Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats
Introduction
Rats fed on diets containing a protein level comparable to that of undernourished humans have decreased glucose-induced insulin secretion, but increased insulin sensitivity in peripheral tissues (Escriva et al., 1991, Escriva et al., 1992, Swenne et al., 1987). Furthermore, islets isolated from undernourished rats show a significant decrease in the insulin secretory response to carbamylcholine (CCh) and phorbol 12-myristate 13-acetate (PMA) (Ferreira et al., 2003). This impairment is partially related to a reduction in pancreatic β-cell mass (Swenne et al., 1992), lower responsiveness to nutrients by the remaining β-cells (Cherif et al., 2001, Escriva et al., 1991, Escriva et al., 1992, Ferreira et al., 2003, Swenne et al., 1992), and a decrease in key enzymes acting in the secretory process (Ferreira et al., 2004, Ferreira et al., 2003). Additionally to reduction of both number and mass of pancreatic islets, acinar pancreatic atrophy is also observed in animal models of protein/energy malnutrition, which resembles anorexia nervosa (Brand and Morgan, 1981, Tang et al., 1997, Wagner et al., 1977). In humans, the available data is limited, but a decrease in pancreatic acinar size, and reduced number of zymogen granuli and golgi complex are observed in patients suffering of AIDS with protein/energy deficit (Chehter et al., 2003), indicating that the same pancreatic features occur in human malnutrition states. These characteristics may be consequence of reduced somatomedin levels (Hashizume et al., 2007) that is observed, for instance, in patients suffering from anorexia nervosa.
Several studies associate the control of gene expression with nutritional signals (Foufelle et al., 1998, Pegorier, 1998, Srinivasan et al., 2001, Towle, 1995, Vaulont and Kahn, 1994). An example of a nutritional signalling molecule is leucine, which has been shown to modulate gene expression in mammalian cells (Fafournoux et al., 2000). The availability of free amino acids in the diet is important in maintaining protein homeostasis, and overall protein deficiency or deficiency of any of the essential amino acids can lead to a negative nitrogen balance (Fafournoux et al., 2000) with consequent changes in the amino acid plasma profile and in the gene expression profile of several tissues (Cetin, 2001, Fafournoux et al., 2000, Harper and Rogers, 1965, Jousse et al., 1999, Kilberg et al., 1994, Peng et al., 1975, Richardson et al., 1965).
Insulin secretion by β-cells is modulated by several factors including metabolic fuels, neurotransmitters released from intra-islet nerve endings, paracrine mechanisms and circulating hormones (Araujo et al., 2002, Satin and Kinard, 1998). Glucose stimulates insulin secretion by increasing the levels of ATP in β-cells as a consequence of energy metabolism. Leucine can stimulate insulin release through two different mechanisms, both of which lead to an increase in ATP levels in β-cells (Xu et al., 2001). The first mechanism is the transamination of leucine to α-ketoisocaproate (KIC) and subsequent mitochondrial oxidation. The second promotes insulin release via allosteric activation of glutamate dehydrogenase (GDH) causing glutamate oxidation to the Krebs cycle intermediate α-ketoglutarate, plus ammonia (Li et al., 2003). The utilisation of leucine by mitochondria in β-cells also activates secondary signals that stimulate mTOR, which subsequently phosphorylates and activates S6K-1, changing protein translation in β-cells (Xu et al., 2001). S6K-1 is a serine threonine kinase that directly phosphorylates ribosomal protein S6, and also regulates the kinase upstream of the translation elongation factor eEF2, which is important for the regulation of translation in mammalian cells (Proud and Denton, 1997). Additionally, there is evidences that insulin by itself controls several physiological parameters in β-cells, including the regulation of protein translation in the cells (Li et al., 2003), through activation of the IR/IRS/PI3K/PKB/mTOR/S6K-1 pathway by an autocrine mechanism. Hence, alterations in S6K-1, coupled to other genetic and environmental factors, may contribute to the development of specific forms of diabetes mellitus (Pende et al., 2000).
Our group has previously shown that protein deficiency in the diet provides a good animal model of impaired glucose-induced insulin secretion (Araujo et al., 2004) and that the malnourishment modulates the expression of several proteins in pancreatic islets (Delghingaro-Augusto et al., 2004, Ferreira et al., 2004, Ferreira et al., 2003). However, none is known about the regulation of S6K-1 enzyme, which acts downstream AKT/mTOR pathway, in pancreatic islets from experimental animal models of malnutrition. Therefore, in this study we examined the effect of glucose and leucine on insulin secretion and on the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K) and S6K-1 proteins in islets isolated from rats fed on low protein diet.
Section snippets
Animals and diet
All experiments described here were approved by the institutional Committee for Ethics in Animal Experimentation. Groups of five male Wistar rats (21-day-old) from the breeding colony at UNICAMP were housed at 24 °C on a 12 h light/dark cycle. The rats were separated at random and maintained on an isocaloric diet containing 6% (low protein diet, LP) or 17% (normal protein diet, NP) protein for 8 weeks. The compositions of the two isocaloric diets are shown in Table 1. At the end of the 8 weeks
Biochemical parameters for the establishment of the malnourished model
To identify whether the malnourishment state was efficiently established, several biochemical parameters were measured. After 8 weeks feeding on low protein diet, the body weight, total serum protein, serum albumin and insulin levels of LP rats were significantly reduced, while the serum FFA level, liver glycogen and fat content were increased compared to NP rats (P < 0.05) (Table 2).
Insulin-secretory responses to glucose and leucine in normal and malnourished rat islets
The effects of glucose on insulin secretion from NP and LP rat islets are shown in Fig. 1. No differences were
Discussion
Rats fed a diet containing low amounts of protein (6%) for 8 weeks display several features similar to those found in malnourished infants and in experimental models of undernourishment, including low body weight, low plasma albumin and insulin levels, and high liver glycogen and fat contents (Table 2). These results are corroborated by previous findings using similar approaches (Claeyssens et al., 1992, Ferreira et al., 2003, Heard, 1978, Heard et al., 1973, Swenne et al., 1987). Insulinaemia
Conclusion
Malnutrition by low protein diet leads to several alterations in pancreatic islets, such as decreased levels of IRS1 and S6K-1 proteins, culminating in lower responsiveness to glucose and to leucine alongside low insulin secretion levels. The reduced glucose metabolism may explain, at least in part, the lower glucose-induced insulin secretion. On the other hand, the lower responsiveness of malnourished rat islets to leucine is not dependent on leucine metabolism, which is maintained in levels
Acknowledgements
The authors thank L. D. Teixeira for technical assistance. This work was supported by the Brazilian foundations FAPESP, CAPES, and CNPq.
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