β cell expression of IGF-I leads to recovery from type 1 diabetes (original) (raw)
Expression of IGF-I in β cells of transgenic mice. The RIP-I/IGF-I chimeric gene was microinjected to mouse embryos, and two founder mice (C57BL/6–SJL genetic background) were obtained. The studies presented below were performed in line 1 of transgenic mice, which carried about 50 intact copies of the transgene, as analyzed by Southern blot (data not shown). Adult mice express very low levels of IGF-I mRNA in pancreas, and four major transcripts of 0.8–1.2, 1.9, 4.7, and 7.5 kb can be detected (43). The pancreata of transgenic mice showed high levels of IGF-I mRNA, and two main transcripts (0.8 and 1.2 kb) were detected when Northern blots were hybridized with IGF-I cDNA (Figure 1). The 1.2-kb transcript came from the expression of the transgene, and it was polyadenylated at the end of the β-globin gene. The 0.8-kb transcript came from the expression of the endogenous gene, and it was present in the pancreata of both nontransgenic and transgenic mice. No expression of the transgene was noted in liver or muscle of the transgenic mice (data not shown). Furthermore, transgenic islets showed high IGF-I immunostaining, while no IGF-I was detected in nontransgenic mice (Figure 2, a and d). However, similar concentrations of serum IGF-I were observed in transgenic (Tg) and nontransgenic (non-Tg) mice (Tg, 182 ± 14 ng/ml; non-Tg, 205 ± 8 ng/ml). These results suggest that IGF-I acted in a paracrine/autocrine manner within the islets. Pancreas from nontransgenic and transgenic mice showed similar levels of insulin mRNA (Figure 1), and no differences were observed in islet insulin (Figure 2, b and e) or glucagon (Figure 2, c and f) immunoreactivity. Moreover, 2-month-old transgenic mice presented similar insulinemia (Tg, 30 ± 1 μU/ml; non-Tg, 31 ± 1.5 μU/ml), glycemia (Tg, 148 ± 6 mg/dl; non-Tg, 147 ± 4 mg/dl), and body weight (Figure 3) to those of nontransgenic mice. Morphometric analysis of pancreata of these transgenic and nontransgenic mice showed similar β cell mass (Tg, 0.83 ± 0.05 mg; non-Tg, 0.80 ± 0.07 mg). Transgenic mice were healthy, did not develop tumors, and had normal lifespan and reproductive life.
Expression of IGF-I and insulin in the pancreas of transgenic mice (C57BL/6–SJL genetic background). Total cellular RNA was obtained from nontransgenic (Con) and transgenic (Tg) mice and analyzed by Northern blot as indicated in Methods. Two transcripts were detected in the pancreas of transgenic mice when hybridized with IGF-I probe. The 0.8-kb transcript came from the expression of the endogenous gene, and the 1.2-kb transcript came from the expression of the transgene and was polyadenylated at the end of the β-globin gene.
Immunohistochemical analysis of IGF-I, glucagon, and insulin expression in islets. IGF-I (a, d, g, and j), insulin (b, e, h, k, m, and n), and glucagon (c, f, i, and l) staining of representative sections of pancreas before (a–f) and 3 months after (g–n) STZ treatment. Nontransgenic mice (Con): a–c and g–i (×400) and m (×40); transgenic mice (Tg): d–f and j–l (×400) and n (×40).
Changes in body weight of nontransgenic (Con; n = 10) and transgenic (Tg; n = 12) mice and STZ-treated nontransgenic (STZ-Con; n = 10) and transgenic (STZ-Tg; n = 15) mice. Results are mean ± SEM of the indicated mice. *P < 0.05 vs. STZ-treated nontransgenic mice.
Counteraction of diabetic hyperglycemia by IGF-I expression in β cells. To determine whether IGF-I expression in β cells counteracts diabetic hyperglycemia, 2-month-old nontransgenic and transgenic mice were intraperitoneally injected with low doses of STZ. Nontransgenic mice showed a blood glucose concentration of over 600 mg/dl, the upper limit of measurement (Figure 4a). Although transgenic mice became hyperglycemic after STZ treatment, the blood glucose levels rose to about 300 mg/dl, remained high for about 2 months, and then returned to normal levels (Figure 4a). Three months after STZ treatment, nontransgenic mice had low levels of serum insulin, while transgenic mice were normoinsulinemic (Figure 5a). Similarly, circulating levels of IGF-I were lower in STZ-treated nontransgenic than in STZ-treated transgenic mice (Figure 5b), which is a feature of diabetes (14). All hyperglycemic nontransgenic mice died, whereas all transgenic mice survived (Figure 4b). Hyperglycemic transgenic mice did not gain body weight. However, body weight rose after normalization of glycemia, and by 3 months it was similar to that of healthy nontransgenic and transgenic mice (Figure 3). In contrast, surviving STZ-treated nontransgenic mice showed lower body weight (Figure 3). Furthermore, 10-month-old STZ-treated transgenic mice and non–STZ-treated transgenic and nontransgenic mice showed similar blood glucose concentrations (STZ-Tg, 154 ± 4 mg/dl; Tg, 157 ± 5 mg/dl; and non-Tg, 155 ± 3 mg/dl) and serum insulin levels (STZ-Tg, 34 ± 1 μU/ml; Tg, 32 ± 3 μU/ml; and non-Tg, 30 ± 5 μU/ml).
(a) Blood glucose levels before and after STZ treatment of mice. Glucose concentration was determined as indicated in Methods. Squares, nontransgenic mice (n = 15); circles, transgenic mice (n = 15). (b) Percent survival of nontransgenic (squares; n = 20) and transgenic (circles; n = 20) mice after STZ treatment. Results are mean ± SEM of the indicated mice.
Serum concentrations of insulin and IGF-I before and after STZ treatment of mice. (a) Insulin and (b) IGF-I levels were determined as indicated in Methods. White bars, nontransgenic mice (n = 15); black bars, transgenic mice (n = 15). Results are mean ± SEM of the indicated mice. *P < 0.05, **P < 0.01 vs. STZ-treated nontransgenic mice.
Histological analysis of nontransgenic pancreas 3 months after STZ treatment showed islets with mild insulitis and loss of insulin-producing cells (Figure 2, g–i), and highly reduced β cell mass (Figure 6a). In contrast, STZ-treated transgenic mice had higher (about fivefold) β cell mass than STZ-treated nontransgenic mice (Figure 6a) due to larger islets; they also had normal morphology (Figure 2, j–l), and small scattered clusters of insulin-producing cells (data not shown). However, β cell mass of STZ-treated transgenic mice was lower than that of non–STZ-treated nontransgenic mice (Figure 6a). Although non–STZ-treated 6-month-old transgenic mice presented an increase of about 1.5-fold in β cell mass compared with nontransgenic mice (Tg, 1.32 ± 0.2 mg; non-Tg, 0.87 ± 0.16 mg; P < 0.05) (Figure 2, m and n), they showed similar pancreatic insulin content (Tg, 6.5 ± 0.5 μg insulin/100 mg pancreas; non-Tg, 6.1 ± 0.4 μg insulin/100 mg pancreas). Furthermore, all these transgenic mice were normoglycemic and normoinsulinemic, indicating that IGF-I overexpression did not lead to hyperinsulinemia or hypoglycemia with age.
(a) β Cell mass and (b) neogenic β cell mass were determined in transgenic (black bars; n = 5) and nontransgenic (white bars; n = 5) pancreas before and 3 months after STZ treatment as described in Methods. (c) Neogenesis in STZ-treated transgenic pancreas. Cells immunostained for insulin (red) can be seen budding from the duct stained for cytokeratin (green) (×400). Results are mean ± SEM of the indicated mice. *P < 0.05, **P < 0.01 vs. STZ-treated nontransgenic mice.
STZ-treated transgenic mice also showed an increase in insulin-positive cells budding from the ductal epithelium (as indicated by cytokeratin staining, Figure 6c), which is the site of neogenesis of new islets from ductal precursor cells (7). In addition, STZ-treated transgenic mice had higher neogenic β cell mass (Figure 6b). Therefore, IGF-I production protected transgenic β cells from destruction after STZ treatment and also increased β cell mass and induced β cell neogenesis.
Counteraction of diabetic alterations by IGF-I expression in β cells of CD-1 mice. To further examine this IGF-I protective effect, transgenic mice were backcrossed to CD-1 mice to obtain transgenic mice in CD-1 genetic background. These mice are highly susceptible to develop insulitis, progressive β cell destruction, and diabetes following multiple injections of subdiabetogenic doses of STZ (35). Two-month-old nontransgenic and transgenic mice of the N4 generation (about 94% CD-1 background) presented similar glycemia (Tg, 139 ± 6 mg/dl; non-Tg, 142 ± 5 mg/dl), insulinemia (Tg, 43 ± 2 μU/ml; non-Tg, 39 ± 4 μU/ml), and body weight (Tg, 38 ± 0.9 g; non-Tg, 38 ± 0.5 g). One month after STZ treatment, nontransgenic and transgenic mice were highly hyperglycemic, reaching blood glucose concentrations of about 600 mg/dl (Figure 7a). Afterward, STZ-treated nontransgenic mice had glycemic levels that surpassed the upper limit of measurement (Figure 7a) and remained very high after starvation (Figure 7b). These mice did not survive longer than 4 months. In contrast, 2 months after STZ treatment, glycemia began to decrease in 12 of 17 transgenic mice. By 3 months these mice showed blood glucose levels of about 200 mg/dl, and thereafter they remained normoglycemic in both fed (Figure 7a) and fasted (Figure 7b) conditions. The other 5 STZ-treated N4 transgenic mice, blood glucose levels decreased more slowly and by 6 months they were 350 ± 20 mg/dl in fed and 110 ± 15 mg/dl in starved conditions. Nevertheless, none of STZ-treated transgenic mice died. Three weeks after STZ treatment, both N4 nontransgenic and transgenic mice showed decreased insulinemia (Figure 7c). Normalization of hyperglycemia in transgenic mice was paralleled by the restoration of insulinemia to normal levels (Figure 7c). Furthermore, 8 months after STZ treatment, transgenic mice, compared with 10-month-old non–STZ-treated transgenic and nontransgenic mice, showed similar glycemia (STZ-Tg, 152 ± 4 mg/dl; Tg, 154 ± 6 mg/dl; and non-Tg, 149 ± 5 mg/dl) and insulinemia (STZ-Tg, 41 ± 5 μU/ml; Tg, 42 ± 5 μU/ml; and non-Tg, 38 ± 6 μU/ml).
Blood glucose concentration of N4 CD-1 mice after STZ treatment. Blood glucose levels were measured in (a) fed nontransgenic (squares; n = 20) and transgenic (circles; n = 12) mice, and in (b) nontransgenic (white bars; n = 10) and transgenic (black bars; n = 12) mice treated for 3 months with STZ and starved overnight. (c) Serum concentration of insulin was determined after STZ treatment in N4 CD-1 nontransgenic (white bars; n = 8) and in transgenic (black bars; n = 6) mice at the times indicated. (d) Intraperitoneal glucose tolerance test. Nontransgenic (squares; n = 6) and transgenic (circles; n = 8) mice treated for 3 months with STZ and starved overnight were given an intraperitoneal injection of 1 mg of glucose/g of body weight. Blood samples were taken at the times indicated from the tail veins of the same animals. Results are mean ± SEM of the indicated mice. *P < 0.05, **P < 0.01 vs. STZ-treated nontransgenic mice.
When an intraperitoneal glucose tolerance test was performed in overnight-starved mice 3 months after STZ treatment, transgenic animals showed lower blood glucose levels than nontransgenic mice (Figure 7d). Furthermore, the increase in circulating glucose levels in transgenic mice was transient and they gradually returned to basal levels, while blood glucose concentration remained high in nontransgenic mice (Figure 7d). These results indicate that STZ-treated mice had a normal response to a glucose load. In addition, 3 months after STZ treatment the concentration of serum β-hydroxybutyrate was normalized in transgenic mice (Table 1). Transgenic mice also showed normal levels of circulating triglycerides and FFAs, which were increased in diabetic nontransgenic mice (Table 1).
One month after STZ treatment, fluid and food intake rose in both nontransgenic and transgenic N4 mice (Figure 8, a and b). These parameters increased progressively in STZ-treated nontransgenic mice, and by 3 months, fluid and food intake were ten- and threefold higher, respectively, indicating the severity of diabetes. However, with normalization of glycemia and insulinemia, normal fluid and food intake were recovered in STZ-treated N4 transgenic mice (Figure 8, a and b).
Fluid (a) and food (b) intake of nontransgenic (white bars) and transgenic (black bars) mice were measured at different times after STZ treatment. Results are mean ± SEM of 12 transgenic and 10 nontransgenic mice. *P < 0.05 vs. STZ-treated nontransgenic mice.
IGF-I expression in β cells of STZ-treated CD-1 mice led to regeneration of endocrine pancreas. Histological analysis of pancreas was performed 2 weeks after STZ treatment, when mice were highly hyperglycemic. In contrast to non–STZ-treated mice, in which islets were spherical, islets from both STZ-treated nontransgenic and transgenic mice showed altered shape and insulitis (Figures 9 and 10). Insulin immunostaining revealed the loss of β cells (Figure 9, a, b, d, and e), whereas glucagon immunostaining indicated α cell preservation (Figure 10, a, b, d, e). Although islets from both STZ-treated nontransgenic and transgenic mice showed lymphocytic infiltration, insulitis was more severe in nontransgenic mice (Figure 9, b and e; and Figure 10, b and e). STZ-treated transgenic mice had a high percentage of noninfiltrated islets and islets with less than 25% infiltration, whereas most of the nontransgenic islets had a high level of infiltration (data not shown), suggesting that IGF-I expression in β cells attenuated insulitis development. Furthermore, to study whether IGF-I expression protected β cells from STZ-induced apoptosis in vivo, we performed double immunohistochemical analysis of insulin and TUNEL to identify apoptotic β cells. Before STZ treatment, no apoptotic β cells were observed in either transgenic or nontransgenic islets, while insulin/TUNEL double positive cells were detected 3 and 8 days after STZ treatment (Figure 11). Although a similar percentage of apoptotic β cells was observed in both groups of mice on day 3, a significant decrease (about 40%) was noted in transgenic mice 8 days after STZ treatment (Figure 11). These results suggest that IGF-I expression partially protected β cells from apoptosis. Nevertheless, soon after STZ treatment, transgenic mice lost a significant number of β cells, which led to the development of hypoinsulinemia and hyperglycemia (Figure 7).
Immunohistochemical analysis of insulin expression in islets from N4 CD-1 mice. (a–f) Pancreata from 2-month-old non–STZ-treated nontransgenic (Con) (a) and transgenic (Tg) (d) mice, nontransgenic (b) and transgenic (e) mice treated for 2 weeks with STZ, and nontransgenic (c) and transgenic (f) mice treated for 4 months with STZ were analyzed. ×400. (g–j) Pancreata from 6-month-old non–STZ-treated nontransgenic (g) and transgenic (h) mice and nontransgenic (i) and transgenic (j) mice treated for 4 months with STZ were analyzed. ×40.
Immunohistochemical analysis of glucagon expression in islets from N4 CD-1 mice. Pancreata from 2-month-old non–STZ-treated nontransgenic (Con) (a) and transgenic (Tg) (d) mice, nontransgenic (b) and transgenic (e) mice treated for 2 weeks with STZ, and nontransgenic (c) and transgenic (f) mice treated for 4 months with STZ were analyzed. ×400.
Analysis of apoptosis in STZ-treated mice. At the indicated times after STZ treatment, the percentage of insulin/TUNEL double positive cells was determined in transgenic (black bars) and nontransgenic (white bars) pancreas as indicated in Methods. Results are mean ± SEM of five transgenic and five nontransgenic mice per group. *P < 0.05 vs. STZ-treated nontransgenic mice.
Four months after STZ treatment, insulin immunostaining of nontransgenic pancreas showed small islets with few β cells (Figure 9, c and i). However, glucagon immunostaining of nontransgenic pancreas revealed numerous α cells (Figure 10c). In contrast, insulin immunostaining of pancreas of normoglycemic STZ-treated N4 transgenic mice revealed a large number of islets without insulitis, and small scattered clusters of insulin-producing cells (Figure 9, f and j). These islets had abnormal distribution of β cells, since several cells in the islet core were not stained (Figure 9f), and glucagon-producing cells were randomly distributed throughout the islet core rather than at the periphery (Figure 10f). IGF-I staining colocalized with insulin-producing cells in these islets (Figure 12). Three months after STZ treatment, transgenic mice showed higher β cell mass (about sevenfold) than diabetic nontransgenic mice (Figure 13a). These STZ-treated transgenic mice presented slightly lower (about 20%) β cell mass than did 6-month-old non–STZ-treated transgenic and nontransgenic mice, which showed similar β cell mass (STZ-Tg, 1.45 ± 0.4 mg; Tg, 1.83 ± 0.6 mg; and non-Tg, 1.9 ± 0.5 mg). This indicated that β cell expression of IGF-I in N4 CD-1 transgenic mice did not lead to islet hyperplasia. Furthermore, the mass of insulin-producing cells budding from pancreatic ducts was also higher in STZ-treated transgenic mice (Figure 13b). In addition, β cell replication was greater (about threefold) in 2-month-old transgenic than in nontransgenic pancreas (Figure 13c). A similar increase in BrdU-positive β cells was noted in transgenic islets 8 days after STZ treatment, but it was lower at 3 months, when mice recovered from hyperglycemia (Figure 13c). All these results suggest that normalization of β cell mass in STZ-treated transgenic mice probably resulted from increased β cell replication and neogenesis.
Colocalization of IGF-I and insulin in β cells from transgenic mice. Four months after STZ treatment, pancreas sections of transgenic mice were coimmunostained for IGF-I (green) and insulin (red) as indicated in Methods. Sections (5 μm) were photographed individually at ×600 magnification and superimposed.
(a) β Cell mass and (b) neogenic β cell mass were determined in transgenic (black bars) and nontransgenic (white bars) pancreas before and 4 months after STZ treatment as described in Methods. (c) Analysis of β cell replication. In sections immunostained for both non-β cells and incorporated BrdU, the frequency of BrdU-positive β cells was determined in nontransgenic and transgenic islets, both before and after STZ treatment, as indicated in Methods. Results are mean ± SEM of five transgenic and five nontransgenic mice. *P < 0.05, **P < 0.01 vs. STZ-treated nontransgenic mice.