Comparative genomics of the vertebrate insulin/TOR signal transduction pathway: A network-level analysis of selective pressures

Complexity of biological function relies on large networks of interacting molecules. However, the evolutionary properties of these networks are not fully understood. It has been shown that selective pressures depend on the position of genes in the network. We have previously shown that in the Drosophila insulin/target of rapamycin (TOR) signal transduction pathway there is a correlation between the pathway position and the strength of purifying selection, with the downstream genes being most constrained. In this study, we investigated the evolutionary dynamics of this well-characterized pathway in vertebrates. More specifically, we determined the impact of natural selection on the evolution of 72 genes of this pathway. We found that in vertebrates there is a similar gradient of selective constraint in the insulin/TOR pathway to that found in Drosophila. This feature is neither the result of a polarity in the impact of positive selection nor of a series of factors affecting selective constraint levels (gene expression level and breadth, codon bias, protein length, and connectivity). We also found that pathway genes encoding physically interacting proteins tend to evolve under similar selective constraints. The results indicate that the architecture of the vertebrate insulin/TOR pathway constrains the molecular evolution of its components. Therefore, the polarity detected in Drosophila is neither specific nor incidental of this genus. Hence, although the underlying biological mechanisms remain unclear, these may be similar in both vertebrates and Drosophila.

Insulin degradation by adipose tissue is increased in human obesity

White adipose tissue samples from obese and lean patients were used for the estimation ofinsulin protease and insulin:glutathione transhydrogenase using 1251-labeled insulin. There was no activity detected in the absence of reduced glutathione, which indicates that insulin is cleaved in human adipose "tissue through reduction of the disulfide bridge between the chains. O bese patients showed higher transhydrogenase activity (per U tissue protein wt, per U tissue wt, and in the total adipose tissue mass) than the lean group. There is a significant correlation between the activity per U tissue wt, and protein and total activity in the whole adipose tissue with respect to body mass index, with a higher activity in obese patients. The potential ofinsulin cleavage by adipose tissue in obese patients was a mean 5.6-fold higher than that in controla. The coexistence of high insulinemia and high cleavage capability implies that insulin secretion and turnover are increased in the o bese. Thus, white adipose tissue may be crucial in the control of energy availability through modulation ofinsulin cleavage.

Rat insulin turnover in vivo

Zucker lean and obese rats were injected under pentobarbital anesthesia with 125I-labeled insulin; at timed intervals from 30 to 120 sec, blood samples were extracted and used for the estimation of insulin levels by RIA. A group of rats from each series was maintained under a constant infusion of noradrenaline. For each insulin determination, a duplicate blood sample containing the same amount of insulin as that used in the RIA, but without the radioactive label, was used as a blank for insulin measurement. The radioactivity in these tubes was then used for the measurement of insulin label per ml blood. From plasma label decay curves and insulin concentrations, the insulin pool size, half-life, and rate of degradation were calculated. Obese rats had higher insulin levels (2.43 nM) and showed less effect of noradrenaline than their lean counterparts, in which insulin distribution volume shrank with noradrenaline treatment. The half-life of plasma insulin was similar in all groups (range, 226-314 sec). Pool size and overall degradation rates were higher in obese (198 femtokatals) than in lean rats (28 femtokatals). It is postulated that obese rats synthesize and cleave much more insulin than lean controls despite their higher circulating levels of insulin.

Utilization of dietary glucose in the metabolic syndrome

This review is focused on the fate of dietary glucose under conditions of chronically high energy (largely fat) intake, evolving into the metabolic syndrome. We are adapted to carbohydrate-rich diets similar to those of our ancestors. Glucose is the main energy staple, but fats are our main energy reserves. Starvation drastically reduces glucose availability, forcing the body to shift to fatty acids as main energy substrate, sparing glucose and amino acids. We are not prepared for excess dietary energy, our main defenses being decreased food intake and increased energy expenditure, largely enhanced metabolic activity and thermogenesis. High lipid availability is a powerful factor decreasing glucose and amino acid oxidation. Present-day diets are often hyperenergetic, high on lipids, with abundant protein and limited amounts of starchy carbohydrates. Dietary lipids favor their metabolic processing, saving glucose, which additionally spares amino acids. The glucose excess elicits hyperinsulinemia, which may derive, in the end, into insulin resistance. The available systems of energy disposal could not cope with the excess of substrates, since they are geared for saving not for spendthrift, which results in an unbearable overload of the storage mechanisms. Adipose tissue is the last energy sink, it has to store the energy that cannot be used otherwise. However, adipose tissue growth also has limits, and the excess of energy induces inflammation, helped by the ineffective intervention of the immune system. However, even under this acute situation, the excess of glucose remains, favoring its final conversion to fat. The sum of inflammatory signals and deranged substrate handling induce most of the metabolic syndrome traits: insulin resistance, obesity, diabetes, liver steatosis, hyperlipidemia and their compounded combined effects. Thus, a maintained excess of energy in the diet may result in difficulties in the disposal of glucose, eliciting inflammation and the development of the metabolic syndrome

The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized g-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment.

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.

Concentrações séricas hormonais em vacas azebuadas submetidas à baixa e alta ingestão alimentar

O objetivo deste trabalho foi avaliar a influência da ingestão alimentar nas concentrações séricas de hormônios reprodutivos e metabólicos em vacas azebuadas. Dezoito vacas foram divididas em dois grupos: 170% (alta ingestão = A) e 66% (baixa ingestão = B) da dieta de manutenção. Com 21 dias nas dietas experimentais, as vacas tiveram o estro sincronizado. Posteriormente, os ovários foram avaliados por ultra-sonografia transretal e sangue foi coletado diariamente até o dia 7 do ciclo (ovulação = dia 1). Na análise estatística, utilizou-se o teste t. As vacas ganharam 1,1 kg por dia no grupo A e perderam 1,5 kg por dia de PV no grupo B. Apesar de não ter havido diferença entre os grupos no diâmetro máximo do folículo ovulatório, o grupo A apresentou pico pré-ovulatório de estradiol sérico menor. Não foi observada diferença entre os grupos quanto ao volume luteal e concentração sérica de progesterona no dia 7 do ciclo e de FSH, IGF-I e insulina séricos no período peri-ovulatório. As dietas experimentais não alteraram a função ovariana e as concentrações séricas de hormônios reprodutivos e metabólicos, com exceção do estradiol, sugerindo que, no grupo de alta ingestão, ocorreu maior metabolismo desse hormônio.

Insulin dysfunction and allostic load in bipolar disorder

Bipolar disorder (BD) is associated with substantial morbidity, as well as premature mortality. Available evidence indicates that 'stress-sensitive' chronic medical disorders, such as cardiovascular disease, obesity and Type 2 diabetes mellitus, are critical mediators and/or moderators of BD. Changes in physiologic systems implicated in allostasis have been proposed to impact brain structures and neurocognition, as well as medical comorbidity in this population. For example, abnormalities in insulin physiology, for example, insulin resistance, hyperinsulinemia and central insulinopenia, are implicated as effectors of allostatic load in BD. Insulin's critical role in CNS physiological (e.g., neurotrophism and synaptic plasticity) and pathophysiological (e.g., neurocognitive deficits, pro-apoptosis and amyloid deposition) processes is amply documented. This article introduces the concept that insulin is a mediator of allostatic load in the BD and possibly a therapeutic target.

Comment on 'The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway'

Letter to the Editor on Wang M, Wang Q, Wang Z, Zhang X, Pan Y. The molecular evolutionary patterns of the insulin/FOXO signaling pathway

Liquid fructose down-regulates liver insulin receptor substrate 2 and gluconeogeneic enzymes by modifying nutrient sensing factors in rats

High consumption of fructose-sweetened beverages has been linked to a high prevalence of chronic metabolic diseases. We have previously shown that a short course of fructose supplementation as a liquid solution induces glucose intolerance in female rats. In the present work, we characterized the fructose-driven changes in the liver and the molecular pathways involved. To this end, female rats were supplemented or not with liquid fructose (10%, w/v) for 7 or 14 days. Glucose and pyruvate tolerance tests were performed, and the expression of genes related to insulin signaling, gluconeogenesis and nutrient sensing pathways was evaluated. Fructose-supplemented rats showed increased plasma glucose excursions in glucose and pyruvate tolerance tests and reduced hepatic expression of several genes related to insulin signaling, including insulin receptor substrate 2 (IRS-2). However, the expression of key gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was reduced. These effects were caused by an inactivation of hepatic forkhead box O1 (FoxO1) due to an increase in its acetylation state driven by a reduced expression and activity of sirtuin 1 (SIRT1). Further contributing to FoxO1 inactivation, fructose consumption elevated liver expression of the spliced form of X-box-binding-protein-1 as a consequence of an increase in the activity of the mammalian target of rapamycin 1 and protein 38-mitogen activated protein kinase (p38-MAPK). Liquid fructose affects both insulin signaling (IRS-2 and FoxO1) and nutrient sensing pathways (p38-MAPK, mTOR and SIRT1), thus disrupting hepatic insulin signaling without increasing the expression of key gluconeogenic enzymes.

El síndrome metabólico en el paciente renal

Se defi ne como síndrome metabólico al conjunto de factores que se dan en un individuo que le llevan a presentar resistencias a la insulina con hiperinsulinismo compensador que se asocia a trastornos del metabolismo hidrocarbonado, hipertensión arterial, alteraciones lipídicas y obesidad. Esta demostrado que este síndrome aumenta la posibilidad de padecer una enfermedad cardiovascular o diabetes mellitus por lo que se considera un factor de riesgo cardiovascular y de mortalidad en la población general. En pacientes...

The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Patterns of nucleotide diversity at the regions encompassing the Drosophila insulin-like peptide (dilp) genes: demography vs positive selection in Drosophila melanogaster.

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events.

Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Comment on 'The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway'

Letter to the Editor on Wang M, Wang Q, Wang Z, Zhang X, Pan Y. The molecular evolutionary patterns of the insulin/FOXO signaling pathway

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.