Role of the evolutionarily conserved starvation response in anorexia nervosa.

This review will summarize recent findings concerning the biological regulation of starvation as it relates to anorexia nervosa (AN), a serious eating disorder that mainly affects female adolescents and young adults. AN is generally viewed as a psychosomatic disorder mediated by obsessive concerns about weight, perfectionism and an overwhelming desire to be thin. By contrast, the thesis that will be developed here is that, AN is primarily a metabolic disorder caused by defective regulation of the starvation response, which leads to ambivalence towards food, decreased food consumption and characteristic psychopathology. We will trace the starvation response from yeast to man and describe the central role of insulin (and insulin-like growth factor-1 (IGF-1))/Akt/ F-box transcription factor (FOXO) signaling in this response. Akt is a serine/threonine kinase downstream of the insulin and IGF-1 receptors, whereas FOXO refers to the subfamily of Forkhead box O transcription factors, which are regulated by Akt. We will also discuss how initial bouts of caloric restriction may alter the production of neurotransmitters that regulate appetite and food-seeking behavior and thus, set in motion a vicious cycle. Finally, an integrated approach to treatment will be outlined that addresses the biological aspects of AN.

Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

Spatial control of gut-specific gene expression during Caenorhabditis elegans development.

The nematode Caenorhabditis elegans was transformed with constructs containing upstream deletions of the gut-specific ges-1 carboxylesterase gene. With particular deletions, ges-1 was expressed, not as normally in the gut, but rather in muscle cells of the pharynx (which belong to a sister lineage of the gut) or in body wall muscle and hypodermal cells (which belong to a cousin lineage of the gut). These observations suggest that gut-specific gene expression in C. elegans involves not only gut-specific activators but also multiple repressors that are present in particular nongut lineages.

The gut esterase gene (ges-1) from the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

The ges-1 gene codes for a non-specific carboxylesterase that is normally expressed only in the intestine of the nematode Caenorhabditis elegans. In the current paper, we describe the cloning and characterization of the ges-1 gene from C. elegans, as well as the homologous gene from the nematode Caenorhabditis briggsae. The ges-1 esterases from the two nematodes are 83% identical at the amino acid level and contain regions of significant similarity to insect and mammalian esterases; these conserved regions can be identified with residues believed to be necessary for esterase function. The ges-1 mRNAs from both C. elegans and C. briggsae are trans-spliced. The coding regions, the codon bias and the splicing signals of the two ges-1 genes are quite similar and most (6/7) of the intron positions are retained precisely. Yet, the flanking sequences of the two ges-1 genes appear to have diverged almost completely. For example, the C. elegans ges-1 5'-flanking region (as well as several introns) contains copies of three different SINE-like sequences, previously identified near the hsp-16 genes, near the unc-22 gene and in a repetitive element CeRep-3; none of these elements are found in the C. briggsae ges-1 gene. We show that: (1) the C. elegans ges-1 gene can be used to transform C. briggsae, whereupon expression of the exogenous ges-1 gene is confined to the C. briggsae intestine; (2) the ges-1 homologue cloned from C. briggsae can be transformed into C. elegans, whereupon it is expressed largely in the C. elegans intestine; and (3) a 5'-deletion of the C. elegans ges-1 gene that we have previously shown to be expressed in the C. elegans pharynx is also expressed in the pharynx of C. briggsae (either in the presence or absence of vector sequences). These results suggest that the ges-1 gene control circuits have been maintained between the two nematode species, despite the divergent 5'-flanking sequences of the gene. This raises the question of the evolutionary distance between C. elegans and C. briggsae and we attempt to estimate the C. elegans-C. briggsae divergence time by analysing the rate of synonymous substitutions in coding regions of ges-1 and six other C. elegans-C. briggsae gene pairs. We propose a new method of analysis, which attempts to remove rate differences found between different genes by extrapolating to zero codon bias.(ABSTRACT TRUNCATED AT 400 WORDS)

Food deprivation and nicotine correct akinesia and freezing in Na(+) -leak current channel (NALCN)-deficient strains of Caenorhabditis elegans.

Mutations in various genes adversely affect locomotion in model organisms, and thus provide valuable clues about the complex processes that control movement. In Caenorhabditis elegans, loss-of-function mutations in the Na(+) leak current channel (NALCN) and associated proteins (UNC-79 and UNC-80) cause akinesia and fainting (abrupt freezing of movement during escape from touch). It is not known how defects in the NALCN induce these phenotypes or if they are chronic and irreversible. Here, we report that akinesia and freezing are state-dependent and reversible in NALCN-deficient mutants (nca-1;nca-2, unc-79 and unc-80) when additional cation channels substitute for this protein. Two main measures of locomotion were evaluated: spontaneous movement (traversal of >2 head lengths during a 5 second observation period) and the touch-freeze response (movement greater than three body bends in response to tail touch). Food deprivation for as little as 3 min stimulated spontaneous movement and corrected the touch-freeze response. Conversely, food-deprived animals that moved normally in the absence of bacteria rapidly reverted to uncoordinated movement when re-exposed to food. The effects of food deprivation were mimicked by nicotine, which suggested that acetylcholine mediated the response. Nicotine appeared to act on interneurons or motor neurons rather than directly at the neuromuscular junction because levamisole, which stimulates muscle contraction, did not correct movement. Neural circuits have been proposed to account for the effects of food deprivation and nicotine on spontaneous movement and freezing. The NALCN may play an unrecognized role in human movement disorders characterized by akinesia and freezing gait.

Association of microtubules and neurofilaments in vitro is not mediated by ATP.

Runge et al. [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435] found that mixtures of microtubules and neurofilaments formed a viscous, sedimentable complex when incubated at 37 degrees C for 20 min in the presence of ATP. They did not observe the high viscosities associated with the complex when the incubation was carried out in the absence of ATP. This paper reports an investigation of the roles of time and ATP in the formation of the complex. Microtubules assembled in a mixture containing GTP and neurofilaments prepared from bovine brain remained assembled for a shorter period of time than they did in similar solutions containing no neurofilaments. Adding ATP to the neurofilament-containing solutions, or doubling their GTP concentration, extended the time during which the microtubules remained assembled. These mixtures then became highly viscous. These phenomena resulted from the action of at least two enzymes present in the neurofilament preparation. A GTPase raised the GDP/GTP ratio, in the mixtures in which ATP was absent, to levels sufficient to cause disassembly of the microtubules. When ATP was present, a nucleotide diphosphokinase catalyzed regeneration of GTP from GDP while converting ATP to ADP. This process kept the GDP/GTP ratio low and delayed the disassembly of the microtubules. These results show that the apparent ATP dependence of formation of the microtubule-neurofilament complex observed by Runge et al. is attributable to a GDP-induced disassembly of microtubules rather than to a disruption of microtubule-neurofilament contacts. Those contacts can form in the absence of ATP.

Microtubule-associated proteins connect microtubules and neurofilaments in vitro.

Neuronal intermediate filaments (neurofilaments) prepared from brain form a viscous sedimentable complex with microtubules under suitable conditions [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435]. Under the same conditions, neurofilaments prepared from spinal cord did not form such a complex. Brain neurofilaments were shown to differ from spinal cord neurofilaments in part by having proteins that resemble microtubule-associated proteins (MAPs) attached to them. MAPs became bound to spinal cord neurofilaments when the two structures were incubated together. The resulting MAP-decorated neurofilaments formed a viscous complex with microtubules, showing that some component of the MAPs mediated the association between the two filamentous organelles. By means of gel filtration, the MAPs were separated into two major fractions. The large Stokes radius fraction was active in producing neurofilament-microtubule mixtures of high viscosity, while the small Stokes radius fraction was not. The dependence of the viscosity of neurofilament-microtubule mixtures upon the concentration of MAPs was found to possess a maximum. This result suggests that the MAPs serve as cross-bridges between the two structures. Neurofilaments, with and without bound MAPs, were allowed to adhere to electron microscope grids. The grids were then exposed to microtubules, fixed, and stained. The grids prepared with MAP-decorated neurofilaments bound numerous microtubules, each in apparent contact with one or more neurofilaments. The grids prepared with untreated neurofilaments lacked microtubules. These results show that one or more of the MAPs mediates association between microtubules and neurofilaments.

Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures.

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans.

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.