The Journal of Nutrition Vol. 127 No. 3 March 1997, pp. 403-410
Copyright ©1997 by the American Society for Nutritional Sciences

Glucose-Induced Insulin Secretion Is Impaired and Insulin-Induced Phosphorylation of the Insulin Receptor and Insulin Receptor Substrate-1 Are Increased in Protein-Deficient Rats¹

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

INTRODUCTION

MATERIAL AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

LITERATURE CITED

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 dose-response 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.

INTRODUCTION

Nutritional deficiency has been implicated as one of the pathogenic factors involved in J-type (Hugh-Jones 1955) or malnutrition-related diabetes (according to the 1985 WHO classification of diabetes). This rare type of diabetes occurs in tropical, developing countries and is related to dietary protein deficiency (Abu-Bakare et al. 1986). The clinical features of the syndrome include incidence in lean subjects, early onset, rare development of ketosis and requirement of large amounts of insulin for achieving blood glucose control (Bajaj 1986, Rao 1988, Rao et al. 1983). Protein deficiency may lead to a disruption in the proper function of the pancreatic -cell or render it susceptible to a viral or autoimmune assault (Rao 1988). Chronic malnutrition leading to pancreatitis is also involved in the pathogenesis of tropical diabetes, a syndrome characterized by exocrine and endocrine pancreatic insufficiency (Rao 1988). Although malnutrition may be associated with an increasing incidence of diabetes in several developing countries, little is known about its pathogenic mechanisms.

Protein deficiency is associated with a decreased glucose tolerance and reduced insulin secretion (Smith et al. 1975, Weinkove et al. 1977). Recently, we have demonstrated impaired glucose-induced insulin secretion and Ca²⁺ uptake by isolated pancreatic islets from rats fed a low protein diet (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 in the early steps of insulin signaling in muscle and liver respond with an increased phosphorylation after insulin treatment in vivo.

---

MATERIAL AND METHODS

Antibodies and chemicals. Antisera against the insulin receptor substrate-1 (IRS-1)³ and the insulin receptor (IR) were previously described (Sun et al. 1991). Antibodies against the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were obtained from Santa Cruz (Santa Cruz, CA). Antibodies against phosphotyrosine were from UBI (Lake Placid, NY). ¹²⁵I-Protein A was from Amersham (Buckinghamshire, UK). Protein A Sepharose 6MB was from Pharmacia (Uppsala, Sweden). Chemicals were from Sigma (St. Louis, MO).

Buffers. Buffer A consisted of 100 mmol/L Tris, 10 g/L SDS, 50 mmol/L HEPES (pH 7.4), 100 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 10 mmol/L EDTA and 10 mmol/L sodium vanadate. Buffer B was similar to buffer A except that 10 g/L Triton X-100 replaced 10 g/L SDS, and 2 mmol/L phenylmethylsulfonyl fluoride (PMSF) and 0.1 mg/mL aprotinin were added. Buffer C contained 100 mmol/L Tris, 10 mmol/L sodium vanadate, 10 mmol/L EDTA and 10 g/L Triton X-100.

Animals. Male Wistar rats (28 d old, 90-100 g), bred at the animal facilities of the University of Campinas, were distributed into two groups and maintained for 8 wk on isocaloric diets containing 6% protein (low protein diet) or 17% protein (control diet) as described in Table 1. At the end of the experimental period, the nutritional status of the rats was evaluated by the determination of their body weight, total serum protein (Lowry et al. 1951), serum albumin (Doumas et al. 1971), serum glucose (Nogueira et al. 1990), plasma free fatty acids (Falholt et al. 1983) and liver glycogen content (Hassid and Abrahan 1957). Rats were anesthetized with sodium amobabital (15 mg/kg body weight) and used in the experiments as soon as anesthesia was assured by loss of pedal and corneal reflexes. Blood and tissue samples were collected as previously described (Saad et al. 1995b, Nogueira et al. 1990). A group of rats was continuously fed a low protein diet until mean weight was similar to that of controls (these rats were treated with the low protein diet ~4 wk longer, until reaching the same mean weight of rats fed the control diet for 8 wks), and then used in paired experiments with 12-wk-old controls.

Table 1. Composition of control (17% protein) and low protein (6% protein) diets [View Table]

All experiments involving animals were approved by the University of Campinas ethical committee.

Glucose-tolerance test. Oral glucose-tolerance test (OGTT) was performed when rats were 12 wk old, following 8 wk of diet treatment, and after 15 h of food deprivation. Glucose (400 g/L in water) was introduced into the stomach of the rats through a gastric catheter at a final dose of 2 g/kg body weight. Blood samples were collected at 0, 30, 60, 90, 120 and 180 min. Serum glucose concentration was determined by the glucose oxidase method (Nogueira et al. 1990). Results are expressed in millimoles per liter. Serum insulin levels were determined by RIA as previously described (Eizirik et al. 1994).

Islet isolation and insulin secretion. Islets were isolated by hand-picking after collagenase digestion of the pancreas, following a technique previously described (Boschero et al. 1995). After isolation, 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 incubator with 5% CO₂. 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 supernatant of each group of islets was determined by RIA as previously described (Eizirik et al. 1994).

Tissue extraction, immunoblot and immunoprecipitation. In vivo exposition of the hind-limb muscle (Musculus gastrocnemius) to isovolumetric (500 µL) solutions containing either insulin (10⁵ mol/100 g body weight) or saline was performed by abdominal cava vein injection. A fragment of the muscle was excised after 90 s and immediately homogenized in freshly prepared boiling buffer A for immunobloting, or freshly prepared ice-cold buffer B for immunoprecipitations. Insoluble material was removed by centrifugation for 45 min at 50,000 × 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 were incubated with 15 µL of antisera anti-IRS-1 or anti-IR at 4°C overnight, followed by the addition of Protein A Sepharose 6MB for 1 h. The pellets were repeatedly washed in buffer C (five times), resuspended in 50 µL of Laemmli sample buffer, and boiled for 5 min prior to loading onto the gel. For immunoblotting, samples of 150 µg of total protein were suspended in 50 µL of Laemmli sample buffer and boiled for 5 min before loading onto 6% SDS-PAGE in a miniature slab gel apparatus (Bio-Rad, Richmond, CA). Electrotransfer, blotting and signal detection were as previously described (Saad et al. 1995a and 1995b, Velloso et al. 1993).

Statistics. Results are presented as means ± SEM followed by the number of rats per experimental condition (n). When working with islets, n refers to the number of groups of islets (200 islets per group) per experimental condition. Student's t tests for paired data were used for evaluating direct comparisons between rats fed a low protein diet and rats fed a control diet. When analyzing two variables as in dose-response curves, the method of least squares (Colton 1974) was employed.

---

RESULTS

Characteristics of the experimental animals. At the completion of the 8-wk treatment period, there were significant differences between the control and low protein diet groups in total body weight, 248 ± 36 vs. 132 ± 42 g, respectively (P < 0.05, n = 16), liver glycogen concentration, 0.7 ± 0.4 vs. 1.3 ± 0.7 mg/100 g, respectively (P < 0.05, n = 10), total serum albumin, 34.6 ± 0.7 vs. 32.7 ± 1.4 g/L, respectively (P < 0.05, n = 10), serum insulin levels of food-deprived rats and rats allowed free access to food, 170 ± 23 and 351 ± 109 pmol/L vs. 129 ± 32 and 121 ± 36 pmol/L, respectively (P < 0.05, n = 10). No significant differences were detected in glucose concentrations, plasma free fatty acids, total serum protein levels and total food intake of food-deprived rats.

Glucose-induced insulin secretion. Islets isolated from rats fed the control or low diet showed an S-shaped pattern of glucose-induced insulin secretion. The comparison of insulin accumulation in the supernatants of islets exposed to increasing concentrations of glucose consistently revealed a significantly 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, pancreatic islets isolated from rats fed a low protein diet have an impaired insulin secretion under both glucose-stimulated and basal conditions.

---

Fig. 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 = 6; *significantly different from controls P < 0.05. 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 protein-deprived rats (P < 0.05).
[View Larger Version of this Image (28K GIF file)]

---

OGTT. No major differences were detected in peripheral glucose homeostasis between the two groups when tested by a regular OGTT (Fig. 2a). During the OGTT, the serum insulin concentrations were significantly lower in the protein malnourished rats than in the controls (Fig. 2b).

---

Fig. 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 = 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 = 5; *significantly different from controls P < 0.05.
[View Larger Version of this Image (25K GIF file)]

---

Effect of a low protein diet on IR and IRS-1 phosphorylation in response to insulin in rat muscle. In vivo stimulation with insulin induced the phosphorylation of proteins present in at least two distinct bands in immunoblots from rat muscle total extracts (Fig. 3a). The upper band migrating at 165-185 kDa corresponds partially to the intracellular substrate of the IR, the IRS-1 (White et al. 1985). The lower band migrating at 95 kDa corresponds to the  subunit of the IR (Kasuga et al. 1982). By densitometric scanning, greater insulin-induced phosphorylation of both bands was detected in the rats fed the low protein diet. Thus, there were 7.1- and 4.9-fold increases above basal (saline-stimulated rats) for the 165-185 kDa and 95 kDa bands, respectively, in rats fed the low protein diet, whereas in the control rats, the stimulation by insulin induced increases in phosphorylation corresponding to 4.3- and 3.6-fold above basal for the 165-185 kDa and 95 kDa bands, respectively, (Fig. 3a). Reblot of filters with anti-IRS-1 and anti-IR antibodies confirmed the identity of the bands and showed no differences in the amount of the specific proteins between the two groups of rats, after either saline or insulin treatment (Fig. 3b and 3c).

---

Fig. 3. Fluorographs of SDS-PAGE of total extracts of hind-limb muscle from rats fed control (C) or low protein (LP) diet after saline () or insulin (+) infusion through the vena cava. Insulin stimulated the tyrosine phosphorylation of proteins in bands migrating at 165-185 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 165-185 kDa phosphorylated band corresponds at least in part to the IRS-1, whereas the 95 kDa band corresponds to the  subunit of the IR. The insulin-induced phosphorylation of the 165-185 kDa band was 50% higher in muscle from rats fed the low protein diet compared with control rats (n = 6; *significantly different than controls, P < 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 = 6; **significantly different than controls, P < 0.001) (a). No differences were detected in the total content of IRS-1 and IR between the two groups studied (b and c).
[View Larger Version of this Image (38K GIF file)]

---

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 9.2- 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-fold above basal (Fig. 4a,b). Moreover, greater insulin-stimulated IRS-1-p85/PI 3-kinase association was detected in the muscle of rats fed the low protein diet compared with that from rats fed the control diet (Fig. 4c). Thus, after insulin stimulation, there was a 8.3-fold increase in IRS-1-p85 association in the muscle of low protein diet-fed rats, whereas in the control diet-fed rats the association of IRS-1 with p85 was only 6.8-fold above basal (Fig. 4c).

---

Fig. 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 = 8; *significantly different than controls, P < 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 = 6; **significantly different than controls, P < 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 = 6; ***significantly different than controls, P = 0.05) (c).
[View Larger Version of this Image (45K GIF file)]

---

Although skeletal muscle represents the main tissue involved in the clearance of serum glucose, we also performed experiments with liver total extracts and liver homogenate IR and IRS-1 immunoprecipitates. Similar in the liver to what was found in the hind-limb muscle, there were no major differences in the amount of IR and IRS-1 protein between the malnourished and control rats; however, the rate of IR and IRS-1 phosphorylation as well as the IRS-1-p85 association after insulin stimulation was greater in rats fed the low protein diet than in those fed the control diet (data not shown).

To exclude a bias due to differences in final weight between control and experimental groups, 10 rats were fed the protein-deficient diet for 12 wk until reaching a mean weight similar to that of controls treated for 8-wk. The amount of IR and IRS-1 and the rate of IR and IRS-1 phosphorylation in response to insulin, as well as the rate of IRS-1-p85 association, were identical to those described in the original set of experiments for rats fed the low protein diet.

---

DISCUSSION

Several lines of evidence suggest that malnutrition early in life or low birthweight may be associated with glucose intolerance. In Hertfordshire, England, noninsulin-dependent diabetes mellitus (NIDDM) or impaired glucose tolerance was correlated with low birthweight, or low weight at 1 y of age (Phillips et al. 1994). This association was present only when babies were small for their gestational age, and not when they were born prematurely (Phipps et al. 1993). Data from the Pima Indians and from Hispanic Americans confirmed this correlation (Athens et al. 1993, McCance et al. 1993). In addition, chronic malnutrition has been associated with the development 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).

Little is known about the pathways leading to malnutrition-related diabetes. Several studies have focused on the function of the pancreatic -cell in malnourished subjects or animal models of malnutrition-induced diabetes. According to Phillips 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 groups of undernourished subjects with diabetes and obese subjects with diabetes, concluded that -cell dysfunction played a major role in glucose intolerance, in agreement with most of the studies in both humans and animal models of malnutrition-induced diabetes (Crace et al. 1990, Okitolonda et al. 1987 and 1988). Apparently, the morphology of the pancreatic islet is changed in animal models of malnutrition (Okitolonda et al. 1988, Rao 1990). The diminished number of -cells per islet and the decreased insulin levels per -cell may be some of the factors influencing the hypoinsulinemia observed in protein malnutrition.

On the other hand, few studies of peripheral insulin action in malnourished diabetic subjects or animal models exist. Thus, Okitolonda and co-workers (1987) confirmed the impaired -cell function in malnourished rats, but found only a mildly diminished glucose tolerance, probably related to an increased peripheral sensitivity to insulin. The same group further demonstrated the linkage between the change in glucose homeostasis and protein deprivation (Okitolonda et al. 1988). In a recent study, Rao (1995) showed that the equilibrium in peripheral glucose concentration observed in malnourished subjects is at least in part controlled by the sum of changes occurring in both the rate of secretion and peripheral action of the main glucose regulatory and counterregulatory hormones, namely, insulin and glucagon.

In this study, we used an animal model of malnutrition that matches all of the metabolic variables of most of the models presented previously (Carneiro et al. 1995, Crace et al. 1990, Heard et al. 1977, Okitolonda et al. 1987 and 1988, Rao 1995, Weinkove et al. 1977). Although demonstrating normal glucose levels after food deprivation and unchanged response to an OGTT, the rats fed the low protein diet presented lower levels of serum insulin than the control rats during the OGTT. This was further confirmed by the detection of glucose-induced insulin secretion in isolated islets. The mechanism by which malnutrition decreases glucose-induced insulin secretion is not completely known but may be related to a defect in the ability of glucose to increase Ca²⁺ uptake and/or to reduce Ca²⁺ efflux from the -cell (Carneiro et al. 1995).

The low level of insulin secretion during the OGTT, in spite of serum glucose levels similar to controls, suggested an increase in peripheral insulin sensitivity in the protein-malnourished rats. As previously mentioned, Okitolonda and co-workers (1987), had similar conclusions. Because the molecular mechanisms responsible for the observed increase were not established, we decided to analyze the effect of insulin on the phosphorylation of the IR and IRS-1, as well as on the rate of IRS-1-PI 3-kinase association after insulin stimulation in hind-limb muscle of malnourished rats. Although the amounts of IR and IRS-1 protein were similar in the muscles of the rats fed the low protein or the control diet, the insulin-induced phosphorylation of the -subunit of the IR and of its main intracellular substrate was greater in the malnourished rats. These phenomena were accompanied by an increase in the association of IRS-1 with the p85 subunit of the lipid-metabolizing enzyme PI 3-kinase in rats fed the low protein diet. Similar results were observed in the liver.

Insulin action in target tissues is mediated by the heterotetrameric IR, which is rapidly autophosphorylated on its  subunit in response to insulin binding (Kasuga et al. 1982). The phosphorylation-activation of the IR engages the intracellular proteins IRS which act as docking proteins for src homology-2 (SH2) domain-containing proteins (Myers and White 1995, White et al. 1985). The SH2 proteins are the link between upstream tyrosine kinases and downstream signaling elements (Koch et al. 1991). One of the substrates of the activated IRS proteins is the lipid metabolizing enzyme, PI 3-kinase (Sun et al. 1991). In addition to its roles in the regulation of mitogenesis, cellular transformation and differentiation, chemotaxis and membrane ruffling (Myers and White 1995), the activation of PI 3-kinase is involved in insulin-stimulated glucose uptake by peripheral tissues (Okada et al. 1994). Thus, the translocation of GLUT 4 from its intracellular pool to the cell surface in muscle and adipose tissue is dependent on PI 3-kinase activation and may be inhibited in the presence of wortmannin, an inhibitor of PI 3-kinase (Okada et al. 1994). Hence, at least in part, the pathway involving the IR, the IRS proteins and PI 3-kinase plays a role in glucose clearance. Therefore, our findings suggest that one of the mechanisms responsible for glucose homeostasis observed in the protein-malnourished rat may be an increased activity of elements involved in the early steps of insulin action in target tissues.

FOOTNOTES

Manuscript received 8 April 1996. Initial reviews completed 21 May 1996. Revision accepted 6 November 1996.

LITERATURE CITED

• Abu-Bakare A., Taylor R., Gill G. V., Alberti K.G.M.M. Tropical or malnutrition-related diabetes: a real syndrome? Lancet 1986; 1:1135-1138 [Medline]
• Athens, M., Valdez, R. & Stern, M. (1993) Effect of birthweight on future development of Syndrome X in adult life. Diabetes 42 (Suppl. 1): 61 a (abs.).
• Bajaj, S. J. (1986) Malnutrition-related, ketosis-resistant diabetes mellitus: classification causes and mechanisms. In: World Book of Diabetes in Practice (Krall, L. P., ed.), pp. 276-280. Elselvier Science Publishers, Amsterdam, The Netherlands.
• Boschero, A. C., Szpak-Glasman, M., Carneiro, E. M., Bordin, S., Paul, I., Rojas, E. & Atwater, I. (1995) Oxotremorine-m potentiation of glucose-induced insulin release from rat islets involves M3 muscarinic receptors. Am. J. Physiol. 268: E336-E342.
• Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. 1976; 72:248-254 [Medline]
• Carneiro E. M., Mello M.A.R., Gobatto C. A., Boschero A. C. Low protein diet impairs glucose-induced insulin secretion from and ⁴⁵Ca uptake by pancreatic rat islets. J. Nutr. Biochem. 1995; 6:314-318
• Colton, T. (1974) Regression and correlation. In: Statistics in Medicine (Colton, T., ed.), pp 189-218. Little, Brown and Company, Boston, MA.
• Crace C. J., Swenne I., Kohn P. G., Strain A. J., Milner R.D.G. Protein-energy malnutrition induces changes in insulin sensitivity. Diabetes & Metab. 1990; 16:484-491
• Doumas B. T., Watson W. B., Briggs H. G. Albumin standards and the measurement of serum albumin with bromocresol green. Clin. Chim. Acta 1971; 31:87-96 [Medline]
• Eizirik D. L., Welsh N., Nieman A., Velloso L. A., Malaisse W. J. Succinic acid monomethyl ester protects rat pancreatic islet secretory potential against interleukin-1 (IL-1) without affecting glutamate decarboxylase expression or nitric oxide production. FEBS Lett. 1994; 337:298-302 [Medline]
• Falholt K., Loud B., Falholt W. An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chim. Acta 1983; 46:105-111
• Hassid W. Z., Abrahan S. Chemical precedures for analysis of polisaccharides. Methods Enzymol. 1957; 3:34-36
• Heard C.R.C., Franji S. M., Wright P. M., McCartney P. R. Biochemical characteristics of different forms of malnutrition: an experimental model using young rats. Br. J. Nutr. 1977; 37:1-21 [Medline]
• Hugh-Jones P. Diabetes in Jamaica. Lancet 1955; 2:891-897
• Kasuga, M., Karlsson, F. A. & Kahn, C. R. (1982) Insulin stimulates the phosphorylation of the 95,000 dalton subunit of its own receptor. Science (Washington, DC) 215: 185-187.
• Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. & Pawson, T. (1991) SH2 and SH3 domains: elements that control interactions of cytoplasmic signalling proteins. Science (Washington, DC) 252: 668-674.
• Lowry O. H., Rosenbrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193:265-275 [Free Full Text]
• McCance, D. R., Pettitt, D. J., Hanson, R. L., Jacobsson, L.T.H., Knowler, W. C. & Bennett, P. H. (1993) Low birthweight and type 2 diabetes in Pima Indians. Diabetologia 36 (Suppl 1.):A4 (abs.).
• Myers M. G. Jr., White M. F. New frontiers in insulin receptor substrate signalling. Trends Endocrinol. Metab. 1995; 6:209-215
• Nogueira, D. M., Strufaldi, B., Hirata, M. H., Abdalla, D.S.P. & Hirata, R.D.C. (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. 153-168. Pancast, So Paulo, Brazil.
• Okada T., Kawano Y., Sakakibara T., Hazeki O., Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes: studies with a selective inhibitor wortmannin. J. Biol. Chem. 1994; 269:3568-3573 [Abstract/Free Full Text]
• Okitolonda W., Brichard S. M., Henquin J. C. Repercussions of chronic protein-calorie malnutrition on glucose homeostasis in the rat. Diabetologia 1987; 30:946-951 [Medline]
• Okitolonda W., Brichard S. M., Pottier A. M., Henquin J. C. Influence of low- and high-protein diets on glucose homeostasis in the rat. Br. J. Nutr. 1988; 60:509-516 [Medline]
• Phillips D.I.W., Hirst S., Clark P.M.S., Hales C. N., Osmond C. Fetal growth and insulin secretion in adult life. Diabetologia 1994; 37:592-596 [Medline]
• Phipps K., Barker D.J.P., Hales C. N., Fall C.H.D., Osmond C., Clark P.M.S. Fetal growth and impaired glucose tolerance in men and women. Diabetologia 1993; 36:225-228 [Medline]
• Rao R. H. Diabetes in the undernourished: coincidence or consequence? Endocrine Rev. 1988; 9:67-87 [Medline]
• Rao R. H. Chronic undernutrition may accentuate the  cell dysfunction of type 2 diabetes. Diabetes Res. Clin. Pract. 1990; 8:125-130 [Medline]
• Rao, R. H. (1995) Fasting glucose homeostasis in the adaptation to chronic nutritional deprivation in rats. Am. J. Physiol. 268: E873-E879.
• Rao R. H., Vigg B. L., Rao K.S.J. Suppressible glucagon secretion in young, ketosis-resistant type J diabetics in India. Diabetes 1983; 32:1163-1169
• Reeves P. G., Nielsen F. H., Fahey G. C. Jr. AIN-93 purified diets for laboratory rodents: report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 1993; 123:1939-1951
• Saad M.J.A., Hartmann L.G.C., Carvalho D. S., Galoro C.A.O., Brenelli S. L., Carvalho C.R.O. Effect of glucagon on insulin receptor substrate-1 (IRS-1) phosphorylation and association with phosphatidylinositol 3-kinase (PI 3-kinase). FEBS Lett. 1995a; 370:131-134 [Medline]
• Saad M.J.A., Velloso L. A., Carvalho C.R.O. Angiotensin II induces tyrosine phosphorylation of insulin receptor substrate 1 and its association with phosphatidylinositol 3-kinase in rat heart. Biochem. J. 1995b; 310:741-744
• Smith S. P., Edgar P. J., Pozefsky T., Cheetri M. K., Prout T. E. Insulin secretion and glucose tolerance in adults with protein-calorie malnutrition. Metab. Clin. Exp. 1975; 24:1073-1084
• Sun X. J., Rothemberg P., Kahn C. R., White M. F. The structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature (Lond.) 1991; 352:73-77 [Medline]
• Velloso L. A., Kämpe O., Hallberg A., Christmanson L., Betsholtz C., Karlsson F. A. Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression. J. Clin. Invest. 1993; 91:2084-2090
• Weinkove C., Weinkove E., Timme A., Pimstone B. L. Pancreatic islets in malnourished rats. Arch. Pathol. Lab. Med. 1977; 1012:266-269
• White M. F., Maron R., Kahn C. R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells. Nature (Lond.) 1985; 318:183-186 [Medline]

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences