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Introduction

    As we enter the 21^st century and the new millennium, malnutrition remains the single most important factor impairing health and production of large human populations. In Brazil, 30.7% of the children under 5 years suffer from some type of malnutrition; in the south east region, the more affluent of the country, the prevalence of malnutrition is around 21.7%¹.

    There are a great number of forms of malnutrition; marasmus and kwashiorkor are clinical manifestations of severe protein-calorie malnutrition. Marasmus is characterized by growth arrest, muscular wasting and absence of subcutaneous fat. Kwashiorkor is associated with extreme protein deficiency, which leads to hypoalbuminemia, pitting edema, muscle wasting and enlarged fatty liver. Even the widely prevalent marginal protein-malnutrition results in growth impairment and physiological alterations².

    Poverty, ignorance, infections, cultural customs, climatic conditions and natural and man-made disasters are among the main causes of malnutrition. Therefore, its control and prevention requires multisectional approaches that include food production and distribution, preventive medicine, education, social development and economic improvement However, even if these conditions are achieved, one can not guarantee that the preexisting cases will be cured. Malnutrition has deleterious effects in a great number of organ systems, leading to accentuate metabolic alterations, which may alter food processing when feeding resumes³. Aware of this, it is important the development of studies aiming a better understanding of the physiological alterations imposed by malnutrition. This may offer a contribution in the prevention and control of this disease.

Malnutrition, obesity and diabetes mellitus: the nutrition transition

    The ongoing demographic and developmental transition has brought about marked changes in the profile of malnutrition, especially in the later half of the previous century. This has been especially noticeable in developing countries^4-7. Until approximately 50 years ago, malnutrition was largely considered a problem of the poor. Although undernutrition continues to be a problem of the poor today, especially among children and women in developing countries, there have been evidences of escalation of the incidence of nutrition-related chronic diseases such as obesity and type 2 diabetes mellitus among adults of the relatively affluent sections of developed and developing nations. As a result, developing countries in particular, face a double burden of undernutrition of poor children and women at one end of the socioeconomic spectrum and of malnutrition among the relatively affluent adults at the other end⁸.

    There is a growing evidence that populations of the third world emerging from poverty into affluence with consequent changes in diets and lifestyles, may be more vulnerable to such chronic degenerative diseases^4-7.

    The interesting works of Hales & Baker^3,9 suggest that manifestations of malnutrition at the two ends of the socioeconomic spectrum may indeed be casually and metabolic related. Their observations indicate that intrauterine growth retardation imposed by malnutrition could "program" the fetal tissues to render them to be more vulnerable to nutritional disorders such as syndrome X and chronic degenerative diseases later in adult life. According to their hypothesis, named "the thrifty phenotype hypothesis", nutrition-related chronic diseases of adulthood could be the "late" effect of early fetal malnutrition.

The thrifty phenotype hypothesis

    This hypothesis was proposed to suggest a mechanism to explain the statistical correlation found between low birth weight and subsequent development of type 2 diabetes mellitus and the insulin resistance syndrome^3,9. It was proposed that when a fetus becomes malnourished, it adapts its metabolism to aid both short and long-term survival. However, these adaptations were foreseen as being detrimental for health if the future nourishment was to be adequate or excessive. While fetal and early post-natal malnutrition were considered to increase the risk of developing diabetes mellitus, the onset and the severity of the illness were suggested as being influenced by factors in the adult life, particularly the development of obesity^3,9.

    Protein deficiency may be one important factor mediating the effects pertaining to the thrifty phenotype hypothesis since children who were severely malnourished, show prolonged impairment of insulin secretion and glucose intolerance, even after nutritional recovery^10,11. These findings led Milner¹¹ to suggest that if this defect in insulin secretion is permanent, the previously malnourished children may be predisposed do the development of diabetes mellitus later in life. In human subjects, around half of the adult mass of B pancreatic cells is already present at one year of age¹². Malnutrition prior to this age during the time when B cell are rapidly dividing may therefore cause deficit in B cell number that is irreversible in later life.

Experimental models for the study of glucose homeostasis in malnutrition

    The effects of protein malnutrition have been modeled in rats fed protein-restricted diets. These animal models have provided important insights into the adaptive mechanisms involved in glucose homeostasis in malnutrition, since there are obvious limitations in investigations with human subjects.

    In one rat model¹³, weanling animals were fed a low (5%) protein diet and had a 45% reduction in body weight after 24 weeks of experiment. Serum protein, liver weight and protein content as well as pancreas insulin levels were significantly reduced when compared to controls, whereas liver glycogen store was increased. In this model, the authors did not observe alterations in fasting serum glucose levels, however both basal and glucose stimulated insulin secretion were reduced. During insulin tolerance tests, it was observed a fast decrease in serum glucose and a slow recovery.

    In another model¹⁴, weanling rats also fed a 5% protein diet showed a greater and more prolonged fall in serum glucose in response to insulin injection than control rats after 4 weeks of diet treatment. After 12 weeks, the differences between the two groups disappeared.

    Other study¹⁵ demonstrated that rats submitted to fetal/neonatal protein restriction (8% protein diet) had a better glucose tolerance and lower pancreatic insulin content than normal protein rats. After feeding a cafeteria style diet from the 63^th to the 133^th day of life the previously protein restricted rats showed worse glucose tolerance than control rats.

    On the other hand, an important study was recently performed by Ozanne et al.¹⁶. These authors reported that early programming of weight gain in mice submitted to protein restriction prevents the induction of obesity by a high palatable diet (cafeteria), depending on the age of the animal at which the higher palatable diet is imposed. These results suggest that the early environment has long-term consequences for weight gain. The programmed responses are powerful enough to block excess weight gain from a highly palatable diet and, thus, have major implications for the drug-free regulation of food intake and obesity. The potential implications of these findings for the voluntary drug-free control of weight gain and for the obesity-inducing effects of early nutrition are of great interest and importance.

    During the last 20 years our research group also developed a model of protein malnutrition using rats fed a low protein diet. The normal protein diet contains amounts of carbohydrate (39.7% cornstarch, 13.2% dextrin, and 10% sugar), fat (7.0% soy oil), fibers (5.0% cellulose micro fiber), salt (3.5%), and vitamin mix (1.0%), in accordance with the 1993 recommendations of the American Institute of Nutrition (AIN-G93)¹⁷. The low protein diet contains more carbohydrate (44.4% cornstarch, 17.8% dextrin, and 14.9% sugar) but the same amounts of fat, fibers, salt, and vitamin mix as the balanced standard protein diets¹⁸.

    In our initial studies, we observed that 50 days old rats fed the low protein diet for 3 weeks developed characteristics often seen in the infantile kwashiorkor such as reduced body weight and length, hypoproteinemia, hypoalbuminemia, hypoglycemia and fatty liver¹⁹. These data indicated that the experimental model proved adequate for the study of metabolism in malnutrition. As the protein malnourished rats are very small animals, we had to adapt some laboratory procedures for the conditions of these animals²⁰ before proceeding our investigations focused on glucose homeostasis.

    First, we verified that the protein restricted rats showed lower basal serum glucose and insulin levels than controls and also showed a greater increase in serum glucose during oral glucose tolerance test²¹. In the following evaluations, we noticed impairment pancreatic function in protein restricted pregnant and their newborn offspring. The pregnant protein restricted rat did not show the characteristic pregnancy-induced hyperinsulinemia²¹. The newborn offspring showed low body weight associated to hypoglycemia and hypoinsulinemia¹⁹.

    Together the results obtained using rat models of protein malnutrition indicate that the protein-restricted animal is able to maintain serum glucose at low or normal values in spite of the hypoinsulinism. However, in some cases, the inability to respond to overfeeding may tip the balance towards glucose intolerance and hyperglycemia may occur. Some questions arise from these data:

• Which are the compensatory mechanisms that guarantee glucose homeostasis in the protein restricted rats?

• Which facts may interfere with these mechanisms and allow the development of glucose intolerance and diabetes mellitus?

• Are de alterations in glucose homeostasis imposed by protein malnutrition reversible?

• Is physical exercise beneficial during recovery from impaired glucose homeostasis imposed by protein malnutrition?

    The main results obtained by our group and by others using protein deficient animal models of malnutrition are discussed in the next sections.

Insulin secretion in protein malnourished animals

    Pancreas weight may be reduced in until 50% in animals submitted to protein restriction when compared to controls. However, this difference is not noticed if pancreas weight is expressed as fraction of body weight¹³.

    Morphological characteristics of protein restricted pancreatic islets had also been described. Apparently, there is an initial hypertrophy of the islets which is substituted by gradual atrophy in long-term exposure to protein deficient diets. Not only the volume of the B cells is reduced but also the number of B cells per islet is decreased. Ultrastructual analysis of B cells of protein malnourished rats showed cells with reduced number of secretory granules which appeared pale, indicating low concentration of insulin per granule²².

    Physiological aspects of pancreatic islets are also impaired in rats submitted to protein restriction. Swenne et al.²³ reported reduced insulin secretion by pancreatic islets from animals submitted to short-term protein deprivation. Others have demonstrated that not only glucose-induced insulin-secretion is impaired but also the response to other secretagogues such as amino acids, protein and glucagon^22,24.

    Rats fed our protein deficient diet from weaning to the adult age were also evaluated with respect to glucose-stimulated insulin secretion²⁵. We observed that glucose induced insulin secretion by the protein restricted islets was reduced in the presence of all glucose concentrations tested (2.8 to 33.4 mmol/L). The dose-response curves to glucose of the islets isolated from the protein-restricted animals were also shifted to the right when compared to controls²⁵.

    Aiming a better characterization of the mechanisms involved in the genesis of the impairment in insulin secretion by protein restricted pancreatic islets. We²⁵ evaluated Ca⁺⁺ uptake by isolated islets incubated in presence of different glucose concentrations (2.8, 8.3 and 16.7 mmol/L). Ca⁺⁺ appeared reduced when compared to controls, in all glucose concentrations. This indicates that the impairment in glucose induced insulin secretion by the protein deficient islet may be, at least in part, a consequence of altered calcium handling by these islets.

    After an oral glucose load, we observed that serum glucose levels of protein restricted animals did not differ from those of control animals. However, serum insulin levels were significantly lower in the protein-restricted animals in relation to controls²⁵. These observations clearly indicated that it was necessary to investigate the mechanisms involved in the peripheral response to insulin in protein restricted animals to understand glucose homeostasis in this condition.

Insulin action in protein malnourished animals

    The first step in insulin action at the cellular level is binding of the hormone to its cellular receptor (IR). IR itself sits at the cell membrane and is a tetrameric protein composed by two α-subunits and two β-subunits. Functionally, the IR behaves like a classical allosteric enzyme with a regulatory α-subunit and a catalytic β-subunit. When insulin binds the α-subunit, there is a conformational change in the receptor and the tyrosine-kinase activity present in the β-subunit of the IR is stimulated²⁶. Once activated by insulin binding, the receptor undergoes phosphorylation and initiates a cascade of events which culminate with the final effects of the insulin, metabolic or mitogenic, at the cellular level²⁶.

    In our model, we have investigated the early steps of insulin action in the skeletal muscle, aiming a better understanding of insulin signaling pathways in tissues submitted to protein restriction. We evaluated the ability of insulin to phosphorylate the IR and the insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) and to promote the association of the latter with phosphatidylinositol 3-kinase (PI3-kinase)²⁷. IR, IRS-1, IRS-2 or p85 subunit of PI3-kinase proteins were not altered in the protein restricted animals. On the other hand, IR, IRS-1 and 1RS-2 showed a greater phosphorylation in protein restricted compared to control rats. Greater insulin stimulated IRS-1-P85/PI3kinase association was detected in the protein restricted muscles than in controls²⁷. These results indicate that the increased phosphorylation of the IR and of two of its major substrates (IRS-1 and IRS-2) and also the increased association of the latter with the enzyme PI3-kinase may play an important role in glucose homeostasis maintenance in this animal model.

Insulin secretion and action in protein restricted recovered animals

    In another series of experiments, we evaluated the secretion and the action of insulin in adult rats exposed to protein deficiency during gestation and lactation and examined the influence of nutritional recovery with a balanced diet (AIN-93) on these responses. We investigated the secretion of insulin by isolated pancreatic islets in response to glucose and the ability of insulin to phosphorylate IR and IRS-1 and to promote the association between IRS-1 and PI3-kinase in protein deprived and recovered rats¹⁸. We also examined the effects of insulin on glucose uptake, glycogen synthesis and lactate production by the skeletal muscle of these animals²⁸.

    The basal and the maximal glucose -stimulated insulin secretion by the islets from the protein deficient rats and the basal secretion by the islets of the recovered rats were significantly lower than of control rats. The dose response curves to glucose of islets from protein deficient and recovered rats were shifted to the right to control islets¹⁸.

    The levels of IR as well as IRS-1 and phosphorylation and association between IRS-1 and PI3-kinase were greater in protein restricted animals than in controls¹⁸. Muscle glucose uptake was slightly increased while glycogen synthesis was significantly reduced and lactate production was significantly increased²⁸. In the recovered rats, the variables related to the early steps in insulin intracellular signalization were not significantly different from controls nor from protein restricted rats.

    These results indicate that in our model, glucose homeostasis was not altered in recovered rats. However, even in absence of a significant improvement in insulin secretion when compared protein restricted and recovered rats, there was deterioration in the peripheral response to the hormone.

Nutritional recovery, glucose homeostasis and physical exercise

    It is known that regular physical activity promotes beneficial effects for health, such as increase of the oxidative capacity and of muscle growth, improvement in cardio respiratory condition and bone mineralization facilitation, among others²⁹.

    Exercise can also be beneficial to nutritional recovery. Comparing the growth rhythm of children between two and four years of age, recovering from malnutrition in hospitals, it was observed that active children, participating in games involving moderated energy expenditure, showed higher lean mass indices and higher linear growth compared to children that followed the routine physical activity of the hospitals (sedentary games)³⁰.

    In laboratory animals, the effects of exercise on body growth are dependent on the mechanic properties of exercise and animal species used³¹. The results referring to studies performed with animal models of malnutrition are conflicting. Some studies indicate positive effects of exercise on growth and development^28,32, while others do not demonstrate any effect^33,34. In these studies, different species of animals were used, such as mice³⁴ and rats^28,33,35, different procedures to induce malnutrition, for example, prolonged fasting³⁵ and protein intake restriction^28,33,34,36 and, yet, different protocols of physical exercise, for instance, free swimming³⁴ and swimming supporting overloads of 5% of body weight^28,33. These aspects make complicated the comparison of results.

    There are many epidemiological evidences of chronic degenerative illnesses related to early malnutrition and of the protection supplied not exclusively by balanced diet, but also by healthful habits, including moderated and regular physical activity. Insulin resistance is aggravated by obesity, sedentary-life and aging in a way that pancreas is not able to supply the elevated demand of insulin, and diabetes mellitus is installed. Obesity has an important role in the Thrifty phenotype hypothesis^3,9, as it has already been described. On the other hand, a regular physical activity (active style of life), beginning early in life, may be an important tool in order to prevent the occurrence of obesity and the installation of many other metabolic complications, as the impairment in glucose homeostasis in adult life³⁷.

    Further studies are required in order to clarify the effects of physical exercise on nutritional recovery and glucose homeostasis. In this case, the protocol for malnutrition-induction and for exercise training (ergometer) are important points to be considered.

    Nowadays, in our laboratory, studies are in course aiming the evaluation of the effects of different sources of protein associated or not to aerobic exercise³⁸ on glucose homeostasis during recovery from protein malnutrition, and the preliminary results are quite satisfactory³⁹.

Concluding remarks

    Animal models have provided important insights into the adaptative mechanisms in glucose homeostasis in malnutrition. It has been demonstrated that protein deficient animals are able to maintain serum glucose at low or normal values in spite of being hypoinsulinemic. The hypoinsulinism is a consequence of B cell dysfunction which is not fully reversed by nutritional recovery. Glucose homeostasis is maintained, at least in part, at expense of increased peripheral sensivity to insulin, determined by increase in the initial steps in the hormone signal transition. Physical exercise may be beneficial to the recovery from protein malnutrition but further studies are required in order to clarify its effects on glucose homeostasis.

Acknowledgements

    The authors wish to thank Clarice Y. Sibuya, Eduardo Custódio and José Roberto R. Silva for technical assistance in all researches cited in the present review.

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