Type 2 diabetes is characterized by insulin secretory

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1 Insulin Effect During Embryogenesis Determines Fetal Growth A Possible Molecular Link Between Birth Weight and Susceptibility to Type 2 Diabetes Yasuo Terauchi, Naoto Kubota, Hiroyuki Tamemoto, Hiroshi Sakura, Ryozo Nagai, Yasuo Akanuma, Satoshi Kimura, and Takashi Kadowaki Low birth weight has been reported to be associated with impaired insulin secretion and insulin resistance. It has been proposed that this association results from fetal programming in response to the intrauterine environment (the thrifty phenotype hypothesis). To elucidate the relationship between birth weight and genetically determined defects in insulin secretion, we measured the birth weights of neonates derived from crosses of male pancreatic -cell type glucokinase knockout (G c k + / ) mice and female wild-type (WT) or G c k + / mice. In 135 offspring, birth weights were lower in the presence of a fetal heterozygous mutation and higher in the presence of a maternal heterozygous mutation. Moreover, G c k / neonates had significantly smaller birth weights than WT or G c k + / n e o n a t e s (means ± SE 1.49 ± 0.03 [n = 30] vs ± 0.03 [n = 30] or 1.63 ± 0.02 [n = 50] g, respectively; P < 0.01). Thus, G c k mutations in -cells may impair insulin response to glucose and alter intrauterine growth as well as glucose metabolism after birth. This study has confirmed the results of a previous report that human subjects carrying mutations in G c k had reduced birth weights and has provided direct evidence for a link between insulin and fetal growth. Moreover, birth weights were reduced in insulin receptor substrate-1 knockout mice despite normal insulin levels. Taken together, these results suggest that a genetically programmed insulin e ffect during embryogenesis determines fetal growth and provides a possible molecular link between birth weight and susceptibility to type 2 diabetes. D i a b e t e s 49 :82 86, 2000 From the Department of Internal Medicine (Y. T., N.K., R.N., S.K., T. K. ), Graduate School of Medicine, University of Tokyo; the Department of Molecular Medicine (H.T.), Institute for Molecular and Cellular Regulation, Gunma University; the Diabetes Center (H.S.), Tokyo Wo m e n s Medical University; and Institute for Diabetes Care and Research (Y.A.), Asahi Life Foundation, Tokyo, Japan. Address correspondence and reprint requests to Takashi Kadowaki, MD, PhD, Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan. k a d o w a k i - 3 i h. u - t o k y o. a c. j p. Received for publication 22 June 1999 and accepted in revised form 5 October Gck, glucokinase; IRI, immunoreactive insulin; Irs, insulin receptor substrate; PCR, polymerase chain reaction. Type 2 diabetes is characterized by insulin secretory dysfunction and peripheral insulin resistance (1,2). These abnormalities are either genetically d e fined or augmented by hyperglycemia itself and may interact in a complex manner to cause and sustain h y p e r g l y c e m i a. Low birth weight has been reported to be associated with impaired insulin secretion and insulin resistance (3,4). These observations have led to the hypothesis that growth retardation affecting the development of particular organs at the fetal stage will predispose the individual to impaired organ function with consequent disease in later life (5). Hales et a l. (6) have hypothesized that low birth weight, which is a r e flection of nutritional deprivation in utero, predisposes individuals to diabetes in later life. The researchers have suggested that type 2 diabetes is mainly the result of intrauterine environmental factors and that genetic factors play little or no role in its development, and they refer to this phenomenon as the thrifty phenotype hypothesis (6). On the other hand, many diabetes geneticists have assumed that individual susceptibility to type 2 diabetes is in large part genetically determined. In the early formulation of the thrifty genotype hypothesis, Neel (7) proposed that the exploding prevalence of diabetes in societies undergoing rapid Westernization resulted from selection for metabolically thrifty genes. These genes may have enhanced survival during prehistory but predispose individuals to diabetes given dietary abundance and a sedentary lifestyle. Thus, both environmental and genetic factors are considered to be involved in the pathogenesis of human type 2 diabetes. The same genetic factors that cause impaired insulin secretion and/or insulin resistance may alter both intrauterine growth and glucose tolerance in adulthood, thereby providing a link between them. Thus, we investigated whether birth weight can be modified in genetically engineered mice and cause impaired insulin secretion or insulin resistance. Glucose is the primary regulator of insulin secretion, and glucose-induced insulin secretion serves as a basic mechanism whereby mammals maintain their blood glucose levels within narrow limits. Glucokinase (Gck), which is mainly expressed in pancreatic -cells and in the liver, constitutes a rate-limiting step in glucose metabolism in these tissues (8,9). Thus, Gck has been proposed to act as a glucose sen- 82 DIABETES, VOL. 49, JANUARY 2000

2 Y. TERAUCHI AND ASSOCIATES TA B L E 1 Birth weight variation, blood glucose levels, and serum insulin levels according to the presence of a fetal G c k gene mutation in offspring derived from crosses between G c k + / father and WT mother or crosses between G c k + / father and G c k + / m o t h e r Genotype of fetus W T G c k + / G c k / P v a l u e * P v a l u e P v a l u e G c k + / father and WT mother Birth weight (g) 1.62 ± 0.02 (29) 1.54 ± 0.02 (26) < Blood glucose (mg/dl) 52 ± 4 (29) 61 ± 6 (26) Serum IRI (ng/ml) 1.81 ± 0.25 (13) 1.28 ± 0.20 (16) IRI/blood glucose (ng/mg) 3.27 ± 0.54 (13) 1.91 ± 0.30 (16) Insulin content per total 13.0 ± 2.0 (13) 13.7 ± 1.3 (16) pancreas (µg of insulin/mg of protein) G c k + / + father and G c k + / m o t h e r Birth weight (g) 1.63 ± 0.03 (30) 1.63 ± 0.02 (50) 1.49 ± 0.03 (30) < < Blood glucose (mg/dl) 41 ± 5 (21) 51 ± 6 (29) 61 ± 8 (11) IRI (ng/ml) 3.06 ± 0.51 (16) 2.15 ± 0.28 (18) 1.51 ± 0.20 (15) Data are means ± SE (n). *P value for WT vs. G c k + / ; P value for WT vs. G c k / ; and P value for G c k + / vs. G c k / ; neonates derived from crosses between male G c k + / and female WT mice were analyzed; blood glucose levels were 118 ± 4 mg/dl (n = 10) in WT pregnant mothers; neonates derived from crosses between male G c k + / and female G c k + / were analyzed; blood glucose levels were 185 ± 1 0 mg/dl (n = 10) in G c k + / pregnant mothers, which were significantly higher than those in WT pregnant mothers. sor in the pancreatic -cell (8,10). Recently, Hattersley et al. (11) reported that mutations in G c k of the fetus resulted in reduced birth weight, in addition to causing hyperglycemia after birth, which can be explained by a reduction of fetal insulin secretion in response to maternal blood glucose levels (the fetal insulin hypothesis). Because measuring insulin levels of human neonates is not easy for practical and ethical reasons, actual insulin levels of human neonates were not reported in the article (11). In addition, because human G c k + / fetuses had decreased Gck activity in the liver, the possibility that the resultant impaired hepatic glycogen accumulation (12) may lead to alterations in fetal development cannot be excluded. In this regard, we were able to eliminate selectively the expression of the -cell isoform of Gck without affecting the expression of the liver isoform in mice by disrupting the first exon specific to the -cell type Gck (13). To understand the relationship between birth weight and fetal insulin secretion, we measured birth weight and insulin levels of neonates derived from crosses of male pancreatic -cell type Gck knockout (G c k + / ) mice (13) and female wildtype (WT) or G c k + / m i c e. Low birth weight can also be associated with insulin resistance resulting from genetic defects that influence insulin action during embryogenesis. Insulin receptor substrate-1 (Irs-1) is the major substrate of the insulin receptor tyrosine kinase. We have previously generated I r s - 1 / mice and showed that they had retarded growth (14). They are also characterized by peripheral insulin resistance, which is a result of a defect in insulin action in organs such as skeletal muscles and adipose tissues (15,16). In the present study, we examined birth weight, blood glucose, and serum immunoreactive insulin (IRI) levels at birth in I r s - 1 / mice to understand the relationship between birth weight and genetic factors causing insulin resistance in mice. RESEARCH DESIGN AND METHODS Animals. G c k + / (129 ICR background) and I r s - 1 / mice (C57Bl/6J C B A background) were generated as described (13,14). Mice were fed normal laboratory chow and water ad libitum and were maintained with standard husbandry p r o c e d u r e s. Genotyping. The genotype of mice on G c k or I r s - 1 was determined by polymerase chain reaction (PCR). The genomic DNA was extracted from the tip of the tail. The primers and PCR conditions for genotyping have been described previously ( 1 4, 1 7 ). Measurement of serum parameters. Mouse neonates were analyzed immediately after birth before sucking milk to exclude the possibility that blood glucose or insulin levels may be altered by lactation. Serum samples were collected by decapitation. Glucose levels were measured with the Glucose Test Sensor (SKK, Nagoya, Japan). Insulin levels were determined with an insulin kit (BIOTRAK; Amersham Life Science, Amersham, U.K.) with rat insulin as the standard. Isolated pancreases were suspended in acid ethanol solution (74% ethanol, 1.4% HCl). The pancreases were sonicated and incubated overnight at 4 C. An aliquot of the islet extract was then assayed for insulin content with the insulin kit and for protein concentration with an assay kit (Bio-Rad Protein Assay; Bio-Rad, Richmond, CA); the results were expressed as micrograms of insulin per milligram of protein. Statistical analysis. Results were expressed as means ± SE (n). Statistical analysis was performed with the Statview software system (Abacus Concepts, B e r k e l e y, CA). Statistical differences were analyzed with Student s t test for unpaired comparisons. A P value of <0.05 was considered statistically signific a n t. R E S U LT S E ffect of the fetal G c k mutation in the offspring of WT mothers. In 55 offspring derived from crosses of male G c k + / and female WT mice, birth weights were significantly lower in the presence of a fetal G c k mutation (P < 0.01) (Ta b l e 1 ). H o w e v e r, the BMI (body weight in grams/length from nose to hip in centimeters squared) was not different between WT (0.164 ± g/cm 2 [n = 13]) and G c k + / mice (0.162 ± g / c m 2 [n = 17]) (P = 0.46), which suggests that G c k + / a n i m a l s had a small body size proportionate to body weight. Although no difference in blood glucose levels was evident immediately after birth before sucking milk between the two mouse groups, IRI levels were lower in the G c k + / mice than in the WT mice (Ta b l e 1). IRI/blood glucose levels were significantly lower in G c k + / mice than in WT mice (P = 0.03). Insulin content per whole pancreas was indistinguishable between the two mouse groups, which suggests that the development of pancreatic -cells was normal in G c k + / m i c e born to a WT mother. DIABETES, VOL. 49, JANUARY

3 FETALINSULIN EFFECT AND BIRTH WEIGHT E ffect of maternal G c k mutation on birth weight of neonates. The body weights of G c k + / neonates derived from a G c k + / mother with hyperglycemia (185 ± 10 mg/dl [n = 10]) were significantly higher than that of G c k + / neonates from a normoglycemic (118 ± 4 mg/dl [n = 10]) WT mother (1.63 ± 0.02 [n = 50] vs ± 0.02 g [n = 26], respectively; P < 0.01). Howe v e r, this effect of maternal hyperglycemia was not apparent between WT neonates derived from G c k + / and WT mothers. E ffect of maternal G c k mutation on IRI levels in neonates. IRI levels in WT neonates derived from a G c k + / mother with hyperglycemia were significantly higher than those in WT neonates from a normoglycemic WT mother (3.06 ± 0.51 [n = 16] vs ± 0.25 ng/ml [n = 13], respectively; P < 0.05). This effect of maternal hyperglycemia was also apparent between G c k + / neonates derived from G c k + / and WT mothers (2.15 ± 0.28 [n = 18] vs ± 0.20 ng/ml [n = 16], respectively; P = 0.02). Characterization of G c k / mouse neonates. H o m o z y- gous G c k- d e ficient mice showed severe diabetes shortly after birth because of a lack of glucose-induced insulin secretion (13). Thus, all humans homozygous for this mutation (G c k / ) would also die shortly after birth. We next characterized G c k / mouse neonates in offspring derived from crosses between heterozygotes. G c k / neonates had significantly lower birth weights than WT or G c k + / neonates (Ta b l e 1 ). Although no difference in blood glucose levels was evident at birth among the three mouse groups, IRI levels were lower in G c k / than in WT or G c k + / a n i m a l s. Characterization of I r s - 1 / mouse neonates. Low birth weight can also be associated with insulin resistance caused by genetic defects that impair insulin action during embryogenesis. We examined blood glucose and IRI levels at birth in addition to birth weight in I r s - 1 / mice and in WT controls. I r s - 1 / mice showed a 22% lower birth weight than WT mice with the same background (C57Bl/6J CBA) (P < ) ( Ta b l e 2). Both blood glucose and IRI levels at birth were indistinguishable between I r s - 1 / and WT mice. Thus, the reduced body weight was associated with normal insulin levels at birth in I r s - 1 / m i c e. D I S C U S S I O N In this study, we have shown that birth weight was significantly lower in the presence of a fetal G c k mutation, that the body weights of G c k + / neonates derived from a G c k + / m o t h e r were significantly higher than that of G c k + / neonates from a normoglycemic WT mother, and that G c k / neonates had s i g n i ficantly lower birth weights than WT or G c k + / n e o n a t e s. These results suggest that both the maternal and fetal G c k genotypes interact to influence birth weight. Moreover, the TABLE 2 Birth weight variation, blood glucose levels, and serum insulin levels in WT and I r s - 1 / offspring derived from I r s / i n t e r c r o s s e s W T I r s - 1 / P v a l u e Birth weight (g) 1.44 ± 0.04 (10) 1.14 ± 0.03 (12) < Blood glucose (mg/dl) 28 ± 4 (10) 38 ± 7 (12) Serum insulin (ng/ml) 2.11 ± 0.63 (10) 1.78 ± 0.53 (12) Data are means ± SE (n). Neonates derived from I r s / i n t e r- crosses were analyzed. reduced birth weight was indeed associated with the lower insulin levels. In humans carrying G c k mutations, the effect of fetal G c k mutations and maternal G c k mutations was additive (11). In the presence of maternal hyperglycemia resulting from the heterozygosity of the G c k mutation, however, WT mice neonates were not heavier than WT mice born of normoglycemic mothers. This appears to be related to the IRI levels of WT neonates derived from a G c k + / m o t h e r, which were not high enough to make the fetuses heavier. Moreover, although maternal hyperglycemia has been reported to cause high birth weight in humans (18), this effect has not necessarily been reproduced in rodent models of diabetes (19). We speculate that birth weight and size tend to be limited in mice because mice usually yield more than 10 pups, and the pregnancy period for mice is only 20 days, factors that are quite different from those in humans. According to studies performed with human fetal pancreatic tissues in vitro (20), islets begin to release insulin in response to nutrients as early as weeks of development. A study reported that Gck was expressed in human 13-week fetal pancreases, which suggests the expression of Gck at the fetal stage (21). Previous reports on fetal islets indicated that insulin responses to glucose matured slowly in normal pregnancies and remained poor relative to the adult situation throughout the early neonatal period (19). However, our data at least in part suggest that murine islets responded to glucose at the fetal stage, although its glucose response may be poorer than at adult stages. Taken together with the fact that G c k mutation was associated with the lower insulin levels in mice (Ta b l e 1), these results suggest that G c k mutations in -cells may impair insulin response to glucose even at the fetal stage and alter intrauterine growth. Compared with human neonates carrying mutations in G c k (11), the changes in birth weights from either a maternal or fetal effect were small in mice. In fact, mice lacking two mouse insulin genes, namely I n s - 1 and I n s - 2, exhibited birth weight reduced by only 22% compared with their siblings (22), which can presumably be explained by the supposition that IGF-I plays a greater role in the fetal development of mice than of humans. I r s - 1 / mice showed birth weights reduced by 22% compared with their WT siblings, which is a similar result to our previous report (14). The reduced body weight associated with normal insulin levels at birth in I r s - 1 / mice suggests that defects in insulin action during fetal development lead to alterations in birth weight. Thus, although G c k + / and I r s - 1 / mice represented quite different genetic defects, both of them showed reduced birth weights that seemed to be associated with insufficient insulin effect during embryogenesis. Although G c k + / mice exhibited hypoinsulinemia at birth because of impaired insulin secretion and developed overt diabetes in the presence of peripheral insulin resistance (17), I r s-1 / mice exhibited normoinsulinemia despite insulin resistance at birth and developed syndrome X alone (23) and overt diabetes when combined with impaired insulin secretion (17). Moreover, homozygous mutant mice for I r s - 2 / showed birth weights reduced by 10% compared with their WT siblings, and the difference persisted into adult life (24). These mouse studies suggest that a genetically determined insufficient insulin effect in human fetuses, regardless of whether it is caused by low insulin secretion or action, leads to low birth weight and increased susceptibility to type 2 84 DIABETES, VOL. 49, JANUARY 2000

4 Y. TERAUCHI AND ASSOCIATES A C K N O W L E D G M E N T S This work was supported by a Grant-in-Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports, and Culture of Japan (T. K. ). We thank Eri Yoshida, Yuko Muto, and Hiroshi Chiyonobu for their excellent technical assistance and mouse husbandry. FIG. 1. A link between low birth weight and susceptibility to type 2 diabetes is determined by genetic and environmental factors. diabetes or syndrome X after birth. In contrast, homozygous mutant mice for the insulin receptor showed similar birth weights to other WT littermates (25,26), which can presumably be explained by the supposition that the IGF-I receptor plays a compensatory role for insulin receptor deficiency in the fetal development of mice. However, because Irs-1 and I r s-2 are common intracellular substrates for both the insulin receptor and the IGF-I receptor, the signal transduction pathway via the IGF-I receptor in addition to that via insulin receptor is impaired in I r s - 1 / and I r s-2 /, which leads to reduced birth weights. This study has confirmed the results of a previous report that showed that human subjects carrying mutations in G c k had reduced birth weights (11) and has provided direct evidence of a link between insulin and fetal growth. Changes in insulin action during embryogenesis also affect glucose levels and other parameters in addition to birth weight. Thus, we propose that a genetically programmed insulin effect during embryogenesis determines fetal growth and provides a possible molecular link between birth weight and susceptibility to type 2 diabetes. This association should provide an explanation for the thrifty genotype hypothesis. This proposal is consistent with the hypothesis based on human subjects with genetically determined impaired insulin secretion or insulin resistance (27). With this insulin effect and the thrifty phenotype hypothesis (6), we are able to provide two alternative explanations (environmental factors and genetic factors) for a link between low birth weight and susceptibility to type 2 diabetes (Fig. 1). This study also suggests that genetically determined effects on birth weight are altered by the maternal environment because the body weight of G c k + / n e o n a t e s derived from a hyperglycemic G c k + / mother was signific a n t l y higher than that of G c k + / neonates from a normoglycemic WT m o t h e r. In human subjects, external factors such as smoking, maternal nutrition status, and obstetrical practice influ e n c e fetal growth and the timing of birth (28). Thus, we must understand the complex mix of genetic and environmental effects on birth weight and size. 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