Biochemical and Molecular Roles of Nutrients

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1 Biochemical and Molecular Roles of Nutrients Glucose-Induced Insulin Secretion Is Impaired and Insulin-Induced Phosphorylation of the Insulin Receptor and Insulin Receptor Substrate-1 Are Increased in Protein-Deficient Rats 1 Marise A. B. Reis, Everardo M. Carneiro,* Maria A. R. Mello,* A. Carlos Boschero, Mario J. A. Saad and Licio A. Velloso 2 Department of Physiology and Biophysics, University of Campinas (UNICAMP), Brazil; *Department of Physical Education, State University of São Paulo (UNESP), Brazil; and Department of Internal Medicine, University of Campinas (UNICAMP), Brazil ABSTRACT Malnutrition is related to diabetes in tropical countries. In experimental animals, protein deficiency may affect insulin secretion. However, the effect of malnutrition on insulin receptor phosphorylation and further intracellular signaling events is not known. Therefore, we decided to evaluate the rate of insulin secretion and the early molecular steps of insulin action in insulin-sensitive tissues of an animal model of protein deficiency. Pancreatic islets isolated from rats fed a standard (17%) or a low (6%) protein diet were studied for their secretory response to increasing concentrations of glucose in the culture medium. Basal as well as maximal rates of insulin secretion were significantly lower in the islets isolated from rats fed a low protein diet. Moreover, the doseresponse curve to glucose was significantly shifted to the right in the islets from malnourished rats compared with islets from control rats. During an oral glucose tolerance test, there were significantly lower circulating concentrations of insulin in the serum of rats fed a low protein diet in spite of no difference in serum glucose concentration between the groups, suggesting an increased peripheral insulin sensitivity. Immunoblotting and immunoprecipitation were used to study the phosphorylation of the insulin receptor and the insulin receptor substrate-1 as well as the insulin receptor substrate-1-p85 subunit of phosphatidylinositol 3-kinase association in response to insulin. Values were greater in hind-limb muscle from rats fed a low protein diet compared with controls. No differences were detected in the total amount of protein corresponding to the insulin receptor or insulin receptor substrate-1 between muscle from rats fed the two diets. Therefore, we conclude that a decreased glucose-induced insulin secretion in pancreatic islets from protein-malnourished rats is responsible, at least in part, for an increased phosphorylation of the insulin receptor, insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase. These might represent some of the factors influencing the equilibrium in glucose concentrations observed in animal models of malnutrition and undernourished subjects. J. Nutr. 127: , KEY WORDS: insulin insulin receptor substrate-1 phosphatidylinositol 3-kinase rats protein malnutrition Nutritional deficiency has been implicated as one of the assault (Rao 1988). Chronic malnutrition leading to pancrepathogenic factors involved in J-type (Hugh-Jones 1955) or atitis is also involved in the pathogenesis of tropical diabetes, malnutrition-related diabetes (according to the 1985 WHO a syndrome characterized by exocrine and endocrine pancre- classification of diabetes). This rare type of diabetes occurs atic insufficiency (Rao 1988). Although malnutrition may in tropical, developing countries and is related to dietary be associated with an increasing incidence of diabetes in protein deficiency (Abu-Bakare et al. 1986). The clinical several developing countries, little is known about its patho- features of the syndrome include incidence in lean subjects, genic mechanisms. early onset, rare development of ketosis and requirement of Protein deficiency is associated with a decreased glucose large amounts of insulin for achieving blood glucose control tolerance and reduced insulin secretion (Smith et al. 1975, (Bajaj 1986, Rao 1988, Rao et al. 1983). Protein deficiency Weinkove et al. 1977). Recently, we have demonstrated may lead to a disruption in the proper function of the pancreby isolated pancreatic islets from rats fed a low protein diet impaired glucose-induced insulin secretion and Ca 2/ uptake atic b-cell or render it susceptible to a viral or autoimmune (Carneiro et al. 1995). In the present report, we show that islets from rats fed a low protein diet have an impaired secretory response to glucose, and that elements involved 1 The costs of publication of this article were defrayed in part by the payment in the early steps of insulin signaling in muscle and liver of page charges. This article must therefore be hereby marked advertisement in accordance with 18 USC section 1734 solely to indicate this fact. respond with an increased phosphorylation after insulin 2 To whom correspondence should be addressed. treatment in vivo /97 $ American Society for Nutritional Sciences. Manuscript received 8 April Initial review completed 21 May Revision accepted 6 November

2 404 REIS ET AL. TABLE 1 0, 30, 60, 90, 120 and 180 min. Serum glucose concentration was determined by the glucose oxidase method (Nogueira et al. 1990). Composition of control (17% protein) and low protein Results are expressed in millimoles per liter. Serum insulin levels were determined by RIA as previously described (Eizirik et al. 1994). (6% protein) diets Islet isolation and insulin secretion. Islets were isolated by handpicking Composition after collagenase digestion of the pancreas, following a tech- nique previously described (Boschero et al. 1995). After isolation, Ingredient Control diet Low protein diet the islets were pre-incubated in Krebs-bicarbonate solution containing 5.6 mmol/l glucose for 30 min at 37 C in a humidified air g/kg incubator with 5% CO 2. The solution was then replaced by fresh buffer, and the islets were further incubated for 60 min under the various experimental conditions. The insulin concentration in the Casein supernatant of each group of islets was determined by RIA as pre- Cornstarch viously described (Eizirik et al. 1994). Sucrose Fiber Tissue extraction, immunoblot and immunoprecipitation. In Dextrinized cornstarch vivo exposition of the hind-limb muscle (Musculus gastrocnemius) to L-Cystine isovolumetric (500 ml) solutions containing either insulin (10 05 mol/ Soybean oil g body weight) or saline was performed by abdominal cava vein Mineral mix (AIN-93G-MX) injection. A fragment of the muscle was excised after 90 s and imme- Vitamin mix (AIN-93-VX) diately homogenized in freshly prepared boiling buffer A for immu- Choline chlorhydrate nobloting, or freshly prepared ice-cold buffer B for immunoprecipitations. Insoluble material was removed by centrifugation for 45 min 1Reeves et al at 50,000 1 g at 4 C. Protein concentration in the supernatants was determined by the Bradford method (Bradford 1976). For immunoprecipitations, samples containing 3 mg of total protein MATERIAL AND METHODS were incubated with 15 ml of antisera anti-irs-1 or anti-ir at 4 C overnight, followed by the addition of Protein A Sepharose 6MB Antibodies and chemicals. Antisera against the insulin receptor for 1 h. The pellets were repeatedly washed in buffer C (five times), substrate-1 (IRS-1) 3 and the insulin receptor (IR) were previously de- resuspended in 50 ml of Laemmli sample buffer, and boiled for 5 min scribed (Sun et al. 1991). Antibodies against the p85 subunit of phos- prior to loading onto the gel. For immunoblotting, samples of 150 phatidylinositol 3-kinase (PI 3-kinase) were obtained from Santa Cruz mg of total protein were suspended in 50 ml of Laemmli sample (Santa Cruz, CA). Antibodies against phosphotyrosine were from UBI buffer and boiled for 5 min before loading onto 6% SDS-PAGE in (Lake Placid, NY). 125 I-Protein A was from Amersham (Buckingham- a miniature slab gel apparatus (Bio-Rad, Richmond, CA). Electroshire, UK). Protein A Sepharose 6MB was from Pharmacia (Uppsala, transfer, blotting and signal detection were as previously described Sweden). Chemicals were from Sigma (St. Louis, MO). (Saad et al. 1995a and 1995b, Velloso et al. 1993). Buffers. Buffer A consisted of 100 mmol/l Tris, 10 g/l SDS, 50 Statistics. Results are presented as means { SEM followed by the mmol/l HEPES (ph 7.4), 100 mmol/l sodium pyrophosphate, 100 number of rats per experimental condition (n). When working with mmol/l sodium fluoride, 10 mmol/l EDTA and 10 mmol/l sodium islets, n refers to the number of groups of islets (200 islets per group) vanadate. Buffer B was similar to buffer A except that 10 g/l Triton per experimental condition. Student s t tests for paired data were used X-100 replaced 10 g/l SDS, and 2 mmol/l phenylmethylsulfonyl for evaluating direct comparisons between rats fed a low protein diet fluoride (PMSF) and 0.1 mg/ml aprotinin were added. Buffer C con- and rats fed a control diet. When analyzing two variables as in dosetained 100 mmol/l Tris, 10 mmol/l sodium vanadate, 10 mmol/l response curves, the method of least squares (Colton 1974) was em- EDTA and 10 g/l Triton X-100. ployed. Animals. Male Wistar rats (28 d old, g), bred at the animal facilities of the University of Campinas, were distributed into RESULTS two groups and maintained for 8 wk on isocaloric diets containing 6% protein (low protein diet) or 17% protein (control diet) as described Characteristics of the experimental animals. At the comtional in Table 1. At the end of the experimental period, the nutri- pletion of the 8-wk treatment period, there were significant status of the rats was evaluated by the determination of their differences between the control and low protein diet groups body weight, total serum protein (Lowry et al. 1951), serum albumin in total body weight, 248 { 36 vs. 132 { 42 g, respectively (Doumas et al. 1971), serum glucose (Nogueira et al. 1990), plasma (P õ 0.05, n Å 16), liver glycogen concentration, 0.7 { free fatty acids (Falholt et al. 1983) and liver glycogen content (Has- 0.4 vs. 1.3 { 0.7 mg/100 g, respectively (P õ 0.05, n Å sid and Abrahan 1957). Rats were anesthetized with sodium amobabital (15 mg/kg body weight) and used in the experiments as soon as 10), total serum albumin, 34.6 { 0.7 vs { 1.4 g/l, anesthesia was assured by loss of pedal and corneal reflexes. Blood respectively (P õ 0.05, n Å 10), serum insulin levels of and tissue samples were collected as previously described (Saad et al. food-deprived rats and rats allowed free access to food, b, Nogueira et al. 1990). A group of rats was continuously fed { 23 and 351 { 109 pmol/l vs. 129 { 32 and 121 { 36 a low protein diet until mean weight was similar to that of controls pmol/l, respectively (P õ 0.05, n Å 10). No significant (these rats were treated with the low protein diet Ç4 wk longer, until differences were detected in glucose concentrations, plasma reaching the same mean weight of rats fed the control diet for 8 wks), free fatty acids, total serum protein levels and total food and then used in paired experiments with 12-wk-old controls. intake of food-deprived rats. All experiments involving animals were approved by the Univer- Glucose-induced insulin secretion. Islets isolated from rats sity of Campinas ethical committee. Glucose-tolerance test. Oral glucose-tolerance test (OGTT) was fed the control or low diet showed an S-shaped pattern of performed when rats were 12 wk old, following 8 wk of diet treatment, glucose-induced insulin secretion. The comparison of insulin and after 15 h of food deprivation. Glucose (400 g/l in water) was accumulation in the supernatants of islets exposed to increasintroduced into the stomach of the rats through a gastric catheter at ing concentrations of glucose consistently revealed a signifi- a final dose of 2 g/kg body weight. Blood samples were collected at cantly lower secretory response of the islets from rats fed a low protein diet (Fig. 1). Moreover, there was a significant shift to the right in the slope of the S-shaped curve (Fig. 1). Thus, 3 Abbreviation used: IR, insulin receptor; IRS-1, insulin receptor substrate-1; pancreatic islets isolated from rats fed a low protein diet have NIDDM, noninsulin-dependent diabetes mellitus; OGTT, oral glucose tolerance test; PI 3-kinase, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; SH2, src homology-2 and basal an impaired insulin secretion under both glucose-stimulated conditions.

3 INSULIN SECRETION AND ACTION IN PROTEIN MALNUTRITION 405 FIGURE 1 Glucose-induced insulin secretion by pancreatic islets isolated from rats fed a control or low protein diet. The comparison between islets isolated from control and protein-deprived rats shows a dose-independent impairment, and a shift to the right in the secretory response in the islets from the rats fed the low protein diet. Values are means { SEM, n Å 6; *significantly different from controls P õ The half-maximal release of insulin occurred at 8.5 mmol/l glucose in the islets isolated from control rats, and at 14.4 mmol/l in the islets isolated form proteindeprived rats (P õ 0.05). OGTT. No major differences were detected in peripheral fold above basal (Fig. 4a,b). Moreover, greater insulin-stimuglucose homeostasis between the two groups when tested by lated IRS-1-p85/PI 3-kinase association was detected in the a regular OGTT (Fig. 2a). During the OGTT, the serum muscle of rats fed the low protein diet compared with that from insulin concentrations were significantly lower in the protein rats fed the control diet (Fig. 4c). Thus, after insulin stimulation, malnourished rats than in the controls (Fig. 2b). there was a 8.3-fold increase in IRS-1-p85 association in the Effect of a low protein diet on IR and IRS-1 phosphorylation muscle of low protein diet fed rats, whereas in the control in response to insulin in rat muscle. In vivo stimulation diet fed rats the association of IRS-1 with p85 was only 6.8- with insulin induced the phosphorylation of proteins present fold above basal (Fig. 4c). in at least two distinct bands in immunoblots from rat muscle Although skeletal muscle represents the main tissue involved total extracts (Fig. 3a). The upper band migrating at 165 in the clearance of serum glucose, we also performed 185 kda corresponds partially to the intracellular substrate of experiments with liver total extracts and liver homogenate IR the IR, the IRS-1 (White et al. 1985). The lower band migrating and IRS-1 immunoprecipitates. Similar in the liver to what at 95 kda corresponds to the b subunit of the IR (Kasuga et was found in the hind-limb muscle, there were no major differ- al. 1982). By densitometric scanning, greater insulin-induced ences in the amount of IR and IRS-1 protein between the phosphorylation of both bands was detected in the rats fed the malnourished and control rats; however, the rate of IR and low protein diet. Thus, there were 7.1- and 4.9-fold increases IRS-1 phosphorylation as well as the IRS-1-p85 association above basal (saline-stimulated rats) for the kda and after insulin stimulation was greater in rats fed the low protein 95 kda bands, respectively, in rats fed the low protein diet, diet than in those fed the control diet (data not shown). whereas in the control rats, the stimulation by insulin induced To exclude a bias due to differences in final weight between increases in phosphorylation corresponding to 4.3- and 3.6- control and experimental groups, 10 rats were fed the protein- fold above basal for the kda and 95 kda bands, deficient diet for 12 wk until reaching a mean weight similar respectively, (Fig. 3a). Reblot of filters with anti-irs-1 and to that of controls treated for 8-wk. The amount of IR and anti-ir antibodies confirmed the identity of the bands and IRS-1 and the rate of IR and IRS-1 phosphorylation in response showed no differences in the amount of the specific proteins to insulin, as well as the rate of IRS-1-p85 association, between the two groups of rats, after either saline or insulin were identical to those described in the original set of experiments treatment (Fig. 3b and 3c). for rats fed the low protein diet. By immunoprecipitation with anti-ir or anti-irs-1 antibodies and immunoblotting with anti-phosphotyrosine antibodies, the insulin-stimulated phosphorylation of IR and IRS-1 were DISCUSSION Several lines of evidence suggest that malnutrition early in life or low birthweight may be associated with glucose intoler- ance. In Hertfordshire, England, noninsulin-dependent diabe and 8.4-fold, respectively, above basal in muscle of the rats fed the low protein diet (Fig. 4a,b), whereas in the muscle of control rats, insulin stimulated phosphorylation 4.9- and 5.6-

4 406 REIS ET AL. FIGURE 2 Serum glucose and serum insulin concentrations during the oral glucose tolerance test (OGTT) in rats fed a control low protein diet. (a) Two g/kg body weight glucose OGTT. No major differences were detected in the serum glucose concentration between the two groups studied suggesting an equilibrium in circulating serum glucose in the protein-deprived rats. Values are means { SEM, n Å 5. (b) Serum insulin concentrations during the OGTT. The levels of insulin were significantly lower in the protein-deficient rats during the OGTT. Values are means { SEM, n Å 5; *significantly different from controls P õ born prematurely (Phipps et al. 1993). Data from the Pima Indians and from Hispanic Americans confirmed this correla- tion (Athens et al. 1993, McCance et al. 1993). In addition, chronic malnutrition has been associated with the develop- tes mellitus (NIDDM) or impaired glucose tolerance was correlated with low birthweight, or low weight at 1yofage(Phillips et al. 1994). This association was present only when babies were small for their gestational age, and not when they were

5 INSULIN SECRETION AND ACTION IN PROTEIN MALNUTRITION 407 FIGURE 3 Fluorographs of SDS-PAGE of total extracts of hind-limb muscle from rats fed control (C) or low protein (LP) diet after saline (0) or insulin (/) infusion through the vena cava. Insulin stimulated the tyrosine phosphorylation of proteins in bands migrating at kda and 95 kda (a). The respective graphic representation of arbitrary scanning units for each band is depicted (a). Reblot of the filter with anti-insulin receptor substrate-1 (IRS-1) (b) or anti-insulin receptor (IR) (c) antibodies shows that the kda phosphorylated band corresponds at least in part to the IRS-1, whereas the 95 kda band corresponds to the b subunit of the IR. The insulin-induced phosphorylation of the kda band was 50% higher in muscle from rats fed the low protein diet compared with control rats (n Å 6; *significantly different than controls, P õ 0.001) (a). The insulin-induced phosphorylation of the 95 kda band was 30% higher in the muscle from rats fed the low protein diet compared with control rats (n Å 6; **significantly different than controls, P õ 0.001) (a). No differences were detected in the total content of IRS-1 and IR between the two groups studied (b and c). Little is known about the pathways leading to malnutritionrelated diabetes. Several studies have focused on the function of the pancreatic b-cell in malnourished subjects or animal models of malnutrition-induced diabetes. According to Phil- lips and co-workers (1994), NIDDM or glucose intolerance in subjects who were malnourished early in life was not due to defective insulin secretion. However, Rao (1990), studying ment of at least two variants of the diabetic syndrome. Type J diabetes is characterized by insulin insufficiency, peripheral insulin resistance and the absence of ketosis (Rao 1988), whereas tropical pancreatic diabetes is present in severe chronic malnutrition with pancreatitis, featuring the insulin insufficiency due to endocrine as well as exocrine, pancreatic destruction (Rao 1988).

6 408 REIS ET AL. FIGURE 4 Fluorographs of SDS-PAGE of immunoprecipitates from hind-limb muscle from rats fed low protein (LP) or control (C) diet. Insulin receptor (IR) immunoprecipitates were blotted with anti-phosphotyrosine antibody, and a 70% increase in the rate of insulin-induced phosphorylation was detected in the muscle of rats fed the low protein diet compared with controls (n Å 8; *significantly different than controls, P õ 0.005) (a). In insulin receptor substrate-1 (IRS-1) immunoprecipitates blotted with anti-phosphotyrosine antibody, a 40% greater rate of insulin-induced phosphorylation of IRS-1 was detected in the muscle of rats fed the low protein diet compared with the control rats (n Å 6; **significantly different than controls, P õ 0.005) (b). IRS-1 immunoprecipitates blotted with anti-p85 antibody showed a 20% greater IRS-1-p85 association after insulin stimulation in the muscle of rats fed the low protein diet compared with controls (n Å 6; ***significantly different than controls, P Å 0.05) (c).

7 INSULIN SECRETION AND ACTION IN PROTEIN MALNUTRITION 409 groups of undernourished subjects with diabetes and obese proteins is the lipid metabolizing enzyme, PI 3-kinase (Sun et subjects with diabetes, concluded that b-cell dysfunction al. 1991). In addition to its roles in the regulation of mitogenesis, played a major role in glucose intolerance, in agreement with cellular transformation and differentiation, chemotaxis and most of the studies in both humans and animal models of membrane ruffling (Myers and White 1995), the activation of malnutrition-induced diabetes (Crace et al. 1990, Okitolonda PI 3-kinase is involved in insulin-stimulated glucose uptake by et al and 1988). Apparently, the morphology of the peripheral tissues (Okada et al. 1994). Thus, the translocation pancreatic islet is changed in animal models of malnutrition of GLUT 4 from its intracellular pool to the cell surface in (Okitolonda et al. 1988, Rao 1990). The diminished number muscle and adipose tissue is dependent on PI 3-kinase activation of b-cells per islet and the decreased insulin levels per b-cell and may be inhibited in the presence of wortmannin, an inhibitor may be some of the factors influencing the hypoinsulinemia of PI 3-kinase (Okada et al. 1994). Hence, at least in part, observed in protein malnutrition. the pathway involving the IR, the IRS proteins and PI 3-kinase On the other hand, few studies of peripheral insulin action plays a role in glucose clearance. Therefore, our findings suggest in malnourished diabetic subjects or animal models exist. that one of the mechanisms responsible for glucose homeostasis Thus, Okitolonda and co-workers (1987) confirmed the impaired observed in the protein-malnourished rat may be an increased b-cell function in malnourished rats, but found only a activity of elements involved in the early steps of insulin action mildly diminished glucose tolerance, probably related to an in target tissues. increased peripheral sensitivity to insulin. The same group further demonstrated the linkage between the change in glucose homeostasis and protein deprivation (Okitolonda et al. LITERATURE CITED 1988). In a recent study, Rao (1995) showed that the equilib- Abu-Bakare, A., Taylor, R., Gill, G. V. & Alberti K.G.M.M. (1986) Tropical or rium in peripheral glucose concentration observed in malnour- malnutrition-related diabetes: a real syndrome? 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(1995) Oxotremorine-m potentiation of glucose-induced presented previously (Carneiro et al. 1995, Crace et al. 1990, insulin release from rat islets involves M3 muscarinic receptors. Am. J. Physiol. Heard et al. 1977, Okitolonda et al and 1988, Rao 268: E336 E342. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of 1995, Weinkove et al. 1977). Although demonstrating normal microgram quantities of protein utilising the principle of protein dye binding. glucose levels after food deprivation and unchanged response Anal. Biochem. 72: to an OGTT, the rats fed the low protein diet presented lower Carneiro, E. M., Mello, M.A.R., Gobatto, C. A., Boschero, A. C. (1995) Low protein diet impairs glucose-induced insulin secretion from and 45 Ca uptake levels of serum insulin than the control rats during the OGTT. by pancreatic rat islets. J. Nutr. Biochem. 6: This was further confirmed by the detection of glucose-induced Colton, T. (1974) Regression and correlation. 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H., Abdalla, D.S.P. & Hirata, R.D.C. phosphorylation-activation of the IR engages the intracellular (1990) Sangue-Parte 1. In: Metodos de Bioquimica Clinica (Nogueira, D. M., Strufaldi, B., Hirata, M. H., Abdalla, D.S.P. & Hirata, R.D.C., eds.), pp. 153 proteins IRS which act as docking proteins for src homology Pancast, Sǎo Paulo, Brazil. (SH2) domain-containing proteins (Myers and White 1995, Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O. & Ui, M. (1994) Essential White et al. 1985). The SH2 proteins are the link between role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes: studies with a selective inhibitor wortmannin. upstream tyrosine kinases and downstream signaling elements J. Biol. Chem. 269: (Koch et al. 1991). One of the substrates of the activated IRS Okitolonda, W., Brichard, S. M. & Henquin, J. C. (1987) Repercussions of

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