Pancreatic Islet-Specific Expression of an Insulin-Like Growth Factor-I Transgene Compensates Islet Cell Growth in Growth Hormone Receptor G
http://www.100md.com
内分泌学杂志 2005年第6期
Fraser Laboratories (Y.G., Y.L., K.R., Z.T., Y.L.L., J.-L.L.), Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 1A1; Transgenic Unit (D.H.), Montreal General Hospital Research Institute, Montreal, Quebec, Canada H3G 1A4; and Edison Biotechnology Institute and Department of Biomedical Sciences (J.J.K.), College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701
Address all correspondence and requests for reprints to: Dr. Jun-Li Liu, Fraser Laboratories, Room M3-15, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: jun-li.liu@mcgill.ca.
Abstract
Both GH and IGF-I stimulate islet cell growth, inhibit cell apoptosis, and regulate insulin biosynthesis and secretion. GH receptor gene deficiency (GHR–/–) caused diminished pancreatic islet cell mass and serum insulin level and elevated insulin sensitivity. Because IGF-I gene expression was nearly abolished in these mice, we sought to determine whether that had caused the islet defects. To restore IGF-I level, we have generated transgenic mice that express rat IGF-I cDNA under the direction of rat insulin promoter 1 (RIP-IGF). Using RNase protection assay and immunohistochemistry, the IGF-I transgene expression was revealed specifically in pancreatic islets of the RIP-IGF mice, which exhibited normal growth and development and possess no abnormalities in glucose homeostasis, insulin production, and islet cell mass. GHR–/– mice exhibited 50% reduction in the ratio of islet cell mass to body weight and increased insulin sensitivity but impaired glucose tolerance. Compared with GHR–/– alone, IGF-I overexpression on a GHR–/– background caused no change in the diminished blood glucose and serum insulin levels, pancreatic insulin contents, and insulin tolerance but improved glucose tolerance and insulin secretion. Remarkably, islet-specific overexpression of IGF-I gene in GHR–/– mice restored islet cell mass, at least partially through cell hypertrophy. Interestingly, double-transgenic male mice demonstrated a transient rescue in growth rates vs. GHR–/– alone, at 2–3 months of age. Our results suggest that IGF-I deficiency is part of the underlying mechanism of diminished islet growth in GHR–/– mice and are consistent with the notion that IGF-I mediates GH-induced islet cell growth.
Introduction
GH AND IGF-I ARE POTENT regulators of cell growth, differentiation, and metabolism and are essential for postnatal growth in mammals (1). Receptors for both GH and IGF-I are expressed in the pancreatic islet cells (2, 3). Acting on their own, both GH and IGF-I promote islet cell growth, inhibit apoptosis, and are potentially involved in normal islet growth and maintenance (4, 5, 6), but it is unclear whether GH and IGF-I interact with each other in regulating pancreatic islet function and how IGF-I is involved in GH-stimulated islet growth and insulin biosynthesis and secretion. IGF-I produced either from the liver or locally within the pancreatic islets might mediate GH actions. We and others have recently demonstrated that GH receptor gene deficiency (GHR–/–) caused diminished pancreatic islet cell mass and serum insulin level and elevated insulin sensitivity (7, 8). Because IGF-I gene expression was nearly abolished in GHR–/– mice, we sought to determine whether that had caused the islet defects (9). For this purpose, we have generated transgenic mice RIP-IGF that express an IGF-I transgene under the direction of rat insulin promoter 1 (RIP) and studied whether the islet defects in GHR–/– mice can be rescued. As a result, local expression of the IGF-I transgene restored pancreatic islet cell mass and improved glucose tolerance in GHR–/– mice, which is consistent with the notion that IGF-I mediates the GH-induced growth-promoting effect on pancreatic islet cells.
Materials and Methods
Creation of RIP-IGF transgenic mice
A transgenic line has been developed to overexpress IGF-I cDNA driven by an insulin promoter (RIP-IGF). The promoter was chosen based on its high level and ?-cell-restricted expression in driving IGF-II and Glut-2 antisense (10, 11). Briefly, a 0.5-kb PvuII/AvaI fragment of a rat prepro-IGF-I cDNA from a pGEM4Z vector (12) was subcloned into a BamHI/EcoRI site downstream of RIP in a pKS-RIP/globin vector (Fig. 1) (11). The integrity of the transgenic construct, pKS-RIP-IGF6, was confirmed by sequencing and restriction analysis before it was injected into the pronucleus of fertilized mouse ova. The manipulated ova were transferred into the oviducts of recipient female mice, which gave birth to founder mice.
FIG. 1. Pancreatic islet-specific IGF-I overexpression: characterization of transgenic lines. A, Diagram of transgenic DNA vector: pKS/RIP-IGF-I. Based on pKS-RIP/globin vector, rat insulin promoter 1 (RIP) was used to drive expression of an intact rat prepro-IGF-I cDNA. The arrows mark the region covered by the pSP72-RIF probe and primers Tg1 and Tg2. B, Detection of transgene expression by RNase protection assay. Total pancreatic RNA prepared from transgenic mice (+) or wild-type littermates (–), determined by PCR, was hybridized to rat IGF-I and mouse ?-actin probes. A protected band with rat IGF-I probe indicates transgene expression. Endogenous mouse IGF-I mRNA was unprotected by the probe thus destroyed by RNase treatment. Results from four representative families (codes 34903 etc.) are illustrated.
To identify founder mice (on a mixed C3H and C57BL/6 background) and offspring that carry the transgene, a PCR, using the primers Tg1 (5'-GGT GAT ATT GGC AGG TGT TCC-3') and Tg2 (5'-CAA ATC GGC AAA GTC CAG G-3'), generated a product of 600 bp corresponding to the entire cDNA. To probe the transgene expression by Northern blots and RNase protection assays, a 0.7-kb PstI/EcoRI fragment that contains the cDNA including the two primer sites was subcloned into pSP72 vector (Promega, Madison, WI). The resulting vector, named pSP72-RIF, was linearized by HindIII to direct synthesis of an antisense RNA probe using T7 polymerase.
Intercross with GHR–/– mice and genotyping
GHR–/– mice carry a targeted disruption of exon 4 of the mouse GHR/BP gene, as previously reported (8, 9). To intercross with RIP-IGF mice, heterozygous (GHR+/–) mice, on a hybrid 129/Ola-BALB/c-C57BL/6 background, were used. To genotype the offspring, genomic DNA was isolated from tail clips using standard methods. Primers In4-1 (5'-CCC TGA GAC CTC CTC AGT TC-3'), In3+1 (5'-CCT CCC AGA GAG ACT GGC TT-3'), and Neo-3 (5'-GCT CGA CAT TGG GTG GAA ACA T-3') were used in PCR, which yield a 390-base band for the wild-type allele, and 290/200 double bands for the GHR knockout allele, as previously reported (13). Offspring of four genotypes were selected: wild type, RIP-IGF (GHR+/+), GHR–/– (no RIP-IGF), and GHR–/– plus RIP-IGF (GHR+RIP). All heterozygous (GHR+/–) animals were excluded.
Animal procedures
The animals were maintained in 12-h dark, 12-h light cycles at room temperature with free access to food and water or when indicated, food deprived for 24 h with free access to water. At the desired age, the mice were anesthetized with a cocktail of ketamine/xylazine/acepromazine, bled via periorbital puncture, and killed by cervical dislocation. Blood was collected for serum preparation, and pancreata were rapidly removed for biochemical or histological analysis. All animal-handling procedures were approved by the McGill University Animal Care Committee.
Serum concentrations of insulin and glucagon were determined using RIA kits obtained from Linco Research Inc. (St. Charles, MO). IGF-I was determined using an RIA kit obtained from Diagnostic Systems Laboratories (Webster, TX). Blood glucose levels were measured using the OneTouch blood glucose meter and strips (LifeScan Canada, Burnaby, British Columbia, Canada). For the insulin tolerance test, animals were injected with human insulin (0.75 IU/kg, ip; Roche Applied Science, Penzberg, Germany), and blood glucose levels were measured at 0, 20, 40, and 60 min after the injection. For the glucose tolerance test, mice were fasted 24 h and injected with glucose (1 g/kg, ip), and blood glucose levels were measured at 0, 15, 30, 60, and 120 min after the injection.
Immunohistochemistry and islet cell mass determination
Pancreata were removed from 2-month-old mice (n = 4 in each group) and fixed, embedded in paraffin, and cut into 5-μm sections (14). The sections were then subjected to immunohistochemical staining for insulin and glucagon with rabbit polyclonal antibodies (Monosan, Uden, The Netherlands) using the avidin-biotin-peroxidase complex technique, which results in a red immunoreactive signal with a nuclear counterstain using methyl green, or diaminobenzidine substrate, which resulted in a brown immunoreactive signal with a hematoxylin counterstain (blue) of cell nuclei (14, 15). Mouse monoclonal IgG against human IGF-I (clone Sm1.2; Upstate USA Inc., Charlottesville, VA) was used to reveal IGF-I overexpression. Images of all pancreatic islets were captured with a Retiga 1300 digital camera (Q Imaging, Burnaby, British Columbia, Canada) at magnifications of x25, x100, or x400. The area of the pancreatic tissue was measured using Northern Eclipse computer software, version 6.0 (Empix imaging, Mississauga, Ontario, Canada). The number of insulin-stained pancreatic islets in each image was manually counted using Adobe Photoshop 7.0 computer software.
The islet cell mass (defined as all cells staining positive for the hormone insulin) was determined by initially weighing the excised pancreatic tissue and then determining the percentage of the excised organ that was insulin positive (16). All insulin-positive ?-cell clusters (islets) were loosely traced, and the insulin-immunoreactive area was determined using the thresholding option. Total tissue area was also quantified using the threshold option to select the stained areas while not selecting unstained areas (white space). Islet cell percent was determined by dividing the total insulin area by the total tissue area for each animal. The islet cell mass for each animal was then derived by multiplying the islet cell percent by the excised pancreas tissue weight. Each mouse pancreas was examined in one slide of approximately 40 fields of view and approximately 12 mm2 of total tissue.
To reflect individual islet cell growth in adult (2 month old) mice, average cell size was calculated in hematoxylin-eosin-stained x400 images using total islet area divided by the number of cell nuclei. For this purpose, a minimum of 10 mature islets were chosen from each genotype group.
Northern blot and RNase protection assay
RNA isolation and Northern blot analysis were as reported except with digoxigenin-labeled probes (Roche) (17, 18). RNase protection assay was as reported using 32P-labeled probes (19, 20). The intensity of the hybridization signals on the autoradiogram was analyzed using a FluorChem 8900 imaging system (Alpha Innotech, San Leandro, CA).
Insulin and glucagon secretion
Mice at age 2–4 months, both male and female, were fasted 24 h and injected with glucose (3 g/kg, ip) (21). At 0 (without stimulation), 5, 15, or 30 min, they were anesthetized, bled via periorbital puncture, and killed. Blood was collected for serum preparation. Insulin and glucagon concentrations were determined by RIA.
Statistics and data plotting
Data are expressed as the mean ± SE. The Student’s t test (unpaired and paired) and one-way ANOVA were performed using InStat software version 3 (GraphPad Software Inc., San Diego, CA). Data were plotted into curves, and the area under curve was calculated using SigmaPlot software version 9 (Systat Software, Inc., Point Richmond, CA).
Results
Pancreatic islet ?-cell-specific IGF-I overexpression in RIP-IGF mice
To overexpress IGF-I in most cells of the pancreatic islets, we have used an insulin promoter to drive the transgenic expression of rat IGF-I cDNA (Fig. 1A). Multiple founder lines were created and screened for genomic integration of the transgene by specific PCR. Using immunohistochemistry and RNase protection, two mouse families (3 and 13) exhibited high levels IGF-I expression, which was specific in pancreatic islets. As shown in Fig. 1B, rat IGF-I mRNA could be detected in the pancreatic RNA prepared from RIP-IGF transgenic mice but not in nontransgenic littermates. As shown by immunohistochemistry in Fig. 2A (top), pancreatic islets in wild-type mice only exhibited scattered IGF-I staining in very few islet cells; the levels of IGF-I staining (brown pigmentation) was drastically elevated in RIP-IGF mice. Judging from the ratio of IGF-I-positive cells, it seems that not all ?-cells express the transgene. Except nonspecific staining of the blood cells, the transgenic expression was relatively specific and not seen in exocrine acinar cells.
FIG. 2. Increased pancreatic islet cell mass and improved body growth caused by islet-specific IGF-I overexpression in male GHR–/– mice. A, Islet-specific transgenic expression revealed by immunohistochemistry. Pancreatic sections prepared from 2-month-old mice of four genotype groups were stained for IGF-I using the diaminobenzidine complex. IGF-I staining was shown as brownpigmentation within the islets. Cell nuclei were counterstained with hematoxylin. Images are representatives of at least 15–20 mature islets from each mouse and have been recorded at x400. B, Postnatal growth rates in mice of various genotypes. Body weight was measured at 1, 2, 3, and 4 months of age and illustrated as mean ± SE. Numbers of animals are shown in parentheses. *, P < 0.05; **, P < 0.01 vs. GHR–/– alone. C, Changes in pancreatic islet cell mass, corrected for total body weight (n = 5). **, P < 0.01 vs. wild-type (WT); #, P < 0.05 vs. GHR–/– alone. ANOVA: P = 0.006; WT vs. GHR–/–, P < 0.05; GHR–/– vs. GHR+RIP, P < 0.05. D, Pancreatic islet-specific overexpression of IGF-I increased the average islet cell size in GHR–/– mice (islet cell hypertrophy). From each genotype group of mice at age 2–3 months, 11–12 mature islets stained with hematoxylin-eosin were analyzed. The islet size and the number of cell nuclei were determined using Northern Eclipse software. ANOVA of four groups: P < 0.001; **, P < 0.01; ***, P < 0.001 vs. WT littermates; ##, P < 0.01 vs. GHR–/– alone. Except that of B, similar results were obtained using female mice (not shown).
Normal growth and islet formation in RIP-IGF mice
RIP-IGF mice exhibited normal growth and development and possessed no abnormalities in blood glucose (fasted or fed), serum insulin, and glucagon levels, as shown in Table 1. Northern blot analysis revealed normal levels of insulin mRNA in transgenic vs. wild-type mice (data not shown). Pancreatic insulin content was unaltered in RIP-IGF mice (Table 2). Immunohistochemistry showed no obvious abnormality in islet morphology and - and ?-cell distribution patterns within the islets (data not shown). As shown in Fig. 2C (columns 1 and 2), RIP-IGF mice had normal islet cell mass. When challenged with a bolus injection of glucose, RIP-IGF mice exhibited an unaltered glucose clearance curve vs. wild-type littermates. Likewise, RIP-IGF mice showed no significant difference in their glucose-lowering response to insulin injection, compared with wild-type littermates (data not shown).
TABLE 1. Changes in body weight and glucose homeostasis in RIP-IGF mice vs. wild-type controls
TABLE 2. Effects of IGF-I overexpression on serum chemistry and pancreatic insulin content in GHR–/– mice
Effects of IGF-I overexpression on animal growth and glucose and insulin levels in GHR–/– mice
GHR–/– mice exhibited severe growth retardation, decreased blood glucose and serum insulin levels, increased insulin sensitivity, and diminished islet cell mass (7, 8). To investigate whether restored IGF-I expression in the pancreatic islets can rescue the islet defects, we intercrossed GHR+/– with RIP-IGF mice and studied second-generation offspring of four genotypes, i.e. wild type, RIP-IGF, GHR–/–, and GHR+RIP. Compared with GHR–/– alone, islet IGF-I expression was indeed significantly increased in GHR+RIP mice as shown by immunohistochemistry (Fig. 2A, bottom). Computer-assisted image analysis indicated that, on average, the IGF-I-stained area increased from 4 ± 1% (n = 10) of total islet area in GHR–/– mice to 21 ± 3% (n = 11; P < 0.05) in GHR+RIP mice. Because the transgenic expression was limited to the islet cells at a moderate level, compared with similar reports (10, 22), and caused no change in serum IGF-I level (Table 2), it was not expected to have an impact on the growth retardation of GHR–/– mice. Nevertheless, we had measured their body weight from 1–4 months of age. As shown in Fig. 2B, RIP-IGF expression alone caused no change in growth vs. wild-type littermates. GHR–/– mice exhibited severe growth retardation with only approximately one half of the wild-type body weight at adult age. Interestingly, the double-transgenic male mice demonstrated a significant partial rescue in growth rates vs. GHR–/– mice at 2–3 months of age (i.e. 19% increased body weight). This effect was transient, was not seen in females, and did not last beyond 4 months of age.
As expected, GHR–/– mice exhibited drastic reductions in serum insulin level (–33%) and pancreatic insulin content (–52%), suggesting reduced insulin production. RIP-IGF expression on this GHR–/– background failed to normalize serum insulin, pancreatic insulin content, and insulin mRNA to wild-type levels (Table 2 and data not shown).
Transgenic IGF-I overexpression restored islet cell mass in GHR–/– mice
Total islet cell mass, determined by insulin staining, was decreased 6.2-fold in GHR–/– mice vs. wild-type littermates. When corrected for body weight, the decrease was 2.9-fold (Fig. 2C, column 3). In double-transgenic GHR–/– mice that express the IGF-I transgene (GHR+RIP, column 4), total islet cell mass was increased 3.8-fold vs. GHR–/– mice alone. When corrected for body weight, the increase had effectively restored the islet cell mass to the level of wild-type mice (GHR+RIP 61 ± 22 vs. WT 46 ± 13 mg/kg; n = 5) (Fig. 2C, column 4).
Average islet cell size is a measure of islet cell hypotrophy (such as in GHR–/– mice) or hypertrophy (such as in ?-cell compensation to type 2 diabetes) and a reflection of cell health and activity (8, 23). As shown in Fig. 2D, compared with wild-type littermates, IGF-I overexpression alone in RIP-IGF mice did not change islet cell size; as previously reported, GHR–/– mice exhibited a 42% reduction in islet cell size (8); in GHR–/– mice that overexpress IGF-I in islet cells, the average islet cell size was increased 29%, exhibiting a partial rescue in pancreatic islet cell growth. Similar results were obtained from both male and female mice.
Transgenic IGF-I overexpression improved glucose tolerance in GHR–/– mice
GHR–/– mice exhibit glucose intolerance and elevated insulin sensitivity, because of specific changes within the pancreatic islets and insulin target tissues (Coschigano, K.T., et al., 1999, 81st Annual Meeting of The Endocrine Society, San Diego, CA) (8). In double-transgenic GHR+RIP mice, with restored IGF-I production, their glucose tolerance was largely restored (Fig. 3A). At 40 and 60 min after glucose injection, GHR+RIP mice exhibited significantly reduced blood glucose level vs. GHR–/– alone. Their rate of glucose disposal was almost as efficient as wild-type littermates (Fig. 3A).
FIG. 3. Improved glucose tolerance but unaffected insulin sensitivity caused by islet-specific IGF-I overexpression in male GHR–/– mice. A, Glucose tolerance test. Mice (9–10 wk old) were fasted for 24 h, and glucose (1 g/kg) was injected ip. Blood glucose was measured at 0, 15, 30, 60, and 120 min after the injection. *, P < 0.05; **, P < 0.01 vs. wild-type (WT) littermates in unpaired t test. The number of animals in each group is indicated in parentheses. B, Insulin tolerance test. Mice (11–12 wk old) were injected with insulin (0.75 U/kg, ip), and blood glucose was measure at 0, 20, 40, and 60 min after. The percentage values relative to 0 time were expressed as mean ± SE. *, P < 0.05; ***, P < 0.001 vs. WT control mice. Similar results were obtained using female mice (not shown).
Animals of the four genotypes were tested for insulin tolerance. As shown in Fig. 3B, compared with wild-type littermates, GHR–/– mice exhibited significantly decreased glucose levels at 20, 40, and 60 min after insulin injection. GHR+RIP mice exhibited less deviation from wild-type mice, but the differences were insignificant from either group. Thus, pancreatic islet-specific IGF-I overexpression did not affect the phenotype of insulin hypersensitivity.
Changes in glucose-stimulated insulin and glucagon secretion
Reduced glucose tolerance in GHR–/– mice, in the face of increased insulin sensitivity, suggests possible defects in insulin secretion. Likewise, improved glucose tolerance in GHR+RIP mice would indicate elevated insulin secretion vs. GHR–/– alone. To verify these speculations, we have measured serum insulin levels after a glucose load (Fig. 4A). Insulin secretion exhibited 2.7- to 4.9-fold increases in wild-type mice at 5 and 15 min after glucose stimulation, which was virtually restored to normal by 30 min. RIP mice exhibited a similar response, except with a delayed return to basal level at 30 min. In contrast, the secretion in GHR–/– mice was drastically diminished and reached to only 2.2- and 2.7-fold at 5 and 15 min vs. 0 min, even after being corrected for their low basal level. Interestingly, at 5 min after glucose stimulation, GHR+RIP mice were able to demonstrate a transient enhancement in serum insulin level vs. GHR–/– mice alone (which did not last till 15 and 30 min), suggesting a certain degree of improvement in insulin secretion associated with islet-specific IGF-I overexpression. In the same experiment, serum glucagon levels exhibited no significant reductions in wild-type and RIP mice within 15 min of glucose injection (Fig. 4B). In GHR–/– and GHR+RIP mice, however, glucagon levels were reduced approximately 40% at 5 min after glucose stimulation, significantly lower than wild-type mice.
FIG. 4. Changes in glucose-stimulated insulin and glucagon secretion caused by islet-specific IGF-I overexpression in GHR–/– mice. A, Serum insulin level. Mice at age 3–4 months, both male and female, were fasted for 24 h before being stimulated with glucose (3 g/kg, ip). Serum insulin concentrations were measured at 0, 5, 15, and 30 min (n = 5–14). *,P < 0.05; **, P < 0.01 vs. wild-type (WT) mice; #, P < 0.05 vs. GHR–/– alone by unpaired t tests. The area under curve values were 56.0 for WT, 69.7 for RIP, 23.5 for GHR–/–, and 29.9 for GHR+RIP. B, Serum glucagon levels. The values at 30 min are not presented because of insufficient samples.
Discussion
Both GH and IGF-I stimulate islet cell growth and inhibit apoptosis and thus are potentially involved in normal islet development. GH stimulated insulin and glucagon secretion and pancreatic islet cell proliferation (24, 25, 26, 27). The stimulation of cell replication in neonatal rat pancreatic monolayer cultures by GH was independent of glucose concentration (28). More recently, in primary cultures of pancreatic islet cells, GH stimulated ?-cell proliferation, insulin gene transcription and insulin secretion (4). Among the various postreceptor substrates, Stat5a/b, Stat1, and Stat3 were found to be activated in pancreatic islet or islet-derived tumor cells (29, 30). GH overexpression in vivo increased pancreatic islet number and volume in transgenic mice (31). In a previous report, we have demonstrated reduced islet cell mass and enhanced insulin sensitivity in GHR–/– mice (8). Because IGF-I gene expression in liver and pancreas was severely affected and IGF-I is known to stimulate islet cell growth, we believe that lack of IGF-I production in GHR–/– mice had contributed to islet growth defect. In this study, we have created transgenic mice (RIP-IGF) that overexpressed the IGF-I gene in pancreatic islet cells and exhibited no obvious effect on islet growth by themselves. Crossing them with GHR–/– mice restored local production of IGF-I in the islet cells and rescued to various degrees islet cell mass, average cell size, glucose tolerance, and even transiently somatic growth. It is consistent with the notion that locally produced IGF-I mediates GH-stimulated islet cell growth. Glucose intolerance in GHR–/– mice is likely caused by insufficient (amount and speed) release of insulin in response to glucose (first), especially so in the face of increased insulin sensitivity. A significant improvement in GHR+RIP mice vs. GHR–/– alone (Fig. 3A) would indicate improvement in insulin secretion (second). Our in vivo insulin secretion study clearly confirmed the first possibility. The improvement in GHR+RIP mice, although marginal because it occurred only at 5 min (Fig. 4A), was supportive of the second possibility as well. On the other hand, it is unlikely that overexpressed IGF-I, coreleased with insulin, would increase hypoglycemic activity because total IGF-I level was unaffected (Table 2) and any increase in free (and active) IGF-I would be first neutralized by IGF binding proteins.
The interaction between GH and IGF-I in regulating growth and development has been well defined by the somatomedin hypothesis (1). More recently, GH has been known to have IGF-I-independent, direct actions, in addition to its interactions with locally produced IGF-I. According to the dual-effectors hypothesis, GH acts directly at the epiphyseal plate to stimulate linear growth, and GH, IGF-I, and IGF-II each has a unique and complementary role in augmenting long-bone growth (1). As recently reported, cortical and longitudinal bone growth and bone turnover were all reduced in GHR–/– mice. Short-term administration of IGF-I substantially reversed many of these defects, suggesting a main mechanism of reduced IGF-I levels in the absence of GHR (32). On the other hand, GH is clearly not essential for the differentiation of adipocytes, which were abundant in GHR gene-deficient mice and humans (1). Also from GHR–/– mice, the actions of GH on follicular growth seem independent of circulating IGF-I (33). Although in isolated pancreatic islets, GH stimulated cell growth partially through IGF-I release, it is unclear under normal conditions how IGF-I is involved in GH-stimulated islet growth and insulin biosynthesis and secretion (34, 35).
Precise colocalization by immunohistochemistry had indicated that IGF-I is normally produced in the - and -cells of pancreatic islets, which perhaps act on the ?-cells in a paracrine manner, whereas IGF-II is coproduced in ?-cells with insulin (36, 37). Northern blot analysis showed that IGF-II is the major IGF expressed in the fetal and neonatal rat pancreas, the expression of which is replaced by IGF-I by the second postnatal week. Isolated rat islet - and ?-cells as well as islet-derived cell lines expressed high-affinity IGF-I receptors (IGF-IRs) (3). Although under intense study, the role of IGF-I in normal islet cell growth is still unclear. Transgenic IGF-II promoted islet growth, whereas IGF-I acted solely during regeneration after islet cell damage (10, 22, 38). At the cellular level, IGF-I induced proliferation of rat insulinoma-1 (INS-1) cells in a glucose-dependent manner via insulin receptor substrate-induced phosphatidylinositol 3-kinase activity and downstream activation of p70S6K (5). On the other hand, total deficiency in IGF-I or IGF-IR genes as well as islet ?-cell-specific inactivation of IGF-IR gene caused no change in ?-cell mass, suggesting that IGF signaling is not essential for normal growth and development of pancreatic islets (39, 40). In our recent studies, liver-specific IGF-I gene-deficient mice exhibited islet hyperplasia and hyperinsulinemia caused by compensatory GH hypersecretion (41, 42, 43). Furthermore, pancreatic-specific IGF-I gene-deficient mice exhibited increased islet cell mass probably because of indirect compensations (44). Of course, there might be possible defects in these studies such as gene redundancy, promoter limitation, indirect effects, and limited sample numbers that need to be addressed in future studies.
Nevertheless, this study does not exclude a direct action of GH on pancreatic islets because, first, the rescue was incomplete and limited to increased islet cell growth that restored islet cell mass and glucose tolerance. Restoring local production of IGF-I in pancreatic islets of GHR–/– mice failed to rescue other defects including serum insulin and glucagon levels, hypoglycemia, and insulin sensitivity. Although endocrine IGF-I is known to increase insulin sensitivity, locally produced IGF-I within the islet cells might be insufficient to cause improved insulin responsiveness in target tissues such as the skeletal muscles (thus unaltered insulin tolerance). Second, the level of IGF-I transgenic overexpression was only moderate compared with similar reports often showing 50-fold increase over endogenous levels (22). The choice of insulin promoter (which is presumably severely inhibited in GHR–/– mice) might be a restriction. Third, islet ?-cells seem to have an intrinsic network capable of responding to GH directly. Activation of Stat5 was sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic ?-cells. Cell-cycle regulatory factor cyclin D2 acts as a growth factor sensor for cell transition from G1 to S phase (45). Fourth, it has been reported that the stimulatory effect of GH on ?-cell proliferation cannot be prevented by IGF-I antiserum and was additive to the IGF-I effect (46, 47). On the other hand, GH antagonizes insulin actions and GHR gene deficiency causes significantly elevated insulin sensitivity and hypoglycemia, which might contribute to an adaptive hypotrophy of islet cells, independent of direct actions of either GH or IGF-I.
The level of IGF-I in GHR–/– mice can also be compensated via short-term administration, which has almost completely rescued all defects on both bone growth and remodeling (premature reduction in chondrocyte proliferation and cortical bone growth as well as reduced trabecular bone turnover), supporting a direct effect of IGF-I on both osteoblasts and chondrocytes (32). Its effect on islet cell growth has not been studied. There has been a previous report on islet-specific IGF-I gene overexpression. As in our study, those transgenic mice exhibited similar islet cell mass, normal insulinemia and glycemia, and similar levels of insulin mRNA to wild-type control mice (22). The IGF-I overexpression was without effect until the mice were challenged with type 1 diabetes.
In summary, we have created a transgenic line in which the IGF-I gene was overexpressed in pancreatic islet ?-cells and crossed the mice on to a GHR–/– background. As previously reported, islet-specific IGF-I overexpression alone caused no obvious change in islet cell growth and insulin production. Compared with GHR–/– mice, IGF-I overexpression on a GHR–/– background increased IGF-I production in the islet cells and caused no change in the diminished blood glucose and serum insulin levels and pancreatic insulin contents but improved glucose tolerance and insulin secretion. More remarkably, islet-specific overexpression of IGF-I gene in GHR–/– mice restored islet cell mass through cell hyperplasia and/or hypertrophy. Our results seem to suggest that IGF-I deficiency is part of the underlying mechanism of diminished islet cell growth in GHR–/– mice, consistent with the notion that IGF-I mediates the islet cell growth effect caused by GH release.
Acknowledgments
Transgenic mice were developed in the core facility of the Research Institute of McGill University Health Centre. Dr. Efren Riu of Universitat Autonoma Barcelona, Spain, provided pKS-RIP/globin vector. Dr. Derek LeRoith of National Institutes of Health, Bethesda, MD, provided the rat prepro-IGF-I cDNA. Dr. Shimon Efrat of Tel Aviv University, Israel, provided mouse insulin I cDNA probe. We also acknowledge contributions made by Dr. Dengshun Miao and Sheila Xi Huang of McGill University.
References
Le Roith D, Bondy C, Yakar S, Liu JL, Butler A 2001 The somatomedin hypothesis: 2001. Endocr Rev 22:53–74
Nielsen JH, Moldrup A, Billestrup N 1990 Expression of the growth hormone receptor gene in insulin producing cells. Biomed Biochim Acta 49:1151–1155
Fehmann HC, Jehle P, Markus U, Goke B 1996 Functional active receptors for insulin-like growth factors-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells. Metabolism 45:759–766
Nielsen JH, Linde S, Welinder BS, Billestrup N, Madsen OD 1989 Growth hormone is a growth factor for the differentiated pancreatic ?-cell. Mol Endocrinol 3:165–173
Hugl SR, White MF, Rhodes CJ 1998 Insulin-like growth factor I (IGF-I)-stimulated pancreatic ?-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 273:17771–17779
Harrison M, Dunger AM, Berg S, Mabley J, John N, Green MH, Green IC 1998 Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas. FEBS Lett 435:207–210
Dominici FP, Arostegui Diaz G, Bartke A, Kopchick JJ, Turyn D 2000 Compensatory alterations of insulin signal transduction in liver of growth hormone receptor knockout mice. J Endocrinol 166:579–590
Liu JL, Coschigano KT, Robertson K, Lipsett M, Guo Y, Kopchick JJ, Kumar U, Liu YL 2004 Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am J Physiol Endocrinol Metab 287:E405–E413
Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ 1997 A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:13215–13220
Devedjian JC, George M, Casellas A, Pujol A, Visa J, Pelegrin M, Gros L, Bosch F 2000 Transgenic mice overexpressing insulin-like growth factor-II in ? cells develop type 2 diabetes. J Clin Invest 105:731–740
Valera A, Solanes G, Fernandez-Alvarez J, Pujol A, Ferrer J, Asins G, Gomis R, Bosch F 1994 Expression of GLUT-2 antisense RNA in ? cells of transgenic mice leads to diabetes. J Biol Chem 269:28543–28546
Neuenschwander S, Schwartz A, Wood TL, Roberts Jr CT, Henninghausen L, LeRoith D 1996 Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. J Clin Invest 97:2225–2232
Chandrashekar V, Bartke A, Coschigano KT, Kopchick JJ 1999 Pituitary and testicular function in growth hormone receptor gene knockout mice. Endocrinology 140:1082–1088
Miao D, Bai X, Panda D, McKee M, Karaplis A, Goltzman D 2001 Osteomalacia in Hyp mice is associated with abnormal Phex expression and with altered bone matrix protein expression and deposition. Endocrinology 142:926–939
Hsu S, Raine L, H F 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580
Bonner-Weir S 2001 ?-Cell turnover: its assessment and implications. Diabetes 50(Suppl 1):S20–S4
Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Vasavada RC, Cavaliere C, D’Ercole AJ, Dann P, Burtis WJ, Madlener AL, Zawalich K, Zawalich W, Philbrick W, Stewart AF 1996 Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia. J Biol Chem 271:1200–1208
Liu JL, Grinberg A, Westphal H, Sauer B, Accili D, Karas M, LeRoith D 1998 Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice. Mol Endocrinol 12:1452–1462
Rosenau C, Kaboord B, Qoronfleh MW 2002 Development of a chemiluminescence-based ribonuclease protection assay. Biotechniques 33:1354–1358
Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR 1999 Tissue-specific knockout of the insulin receptor in pancreatic ? cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96:329–339
George M, Ayuso E, Casellas A, Costa C, Devedjian JC, Bosch F 2002 ?-Cell expression of IGF-I leads to recovery from type 1 diabetes. J Clin Invest 109:1153–1163
Weir GC, Bonner-Weir S 2004 Five stages of evolving ?-cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3):S16–S21
Rhodes CJ 2000 IGF-I and GH post-receptor signaling mechanisms for pancreatic ?-cell replication. J Mol Endocrinol 24:303–311
Dominici FP, Turyn D 2002 Growth hormone-induced alterations in the insulin-signaling system. Exp Biol Med (Maywood) 227:149–157
Okuda Y, Pena J, Chou J, Field JB 2001 Acute effects of growth hormone on metabolism of pancreatic hormones, glucose and ketone bodies. Diabetes Res Clin Pract 53:1–8
Nielsen JH, Galsgaard ED, Moldrup A, Friedrichsen BN, Billestrup N, Hansen JA, Lee YC, Carlsson C 2001 Regulation of ?-cell mass by hormones and growth factors. Diabetes 50:S25–S29
Rabinovitch A, Quigley C, Rechler MM 1983 Growth hormone stimulates islet B-cell replication in neonatal rat pancreatic monolayer cultures. Diabetes 32:307–312
Galsgaard ED, Gouilleux F, Groner B, Serup P, Nielsen JH, Billestrup N 1996 Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene. Mol Endocrinol 10:652–660
Galsgaard ED, Nielsen JH, Moldrup A 1999 Regulation of prolactin receptor (PRLR) gene expression in insulin-producing cells. Prolactin and growth hormone activate one of the rat prlr gene promoters via STAT5a and STAT5b. J Biol Chem 274:18686–18692
Parsons JA, Bartke A, Sorenson RL 1995 Number and size of islets of Langerhans in pregnant, human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones. Endocrinology 136:2013–2021
Sims NA, Clement-Lacroix P, Da Ponte F, Bouali Y, Binart N, Moriggl R, Goffin V, Coschigano K, Gaillard-Kelly M, Kopchick J, Baron R, Kelly PA 2000 Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5. J Clin Invest 106:1095–1103
Bachelot A, Monget P, Imbert-Bollore P, Coshigano K, Kopchick JJ, Kelly PA, Binart N 2002 Growth hormone is required for ovarian follicular growth. Endocrinology 143:4104–4112
Swenne I, Hill DJ 1989 Growth hormone regulation of DNA replication, but not insulin production, is partly mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats. Diabetologia 32:191–197
Hill DJ, Hogg J 1991 Growth factor control of pancreatic B cell hyperplasia. Baillieres Clin Endocrinol Metab 5:689–698
Maake C, Reinecke M 1993 Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistence with classical islet hormones. Cell Tissue Res 273:249–259
Portela-Gomes GM, Hoog A 2000 Insulin-like growth factor II in human fetal pancreas and its co-localization with the major islet hormones: comparison with adult pancreas. J Endocrinol 165:245–251
Hill DJ, Strutt B, Arany E, Zaina S, Coukell S, Graham CF 2000 Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets. Endocrinology 141:1151–1157
Xuan S, Kitamura T, Nakae J, Politi K, Kido Y, Fisher PE, Morroni M, Cinti S, White MF, Herrera PL, Accili D, Efstratiadis A 2002 Defective insulin secretion in pancreatic ?-cells lacking type 1 IGF receptor. J Clin Invest 110:1011–1019
Kulkarni RN, Holzenberger M, Shih DQ, Ozcan U, Stoffel M, Magnuson MA, Kahn CR 2002 ?-Cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter ?-cell mass. Nat Genet 31:111–115
Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD, Mejia W, Le Roith D 2001 Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50:1110–1118
Zhao H, Yakar S, Gavrilova O, Sun H, Zhang Y, Kim H, Setser J, Jou W, LeRoith D 2004 Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 113:96–105
Yu R, Yakar S, Liu YL, Lu Y, LeRoith D, Miao D, Liu JL 2003 Liver-specific IGF-I gene deficient mice exhibit accelerated diabetes in response to streptozotocin, associated with early onset of insulin resistance. Mol Cell Endocrinol 204:31–42
Lu Y, Herrera PL, Guo Y, Sun D, Tang Z, LeRoith D, Liu JL 2004 Pancreatic specific inactivation of IGF-I gene causes enlarged pancreatic islets and significant resistance to diabetes. Diabetes 53:3131–3141
Friedrichsen BN, Richter HE, Hansen JA, Rhodes CJ, Nielsen JH, Billestrup N, Moldrup A 2003 Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic ?-cells. Mol Endocrinol 17:945–958
Billestrup N, Nielsen JH 1991 The stimulatory effect of growth hormone, prolactin, and placental lactogen on ?-cell proliferation is not mediated by insulin-like growth factor-I. Endocrinology 129:883–888
Cousin SP, Hugl SR, Myers Jr MG, White MF, Reifel-Miller A, Rhodes CJ 1999 Stimulation of pancreatic ?-cell proliferation by growth hormone is glucose-dependent: signal transduction via janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) with no crosstalk to insulin receptor substrate-mediated mitogenic signalling. Biochem J 344:649–658(Yubin Guo, Yarong Lu, Dan)
Address all correspondence and requests for reprints to: Dr. Jun-Li Liu, Fraser Laboratories, Room M3-15, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: jun-li.liu@mcgill.ca.
Abstract
Both GH and IGF-I stimulate islet cell growth, inhibit cell apoptosis, and regulate insulin biosynthesis and secretion. GH receptor gene deficiency (GHR–/–) caused diminished pancreatic islet cell mass and serum insulin level and elevated insulin sensitivity. Because IGF-I gene expression was nearly abolished in these mice, we sought to determine whether that had caused the islet defects. To restore IGF-I level, we have generated transgenic mice that express rat IGF-I cDNA under the direction of rat insulin promoter 1 (RIP-IGF). Using RNase protection assay and immunohistochemistry, the IGF-I transgene expression was revealed specifically in pancreatic islets of the RIP-IGF mice, which exhibited normal growth and development and possess no abnormalities in glucose homeostasis, insulin production, and islet cell mass. GHR–/– mice exhibited 50% reduction in the ratio of islet cell mass to body weight and increased insulin sensitivity but impaired glucose tolerance. Compared with GHR–/– alone, IGF-I overexpression on a GHR–/– background caused no change in the diminished blood glucose and serum insulin levels, pancreatic insulin contents, and insulin tolerance but improved glucose tolerance and insulin secretion. Remarkably, islet-specific overexpression of IGF-I gene in GHR–/– mice restored islet cell mass, at least partially through cell hypertrophy. Interestingly, double-transgenic male mice demonstrated a transient rescue in growth rates vs. GHR–/– alone, at 2–3 months of age. Our results suggest that IGF-I deficiency is part of the underlying mechanism of diminished islet growth in GHR–/– mice and are consistent with the notion that IGF-I mediates GH-induced islet cell growth.
Introduction
GH AND IGF-I ARE POTENT regulators of cell growth, differentiation, and metabolism and are essential for postnatal growth in mammals (1). Receptors for both GH and IGF-I are expressed in the pancreatic islet cells (2, 3). Acting on their own, both GH and IGF-I promote islet cell growth, inhibit apoptosis, and are potentially involved in normal islet growth and maintenance (4, 5, 6), but it is unclear whether GH and IGF-I interact with each other in regulating pancreatic islet function and how IGF-I is involved in GH-stimulated islet growth and insulin biosynthesis and secretion. IGF-I produced either from the liver or locally within the pancreatic islets might mediate GH actions. We and others have recently demonstrated that GH receptor gene deficiency (GHR–/–) caused diminished pancreatic islet cell mass and serum insulin level and elevated insulin sensitivity (7, 8). Because IGF-I gene expression was nearly abolished in GHR–/– mice, we sought to determine whether that had caused the islet defects (9). For this purpose, we have generated transgenic mice RIP-IGF that express an IGF-I transgene under the direction of rat insulin promoter 1 (RIP) and studied whether the islet defects in GHR–/– mice can be rescued. As a result, local expression of the IGF-I transgene restored pancreatic islet cell mass and improved glucose tolerance in GHR–/– mice, which is consistent with the notion that IGF-I mediates the GH-induced growth-promoting effect on pancreatic islet cells.
Materials and Methods
Creation of RIP-IGF transgenic mice
A transgenic line has been developed to overexpress IGF-I cDNA driven by an insulin promoter (RIP-IGF). The promoter was chosen based on its high level and ?-cell-restricted expression in driving IGF-II and Glut-2 antisense (10, 11). Briefly, a 0.5-kb PvuII/AvaI fragment of a rat prepro-IGF-I cDNA from a pGEM4Z vector (12) was subcloned into a BamHI/EcoRI site downstream of RIP in a pKS-RIP/globin vector (Fig. 1) (11). The integrity of the transgenic construct, pKS-RIP-IGF6, was confirmed by sequencing and restriction analysis before it was injected into the pronucleus of fertilized mouse ova. The manipulated ova were transferred into the oviducts of recipient female mice, which gave birth to founder mice.
FIG. 1. Pancreatic islet-specific IGF-I overexpression: characterization of transgenic lines. A, Diagram of transgenic DNA vector: pKS/RIP-IGF-I. Based on pKS-RIP/globin vector, rat insulin promoter 1 (RIP) was used to drive expression of an intact rat prepro-IGF-I cDNA. The arrows mark the region covered by the pSP72-RIF probe and primers Tg1 and Tg2. B, Detection of transgene expression by RNase protection assay. Total pancreatic RNA prepared from transgenic mice (+) or wild-type littermates (–), determined by PCR, was hybridized to rat IGF-I and mouse ?-actin probes. A protected band with rat IGF-I probe indicates transgene expression. Endogenous mouse IGF-I mRNA was unprotected by the probe thus destroyed by RNase treatment. Results from four representative families (codes 34903 etc.) are illustrated.
To identify founder mice (on a mixed C3H and C57BL/6 background) and offspring that carry the transgene, a PCR, using the primers Tg1 (5'-GGT GAT ATT GGC AGG TGT TCC-3') and Tg2 (5'-CAA ATC GGC AAA GTC CAG G-3'), generated a product of 600 bp corresponding to the entire cDNA. To probe the transgene expression by Northern blots and RNase protection assays, a 0.7-kb PstI/EcoRI fragment that contains the cDNA including the two primer sites was subcloned into pSP72 vector (Promega, Madison, WI). The resulting vector, named pSP72-RIF, was linearized by HindIII to direct synthesis of an antisense RNA probe using T7 polymerase.
Intercross with GHR–/– mice and genotyping
GHR–/– mice carry a targeted disruption of exon 4 of the mouse GHR/BP gene, as previously reported (8, 9). To intercross with RIP-IGF mice, heterozygous (GHR+/–) mice, on a hybrid 129/Ola-BALB/c-C57BL/6 background, were used. To genotype the offspring, genomic DNA was isolated from tail clips using standard methods. Primers In4-1 (5'-CCC TGA GAC CTC CTC AGT TC-3'), In3+1 (5'-CCT CCC AGA GAG ACT GGC TT-3'), and Neo-3 (5'-GCT CGA CAT TGG GTG GAA ACA T-3') were used in PCR, which yield a 390-base band for the wild-type allele, and 290/200 double bands for the GHR knockout allele, as previously reported (13). Offspring of four genotypes were selected: wild type, RIP-IGF (GHR+/+), GHR–/– (no RIP-IGF), and GHR–/– plus RIP-IGF (GHR+RIP). All heterozygous (GHR+/–) animals were excluded.
Animal procedures
The animals were maintained in 12-h dark, 12-h light cycles at room temperature with free access to food and water or when indicated, food deprived for 24 h with free access to water. At the desired age, the mice were anesthetized with a cocktail of ketamine/xylazine/acepromazine, bled via periorbital puncture, and killed by cervical dislocation. Blood was collected for serum preparation, and pancreata were rapidly removed for biochemical or histological analysis. All animal-handling procedures were approved by the McGill University Animal Care Committee.
Serum concentrations of insulin and glucagon were determined using RIA kits obtained from Linco Research Inc. (St. Charles, MO). IGF-I was determined using an RIA kit obtained from Diagnostic Systems Laboratories (Webster, TX). Blood glucose levels were measured using the OneTouch blood glucose meter and strips (LifeScan Canada, Burnaby, British Columbia, Canada). For the insulin tolerance test, animals were injected with human insulin (0.75 IU/kg, ip; Roche Applied Science, Penzberg, Germany), and blood glucose levels were measured at 0, 20, 40, and 60 min after the injection. For the glucose tolerance test, mice were fasted 24 h and injected with glucose (1 g/kg, ip), and blood glucose levels were measured at 0, 15, 30, 60, and 120 min after the injection.
Immunohistochemistry and islet cell mass determination
Pancreata were removed from 2-month-old mice (n = 4 in each group) and fixed, embedded in paraffin, and cut into 5-μm sections (14). The sections were then subjected to immunohistochemical staining for insulin and glucagon with rabbit polyclonal antibodies (Monosan, Uden, The Netherlands) using the avidin-biotin-peroxidase complex technique, which results in a red immunoreactive signal with a nuclear counterstain using methyl green, or diaminobenzidine substrate, which resulted in a brown immunoreactive signal with a hematoxylin counterstain (blue) of cell nuclei (14, 15). Mouse monoclonal IgG against human IGF-I (clone Sm1.2; Upstate USA Inc., Charlottesville, VA) was used to reveal IGF-I overexpression. Images of all pancreatic islets were captured with a Retiga 1300 digital camera (Q Imaging, Burnaby, British Columbia, Canada) at magnifications of x25, x100, or x400. The area of the pancreatic tissue was measured using Northern Eclipse computer software, version 6.0 (Empix imaging, Mississauga, Ontario, Canada). The number of insulin-stained pancreatic islets in each image was manually counted using Adobe Photoshop 7.0 computer software.
The islet cell mass (defined as all cells staining positive for the hormone insulin) was determined by initially weighing the excised pancreatic tissue and then determining the percentage of the excised organ that was insulin positive (16). All insulin-positive ?-cell clusters (islets) were loosely traced, and the insulin-immunoreactive area was determined using the thresholding option. Total tissue area was also quantified using the threshold option to select the stained areas while not selecting unstained areas (white space). Islet cell percent was determined by dividing the total insulin area by the total tissue area for each animal. The islet cell mass for each animal was then derived by multiplying the islet cell percent by the excised pancreas tissue weight. Each mouse pancreas was examined in one slide of approximately 40 fields of view and approximately 12 mm2 of total tissue.
To reflect individual islet cell growth in adult (2 month old) mice, average cell size was calculated in hematoxylin-eosin-stained x400 images using total islet area divided by the number of cell nuclei. For this purpose, a minimum of 10 mature islets were chosen from each genotype group.
Northern blot and RNase protection assay
RNA isolation and Northern blot analysis were as reported except with digoxigenin-labeled probes (Roche) (17, 18). RNase protection assay was as reported using 32P-labeled probes (19, 20). The intensity of the hybridization signals on the autoradiogram was analyzed using a FluorChem 8900 imaging system (Alpha Innotech, San Leandro, CA).
Insulin and glucagon secretion
Mice at age 2–4 months, both male and female, were fasted 24 h and injected with glucose (3 g/kg, ip) (21). At 0 (without stimulation), 5, 15, or 30 min, they were anesthetized, bled via periorbital puncture, and killed. Blood was collected for serum preparation. Insulin and glucagon concentrations were determined by RIA.
Statistics and data plotting
Data are expressed as the mean ± SE. The Student’s t test (unpaired and paired) and one-way ANOVA were performed using InStat software version 3 (GraphPad Software Inc., San Diego, CA). Data were plotted into curves, and the area under curve was calculated using SigmaPlot software version 9 (Systat Software, Inc., Point Richmond, CA).
Results
Pancreatic islet ?-cell-specific IGF-I overexpression in RIP-IGF mice
To overexpress IGF-I in most cells of the pancreatic islets, we have used an insulin promoter to drive the transgenic expression of rat IGF-I cDNA (Fig. 1A). Multiple founder lines were created and screened for genomic integration of the transgene by specific PCR. Using immunohistochemistry and RNase protection, two mouse families (3 and 13) exhibited high levels IGF-I expression, which was specific in pancreatic islets. As shown in Fig. 1B, rat IGF-I mRNA could be detected in the pancreatic RNA prepared from RIP-IGF transgenic mice but not in nontransgenic littermates. As shown by immunohistochemistry in Fig. 2A (top), pancreatic islets in wild-type mice only exhibited scattered IGF-I staining in very few islet cells; the levels of IGF-I staining (brown pigmentation) was drastically elevated in RIP-IGF mice. Judging from the ratio of IGF-I-positive cells, it seems that not all ?-cells express the transgene. Except nonspecific staining of the blood cells, the transgenic expression was relatively specific and not seen in exocrine acinar cells.
FIG. 2. Increased pancreatic islet cell mass and improved body growth caused by islet-specific IGF-I overexpression in male GHR–/– mice. A, Islet-specific transgenic expression revealed by immunohistochemistry. Pancreatic sections prepared from 2-month-old mice of four genotype groups were stained for IGF-I using the diaminobenzidine complex. IGF-I staining was shown as brownpigmentation within the islets. Cell nuclei were counterstained with hematoxylin. Images are representatives of at least 15–20 mature islets from each mouse and have been recorded at x400. B, Postnatal growth rates in mice of various genotypes. Body weight was measured at 1, 2, 3, and 4 months of age and illustrated as mean ± SE. Numbers of animals are shown in parentheses. *, P < 0.05; **, P < 0.01 vs. GHR–/– alone. C, Changes in pancreatic islet cell mass, corrected for total body weight (n = 5). **, P < 0.01 vs. wild-type (WT); #, P < 0.05 vs. GHR–/– alone. ANOVA: P = 0.006; WT vs. GHR–/–, P < 0.05; GHR–/– vs. GHR+RIP, P < 0.05. D, Pancreatic islet-specific overexpression of IGF-I increased the average islet cell size in GHR–/– mice (islet cell hypertrophy). From each genotype group of mice at age 2–3 months, 11–12 mature islets stained with hematoxylin-eosin were analyzed. The islet size and the number of cell nuclei were determined using Northern Eclipse software. ANOVA of four groups: P < 0.001; **, P < 0.01; ***, P < 0.001 vs. WT littermates; ##, P < 0.01 vs. GHR–/– alone. Except that of B, similar results were obtained using female mice (not shown).
Normal growth and islet formation in RIP-IGF mice
RIP-IGF mice exhibited normal growth and development and possessed no abnormalities in blood glucose (fasted or fed), serum insulin, and glucagon levels, as shown in Table 1. Northern blot analysis revealed normal levels of insulin mRNA in transgenic vs. wild-type mice (data not shown). Pancreatic insulin content was unaltered in RIP-IGF mice (Table 2). Immunohistochemistry showed no obvious abnormality in islet morphology and - and ?-cell distribution patterns within the islets (data not shown). As shown in Fig. 2C (columns 1 and 2), RIP-IGF mice had normal islet cell mass. When challenged with a bolus injection of glucose, RIP-IGF mice exhibited an unaltered glucose clearance curve vs. wild-type littermates. Likewise, RIP-IGF mice showed no significant difference in their glucose-lowering response to insulin injection, compared with wild-type littermates (data not shown).
TABLE 1. Changes in body weight and glucose homeostasis in RIP-IGF mice vs. wild-type controls
TABLE 2. Effects of IGF-I overexpression on serum chemistry and pancreatic insulin content in GHR–/– mice
Effects of IGF-I overexpression on animal growth and glucose and insulin levels in GHR–/– mice
GHR–/– mice exhibited severe growth retardation, decreased blood glucose and serum insulin levels, increased insulin sensitivity, and diminished islet cell mass (7, 8). To investigate whether restored IGF-I expression in the pancreatic islets can rescue the islet defects, we intercrossed GHR+/– with RIP-IGF mice and studied second-generation offspring of four genotypes, i.e. wild type, RIP-IGF, GHR–/–, and GHR+RIP. Compared with GHR–/– alone, islet IGF-I expression was indeed significantly increased in GHR+RIP mice as shown by immunohistochemistry (Fig. 2A, bottom). Computer-assisted image analysis indicated that, on average, the IGF-I-stained area increased from 4 ± 1% (n = 10) of total islet area in GHR–/– mice to 21 ± 3% (n = 11; P < 0.05) in GHR+RIP mice. Because the transgenic expression was limited to the islet cells at a moderate level, compared with similar reports (10, 22), and caused no change in serum IGF-I level (Table 2), it was not expected to have an impact on the growth retardation of GHR–/– mice. Nevertheless, we had measured their body weight from 1–4 months of age. As shown in Fig. 2B, RIP-IGF expression alone caused no change in growth vs. wild-type littermates. GHR–/– mice exhibited severe growth retardation with only approximately one half of the wild-type body weight at adult age. Interestingly, the double-transgenic male mice demonstrated a significant partial rescue in growth rates vs. GHR–/– mice at 2–3 months of age (i.e. 19% increased body weight). This effect was transient, was not seen in females, and did not last beyond 4 months of age.
As expected, GHR–/– mice exhibited drastic reductions in serum insulin level (–33%) and pancreatic insulin content (–52%), suggesting reduced insulin production. RIP-IGF expression on this GHR–/– background failed to normalize serum insulin, pancreatic insulin content, and insulin mRNA to wild-type levels (Table 2 and data not shown).
Transgenic IGF-I overexpression restored islet cell mass in GHR–/– mice
Total islet cell mass, determined by insulin staining, was decreased 6.2-fold in GHR–/– mice vs. wild-type littermates. When corrected for body weight, the decrease was 2.9-fold (Fig. 2C, column 3). In double-transgenic GHR–/– mice that express the IGF-I transgene (GHR+RIP, column 4), total islet cell mass was increased 3.8-fold vs. GHR–/– mice alone. When corrected for body weight, the increase had effectively restored the islet cell mass to the level of wild-type mice (GHR+RIP 61 ± 22 vs. WT 46 ± 13 mg/kg; n = 5) (Fig. 2C, column 4).
Average islet cell size is a measure of islet cell hypotrophy (such as in GHR–/– mice) or hypertrophy (such as in ?-cell compensation to type 2 diabetes) and a reflection of cell health and activity (8, 23). As shown in Fig. 2D, compared with wild-type littermates, IGF-I overexpression alone in RIP-IGF mice did not change islet cell size; as previously reported, GHR–/– mice exhibited a 42% reduction in islet cell size (8); in GHR–/– mice that overexpress IGF-I in islet cells, the average islet cell size was increased 29%, exhibiting a partial rescue in pancreatic islet cell growth. Similar results were obtained from both male and female mice.
Transgenic IGF-I overexpression improved glucose tolerance in GHR–/– mice
GHR–/– mice exhibit glucose intolerance and elevated insulin sensitivity, because of specific changes within the pancreatic islets and insulin target tissues (Coschigano, K.T., et al., 1999, 81st Annual Meeting of The Endocrine Society, San Diego, CA) (8). In double-transgenic GHR+RIP mice, with restored IGF-I production, their glucose tolerance was largely restored (Fig. 3A). At 40 and 60 min after glucose injection, GHR+RIP mice exhibited significantly reduced blood glucose level vs. GHR–/– alone. Their rate of glucose disposal was almost as efficient as wild-type littermates (Fig. 3A).
FIG. 3. Improved glucose tolerance but unaffected insulin sensitivity caused by islet-specific IGF-I overexpression in male GHR–/– mice. A, Glucose tolerance test. Mice (9–10 wk old) were fasted for 24 h, and glucose (1 g/kg) was injected ip. Blood glucose was measured at 0, 15, 30, 60, and 120 min after the injection. *, P < 0.05; **, P < 0.01 vs. wild-type (WT) littermates in unpaired t test. The number of animals in each group is indicated in parentheses. B, Insulin tolerance test. Mice (11–12 wk old) were injected with insulin (0.75 U/kg, ip), and blood glucose was measure at 0, 20, 40, and 60 min after. The percentage values relative to 0 time were expressed as mean ± SE. *, P < 0.05; ***, P < 0.001 vs. WT control mice. Similar results were obtained using female mice (not shown).
Animals of the four genotypes were tested for insulin tolerance. As shown in Fig. 3B, compared with wild-type littermates, GHR–/– mice exhibited significantly decreased glucose levels at 20, 40, and 60 min after insulin injection. GHR+RIP mice exhibited less deviation from wild-type mice, but the differences were insignificant from either group. Thus, pancreatic islet-specific IGF-I overexpression did not affect the phenotype of insulin hypersensitivity.
Changes in glucose-stimulated insulin and glucagon secretion
Reduced glucose tolerance in GHR–/– mice, in the face of increased insulin sensitivity, suggests possible defects in insulin secretion. Likewise, improved glucose tolerance in GHR+RIP mice would indicate elevated insulin secretion vs. GHR–/– alone. To verify these speculations, we have measured serum insulin levels after a glucose load (Fig. 4A). Insulin secretion exhibited 2.7- to 4.9-fold increases in wild-type mice at 5 and 15 min after glucose stimulation, which was virtually restored to normal by 30 min. RIP mice exhibited a similar response, except with a delayed return to basal level at 30 min. In contrast, the secretion in GHR–/– mice was drastically diminished and reached to only 2.2- and 2.7-fold at 5 and 15 min vs. 0 min, even after being corrected for their low basal level. Interestingly, at 5 min after glucose stimulation, GHR+RIP mice were able to demonstrate a transient enhancement in serum insulin level vs. GHR–/– mice alone (which did not last till 15 and 30 min), suggesting a certain degree of improvement in insulin secretion associated with islet-specific IGF-I overexpression. In the same experiment, serum glucagon levels exhibited no significant reductions in wild-type and RIP mice within 15 min of glucose injection (Fig. 4B). In GHR–/– and GHR+RIP mice, however, glucagon levels were reduced approximately 40% at 5 min after glucose stimulation, significantly lower than wild-type mice.
FIG. 4. Changes in glucose-stimulated insulin and glucagon secretion caused by islet-specific IGF-I overexpression in GHR–/– mice. A, Serum insulin level. Mice at age 3–4 months, both male and female, were fasted for 24 h before being stimulated with glucose (3 g/kg, ip). Serum insulin concentrations were measured at 0, 5, 15, and 30 min (n = 5–14). *,P < 0.05; **, P < 0.01 vs. wild-type (WT) mice; #, P < 0.05 vs. GHR–/– alone by unpaired t tests. The area under curve values were 56.0 for WT, 69.7 for RIP, 23.5 for GHR–/–, and 29.9 for GHR+RIP. B, Serum glucagon levels. The values at 30 min are not presented because of insufficient samples.
Discussion
Both GH and IGF-I stimulate islet cell growth and inhibit apoptosis and thus are potentially involved in normal islet development. GH stimulated insulin and glucagon secretion and pancreatic islet cell proliferation (24, 25, 26, 27). The stimulation of cell replication in neonatal rat pancreatic monolayer cultures by GH was independent of glucose concentration (28). More recently, in primary cultures of pancreatic islet cells, GH stimulated ?-cell proliferation, insulin gene transcription and insulin secretion (4). Among the various postreceptor substrates, Stat5a/b, Stat1, and Stat3 were found to be activated in pancreatic islet or islet-derived tumor cells (29, 30). GH overexpression in vivo increased pancreatic islet number and volume in transgenic mice (31). In a previous report, we have demonstrated reduced islet cell mass and enhanced insulin sensitivity in GHR–/– mice (8). Because IGF-I gene expression in liver and pancreas was severely affected and IGF-I is known to stimulate islet cell growth, we believe that lack of IGF-I production in GHR–/– mice had contributed to islet growth defect. In this study, we have created transgenic mice (RIP-IGF) that overexpressed the IGF-I gene in pancreatic islet cells and exhibited no obvious effect on islet growth by themselves. Crossing them with GHR–/– mice restored local production of IGF-I in the islet cells and rescued to various degrees islet cell mass, average cell size, glucose tolerance, and even transiently somatic growth. It is consistent with the notion that locally produced IGF-I mediates GH-stimulated islet cell growth. Glucose intolerance in GHR–/– mice is likely caused by insufficient (amount and speed) release of insulin in response to glucose (first), especially so in the face of increased insulin sensitivity. A significant improvement in GHR+RIP mice vs. GHR–/– alone (Fig. 3A) would indicate improvement in insulin secretion (second). Our in vivo insulin secretion study clearly confirmed the first possibility. The improvement in GHR+RIP mice, although marginal because it occurred only at 5 min (Fig. 4A), was supportive of the second possibility as well. On the other hand, it is unlikely that overexpressed IGF-I, coreleased with insulin, would increase hypoglycemic activity because total IGF-I level was unaffected (Table 2) and any increase in free (and active) IGF-I would be first neutralized by IGF binding proteins.
The interaction between GH and IGF-I in regulating growth and development has been well defined by the somatomedin hypothesis (1). More recently, GH has been known to have IGF-I-independent, direct actions, in addition to its interactions with locally produced IGF-I. According to the dual-effectors hypothesis, GH acts directly at the epiphyseal plate to stimulate linear growth, and GH, IGF-I, and IGF-II each has a unique and complementary role in augmenting long-bone growth (1). As recently reported, cortical and longitudinal bone growth and bone turnover were all reduced in GHR–/– mice. Short-term administration of IGF-I substantially reversed many of these defects, suggesting a main mechanism of reduced IGF-I levels in the absence of GHR (32). On the other hand, GH is clearly not essential for the differentiation of adipocytes, which were abundant in GHR gene-deficient mice and humans (1). Also from GHR–/– mice, the actions of GH on follicular growth seem independent of circulating IGF-I (33). Although in isolated pancreatic islets, GH stimulated cell growth partially through IGF-I release, it is unclear under normal conditions how IGF-I is involved in GH-stimulated islet growth and insulin biosynthesis and secretion (34, 35).
Precise colocalization by immunohistochemistry had indicated that IGF-I is normally produced in the - and -cells of pancreatic islets, which perhaps act on the ?-cells in a paracrine manner, whereas IGF-II is coproduced in ?-cells with insulin (36, 37). Northern blot analysis showed that IGF-II is the major IGF expressed in the fetal and neonatal rat pancreas, the expression of which is replaced by IGF-I by the second postnatal week. Isolated rat islet - and ?-cells as well as islet-derived cell lines expressed high-affinity IGF-I receptors (IGF-IRs) (3). Although under intense study, the role of IGF-I in normal islet cell growth is still unclear. Transgenic IGF-II promoted islet growth, whereas IGF-I acted solely during regeneration after islet cell damage (10, 22, 38). At the cellular level, IGF-I induced proliferation of rat insulinoma-1 (INS-1) cells in a glucose-dependent manner via insulin receptor substrate-induced phosphatidylinositol 3-kinase activity and downstream activation of p70S6K (5). On the other hand, total deficiency in IGF-I or IGF-IR genes as well as islet ?-cell-specific inactivation of IGF-IR gene caused no change in ?-cell mass, suggesting that IGF signaling is not essential for normal growth and development of pancreatic islets (39, 40). In our recent studies, liver-specific IGF-I gene-deficient mice exhibited islet hyperplasia and hyperinsulinemia caused by compensatory GH hypersecretion (41, 42, 43). Furthermore, pancreatic-specific IGF-I gene-deficient mice exhibited increased islet cell mass probably because of indirect compensations (44). Of course, there might be possible defects in these studies such as gene redundancy, promoter limitation, indirect effects, and limited sample numbers that need to be addressed in future studies.
Nevertheless, this study does not exclude a direct action of GH on pancreatic islets because, first, the rescue was incomplete and limited to increased islet cell growth that restored islet cell mass and glucose tolerance. Restoring local production of IGF-I in pancreatic islets of GHR–/– mice failed to rescue other defects including serum insulin and glucagon levels, hypoglycemia, and insulin sensitivity. Although endocrine IGF-I is known to increase insulin sensitivity, locally produced IGF-I within the islet cells might be insufficient to cause improved insulin responsiveness in target tissues such as the skeletal muscles (thus unaltered insulin tolerance). Second, the level of IGF-I transgenic overexpression was only moderate compared with similar reports often showing 50-fold increase over endogenous levels (22). The choice of insulin promoter (which is presumably severely inhibited in GHR–/– mice) might be a restriction. Third, islet ?-cells seem to have an intrinsic network capable of responding to GH directly. Activation of Stat5 was sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic ?-cells. Cell-cycle regulatory factor cyclin D2 acts as a growth factor sensor for cell transition from G1 to S phase (45). Fourth, it has been reported that the stimulatory effect of GH on ?-cell proliferation cannot be prevented by IGF-I antiserum and was additive to the IGF-I effect (46, 47). On the other hand, GH antagonizes insulin actions and GHR gene deficiency causes significantly elevated insulin sensitivity and hypoglycemia, which might contribute to an adaptive hypotrophy of islet cells, independent of direct actions of either GH or IGF-I.
The level of IGF-I in GHR–/– mice can also be compensated via short-term administration, which has almost completely rescued all defects on both bone growth and remodeling (premature reduction in chondrocyte proliferation and cortical bone growth as well as reduced trabecular bone turnover), supporting a direct effect of IGF-I on both osteoblasts and chondrocytes (32). Its effect on islet cell growth has not been studied. There has been a previous report on islet-specific IGF-I gene overexpression. As in our study, those transgenic mice exhibited similar islet cell mass, normal insulinemia and glycemia, and similar levels of insulin mRNA to wild-type control mice (22). The IGF-I overexpression was without effect until the mice were challenged with type 1 diabetes.
In summary, we have created a transgenic line in which the IGF-I gene was overexpressed in pancreatic islet ?-cells and crossed the mice on to a GHR–/– background. As previously reported, islet-specific IGF-I overexpression alone caused no obvious change in islet cell growth and insulin production. Compared with GHR–/– mice, IGF-I overexpression on a GHR–/– background increased IGF-I production in the islet cells and caused no change in the diminished blood glucose and serum insulin levels and pancreatic insulin contents but improved glucose tolerance and insulin secretion. More remarkably, islet-specific overexpression of IGF-I gene in GHR–/– mice restored islet cell mass through cell hyperplasia and/or hypertrophy. Our results seem to suggest that IGF-I deficiency is part of the underlying mechanism of diminished islet cell growth in GHR–/– mice, consistent with the notion that IGF-I mediates the islet cell growth effect caused by GH release.
Acknowledgments
Transgenic mice were developed in the core facility of the Research Institute of McGill University Health Centre. Dr. Efren Riu of Universitat Autonoma Barcelona, Spain, provided pKS-RIP/globin vector. Dr. Derek LeRoith of National Institutes of Health, Bethesda, MD, provided the rat prepro-IGF-I cDNA. Dr. Shimon Efrat of Tel Aviv University, Israel, provided mouse insulin I cDNA probe. We also acknowledge contributions made by Dr. Dengshun Miao and Sheila Xi Huang of McGill University.
References
Le Roith D, Bondy C, Yakar S, Liu JL, Butler A 2001 The somatomedin hypothesis: 2001. Endocr Rev 22:53–74
Nielsen JH, Moldrup A, Billestrup N 1990 Expression of the growth hormone receptor gene in insulin producing cells. Biomed Biochim Acta 49:1151–1155
Fehmann HC, Jehle P, Markus U, Goke B 1996 Functional active receptors for insulin-like growth factors-I (IGF-I) and IGF-II on insulin-, glucagon-, and somatostatin-producing cells. Metabolism 45:759–766
Nielsen JH, Linde S, Welinder BS, Billestrup N, Madsen OD 1989 Growth hormone is a growth factor for the differentiated pancreatic ?-cell. Mol Endocrinol 3:165–173
Hugl SR, White MF, Rhodes CJ 1998 Insulin-like growth factor I (IGF-I)-stimulated pancreatic ?-cell growth is glucose-dependent. Synergistic activation of insulin receptor substrate-mediated signal transduction pathways by glucose and IGF-I in INS-1 cells. J Biol Chem 273:17771–17779
Harrison M, Dunger AM, Berg S, Mabley J, John N, Green MH, Green IC 1998 Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas. FEBS Lett 435:207–210
Dominici FP, Arostegui Diaz G, Bartke A, Kopchick JJ, Turyn D 2000 Compensatory alterations of insulin signal transduction in liver of growth hormone receptor knockout mice. J Endocrinol 166:579–590
Liu JL, Coschigano KT, Robertson K, Lipsett M, Guo Y, Kopchick JJ, Kumar U, Liu YL 2004 Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am J Physiol Endocrinol Metab 287:E405–E413
Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ 1997 A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:13215–13220
Devedjian JC, George M, Casellas A, Pujol A, Visa J, Pelegrin M, Gros L, Bosch F 2000 Transgenic mice overexpressing insulin-like growth factor-II in ? cells develop type 2 diabetes. J Clin Invest 105:731–740
Valera A, Solanes G, Fernandez-Alvarez J, Pujol A, Ferrer J, Asins G, Gomis R, Bosch F 1994 Expression of GLUT-2 antisense RNA in ? cells of transgenic mice leads to diabetes. J Biol Chem 269:28543–28546
Neuenschwander S, Schwartz A, Wood TL, Roberts Jr CT, Henninghausen L, LeRoith D 1996 Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. J Clin Invest 97:2225–2232
Chandrashekar V, Bartke A, Coschigano KT, Kopchick JJ 1999 Pituitary and testicular function in growth hormone receptor gene knockout mice. Endocrinology 140:1082–1088
Miao D, Bai X, Panda D, McKee M, Karaplis A, Goltzman D 2001 Osteomalacia in Hyp mice is associated with abnormal Phex expression and with altered bone matrix protein expression and deposition. Endocrinology 142:926–939
Hsu S, Raine L, H F 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580
Bonner-Weir S 2001 ?-Cell turnover: its assessment and implications. Diabetes 50(Suppl 1):S20–S4
Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Vasavada RC, Cavaliere C, D’Ercole AJ, Dann P, Burtis WJ, Madlener AL, Zawalich K, Zawalich W, Philbrick W, Stewart AF 1996 Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia. J Biol Chem 271:1200–1208
Liu JL, Grinberg A, Westphal H, Sauer B, Accili D, Karas M, LeRoith D 1998 Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice. Mol Endocrinol 12:1452–1462
Rosenau C, Kaboord B, Qoronfleh MW 2002 Development of a chemiluminescence-based ribonuclease protection assay. Biotechniques 33:1354–1358
Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR 1999 Tissue-specific knockout of the insulin receptor in pancreatic ? cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96:329–339
George M, Ayuso E, Casellas A, Costa C, Devedjian JC, Bosch F 2002 ?-Cell expression of IGF-I leads to recovery from type 1 diabetes. J Clin Invest 109:1153–1163
Weir GC, Bonner-Weir S 2004 Five stages of evolving ?-cell dysfunction during progression to diabetes. Diabetes 53(Suppl 3):S16–S21
Rhodes CJ 2000 IGF-I and GH post-receptor signaling mechanisms for pancreatic ?-cell replication. J Mol Endocrinol 24:303–311
Dominici FP, Turyn D 2002 Growth hormone-induced alterations in the insulin-signaling system. Exp Biol Med (Maywood) 227:149–157
Okuda Y, Pena J, Chou J, Field JB 2001 Acute effects of growth hormone on metabolism of pancreatic hormones, glucose and ketone bodies. Diabetes Res Clin Pract 53:1–8
Nielsen JH, Galsgaard ED, Moldrup A, Friedrichsen BN, Billestrup N, Hansen JA, Lee YC, Carlsson C 2001 Regulation of ?-cell mass by hormones and growth factors. Diabetes 50:S25–S29
Rabinovitch A, Quigley C, Rechler MM 1983 Growth hormone stimulates islet B-cell replication in neonatal rat pancreatic monolayer cultures. Diabetes 32:307–312
Galsgaard ED, Gouilleux F, Groner B, Serup P, Nielsen JH, Billestrup N 1996 Identification of a growth hormone-responsive STAT5-binding element in the rat insulin 1 gene. Mol Endocrinol 10:652–660
Galsgaard ED, Nielsen JH, Moldrup A 1999 Regulation of prolactin receptor (PRLR) gene expression in insulin-producing cells. Prolactin and growth hormone activate one of the rat prlr gene promoters via STAT5a and STAT5b. J Biol Chem 274:18686–18692
Parsons JA, Bartke A, Sorenson RL 1995 Number and size of islets of Langerhans in pregnant, human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones. Endocrinology 136:2013–2021
Sims NA, Clement-Lacroix P, Da Ponte F, Bouali Y, Binart N, Moriggl R, Goffin V, Coschigano K, Gaillard-Kelly M, Kopchick J, Baron R, Kelly PA 2000 Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5. J Clin Invest 106:1095–1103
Bachelot A, Monget P, Imbert-Bollore P, Coshigano K, Kopchick JJ, Kelly PA, Binart N 2002 Growth hormone is required for ovarian follicular growth. Endocrinology 143:4104–4112
Swenne I, Hill DJ 1989 Growth hormone regulation of DNA replication, but not insulin production, is partly mediated by somatomedin-C/insulin-like growth factor I in isolated pancreatic islets from adult rats. Diabetologia 32:191–197
Hill DJ, Hogg J 1991 Growth factor control of pancreatic B cell hyperplasia. Baillieres Clin Endocrinol Metab 5:689–698
Maake C, Reinecke M 1993 Immunohistochemical localization of insulin-like growth factor 1 and 2 in the endocrine pancreas of rat, dog, and man, and their coexistence with classical islet hormones. Cell Tissue Res 273:249–259
Portela-Gomes GM, Hoog A 2000 Insulin-like growth factor II in human fetal pancreas and its co-localization with the major islet hormones: comparison with adult pancreas. J Endocrinol 165:245–251
Hill DJ, Strutt B, Arany E, Zaina S, Coukell S, Graham CF 2000 Increased and persistent circulating insulin-like growth factor II in neonatal transgenic mice suppresses developmental apoptosis in the pancreatic islets. Endocrinology 141:1151–1157
Xuan S, Kitamura T, Nakae J, Politi K, Kido Y, Fisher PE, Morroni M, Cinti S, White MF, Herrera PL, Accili D, Efstratiadis A 2002 Defective insulin secretion in pancreatic ?-cells lacking type 1 IGF receptor. J Clin Invest 110:1011–1019
Kulkarni RN, Holzenberger M, Shih DQ, Ozcan U, Stoffel M, Magnuson MA, Kahn CR 2002 ?-Cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter ?-cell mass. Nat Genet 31:111–115
Yakar S, Liu JL, Fernandez AM, Wu Y, Schally AV, Frystyk J, Chernausek SD, Mejia W, Le Roith D 2001 Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50:1110–1118
Zhao H, Yakar S, Gavrilova O, Sun H, Zhang Y, Kim H, Setser J, Jou W, LeRoith D 2004 Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1-deficient mice. J Clin Invest 113:96–105
Yu R, Yakar S, Liu YL, Lu Y, LeRoith D, Miao D, Liu JL 2003 Liver-specific IGF-I gene deficient mice exhibit accelerated diabetes in response to streptozotocin, associated with early onset of insulin resistance. Mol Cell Endocrinol 204:31–42
Lu Y, Herrera PL, Guo Y, Sun D, Tang Z, LeRoith D, Liu JL 2004 Pancreatic specific inactivation of IGF-I gene causes enlarged pancreatic islets and significant resistance to diabetes. Diabetes 53:3131–3141
Friedrichsen BN, Richter HE, Hansen JA, Rhodes CJ, Nielsen JH, Billestrup N, Moldrup A 2003 Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic ?-cells. Mol Endocrinol 17:945–958
Billestrup N, Nielsen JH 1991 The stimulatory effect of growth hormone, prolactin, and placental lactogen on ?-cell proliferation is not mediated by insulin-like growth factor-I. Endocrinology 129:883–888
Cousin SP, Hugl SR, Myers Jr MG, White MF, Reifel-Miller A, Rhodes CJ 1999 Stimulation of pancreatic ?-cell proliferation by growth hormone is glucose-dependent: signal transduction via janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) with no crosstalk to insulin receptor substrate-mediated mitogenic signalling. Biochem J 344:649–658(Yubin Guo, Yarong Lu, Dan)