic stage of life and regulation of aqueous humor flow during adult life (18, 38).
The role of insulin on the ocular surface is also inferred by tear film and corneal epithelial cell disorders in diabetic patients (9, 11, 14). From in vitro work, it is known that insulin is necessary for corneal and lachrymal gland cell culture (13, 20, 27). Some signaling elements that mediate the action of insulin on the lachrymal gland (LG) and ocular surface in various conditions have been recently studied (3032). Other proteins under the influence of insulin signaling are the glucose transporter (GLUT) family. GLUTs are responsible for glucose transfer trough the cell membranes lipid layer and may also be present in the LG (21, 40).
Classically, pancreatic islet -cells are responsible for insulin production; however, various reports support the hypothesis of a capacity of insulin storage or local production in exocrine glands and other tissues in pathological conditions, (24, 35) or in normal situations (1, 68, 33, 39, 45), although others do not accept this possibility, maintaining that some tissues actually have a unique capacity for insulin storage and concentration (29, 44).
The hypothesis of the present study was that insulin is a key element in the tear film and that its secretion is sensitive to systemic and local influence.
Our objective was to characterize insulin secretion in the rat tear film. More specifically, we wanted to investigate the effects of fasting, acute glucose injection, and other secretagogues (i.e., KCl and carbachol) on insulin secretion by the LG as well as to obtain a better understanding of the possibility of extrapancreatic insulin production.
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MATERIALS AND METHODS |
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Sample collection. Rats were anesthetized with thiopental sodium (100 µg/kg body wt ip; Itapira, Cristália, Sao Paulo, SP, Brazil). For the analysis of the effect of glucose on insulin secretion in the tear film, tears and blood samples were collected from 0 to 60 min after glucose injection (1 g/kg body wt). Blood was collected from the caudal vein with heparinized hematocrit tubes. Tears were collected with graduated Pasteur pipettes from the ocular surface with minor manipulation and transferred to Eppendorf tubes containing 50 µl of 0.9% NaCl. The samples were frozen at 75°C until the time for use.
In vitro insulin secretion. Under anesthesia, the pancreas and lachrymal gland (LG) were removed from the animals. The tissues were immersed in Krebs-bicarbonate buffer on separate Petri dishes for each experimental group and tissue.
The pancreatic tissue was digested with collagenase as described (4), and LG tissue was cut with fine scissors into fragments with a mean volume of 1 mm3 under a light microscope. LG samples consisted of groups of two LG fragments that were first incubated in cell strainers for 45 min at 37°C in Krebs-bicarbonate buffer containing 5.6 mmol glucose/l and equilibrated with 95% O2-5% CO2, pH 7.4; samples of five islets (0.1 mm3) were run in parallel for comparison. The solution was then replaced with fresh Krebs-bicarbonate buffer, and the islets were further incubated for 1 h in medium of the following composition: 2.8, 8.3, and 16.7 mM glucose, 16.7 mM glucose combined with 20 µg/ml diazoxide, 200 µM carbachol, and 200 µM carbachol combined with 66 µg/ml atropine or 40 mM potassium. The incubation medium contained 115 mM NaCl, 5 mM KCl, 24 mM NaHCO3, 2.56 mM CaCl2, 1 mM MgCl2, and 3 g/l bovine serum albumin (BSA).
After the incubation period, the supernatant of each experimental preparation (n = 5 per preparation) and negative controls were collected and processed by radioimmunoassay (RIA).
To measure the total content of insulin in LG and isolated islets, samples were homogenized with a polytron PT1200C (Brinkmann Instruments, Westbury, NY) in alcohol-acid solution (20% ethanol + 0.2 N HCl) and also processed by RIA.
Insulin and C-peptide quantification. The insulin content in the tears, plasma, supernatant of in vitro experiments, and homogenized tissues was measured by RIA. The C-peptide content in homogenized LG and pancreatic islets was also evaluated by RIA.
To ensure sensitivity, specificity, and reproducibility of the method, the following procedures were performed: 1) curves with triplicate samples of commercially available insulin and C-peptide (Amersham, Aylesbury, UK, and Linco, St. Charles, MO, respectively) were run in parallel; 2) samples with similar dilutions of IGF-I (Sigma, St. Louis, MO) or containing only buffer were also analyzed; and 3) assay samples were run in duplicate. Sensitivity ranged from 0.1 to 20 ng/ml for insulin and from 25 pM to 1.6 nM for C-peptide, and interassay and intra-assay coefficients of variation were estimated at 0.12 and 0.075 for insulin and 0.10 and 0.063 for C-peptide, respectively.
Glucose metabolism. Glucose oxidation was measured in isolated cornea samples as previously described (3). Corneas excised from euthanized 8-wk-old male Wistar rats were placed in wells containing Krebs-bicarbonate buffered medium (50 µl) supplemented with trace amounts of D-[U-14C] glucose (10 µCi/ml). Four experimental conditions were compared: 1) 5.6 mM glucose without insulin or 2) with 6.0 nM insulin, 3) 11.2 mM glucose, and 4) 5.6 mM glucose with 60 nM insulin, to complement the fixed amounts of radioactive glucose present in each sample (n = 5/group). The content of the wells was suspended in 20-ml scintillation vials, which were gassed with 5% O2-95% CO2 and capped airtight with a rubber membrane. The vials were shaken continuously for 2 h at 37°C in a water bath. After incubation, 0.1 ml of 0.2 N HCl and 0.2 ml of hyamine hydroxide were injected through the rubber cap into the glass cup containing the incubation medium and into the counting vial, respectively, to stop the oxidative reaction. After 1 h at room temperature, the internal flask containing the tissue and the solutions was separated, 6 ml of scintillation fluid were added to the external flask, and the radioactivity present in 14CO2 molecules resulting from glucose oxidation was counted. Glucose utilization was measured by the generation of 14CO2 in a scintillator analyzer, in parallel with identically treated blank incubations. The rate of glucose oxidation (i.e., production of 14CO2) was directly proportional to the radioactivity counted in the flasks and is expressed as picomoles per cornea per hour.
RT-PCR for insulin and pancreatic duodenal homeobox-1. Reverse transcription-polymerase chain reaction (RT-PCR) was used to identify insulin II (rats and mice have two insulin genes) and pancreatic duodenal homeobox-1 (PDX-1) mRNA in rat LG. In addition, RPS-29 mRNA, a sequence that determines the ribosomal protein subunit 29 expressed in eukaryotic cells, was used as control. Total RNA was purified from LG and pancreatic islet samples (positive control) using TRIzol (Invitrogen, San Diego, CA) and treated with deoxyribonuclease I (GIBCO-BRL, Gaithersburg, MD) to eliminate DNA contamination. Samples of the resulting RNA were quantified by spectrophotometry at 260 nm and evaluated in 6.6% formaldehyde, 1% agarose (GIBCO-BRL) gels to ensure RNA integrity. Reverse transcriptase, oligo(dT) priming, and the Advantage RT-for-PCR kit from Clontech Laboratories (Palo Alto, CA) were used for cDNA transcription.
PCR amplification of cDNA was performed with a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) using 1.5 units of Taq DNA polymerase (GIBCO-BRL), 0.3 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen), PCR buffer [60 mM Tris·HCl, 1.5 mM MgCl2, 15 mM NH4SO4, pH 10 (Invitrogen)], and 10 mM of 5' and 3' primers corresponding to rat insulin II, PDX-1, and RPS-29 cDNA. The primers corresponding to rat insulin II (acc. no. NM 019130, sense 5'-TTG CAG TAG TTC TCC AGT T-3' and antisense 5'-ATT GTT CCA ACA TGG CCC TGT-3'), rat PDX-1 (acc. no. NM 022852, sense 5'-AAC CGG AGG AGA ATA AGA GG-3' and antisense 5'-GTT GTC CCG CTA CTA CGT TT-3'), and rat RPS-29 (acc. no. BC 058150, sense 5'-AGG CAA GAT GGG TCA CCA GC-3' and antisense 5'-AGT CGA ATC ATC CAT TCA GGT CG-3') were designed by reference to GenBank sequences and synthesized by Life Technologies (Gaithersburg, MD).
The PCR program for insulin, PDX-1, and RPS-29 mRNA involved the following cycle profile: 32 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 57°C, extension for 1.5 min at 72°C, and maximization of strand completion for 7 min at 72°C. After amplification, the cDNA fragments were analyzed on 1% agarose gels containing a 100-bp DNA molecular weight leader (GIBCO-BRL) and poststained with ethidium bromide to confirm the anticipated 340-, 225-, and 202-bp sizes for insulin, PDX-1, and RPS-29 products, respectively. Preliminary assays were performed to ensure products in the linear range. In all PCR procedures, positive and negative control cDNAs were run in parallel, but separate, tubes. Positive controls for insulin and PDX-1 mRNAs included cDNA prepared from rat pancreatic islets. Negative controls included samples without reverse trasncriptase or without cDNA samples. The results were registered in Gel Doc (Bio-Rad, Hercules, CA).
cDNA sequencing. To confirm that insulin and PDX-1 mRNA were obtained by RT-PCR, cDNA samples were cloned in vector pGEM T-easy (Promega, Madison, WI), and PCR was carried out in a thermal cycler PTC-200 Peltier Thermal Cycler (PerkinElmer, Boston, MA) with an initial denaturation step at 95°C for 3 min, subjected to 25 cycles of denaturation at 95°C for 10 s, annealing at 50°C for 5 s, and elongation at 60°C for 4 min. The reactions included 11 µl of water milliQ, 2 µl of BigDye buffer, 3 µl of 5x buffer, 1 µl of primer, and 3 µl of each template. The sequencing was performed in a 310 Genetic Analyzer ABI Prism (PerkinElmer). Sequence specificity for insulin and PDX-1 was evaluated by using BLAST analysis.
Immunohistochemistry. Exorbital LGs and pancreas were excised and fixed in Bouins solution for 24 h. Tissue samples were embedded in paraffin, cut into 6-µm sections, and transferred to poly-L-lysine (Sigma)-precoated glass slides (Perfecta, Sao Paulo, SP, Brazil). The slides were incubated in 0.1% H2O2 for 5 min, washed in PBS (0.05 M sodium phosphate + 0.15 M sodium chloride, pH 7.3) and exposed to 2% normal goat serum solution (Vector, Burlingame, CA) for 20 min at 4°C. The sections were then overlaid with an aliquot of purified mouse monoclonal anti-insulin (Dako, Carpinteria, CA) at a final concentration of 2 µg/ml, prepared using 10 µl of antibody stock solution (200 µg/ml) diluted in 990 µl of 0.3% BSA (GIBCO-BRL) in PBS, or negative control solutions. The controls included 0.1% BSA in PBS and preimmune IgG (Sigma) and sections of pancreatic islets.
After incubation for 4 h with primary antibody in a humidified chamber at 4°C, the sections were washed in PBS and incubated with a biotinylated goat anti-rabbit IgG antibody (Vector). After incubation with the second antibody, sections were again washed in PBS and incubated with an avidin-biotin complex (Vector) for 30 min at 25°C before being developed with a diaminobenzidine substrate kit (Vector).
For histological correlation, conventional hematoxylin (Sigma) counterstaining was performed on tissue sections, and the slides were covered with Entellan (Merck, Darmstadt, Germany) and a coverslip. Photographic documentation was done using a Leica DMLS microscope at x100 and x400 magnification and ASA 100 Kodak film.
Western blotting for GLUTs and PDX-1. The tissues were excised under anesthesia, as described above, and homogenized in 400 µl of solubilization buffer (10% Triton X-100, 100 mM Tris, pH 7.4, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PSMF, and 0.1 mg/ml aprotinin) for 30 s using a polytron PT1200C (Brinkmann Instruments). The tissue extracts were centrifuged at 12,000 rpm at 4°C for 20 min. The supernatant was added to 18 µl of Laemmli sample buffer and boiled for 5 min before loading for 12% SDS-polyacrylamide gel electrophoresis in a Mini-Protean miniature lab gel apparatus (Bio-Rad). The electrotransfer of proteins from the gel to nitrocellulose was performed for 2 h at 120 V using a Bio-Rad miniature transfer apparatus. Nonspecific protein binding to nitrocellulose was reduced by preincubating the filter for 2 h at 22°C in blocking buffer (3% BSA, 10 mM Tris, 150 mM NaCl, and 0.02% Tween-20). The nitrocellulose blot was incubated for 2 h at 22°C with polyclonal antibodies, anti-PDX-1 (Chemicon, Temecula, CA), GLUT1, GLUT2, and GLUT4 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in blocking buffer, and subsequently washed for 30 min in blocking buffer without BSA. The blots were then incubated with 2 µCi of 125I-labeled protein A (30 µCi/µg) in 10 ml of blocking buffer for 1 h at 22°C and washed again as described above for 2 h. 125I-protein A bound to the antibodies was detected by autoradiography using preflashed Kodak film at 70°C for 2460 h. Images of the developed autoradiographs were scanned with a ScanJet 5p scanner (Hewlett-Packard, Boise, ID) into Adobe Photoshop 7.0 on a personal computer.
Electron microscopy. For transmission electron microscopy (EM), LG and pancreas were removed and fixed in 2% glutaraldehyde and 2% paraformaldehyde (Ladd Research Industries, Burlington, VT) in 0.1 M cacodylate buffer, pH 7.4, containing 0.05% CaCl2 for 40 min at room temperature. Tissues were postfixed in 2% OsO4 (EM Sciences, Hatfield, PA) for 1 h at room temperature, rinsed in distilled water, dehydrated through a graded ethanol series, rinsed in acetone, and embedded in Embed 812 (EM Sciences). Thin sections (8090 nm) were cut with a diamond knife and stained for 10 min each in Reynolds lead citrate and 2% uranyl acetate. Sections were examined with a Phillips EN 208 electron microscope. After photographic documentation, negatives were scanned and stored as digital files.
Statistical analysis. Data are reported as means ± SE. Insulin concentrations were compared by the Mann-Whitney U-test (Statview software; Abacus, Berkeley, CA). Glucose consumption by corneal tissue was compared among different media by ANOVA and by Fishers paired least significant difference test. The level of significance was set at P < 0.05 in all analyses.
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RESULTS |
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C-peptide was evaluated in LG and pancreatic islets to verify the possibility of local production of insulin in LG tissue. The RIA revealed that C-peptide concentration is 5.77 ± 0.38 nM in pancreatic islets but is undetectable in LG (n = 3/tissue).
To evaluate the influence of insulin on glucose uptake and metabolism by rat corneal tissues, glucose oxidation was measured in isolated corneal samples to infer about the action of insulin in the tear film. Corneal metabolism was measured by comparing glucose utilization by rat corneal tissues (n = 5 corneas/condition). In the basal condition (5.6 mM glucose), glucose consumption was 24.07 ± 0.61 nmol·cornea1·2 h1. In condition two, in which 6 nM insulin was added to the medium containing 5.6 mM glucose, glucose consumption was 31.63 ± 3.15 nmol·cornea1·2 h1, a 32% rise compared with control (P = 0.033). In a third condition, in which the glucose concentration was doubled to 11.2 mM and the assays were conducted without insulin, glucose consumption was 37.5 ± 3.7 nmol·cornea1·120 min1, i.e., a 56% rise compared with control (P = 0.015), but not significantly higher than condition two (P = 0.14). However, in a fourth condition, in the presence of basal glucose levels (5.6 mM) and 60 nM insulin, glucose consumption was 31.1 ± 1.76 nmol·cornea1·120 min1 (n = 5 corneas/condition), which was 29% higher than the basal situation (P = 0.005) but similar to that observed in the presence of physiological levels of insulin (condition 2, P = 0.88; Fig. 5).
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Immunohistochemical analysis indicated the presence of insulin on the apical side of acinar cells of the rat LG and also in epithelial cells and ductal cells (Fig. 7A). Marked staining was also obtained in samples from pancreatic islets (Fig. 7B), although no specific staining was found in negative control tissues (Fig. 7, C and D).
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DISCUSSION |
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The present findings indicate that fasting reduces insulin levels in the tear film and that this phenomenon is reversed by intravenous glucose injection in vivo, in agreement with our findings in human tears and with observations of a systemic influence on insulin release from salivary secretion in diabetic and nondiabetic humans (31, 22).
Higher glucose levels also enhance the concentration of insulin secreted by LG in vitro. Similarly, the presence of the cholinergic agonist carbachol or the depolarizing action of K+ increased the levels of insulin secretion in a similar way but at much lower concentrations than observed in pancreatic islets and was reversed by specific inhibitors, such as atropine for carbachol and diazoxide as a GLUT inhibitor (12). This information indicates that LG insulin secretion is directly sensitive to environmental and systemic conditions and may not just reflect the rise in blood insulin levels.
The role of insulin in the tear film may involve promotion of metabolic events and cell proliferation in the tissues related to the ocular surface, as addressed by previous experimental studies (13, 27, 34, 46, 47). These previous data indicate that insulin is an adjuvant in corneal epithelium replacement in vivo.
The mechanism involved in the internal recognition of extracellular glucose levels and of glucose uptake was previously attributed to GLUTs (2, 40). GLUT1 was identified on the ocular surface, but its expression in corneal wound repair in diabetic and normal rats was not changed (43). Our finding of limited enhancement of glucose metabolism by corneal tissues in response to insulin compared with higher levels of extracellular glucose without insulin confirms that this hormone enhances the glucose metabolism by corneal tissues but is not crucial for glucose intake, as characteristically mediated by GLUT1.
The present study also identified only GLUT1 in LG, which may represent the major facilitatory GLUT in LG and other exocrine glands and may play a role in the glucose content of exocrine secretion. This finding confirms the predominance of this isoform in epithelial cells and adult ocular tissues and indicates that the input of glucose to these glands is independent of insulin (21).
The facilitatory transport of glucose mediated by GLUT1 may explain the finding of a limited capacity to release insulin in response to higher levels of glucose in LG, which could be attributed to the saturation of glucose influx through GLUT1. Conversely, the capacity of islet -cells to respond to 16.7 mM glucose was much higher than their capacity to respond to carbachol or K+, which also indicates a specialization of this tissue in metabolic control, attributed in part to the presence of GLUT2 (40).
The expression of insulin and PDX-1 mRNA, a transcription factor involved in pancreatic islet cell differentiation and insulin expression (17, 23), without the presence of C-peptide and PDX-1 protein in detectable levels, indicates that extrapancreatic insulin production, as previously reported in other tissues, including rat retina (6, 8, 16), may occur, but not in meaningful levels in LG or is inhibited in its further steps in physiological conditions.
Moreover, the presence of insulin and PDX-1 mRNA in the LG does not ensure that the identified mRNA sequences will be used in the transcription process. Rather, it is possible to speculate that insulin and PDX-1 mRNA may work on the RNA and protein process and on the transport of linked genes, as indicated by other studies with different gene products (5, 10, 37). For PDX-1 mRNA, two homologous sequences previously described (acc. nos. S76307 [GenBank] and S67435 [GenBank] ) but not assigned to any gene would match with our sequencing product, and that may indicate that it is cross-reacting with our PDX-1 sequence.
A recent study in which insulin and other -cell transcripts were detected in extrapancreatic tissues of diabetic mice revealed, however, a much fainter signal compared with normal
-cell samples and variable expression of these elements depending on the DM model (16). In addition, previous work described embryonic stem cells expressing insulin without typical
-cell granules and negative for C-peptide (36).
Further studies would open perspectives into the understanding of regulation of insulin gene expression and to clarify whether extrapancreatic insulin production upregulated in other tissues would be helpful in DM (16, 36).
The role of insulin on the ocular surface also needs to be addressed in physiological and pathological conditions to determine whether DM or other conditions related to dry eye present with reduced insulin secretion in the tear film. In addition, topical replacement of this hormone may be helpful to attenuate ocular surface complications in these cases.
In conclusion, our experiments raise the possibility that insulin secretion in the tear film is systemically controlled and may be sensitive to local influences able to mobilize this hormone from lachrymal gland acinar cells. These observations confirm the ubiquitous distribution and actions of insulin and provide evidence of an additional role for the lachrymal gland in the maintenance of the ocular surface.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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