Metabolic Programming: Causes and Consequences

high carbohydrate rat raised on a high carbohydrate milk formula pancreatic duodenal homeobox factor-1 stress-activated protein kinase-2 phosphatidylinositol 3-kinase glucagon-like peptide-1 The normal programmed development of a multicellular organism from the germ cell is a synchronized series of events driven by genetic instructions acquired during conception. During the early critical periods in life the organism also has the ability to respond to environmental situations that are alien to normal development by adaptations at the cellular, molecular, and biochemical levels. Such early adaptations to a nutritional stress/stimulus permanently change the physiology and metabolism of the organism and continue to be expressed even in the absence of the stimulus/stress that initiated them, a process termed “metabolic programming” (1Lucas A. CIBA Found. Symp. 1991; 156: 38-50PubMed Google Scholar). A brief summary of the findings from human epidemiological and animal studies is presented below in support of the concept of metabolic programming induced by nutritional experiences during critical periods in development with consequences later in adulthood. For detailed accounts the reader is referred to excellent reviews on this subject (2Whitaker R.C. Dietz W.H. J. Pediatr. 1998; 132: 768-776Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3Hoet J.J. Hanson M.A. J. Physiol. (Lond.). 1999; 514: 617-627Crossref Scopus (194) Google Scholar, 4Godfrey K.M. Barker D.J.P. Am. J. Clin. Nutr. 2000; 71: 1344S-1352SCrossref PubMed Scopus (1032) Google Scholar, 5Waterland R.A. Garza C. Am. J. Clin. Nutr. 1999; 69: 179-197Crossref PubMed Scopus (466) Google Scholar). This minireview will focus on metabolic programming with reference to a novel rat model developed in our laboratory. Extensive epidemiological findings indicate that metabolic programming occurs in humans. Barker (6Barker D.J.P. Br. Med. J. 1995; 311: 171-174Crossref PubMed Scopus (2590) Google Scholar) was the first to suggest from epidemiological studies that the disproportionate size of the newborn resulting from maternal malnutrition correlated with an increased risk for adverse health outcomes (type II diabetes, hypertension, and cardiovascular diseases) later in adult life. These primary observations resulted in the now widely recognized “fetal origins” hypothesis emphasizing the importance of adequate maternal nutrition during pregnancy (4Godfrey K.M. Barker D.J.P. Am. J. Clin. Nutr. 2000; 71: 1344S-1352SCrossref PubMed Scopus (1032) Google Scholar). Nutritional programming has been demonstrated in animal studies. In pioneering studies with rodents, McCance (7McCance R.A. Lancet. 1962; ii: 271-272Google Scholar) demonstrated by adjusting litter size that the quantity of food consumed during early periods of postnatal life has long term consequences on growth. The consequences of maternal malnutrition induced by either a low protein diet or caloric restriction during gestation and lactation cause major changes in the structure and function of several organs in the offspring. Pregnant rats fed a low protein diet produced pups with alterations in pancreatic islets (8Berney D.M. Desai M. Palmer D.J. Greenwald S. Brown A. Hales C.N. Berry C.L. J. Pathol. 1997; 183: 109-115Crossref PubMed Scopus (71) Google Scholar, 9Latorraca M.Q. Reis M.A.B. Carneiro E.M. Mello M.A.R. Velloso L.A. Saad M.J.A. Boschero A.C. J. Nutr. 1998; 128: 1643-1649Crossref PubMed Scopus (69) Google Scholar). These include reduced islet vascularization, β cell proliferative capacity, and islet size with rightward shift (decreased sensitivity) in glucose-stimulated insulin secretion and altered sensitivity to insulin in muscle (8Berney D.M. Desai M. Palmer D.J. Greenwald S. Brown A. Hales C.N. Berry C.L. J. Pathol. 1997; 183: 109-115Crossref PubMed Scopus (71) Google Scholar, 9Latorraca M.Q. Reis M.A.B. Carneiro E.M. Mello M.A.R. Velloso L.A. Saad M.J.A. Boschero A.C. J. Nutr. 1998; 128: 1643-1649Crossref PubMed Scopus (69) Google Scholar). Furthermore, hypothalamic nuclei are malformed in these weanling rats; this is accompanied by reduced vascularization of the cerebral cortex in the progeny (10Bennis-Taleb N. Remacle C. Hoet J.J. Reusens B. J. Nutr. 1999; 129: 1613-1619Crossref PubMed Scopus (89) Google Scholar). Metabolic capacities of the liver, muscle, and adipose tissue are compromised by maternal protein restriction during gestation and lactation with adverse adult onset outcomes (11Ozanne S.E. Hales C.N. Proc. Nutr. Soc. 1999; 58: 615-619Crossref PubMed Scopus (157) Google Scholar). Elevated insulin concentrations during critical periods of development, as occurs perinatally in the offspring of gestationally diabetic mothers, lead to permanent malorganization of the ventromedial hypothalamic nuclei followed by glucose intolerance in adult life (12Harder T. Plagemann A. Rohde W. Dorner G. Metabolism. 1998; 47: 855-862Abstract Full Text PDF PubMed Scopus (56) Google Scholar). Collectively, the results from animal models indicate that even brief periods of dietary manipulation in early life have implications for ill health outcomes, which become evident only in adulthood. Most of the above cited animal studies deal with maternal malnutrition during pregnancy leading to intrauterine growth retardation and accompanying adaptations in the fetus for survival under adverse conditions. Except for the effect of overnourishment caused by reducing litter size, the effect of an altered nutrition during the suckling period has not been investigated extensively in the rat. This is because of experimental difficulties in successfully rearing rat pups on a modified milk formula away from nursing mothers. The artificial rearing technique as described by Hall (13Hall W.G. Science. 1975; 190: 1313-1315Crossref PubMed Scopus (186) Google Scholar) provides a means to study the effects of altered nutrition during the suckling period in the rat. We have adapted this technique to evaluate the consequences of a switch in the “quality” of nutrition (from fat-rich rat milk to high carbohydrate (HC)1 milk formula) during the suckling period in the rat. The result is the “pup in a cup” rat model (also referred to as the HC rat) where the newborn rats are reared in Styrofoam cups floating in a temperature-controlled water bath. Four-day-old rats are fed via intragastric cannulas, introduced nonsurgically, raised on a HC milk formula (56% of the calories derived from carbohydrate compared with 8% in rat milk) up to day 24, and then weaned onto laboratory chow. Four-day-old rats artificially reared on a high fat milk formula (macronutrient composition similar to rat milk to show that the artificial rearing technique per se has no metabolic programming effects) and pups nursed by their own mothers served as controls. The detailed protocol followed by our laboratory for the experiments performed using the “pup in a cup” rat model is described elsewhere (14Hiremagalur B.K. Johanning G.L. Kalhan S.C. Patel M.S. J. Nutr. Biochem. 1992; 3: 474-480Crossref Scopus (16) Google Scholar, 15Hiremagalur B.K. Vadlamudi S. Johanning G.L. Patel M.S. Int. J. Obes. 1993; 17: 495-502PubMed Google Scholar). Fig.1A depicts metabolic adaptations in the first generation HC rats. An immediate outcome of the HC dietary intervention is the onset of hyperinsulinemia within 24 h; this persists throughout the suckling period and into adulthood even after the withdrawal of the HC milk formula at the time of weaning (15Hiremagalur B.K. Vadlamudi S. Johanning G.L. Patel M.S. Int. J. Obes. 1993; 17: 495-502PubMed Google Scholar, 16Haney P.M. Raefsky-Estrin C. Caliendo A. Patel M.S. Arch. Biochem. Biophys. 1986; 244: 787-794Crossref PubMed Scopus (35) Google Scholar) (Figs. 1Aand 2A). During the suckling period there are no differences in body weights and in plasma glucose levels between the HC and age-matched control groups (17Vadlamudi S. Hiremagalur B.K. Tao S. Kalhan S.C. Kalaria R.N. Kaung H.C. Patel M.S. Am. J. Physiol. 1993; 265: E565-E571PubMed Google Scholar). The overlap of the critical window for postnatal pancreatic development with the high carbohydrate nutritional intervention in the HC rat suggests that the endocrine pancreas is a target organ for significant adaptations. We have observed significant alterations at the cellular, molecular, and biochemical levels in islets isolated from neonatal HC rats and have also observed the programming of these adaptations into adulthood. Cellular adaptations induced by dietary intervention during the suckling period include an increased number of smaller sized islets in the HC pancreas compared with controls (18Petrik J. Srinivasan M. Aalinkeel A. Coukell S. Arany E. Patel M.S. Hill D.J. Pediatr. Res. 2001; 49: 84-92Crossref PubMed Scopus (38) Google Scholar). Such HC islets have a larger immunopositive area for insulin resulting in a net increase in the insulin-producing mass in HC islets (18Petrik J. Srinivasan M. Aalinkeel A. Coukell S. Arany E. Patel M.S. Hill D.J. Pediatr. Res. 2001; 49: 84-92Crossref PubMed Scopus (38) Google Scholar). Additionally, the rate of apoptosis and the expression of proliferating cell nuclear antigen are inversely altered in islets and the ductal epithelium of the HC rat (18Petrik J. Srinivasan M. Aalinkeel A. Coukell S. Arany E. Patel M.S. Hill D.J. Pediatr. Res. 2001; 49: 84-92Crossref PubMed Scopus (38) Google Scholar). One of the factors contributing to the altered ontogeny has been attributed to the change in the expression of insulin-like growth factor II (18Petrik J. Srinivasan M. Aalinkeel A. Coukell S. Arany E. Patel M.S. Hill D.J. Pediatr. Res. 2001; 49: 84-92Crossref PubMed Scopus (38) Google Scholar). Several molecular adaptations are observed in neonatal islets in response to the HC dietary intervention. Increases in insulin biosynthesis and in gene expression of preproinsulin are observed in islets of neonatal HC rats (19Srinivasan M. Song F. Aalinkeel R. Patel M.S. J. Nutr. Biochem. 2001; 12: 575-584Crossref PubMed Scopus (24) Google Scholar) (Fig. 2B). Additionally, mRNA levels of transcription factors such as pancreatic duodenal homeobox factor-1 (PDX-1, also known as somatostatin transcription factor-1) (Fig. 2B), islet factor-1 (Isl-1), upstream stimulatory factor-1, regenerating factor-3 (reg-3) (19Srinivasan M. Song F. Aalinkeel R. Patel M.S. J. Nutr. Biochem. 2001; 12: 575-584Crossref PubMed Scopus (24) Google Scholar, 20Song F. Srinivasan M. Aalinkeel R. Patel M.S. Diabetes. 2001; 50: 2053-2060Crossref PubMed Scopus (19) Google Scholar), and Beta2/NeuroD and hepatocyte nuclear factor β-3 2M. S. Patel, M. Srinivasan, F. Song, and R. Aalinkeel, unpublished observations. are significantly increased in neonatal HC islets. mRNA levels of stress-activated protein kinase-2 (SAPK-2), phosphatidylinositol 3-kinase (PI3-kinase), acetyl-CoA carboxylase, glucose transporter 2, and insulin receptor substrate-1 and -2 are also significantly higher in these HC islets (19Srinivasan M. Song F. Aalinkeel R. Patel M.S. J. Nutr. Biochem. 2001; 12: 575-584Crossref PubMed Scopus (24) Google Scholar, 20Song F. Srinivasan M. Aalinkeel R. Patel M.S. Diabetes. 2001; 50: 2053-2060Crossref PubMed Scopus (19) Google Scholar). PDX-1 is an important transactivator of the insulin gene and is an essential component of the mechanisms whereby glucose modulates insulin promoter activity (21Macfarlane W.M. Smith S.B. James R.F.L. Clifton A.D. Doza Y.N. Cohen P. Docherty K. J. Biol. Chem. 1997; 272: 20936-20944Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). DNA binding activity of PDX-1 has been proposed to be modulated by glucose via a phosphorylation cascade(s) involving SAPK-2 and PI3-kinase, which facilitates the translocation of the 46-kDa, phosphorylated active form to the nucleus resulting in increased insulin gene transcription (21Macfarlane W.M. Smith S.B. James R.F.L. Clifton A.D. Doza Y.N. Cohen P. Docherty K. J. Biol. Chem. 1997; 272: 20936-20944Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) (Fig. 3). The possible correlation between the observed effects of the HC dietary intervention in neonatal HC rats and preproinsulin gene transcription is shown in Fig. 3. In addition PDX-1 is an important transcription factor that commits the progression of the pluripotent ductal cell into an endocrine cell (22Huang H-P. Tsai M-L. J. Biomed. Sci. 2000; 7: 27-34Crossref PubMed Google Scholar). The significant increase in the gene expression, DNA binding activity, and protein content of PDX-1 in neonatal HC islets suggests that it plays a pivotal role in the cellular adaptations as well as in the onset and maintenance of hyperinsulinemia in this rat model (19Srinivasan M. Song F. Aalinkeel R. Patel M.S. J. Nutr. Biochem. 2001; 12: 575-584Crossref PubMed Scopus (24) Google Scholar). Transcription factors like Isl-1, Beta2/NeuroD, reg-3, and hepatocyte nuclear factor β-3 also contribute to pancreatic organogenesis (22Huang H-P. Tsai M-L. J. Biomed. Sci. 2000; 7: 27-34Crossref PubMed Google Scholar, 23Okamoto H. J. Hepato-Biliary-Pancreatic Surgery. 1999; 6: 254-262Crossref PubMed Scopus (171) Google Scholar, 24Wu K-L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V.E. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Crossref PubMed Google Scholar). The increases in the gene expression of these factors suggest that they too contribute to the modification of the islet architecture in the neonatal HC rats. cDNA array analysis has indicated significant changes in global gene expression patterns in neonatal HC islets as well as in adult HC islets suggesting that a wide range of molecular alterations is essential for the onset and persistence of the HC phenotype in HC rats (20Song F. Srinivasan M. Aalinkeel R. Patel M.S. Diabetes. 2001; 50: 2053-2060Crossref PubMed Scopus (19) Google Scholar). Significant biochemical adaptations summarized below initiate and sustain hyperinsulinemia in neonatal HC rats. A characteristic feature of neonatal HC rats is about a 6-fold increase in the concentration of circulating insulin (16Haney P.M. Raefsky-Estrin C. Caliendo A. Patel M.S. Arch. Biochem. Biophys. 1986; 244: 787-794Crossref PubMed Scopus (35) Google Scholar, 25Aalinkeel R. Srinivasan M. Kalhan S.C. Laychock S.G. Patel M.S. Am. J. Physiol. 1999; 277: E1061-E1069PubMed Google Scholar) (Fig. 2A). There is a distinct leftward shift (increased sensitivity) in the glucose-stimulated insulin secretory response in HC islets (Fig. 2C), and this is associated with increases in the low Kmhexokinase activity (Fig. 2D) and an increase in glucose transporter 2 protein content (25Aalinkeel R. Srinivasan M. Kalhan S.C. Laychock S.G. Patel M.S. Am. J. Physiol. 1999; 277: E1061-E1069PubMed Google Scholar). Insulin secretion by islets is regulated by three pathways (the KATPchannel-dependent pathway, the KATPchannel-independent augmentation pathway, and the Ca2+channel-independent augmentation pathway (26Komatsu M. Schermerhorn T. Noda M. Straub S.G. Aizawa T. Sharp G.W.G. Diabetes. 1997; 46: 1928-1938Crossref PubMed Scopus (94) Google Scholar)), and these pathways are up-regulated in HC islets (27Srinivasan M. Aalinkeel R. Song F. Lee B. Laychock S.G. Patel M.S. Am. J. Physiol. 2000; 279: E1347-E1357PubMed Google Scholar). Circulating levels of glucagon-like peptide-1 (GLP-1) are significantly higher in HC rats suggesting that GLP-1-mediated events contribute significantly to the hyperinsulinemia of HC rats (27Srinivasan M. Aalinkeel R. Song F. Lee B. Laychock S.G. Patel M.S. Am. J. Physiol. 2000; 279: E1347-E1357PubMed Google Scholar). In addition to its insulinotropic effects, GLP-1 stimulates transcription of the preproinsulin gene and PDX-1 gene and also the proliferation and neogenesis of β cells from ductal epithelium in rodents (28Fehmann H.C. Habener J.F. Endocrinology. 1992; 130: 159-166Crossref PubMed Scopus (258) Google Scholar, 29Stoffers D.A. Kieffer T.J. Hussain M.A. Drucker D.J. Bonner-Weir S. Habener J.F. Egan J.M. Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (516) Google Scholar, 30Xu G. Stoffers D.A. Habener J.F. Bonner-Weir S. Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1094) Google Scholar). In light of the above, the significant increase in circulating levels of GLP-1 in 12-day-old HC rats has important implications for the HC phenotype in suckling HC rats. Because of chronic hyperinsulinemia the capacity for hepatic lipogenesis is enhanced in neonatal HC rats (16Haney P.M. Raefsky-Estrin C. Caliendo A. Patel M.S. Arch. Biochem. Biophys. 1986; 244: 787-794Crossref PubMed Scopus (35) Google Scholar). Enzymes such as glucokinase and malic enzyme that normally appear in the liver at weaning are precociously induced by this dietary treatment suggesting that their appearance is not development-dependent but is controlled by the presence of the stimulus (diet-induced hyperinsulinemia) (16Haney P.M. Raefsky-Estrin C. Caliendo A. Patel M.S. Arch. Biochem. Biophys. 1986; 244: 787-794Crossref PubMed Scopus (35) Google Scholar). It is evident from the above that feeding rats an HC milk formula, instead of mother's during the suckling period significant alterations in islet as well as in metabolic of which are important for the development of persists into adulthood of HC rats withdrawal of the HC nutritional intervention on day 24 in the insulin secretory pathway observed in the neonatal HC islets are programmed into adult islets S.G. Vadlamudi S. Patel M.S. Am. J. Physiol. 1995; PubMed Google Scholar, R. Srinivasan M. Song F. Patel M.S. Am. J. Physiol. 2001; Google Scholar). The molecular changes in the preproinsulin gene observed in islets from neonatal HC rats are also observed in adult HC islets R. Srinivasan M. Song F. Patel M.S. Am. J. Physiol. 2001; Google Scholar). There is an increase in the insulin-producing mass of the adult HC pancreas (17Vadlamudi S. Hiremagalur B.K. Tao S. Kalhan S.C. Kalaria R.N. Kaung H.C. Patel M.S. Am. J. Physiol. 1993; 265: E565-E571PubMed Google Scholar). hyperinsulinemia is accompanied by an increase in the body of the from day and by day (15Hiremagalur B.K. Vadlamudi S. Johanning G.L. Patel M.S. Int. J. Obes. 1993; 17: 495-502PubMed Google Scholar) (Fig. the adult HC rats an response to an glucose is observed in HC on day In liver and adipose tissue show increased capacities in adulthood (15Hiremagalur B.K. Vadlamudi S. Johanning G.L. Patel M.S. Int. J. Obes. 1993; 17: 495-502PubMed Google Scholar). An increase in the cell size in adipose tissue the observed in adulthood (15Hiremagalur B.K. Vadlamudi S. Johanning G.L. Patel M.S. Int. J. Obes. 1993; 17: 495-502PubMed Google Scholar). content and the are in liver and muscle M. Vadlamudi S. Patel M.S. Int. J. Obes. Scholar) and up-regulated in adipose tissue of adult HC rats M. Patel M.S. J. Nutr. Biochem. 1998; Scopus Google Scholar). these findings indicate that early adaptations are programmed and are accompanied by changes by factors (Fig. An important and of the induced metabolic programming above is the of the phenotype from the HC to the progeny Fig. rats fed an HC milk formula during their suckling period their metabolic to their progeny the pups to nutritional treatment S. Kalhan S.C. Patel M.S. Am. J. Physiol. 1995; Google Scholar). The generation HC rats hyperinsulinemia and an altered insulin secretory within after weaning onto laboratory on day adaptations in mRNA levels of upstream stimulatory factor-1, and are also programmed in generation HC The growth of HC rats in the generation that of first generation HC rats S. Kalhan S.C. Patel M.S. Am. J. Physiol. 1995; Google Scholar) (Fig. experiments have demonstrated that only HC these to the suggesting that the intrauterine be essential for the (Fig. Metabolic programming is an that occurs in response to a nutritional during a period of early in life. for mechanisms for metabolic programming is not mechanisms have been by (1Lucas A. CIBA Found. Symp. 1991; 156: 38-50PubMed Google Scholar) and and Garza R.A. Garza C. Am. J. Clin. Nutr. 1999; 69: 179-197Crossref PubMed Scopus (466) Google Scholar). The immediate effects of a nutritional on development with long term consequences have been demonstrated in rats (8Berney D.M. Desai M. Palmer D.J. Greenwald S. Brown A. Hales C.N. Berry C.L. J. Pathol. 1997; 183: 109-115Crossref PubMed Scopus (71) Google Scholar, N. Remacle C. Hoet J.J. Reusens B. J. Nutr. 1999; 129: 1613-1619Crossref PubMed Scopus (89) Google Scholar). induced hyperinsulinemia in neonatal rats during the critical period of development results in alterations in body and glucose metabolism in adult life because of of the ventromedial hypothalamic nuclei (12Harder T. Plagemann A. Rohde W. Dorner G. Metabolism. 1998; 47: 855-862Abstract Full Text PDF PubMed Scopus (56) Google Scholar). The HC milk formula in pancreatic islets and the as by increases in the circulating plasma levels of insulin and GLP-1 in neonatal HC rats. In the of on the effects of a nutritional modification on the and the effects of the HC dietary intervention on the islets and in neonatal HC it is to suggest that the and resulting in the onset and persistence of hyperinsulinemia in HC mechanisms that such as also contribute to programming M.S. Science. 2000; PubMed Scopus Google Scholar). modification is by changes in and in and germ cell during development M. 1995; 17: PubMed Scopus Google Scholar). DNA patterns have been shown to be caused by protein and R.A. Am. J. Clin. Nutr. 2000; PubMed Scopus Google Scholar). Nutritional alterations early in life DNA patterns leading to altered levels of gene expression in In because the altered DNA patterns in cells are to the cells by the are (Fig. The “pup in a cup” rat model is in that it permanently the metabolism of an adult rat by the composition of the milk fed to the animal during its suckling In this of metabolic programming is to the generation by the It is evident from our studies with rats that the and the of the dietary treatment the onset of in the adult that major metabolic in such as and II to hyperinsulinemia as the primary in metabolic programming in the HC rat. In a Dietz W.H. Marks J. Am. Med. 2000; PubMed Scopus Google Scholar) have that the of is a critical health in the of is Because there has not been a significant change in the gene in the in Dietz W.H. Marks J. Am. Med. 2000; PubMed Scopus Google Scholar), it is that to are in the increased in this There has also been a increase in the of in the and this has been correlated with the increase in the of suggesting that is a major risk factor for chronic Dietz W.H. Marks J. Am. Med. 2000; PubMed Scopus Google Scholar). It is that the of chronic are not to only life The results from the HC rat model suggest that nutritional experiences of during the immediate postnatal life such as of formula and early of weaning high in contribute to metabolic programming leading to like and The of metabolic programming an to the of chronic We W. Hanson of for and for critical of the and of this for on the