973 Vol.47, n. 6 : pp. 973-981, November 2004 ISSN 1516-8913 Printed in Brazil BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL Insulin or Insulin-Like Studies on Unicellular Organisms: a Review Alzira Martins Ferreira de Souza1* and Jorge A. López2 1Departamento de Bioquímica, CCB, Universidade Federal de Pernambuco, ex-Professor Av. Morais Rego, s/n Cidade Universitária; 50670-420; Recife - PE - Brazil; 2Departamento de Parasitologia; ICB 2; Universidade de São Paulo; 05508-900; São Paulo - SP - Brazil; e-mail: [email protected] ABSTRACT This paper presents a review of studies about insulin and insulin-like substances in prokaryotes, eukaryotes and fungi have been published over the last two decades, constituting an updating of our previous review (1988) which included references to invertebrate insulin or insulin-like substances both in uni- and pluricellular Monera, Protoctista, Fungii and Animal species. This present article reviews experiments and evidence obtained using modern techniques in the understanding of molecule evolution and behaviour, which confirm its very ancient molecular structure origin. The involvement of insulin-like and related material in signalling biological pathway and modulator effects is also reported. Key words: Unicellular, invertebrate, insulin, insulin-like, insulin receptor INTRODUCTION aiming to include research on protist and prokaryote insulin or insulin-like substances Since the 1970’s investigations in the field of the carried out over the past two decades. In the comparative biochemistry have increased current Biological Classification scheme these exponentially. Nowadays, investigators focus on insulin-like substances correspond to prokaryotes, the possible evolutionary ancestors of the insulin eukaryotes and fungi substances (Margulis, superfamily, e.g, insulins, insulin-related peptides, 1992). The review studies relieves the and the insulin-like growth factor. These peptides evolutionary origins of these insulin or insulin-like are not only present in vertebrates, as other substances, and moves on to compare findings of related proteins have also been found in some metazoan insulin, or insulin-like substances and unicellular organisms (Le Roith et al., 1980; Le vertebrate insulin with respect to molecular Roith et al., 1981; Souza and López-Rodríguez, structure, biological and immunological activities, 1988; Lenard, 1992). and the technical difficulties of defining the Insulin-like occurrence in invertebrates was molecular structure with the insufficient amount identified with the clam tissues of Mya arenaria of material usually available. (Collip, 1923) as far back as eighty years ago. This present paper represents an updating of our previous review of studies on insulin in invertebrates (Souza and López-Rodríguez, 1988), *Author for correspondence Brazilian Archives of Biology and Technology 974 Souza, A. M. F. and López, J. A. THE ORIGINS OF INSULIN Tetrahymena among species belonging to the MOLECULAR STRUCTURE Protoctista Kingdom. Applying the flourescein- isothiocyanate (FICT)-insulin-binding technique to The disclosure that insulin may have originated the detection of cellular membrane dynamics in more than a billion years ago arose with the the eukaryote Amoeba proteus, Köhidai et al. findings of Le Roith et al. (1980) which suggested (1992) verified that 1) the number of insulin that the molecular origins of insulin go at least as binding sites decrease in the control conditions as far back as the simplest univellular eukaryotes. a function of time. The down regulation of binding Evidence of the presence of a substance with sites lasts approximately 48h and only low insulin immunological and biological similarities to concentration can induce a binding site elevation mammalian insulin in the bacterium Escherichia over the long term; 2) hormonal imprinting could coli K12 the year after (Le Roith et al., 1981), be present in Amoeba. According to Le Roith et al. increased the possible time-span of its origins to (1980), immunologically and biologically active two billion years. insulin was found in ciliated protozoa Tetrahymena pyriformis extracts, grown in defined media in the absence of macromolecules. The EVIDENCE OF INSULIN IN extraction was proceeded according to a classical UNICELLULAR ORGANISMS process (Mirsky, 1973), except that albumin was eliminated. The cells acid-ethanol extracts In this updated review, the initial studies with obtained from conditioned and unconditioned prokayryote and eukaryote organisms from the medium, where T. pyriformis has grown, were gel- decade of the 80’s are reported, in particular the filtered and the eluted material, using findings obtained from studies on the radioimmunoassay, was similar to the standard heterotrophic Protist protozoan species, (Monera mammalian insulin. The conditioned medium and Protoctista Kingdom), which refer to the eluate stimulated glucose incorporation into lipids occurence of insulin-related molecules, their when applied to rats, and its bioactivity was characterization and functionality in Monera and largely neutralized in the presence of anti-insulin Protoctista species. From the Monera Kingdom antibody. Another study performed with another the scientific literature presents references only strain of the T. pyriformis extracts and with their from the bacterium Escherichia coli. The result culture medium extracts, purified on DEAE- cellulose or DEAE-Sephadex and HPLC, was obtained with E. coli K12 (Le Roith et al., 1981) cited above, was confirmed when Le Roith et al. conducted submitting the material to (1985) worked with four more strains grown in immunoactivity assays. The results obtained defined medium. The extracts obtained were showed a similar specific reaction to insulin in the purified and the fractions submitted to immunoassay and bioassay for insulin as the immunoassay. The active material obtained was previous, improving the probability that its insulin-like material may act as a signal (intra- or purified again with Sep-Pack (C18) and Sephadex G-50 chromatography and applied to a DEAE- extracellular messenger signal) (Le Roith et al., Sephadex column. The immunoactive material 1985). was recovered as a peak, which overlaps the An autoradiographic study in Tetrahymena was elution position of unlabelled and 125I-labelled conducted by Fulop and Csaba (1991), investigating the turnover and intranuclear porcine insulin and re-purified by HPLC. The 125 purified material was found to have an localization of I-insulin. Cells were shown to have internalised the labelled insulin and the immunoactivity to the anti-insulin antibody and 125 similarity was demonstrated between the insulin had also entered the nucleus of unicellular material and vertebrate insulins. More Tetrahymena, where it was found in a recently, Silva et al. (2002) utilizing modified heterochromatic localization. The insulin- ELISA and Western blotting methods pretreated T. pyroformis retained a greater part of demonstrated the presence of insulin-like antigens internalised insulin in the cytoplasm than the in extracts of the cyanobacterium Spirulina insulin non-pretreated cells. This retention maxima (Monera Kingdom). In the scientific decreased with additional exposure to excessive literature, studies have been published relating to non-labelled insulin. Kovács and Csaba (1991) studied the micronucleus of normal and insulin Brazilian Archives of Biology and Technology Insulin or Insulin-Like Studies on Unicellular Organisms: a Review 975 pre-treated (imprinted) T. pyriformis and T. the binding capacity for lectins and for insulin, malaccensis under the effect of insulin treatment but to enhance insulin imprinting at primary by using Feulgen reaction. They found a different interaction. Kovács et al. (1992a) also researched reaction intensity; the duration of hydrolysis insulin binding and demonstrated that it was not influenced the reaction. The experiments alert us influenced by Na-metavanadate. The ouabain to the possibility of the nuclear effect of hormonal effect prevented insulin imprinting when imprinting. The bacitracin effect on T. pyriformis administered with insulin, but did not affect was also the aim of Kovács and Csaba (1992). imprinting when administered after insulin Their experiments pointed out the role of insulin administration. The results indicate that inhibition degradation in the insulin imprinting of these of Na+K+ - ATPase plays no role in this insulin- protozoa. Bacitracin was observed to inhibit the like effect of vanadate on the unicellular insulin imprinting and direct insulin binding, Tetrahymena. diminishing the glucose activated growth capacity Other aspects of the Tetrahymena insulin binding and increasing lysosomal enzyme exocytosis. have been investigated, with the following results: Several aspects of Tetrahymena insulin binding offspring generations of Tetrahymena pre-treated were observed in the same period, such as the (imprinted) with insulin showed a greater binding results of Hegyesi and Csaba (1992a, 1992b) capacity than that of untreated ones, according to concerning the effect of pre-treatment (imprinting) Csaba and Kovács (1991). The epidermal growth using different concentrations of insulin on insulin factor imprinted for insulin to a greater degree binding of its nuclear envelope and plasma than insulin itself, and vice-versa: insulin membrane, as well the specific insulin binding and imprinted for EGF more efficiently than EGF the imprintability of the nuclear