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,3
* Departments of Medicine and
Anatomy, Cell and Neurobiology, Joan C. Edwards School of Medicine, Marshall University and
** Research and
Medical Services, Huntington VA Medical Center, Huntington, WV
3To whom correspondence should be addressed. E-mail: chertow{at}marshall.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-cell mass, apoptosis, and - and ß-cell replication were measured in rats using a model of marginal vitamin A deficiency. Female rats before and during pregnancy and their offspring postweaning were fed a diet containing retinol as retinyl palmitate at a low marginal (LM, 0.25 mg/kg diet) or a sufficient (SUFF, 4.0 mg/kg diet) level. Fetal islet size, replication, apoptosis, and offspring glucose tolerance were examined. Both ß-cell area and number per islet were reduced 
50% in fetuses from dams fed an LM vitamin A diet compared with those from dams fed the SUFF vitamin A diet. The -cell area and number per fetal islet were not affected by vitamin A deficiency. Apoptosis was not increased. The percentage of newly replicated ß-cells in the LM fetal pancreas was 42% less than that of SUFF offspring, but -cell replication was not affected. To determine whether this decrease in ß-cell area affected adult glucose tolerance and insulin secretion, 65-d-old offspring were subject to glucose tolerance tests. LM rats had a 55% lower plasma insulin level and a 76% higher serum glucose than SUFF rats. The same pattern could be seen in 35-d-old rats. These findings show that vitamin A deficiency decreases ß-cell mass and this reduction can be attributed to a reduced rate of fetal ß-cell replication in LM offspring. This may contribute to impaired glucose tolerance later in adult life.
KEY WORDS: • retinoids • pancreatic ß-cells • insulin secretion • diabetes • ß-cell replication
The development of pancreatic islets and their cells is complex, involving the specification of endoderm to become pancreas, the formation of the dorsal and ventral pancreas, epithelial differentiation, and the integration of newly established endocrine cells with the exocrine pancreas (1–3). The signals that regulate pancreatic development and function include transforming growth factor-ß, notch, sonic hedgehog (SHH),4 fibroblast growth factor (FGF), and epidermal growth factor pathways (4). Early in the development of the pancreas, - and ß-cells in the ductal epithelium of pancreatic tissue develop from and migrate out from stem cells and form aggregates that become islets. Lineage studies suggest that ß- and -cells develop from a common precursor cell containing pancreatic and duodenal homeobox protein (PDX-1) and neurogenin 3 (Ngn3) (4,5).
We are interested in the requirement of vitamin A for insulin secretion and possible islet development. Vitamin A or retinol (ROH) is an essential dietary nutrient and is required for normal growth, reproduction, and vision (3). Intracellularly, ROH is converted to all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA), and a variety of other active metabolites (3). At the tissue level, ATRA has marked effects on morphogenesis, cell proliferation, and differentiation and induces differentiation in a variety of normal and abnormal cell lines (3,6). The effects of retinoids are mediated through the retinoic acid and retinoid-X receptors (RAR and RXR). These receptors belong to a superfamily of nuclear receptors that are ligand-dependent transcription factors. In the nucleus, ATRA binds to RAR, ß, and 
, and 9CRA binds to RAR, ß, and and RXR, ß, and . RXRs specifically bind 9CRA and, as auxiliary proteins in gene transcription, bind to thyroid hormone receptors, vitamin D receptors, and peroxisome proliferator-activated receptors (PPARs) (7). The receptors form heterodimers and homodimers, which bind to DNA response elements, mediating hormone actions through transactivation of cell type–specific genes (3). These nuclear receptors mediate the effects of ATRA on growth and differentiation through regulation of transcription of a set(s) of target genes (3,8) that affect different processes, including morphogenesis.
Retinoids are important morphogens that affect growth and differentiation of epithelial-derived structures including embryonic foregut derivatives (9). The presence of vitamin A–dependent proteins in progenitor ductal and duct-derived islet cells supports the possibility that retinoids could play a critical role in islet cell development (6,10,11). In nonislet developing tissues, vitamin A has a regulatory role in the expression of SHH and FGF and other regulators that are also thought to be involved in - and ß-cell neogenesis and replication (12,13).
Recent findings suggested that type 2 diabetes may have its origin in fetal life (14). Undernutrition in fetal life may give rise to impaired islet development. Defects in fetal ß-cell growth and differentiation may be relevant to the subsequent development of diabetes in later life. Our previous studies showed that vitamin A is required for insulin secretion, both in vivo and in vitro (15–17). In further studies using cell lines, ATRA and 9CRA induced differentiation in an insulin-secreting cell line (18). Because vitamin A has important roles in fetal morphogenesis (19) and our findings showed that vitamin A may affect ß-cell differentiation, we asked whether vitamin A deficiency during intrauterine life may cause impaired islet development and lead to diabetes later in life. To test this possibility, we studied the effects of vitamin A deficiency during fetal life on islet development and subsequent glucose tolerance. Using a model of marginal vitamin A deficiency, the islets of the offspring of marginally vitamin A–deficient rats were examined for defects. Islet number, - and ß-cell area, replication, and apoptosis were measured along with subsequent glucose tolerance and insulin secretion at 35 and 65 d.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol, 90 µg of menadione (ICN Biomedicals), and 4.5 µg of cholecalciferol (Sigma Chemical) 3 times weekly, and soybean oil (Hunt-Wesson) replaced cotton seed oil. At weaning, female pups had free access to powdered diet supplemented with LM or sufficient (SUFF) amounts of retinol as retinyl palmitate. Initial studies indicated that 0.18 µg/g produced marginal vitamin A deficiency, but dams had difficulty delivering pups. Therefore, 0.25 µg/g retinyl palmitate (LM) was used to create marginal deficiency until age 70 d. At this time, rats continued to consume LM or were switched to a SUFF diet (4.0 µg/g). At 100 d of age, the female rats were mated. For fetal pancreas studies, fetuses at 20 d gestation were harvested from pregnant rats. For studies involving 35- or 65-d-old rats, offspring were fed the dams’ diet. For studies of pancreatectomized rats, they were anesthetized with an i.m. dose of ketamine (45 mg/kg; Abbott Laboratories) and xylazine (5 mg/kg; Fort Dodge Laboratories). For studies of glucose tolerance and insulin secretion, ketamine and diazepam (5 mg/kg; Elkins-Sinn) were used to anesthetize the rats. After each procedure, rats were killed by bilateral thoracotomy. Fetuses were killed by decapitation before removal of the pancreas. All procedures were reviewed and approved by the Animal Care and Use Subcommittee of Huntington Veterans Affairs Medical Center Research Service.
ß-Cell and -cell mass.
Fetal pancreata were immediately fixed in 4% paraformaldehyde in PBS overnight. Tissues were dehydrated in increasing concentrations of ethanol and cleared in HemoDe before being infiltrated and embedded in Paraplast Plus (all from Fisher Scientific). Sections (5 µm) were stained immunocytochemically for insulin. Sections were cleared, rehydrated, and permeabilized (22). Slides were incubated for 15 min in 3% H2O2 to block endogenous peroxidase followed by a 15-min incubation in 10% bovine serum albumin (BSA, Fraction V, Sigma) in Tris-buffered saline to block nonspecific binding sites. Then the slides were incubated overnight at 4°C with guinea pig anti-porcine insulin (1:100; ICN Biomedicals) or guinea pig anti-glucagon (1:100, Linco Research). A labeled streptavidin-biotin kit (DAKO) was utilized to begin visualization of the primary antibody, followed by a 15-min incubation in diaminobenzidine (SigmaFast DAB, Sigma). Slides were counterstained with hematoxylin and eosin. After dehydration and clearing, coverslips were mounted with Permount (Fisher). Slides in this experiment and the experiments described below were examined using light microscopy, and 1 slide or the mean of 2 slides was used as an independent observation.
For ß-cell mass, the largest diameter of the insulin-stained portion of the islet and a diameter perpendicular to the first were measured using an eyepiece micrometer, and the ß-cell area was calculated. For -cell mass, islets were stained for glucagon. Two perpendicular diameters of the whole islet and 2 perpendicular diameters of the nonstaining interior of the islet were measured. Total islet area and non--cell area were calculated. The -cell area was calculated by subtracting the nonstained ß-cell area from the total islet area.
Number of ß- and -cells per islet and replication.
Pregnant rats, at 20 d gestation, from both the LM and the SUFF groups, were injected i.p. with 5-bromo-2'deoxyuridine (BrdU, Sigma, 100 mg/kg body weight) (23) 6 h before the experiment. Fetuses were removed from the anesthetized female and decapitated, and the pancreas was excised. The fetal pancreas was fixed, processed, and embedded as described above. Sections (5 µm) of pancreas were adhered to SuperFrost Plus slides and double stained for BrdU and glucagon.
BrdU staining was done using a Cell Proliferation Kit [Amersham BioSciences (23)]. Slides were incubated for 2 h with anti-BrdU and nuclease at room temperature and then for 30 min with peroxidase anti-mouse IgG2a at 10% of recommended strength. BrdU was visualized with 3,3'-diaminobenzidine tetrahydrochloride in the presence of nickel to yield a blue-gray stain of newly replicated nuclei. Nonspecific binding sites were blocked by incubation for 1 h with 10% BSA in PBS. The staining procedure was continued as described above using guinea pig anti-glucagon antibody (1:200), followed by hematoxylin staining. Hematoxylin (Fisher) was filtered and diluted 1:7.5 for a light purple nuclear stain that would not mask the BrdU staining. All antibodies and wash solutions were supplemented with 0.1% Triton X-100.
The islets were identified by a mantle of brown glucagon staining. All ß- and -cells in the islets were identified by light purple nuclear stain and counted. All newly replicated cells among these were identified by BrdU-positive blue-gray nuclei and counted. The number of ß- and -cells per islet and the percentage of new ß- and -cells among all cells counted were calculated.
Apoptosis. To measure apoptosis, 3-µm sections of fetal pancreas were stained using the fluorescein Cell Death Detection kit (Roche Applied Science) to detect fragmented DNA and propidium iodide to detect condensed nuclei. Slides were incubated in 20 mg/L proteinase K (ICN Biomedicals) and 10 mmol/L Tris for 60 min at room temperature followed by permeabilization in sodium citrate, 100 mmol/L, pH 6.0, at 70°C for 30 min. DNA fragments were elongated and labeled by incubation of slides in a terminal UTP nick end labeling reaction mixture followed by insulin staining and propidium iodide staining as described above with no hematoxylin or eosin counterstaining. Slides were examined for positive apoptotic cells. Positive pancreatic control slides were prepared by treatment with DNase I (0.5 g/L; from bovine pancreas; Boehringer Mannheim). Nontreated intestinal slides were used as an additional positive control.
In vivo glucose tolerance and insulin secretion. Anesthetized LM and SUFF offspring at 35 and 65 d underwent glucose tolerance testing. To ensure injection of the total glucose bolus and adequate sample volume without hemolysis that would interfere with our insulin assay, studies measuring insulin release and glucose tolerance were performed separately (16). Glucose (100 mg/kg) was given by intracardiac (IC) injection. For glucose tolerance testing, blood was collected IC at time 0, 1, 3, 5, 15, and 30 min postinjection with a 25-gauge needle. In these and previous studies (16), glucose equilibration occurred by 15 min after injection of intracardiac glucose. Therefore, the blood glucose value at 15 min was used to assess glucose intolerance. To determine insulin secretion, blood was collected IC at time 0, 1, and 3 min using a 25-gauge needle on a syringe rinsed with heparin (porcine, Elkins-Sinn). Blood was collected in microcentrifuge tubes and immediately centrifuged, and the serum or plasma was frozen for analysis at a later date.
Glucose analysis and RIA for insulin. Glucose was measured in serum by the glucose oxidase method using a Glucose Analyzer 2 (Beckman Instruments). Standards and reagents for the assay were also purchased from Beckman. All samples were assayed in duplicate. Intra-assay variation ranged from 1.1 and 2.5% CV and interassay variation ranged from 2.1 and 5.4% CV (low and high pool, respectively).
Plasma samples underwent RIA for insulin as previously described (24,25). The intra-assay variation was 7.9% CV and interassay variation 13.6% CV.
Statistics. Statistical analysis was performed with one-way ANOVA using a computerized statistical package (SPSS) to determine differences between the 2 dietary groups at each specific time point. Each n is a different rat and is an independent observation. Differences with P < 0.05 were considered significant. Results are expressed as the means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-Cell area and number of -cells.
The -cell area of the islets was not affected by marginal vitamin A deficiency (Fig. 2C). The LM fetal offspring had an -cell area per islet of 885 ± 235 µm2 compared with the SUFF -cell area of 1111 ± 254 µm2 (n = 9). In addition, the number of -cells per islet did not differ between the groups (Fig. 2D). The LM fetal offspring had 11.6 ± 1.2 -cells/islet compared with 12.2 ± 1.2.
Replication and apoptosis.
ß-Cell replication in the LM fetal offspring was 42% less than in SUFF fetal offspring. A mean of 2.07 ± 0.26% of the LM ß-cells were newly replicated compared with 3.55 ± 0.65% of the SUFF ß-cells (Fig. 3A; P < 0.05; n = 8–9). -Cell replication was not changed by vitamin A deficiency (Fig. 3B).
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Glucose tolerance and insulin secretion in vivo. In 35-d-old offspring, there was no significant difference between serum glucose levels at 15 min post-IC glucose injection (LM = 22.3 ± 1.8 mmol/L, n = 5; SUFF = 19.7 ± 1.7 mmol/L, n = 13) although SUFF 35-d-old rats had a lower glucose level. LM 35-d-old offspring had lower plasma insulin levels at 0, 1, and 3 min postinjection than SUFF 35-d-old offspring, but this difference was not significant (Figs. 4A and 5A).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The reduced islet ß-cell area and number can be attributed to decreased replication. A 42% decrease in replication rate in LM rats could account for the increased number of small islets with fewer ß-cells and a 51% reduction in ß-cell area compared with SUFF rats. The decrease in replication of ß-cells was observed within the islets, indicating that reduced formation of new ß-cells could be attributed to a defect in replication from preexisting ß-cells.
Decreased replication of islet ß-cells does not exclude the possibility of an additional defect in neogenesis of ß-cells from stem cells. A defect in neogenesis from stem cells would represent an early defect in cell development, and non-ß-cells may have been affected as well as ß-cells. A reduction in neogenesis would likely result in a reduction of both non-ß-cells and ß-cells along with reduced islet formation and number of islets. This was not the case, and considered together with the reduced number of ß-cells within the islets, our findings suggest that the defect is in the replication of preexisting ß-cells in the islets.
Our studies showed that offspring of A-def rats that remain deficient become glucose intolerant later in adult life at 65 d. At 35 d, glucose levels were slightly increased and insulin levels slightly decreased compared with SUFF rats although neither difference was significant. Further studies that test glucose tolerance and insulin secretion between d 35 and 65 are required to confirm the possibility of deterioration of glucose tolerance over time. Nevertheless, considering the 35 and 65 d glucose and insulin data together, the pattern suggests the ß-cells are beginning to fail at 35 d; by 65 d, the islets are no longer able to secrete enough insulin to maintain glucose tolerance. With increasing age, the defect may become evident because of a progressive decrease in ß-cell mass or function. Possible explanations include an effect of continued vitamin A deficiency on islet function or failure of ß-cell replication to replace dying cells. This evolution of glucose intolerance and ß-cell failure with time is reminiscent of the failing islet observed in type 2 diabetes in humans.
Our findings indicated that intrauterine vitamin A deficiency leads to a developmental defect in islet or ß-cell function, which in turn results in impaired glucose intolerance later in life. Vitamin A deficiency could affect specification or maturation. Vitamin A affects the homeobox gene expression of homeodomain proteins that regulate morphogenesis. The origins of the ß-cell are complex, involving many time- and space-dependent transcription factors contributing to the specification and differentiation of the exocrine pancreas and islet cells. Sonic Hedgehog (SHH), a member of the hedgehog family of secreted factors, interferes with pancreatic organogenesis. During pancreatic development, SHH is excluded from endoderm destined to be pancreatic tissue. Its repression permits pancreatic gene expression of proteins that regulate pancreatic development (4). The SHH promoter region contains a retinoic acid response element in the sonic upstream region that can be bound by RAR and RXR heterodimers in vitro and confers retinoic acid inducibility (26). Hox or Hox-related ParaHox gene products are other proteins regulating development. PDX-1 is a ParaHox gene protein highly homologous to the pancreatic protein X1Hbox in Xenopus laevis (27). Early expression of PDX-1 is required for pancreatic exocrine and endocrine development, and late expression is required to maintain the ß-cell pattern of insulin gene expression, insulin production, and Glut 2 expression and function. Hence, in our model of marginal vitamin A deficiency, the lack of vitamin A may fail to suppress SHH expression and lead to increased expression of SHH, which suppresses pancreatic development. Alternatively, vitamin A deficiency may lead to decreased expression of PDX-1 or other transcription factors that impair pancreatic development. These possibilities seem unlikely, however, because the exocrine and endocrine pancreas were developed and the normal topographical arrangement of the -cells peripherally and ß-cells centrally in the islet was maintained.
An alternative possibility is that vitamin A deficiency leads to a defect further downstream in the maturation of the ß-cells. Islets and their cells arise by epithelial differentiation from the pancreatic stem cells in exocrine tissue. Stem cells mature stepwise into specific cell types which, in turn, adhere and aggregate to form islets within the pancreas (1,2). The pancreatic polypeptide (PP) cell appears to be a precursor of the ß-cells; - and ß-cells are identifiable in primordial tissue before the presence of an organized islet. -Cells are the first islet cells to differentiate in the fetus, and they make up a large volume of the islet in early islet development (28–30). The cells within the rat islet are arranged topographically with respect to each other, and the -, 
-, and PP-cells form a peripheral mantle around a core of ß-cells (31,32). In the developing fetal islet cells, proteins (PDX-1, Ngn3, Pax4, Pax6, Isl1, Hlxb9, Hes1, beta2/NeuroD, Nkx2.2) appear to have distinct roles at different steps of islet -and ß-cell differentiation. Ontogenetically, PDX-1/Ngn 3-positive cells are precursors of the PP cell and -cell. Vitamin A may have a role in gene expression of 1 or more of these proteins. Vitamin A deficiency may lead to reduced amounts or altered temporal or spatial expression of PDX-1 or another transcription or regulatory factor that leads to a replication defect in the ß-cell. Vitamin A deficiency could affect the -cell as well as the ß-cell. A defect in both - and ß-cell differentiation would suggest an effect on an early step in development involving a common progenitor cell. In this study and in previous studies (16), we did not see any defects in topographical arrangement of -cells peripherally or in the -cell area. This would support a defect in later development. However, in previous studies using a model in which vitamin A deficiency was created postnatally, we did see a defect in glucagon secretion that appeared to persist into adult life (16,33). Further studies using our model of marginal vitamin A deficiency that assay transcription factors regulating both - and ß-cell development are warranted.
The importance of the intrauterine environment on fetal development is becoming increasingly clear. Our findings are consistent with the Barker hypothesis (14) and the concept that the intrauterine environment, particularly malnutrition and energy deficiency, affects subsequent occurrence of hypertension, hyperlipidemia, insulin resistance, and type 2 diabetes. Our studies would extend the concept to specific deficiencies of vitamins that may affect the state of fetal development and the fetal genome. It is possible that deficiencies in vitamins that affect islet development in utero may affect subsequent islet function later in life. If our model can be translated to humans, our findings suggest that in addition to generalized malnutrition, deficiencies of vitamins that affect gene expression may lead to subsequent disease in adults. Because vitamin A deficiency affects islet development, this effect may be expressed later in life. Vitamin A deficiency in developing countries is a world health problem. High-risk populations exist in the United States and include pregnant women who may be vitamin A–deficient with health consequences to the neonate (34,35). Even marginal vitamin A deficiency may lead to fetal development problems. An understanding of the normal requirements during pregnancy and weaning should enhance child health and nutrition by fostering optimal vitamin A intake for women during pregnancy, weaning, and adulthood.
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FOOTNOTES |
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2 Supported by National Institutes of Health Grant 1 R15 DK57851–01.
4 Abbreviations used: 9CRA, 9-cis-retinoic acid; A-def, vitamin A deficient; ATRA, all-trans-retinoic acid; BSA, bovine serum albumin; BrdU, 5-bromo-2'deoxyuridine; FGF, fibroblast growth factor; IC, intracardiac; LM, Low marginal; Ngn3, neurogenin 3; PDX-1, pancreatic and duodenal homeobox protein; PP, pancreatic polypeptide; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; ROH, retinol; RXR, retinoid-X receptor; SHH, sonic hedgehog; SUFF, sufficient.
Manuscript received 6 April 2004. Initial review completed 26 April 2004. Revision accepted 18 May 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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