Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco-2.

AIM: Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved. METHODS: Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin, and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot. RESULTS: Leptin inhibited 0.1 mm α-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 µm glutamine and 0.1 mm phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na(+) -dependent neutral amino acid transporters ASCT2 and B(0) AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mm proline and ß-alanine uptake in Na(+) medium at pH 6, conditions for optimal activity of the H(+) -dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on ß-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin's regulation of PAT1 activity. CONCLUSION: These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, that is, D-glucose (SGLT1) and amino acids (ASCT2, B(0) AT1 and PAT1).

Inferences on the role of insertion in a mutation accumulation experiment with Drosophila melanogaster using RAPDs.

The genetic variability for RAPDs band pattern was studied in a set of 157 mutation accumulation (MA) lines of Drosophila melanogaster. These MA lines were derived from the same isogenic base population and subsequently maintained by full-sib mating during 132 generations. The ancestral pattern of the original isogenic base can be unambiguously established as the consensus pattern of the MA lines and, because these lines are expected to be homozygous, dominance for band pattern is not a concern. Only repeatable changes in band pattern were considered. The number of ancestral bands detected implies that nine-nucleotide targets are enough for repeatable PCR amplification. Compared with the ancestral pattern, one MA line lost one band and two MA lines gained a new one. These results can be accounted for by the insertion of transposable elements occurring at a rate 0.07 < i < 0.21 per whole haploid genome and generation. This range is typical for Drosophila and consistent with the previously observed mobility for the roo family, supporting the generality of previous estimates of spontaneous mutation rates for morphological and fitness traits based on these MA lines. The sequence of one of the new bands suggests that the Idefix family is also active in the lines.

Lack of nonadditive genetic effects on early fecundity in Drosophila melanogaster.

Fecundity is usually considered as a trait closely connected to fitness and is expected to exhibit substantial nonadditive genetic variation and inbreeding depression. However, two independent experiments, using populations of different geographical origin, indicate that early fecundity in Drosophila melanogaster behaves as a typical additive trait of low heritability. The first experiment involved artificial selection in inbred and non-inbred lines, all of them started from a common base population previously maintained in the laboratory for about 35 generations. The realized heritability estimate was 0.151 +/- 0.075 and the inbreeding depression was very small and nonsignificant (0.09 +/- 0.09% of the non-inbred mean per 1% increase in inbreeding coefficient). With inbreeding, the observed decrease in the within-line additive genetic variance and the corresponding increase of the between-line variance were very close to their expected values for pure additive gene action. This result is at odds with previous studies showing inbreeding depression and, therefore, directional dominance for the same trait and species. All experiments, however, used laboratory populations, and it is possible that the original genetic architecture of the trait in nature was subsequently altered by the joint action of random drift and adaptation to captivity. Thus, we carried out a second experiment, involving inbreeding without artificial selection in a population recently collected from the wild. In this case we obtained, again, a maximum-likelihood heritability estimate of 0.210 +/- 0.027 and very little nonsignificant inbreeding depression (0.06 +/- 0.12%). The results suggest that, for fitness-component traits, low levels of additive genetic variance are not necessarily associated with large inbreeding depression or high levels of nonadditive genetic variance.

The rate of mutation and the homozygous and heterozygous mutational effects for competitive viability: a long-term experiment with Drosophila melanogaster.

The effect of 250 generations of mutation accumulation (MA) on the second chromosome competitive viability of Drosophila melanogaster was analyzed both in homozygous and heterozygous conditions. We used full-sib MA lines, where selection hampers the accumulation of severely deleterious mutations but is ineffective against mildly deleterious ones. A large control population was simultaneously evaluated. Competitive viability scores, unaffected by the expression of mutations in heterozygosis, were obtained relative to a Cy/L(2) genotype. The rate of decline in mean DeltaM approximately 0.1% was small. However, that of increase in variance DeltaV approximately 0.08 x 10(-3) was similar to the values obtained in previous experiments when severely deleterious mutations were excluded. The corresponding estimates of the mutation rate lambda > or = 0.01 and the average effect of mutations E(s) < or = 0.08 are in good agreement with Bateman-Mukai and minimum distance estimates for noncompetitive viability obtained from the same MA lines after 105 generations. Thus, competitive and noncompetitive viability show similar mutational properties. The regression estimate of the degree of dominance for mild-to-moderate deleterious mutations was approximately 0.3, suggesting that the pertinent value for new unselected mutations should be somewhat smaller.

Inter- and intraspecific sexual discrimination in the flour beetles Tribolium castaneum and Tribolium confusum.

In Tribolium castaneum (CS) and T. confusum (CF), intra- and interspecific rates of homosexual mounting have been measured. The intraspecific results are compatible with the hypothesis of both species being sexually indiscriminate. However, the CF intraspecific rates were very high (35%-53% of mountings were homosexual), suggesting a lower sexual attractiveness, or a stronger rejection to being mounted, of CF females relative to conspecific males. CS males discriminate between species but, in interspecific contacts, preferentially mounted CF males rather than CF females. CF males do not discriminate between species, but the loss of sexual attractiveness of CF females, or their rejection to being mounted, may act as a precopulatory isolation mechanism.

Epistasis and the conversion of non-additive to additive genetic variance at population bottlenecks.

The effect of population bottlenecks on the mean and the additive variance generated by two neutral independent epistatic loci has been studied theoretically. Six epistatic models, used in the analysis of binary disease traits, were considered. Ancestral values in an infinitely large panmictic population were compared with their expectations at equilibrium, after t consecutive bottlenecks of equal size N (derived values). An increase in the additive variance after bottlenecks (inversely related to N and t) will occur only if the frequencies of the negative allele at each locus are: (1) low, invariably associated to strong inbreeding depression; (2) high, always accompanied by an enhancement of the mean with inbreeding. The latter is an undesirable property, making the pertinent models unsuitable for the genetic analysis of disease. For the epistatic models considered, it is unlikely that the rate of evolution may be accelerated after population bottlenecks, in spite of occasional increments of the derived additive variance over its ancestral value.

Temporal uniformity of the spontaneous mutational variance of quantitative traits in Drosophila melanogaster.

Spontaneous mutations were allowed to accumulate over 209 generations in more than 100 lines, all of them independently derived from a completely homozygous population of Drosophila melanogaster and subsequently maintained under strong inbreeding (equivalent to full-sib mating). Traits scored were: abdominal (AB) and sternopleural (ST) bristle number, wing length (WL) and egg-to-adult viability (V). On two occasions--early (generations 93-122) and late (generations 169-209)--ANOVA estimates of the mutational variance and the mutational line x generation interaction variance were obtained. Mutational heritabilities of morphological traits ranged from 2 x 10(-4) to 2 x 10(-3) and the mutational coefficient of variation of viability was 0.01. For AB, WL and V, temporal uniformity of the mutational variance was observed. However, a fluctuation of the mutational heritability of ST was detected and could be ascribed to random genotype x environment interaction.

Properties of spontaneous mutations affecting quantitative traits.

Recent mutation accumulation results from invertebrate species suggest that mild deleterious mutation is far less frequent than previously thought, implying smaller expressed mutational loads. Although the rate (lambda) and effect (s) of very slight deleterious mutation remain unknown, most mutational fitness decline would come from moderately deleterious mutation (s approximately 0.2, lambda approximately 0.03), and this situation would not qualitatively change in harsh environments. Estimates of the average coefficient of dominance (h) of non-severe deleterious mutations are controversial. The typical value of h = 0.4 can be questioned, and a lower estimate (about 0.1) is suggested. Estimated mutational parameters are remarkably alike for morphological and fitness component traits (excluding lethals), indicating low mutation rates and moderate mutational effects, with a distribution generally showing strong negative asymmetry and little leptokurtosis. New mutations showed considerable genotype-environment interaction. However, the mutational variance of fitness-component traits due to non-severe detrimental mutations did not increase with environmental harshness. For morphological traits, a class of predominantly additive mutations with no detectable effect on fitness and relatively small effect on the trait was identified. This should be close to that responsible for standing variation in natural populations.

The mutation rate and the distribution of mutational effects of viability and fitness in Drosophila melanogaster.

The empirical distributions of the average viability and fitness of mutation accumulation lines of Drosophila melanogaster were analyzed using minimum distance estimation. Data come from two different experimental designs where mutations were allowed to accumulate: 1) in copies of chromosome II protected from natural selection and recombination (viability: Mukai et al., 1972; Ohnishi, 1977; fitness: Houle et al., 1992), 2) in inbred lines derived from the same isogenic stock (viability: Fernández & López-Fanjul, 1996; fitness: this paper). Information from all data sets converged, indicating that the mutational rates were small, about 1% for viability and 3% for fitness. For both traits, the rate of mutational decline appears to be smaller than suggested by previous studies (about one-fifth of the latter), the average mutational effect was neither severe nor very slight, ranging from -0.1 to -0.3, and the distribution of mutant effects was, at most, slightly leptokurtic. Therefore, the mutational load in natural populations is one to two orders of magnitude smaller than previously thought (as based upon analyses conditional to estimates of the mutational decline of viability or fitness that appear to be biased upward). Over 95% of the mutational variance of each trait was contributed by non-slightly deleterious mutations (absolute homozygous effect larger than 0.03 or 0.1, depending on the data set considered) occurring at a rate not higher than 0.025 per haploid genome and generation. Our data suggest that most deleterious mutations affecting fitness act mainly through a single component-trait.

The lack of mutational variance for fluctuating and directional asymmetry in Drosophila melanogaster.

Starting from a completely homozygous population of Drosophila melanogaster, lines were derived and independently maintained by a single brother-sister mating in each generation. Two bilateral traits--sternopleural bristle number and wing length--were individually scored on the right-(R) and left-hand (L) sides. Directional (DA) and fluctuating (FA) asymmetries were represented by the signed (R-L) and unsigned magnitude of R-L difference, respectively. Mutational variances (the mutational rate of input of genetic variation) and heritabilities (the mutational variance scaled by the environmental variance) of R, L, (R-L) and magnitude of R-L were calculated from the between-line divergence after a number of generations of mutation accumulation (bristle number: 171 lines, 122 generations; wing length: 148 lines, 170 generations). Mutational heritabilities of R and L were all significant, ranging from 0.73 x 10(-3)-2.10 x 10(-3). Those of (R-L) and magnitude of R-L were two orders of magnitude smaller and nonsignificant, ranging from -1.95 x 10(-5)-5.49 x 10(-5). These results imply that mutations affecting the DA or FA of bristle number and wing length have not been fixed in the lines or alternatively, that their effects were too small to be detected. In the population under study, the data strongly suggest that FA reflects only developmental noise due to non-genetic processes.

Spontaneous mutational variances and covariances for fitness-related traits in Drosophila melanogaster.

Starting from a completely homozygous population of Drosophila melanogaster, 176 lines were derived and independently maintained by a single brother-sister mating per generation. Three fitness-related traits were considered (fecundity, egg-to-pupa and pupa-to-adult viabilities). Mutational heritabilities of these traits and genetic correlations between all possible pairs were calculated from the between line divergence (codivergence), after 104-106 generations of mutation accumulation. Mutational heritabilities ranged from 0.60 x 10(-3) to 0.82 x 10(-3) and correlations from -0.11 to 0.25. These values are likely to be underestimates due to selection against deleterious mutations. The distribution of the means of the lines was asymmetric, positive for fecundity and negative for both viability components. The coefficients of asymmetry are also likely to be biased, again due to selection. Extreme lines from the two tails of the distribution were examined in detail. Homozygous line effects were all negative for viability traits but predominantly positive for fecundity, indicating the fixation of mutations with positive effects on the latter. Corresponding heterozygous line effects showed a variable degree of dominance.

Response to selection from new mutation and effective size of partially inbred populations. II. Experiments with Drosophila melanogaster.

Divergent artificial selection for abdominal bristle number in Drosophila melanogaster has been carried out starting from a genetically homogeneous base population. Lines with two different systems of mating, random (P lines) or between full sibs whenever possible (about 50%), random otherwise (I lines) were compared. Responses after 40 generations of selection were mostly due to one or two mutations of large effect (0.2 to 2 phenotypic standard deviations) per line. Ten mutations affecting the selected trait were individually studied (five lethal and five non-lethal, these being predominantly additive). These mutations satisfactorily explain the response attained, although some minor mutations may also be involved. No evidence of epistasis for bristle number was found. The average final divergence was 57% larger in the P lines, but it was mostly due to lethals or highly deleterious mutations. Thus, after relaxation of selection, the ranking reversed and the mean divergence became significantly larger in the I lines (14%). Analysis of inbreeding showed that the very small amount of variation created by spontaneous mutations (a heritability for the selected trait of about 3%) was responsible for a reduction in the effective size of about 50% in the I lines (relative to the case with random selection), but only about 10% in the P lines. Mutational heritabilities estimated from the response to selection (0.05-0.18%) were within the range usually found for this trait in previous experiments. REML estimates account for correlations between relatives, and were much larger in those lines where the response was due to lethal mutations, as these do not contribute to response after reaching maximum frequency.

Spontaneous mutation for a quantitative trait in Drosophila melanogaster. I. Response to artificial selection.

Divergent selection for abdominal bristle number was carried out for 47 generations, starting from a completely homozygous population of Drosophila melanogaster. All lines were selected with the same proportion (20%) but at two different numbers of selected parents of each sex (5 or 25). A significant response to selection was obtained in 25 lines (out of 40). In most cases, it could be wholly attributed to a single mutation of relatively large effect (> 0.3 phenotypic standard deviations). A total number of 30 mutations were detected. In agreement with theory, larger responses in each direction were achieved by those lines selected at greater effective population sizes. A large fraction of mutations were lethals (10/30). Thus, the observed divergence between lines of the same effective size selected in opposite directions was smaller than expected under neutrality. The ratio of new mutational variance to environmental variance was estimated to be (0.52 +/- 0.09) x 10(-3).

Spontaneous mutation for a quantitative trait in Drosophila melanogaster. II. Distribution of mutant effects on the trait and fitness.

Starting from a completely homozygous population of Drosophila melanogaster, lines have been derived and subjected to 47 generations of divergent selection for abdominal bristle number (20 lines selected in each direction) or to 60-67 generations of inbreeding (100 B lines maintained by a single brother-sister mating, 100 C lines maintained by two double first cousin matings). In the selected lines, 25 were identified carrying at least 30 mutations affecting bristle number. A large fraction of these mutations (42%) were lethals. Non-lethal mutations had smaller effects on the trait, were predominantly additive and had no detectable pleiotropic effects on fitness. In the inbred lines, 21 mutations affecting bristles were individually analysed. Deleterious mutations had the largest effects on the trait (irrespective of sign) and showed recessive gene action (complete or incomplete). The rest were predominantly additive and had smaller effects. Thus, both procedures identify a quasi-neutral class of additive mutations which should be close to that responsible for standing variation in natural populations. Moreover, the results indicate a leptokurtic distribution of mutant effects, consistent with a model of natural selection acting on bristles through pleiotropic effects of pertinent loci on fitness. Consequently, neutral additive alleles of considerable effect can be found segregating at intermediate frequencies in natural populations.

The distribution of spontaneous mutations on quantitative traits and fitness in Drosophila melanogaster.

Starting from a completely homozygous population of Drosophila melanogaster, two groups of 100 inbred lines each were established and maintained for 46 generations, by a single brother-sister mating and two double first cousin matings, respectively. Sternopleural bristle number, wing length and wing width were simultaneously scored in all lines every 4-5 generations. The means of four lines in each group departed significantly from the overall mean and, in each case, this was attributed to a single mutation of relatively large effect on at least one trait (0.3-1.4 environmental standard deviations in absolute value). Further analyses revealed widespread pleiotropy, similar gene action of a given mutation for all traits affected, and predominant additive action. No apparent association was found between the magnitudes of mutational effects on the traits and fitness. However, all recessive mutations were deleterious. The distribution of mutant effects was asymmetrical (positive for bristles and negative for wing measurements). Moreover, these distributions had a high variance and may be leptokurtic, due to the presence of major genes. Estimates of the ratio of new mutational variance to environmental variance ranged within (0.7-3.4) x 10(-3), those for wing measurements being generally larger. In agreement with theory, the rate of between-line differentiation was independent of population size.

The genetic properties of homosexual copulation behavior in Tribolium castaneum: diallel analysis.

The rate of homosexual copulation has been defined as the ratio between the number of homosexual mountings and the total number of mountings (homo and heterosexual) performed by a Tribolium castaneum male during a period of 30 min. In a laboratory population, the average rate when a number of males (m) and females (k x m) are tested together has been estimated in each of the six situations defined by m = 2 and 10 and k = 0.5, 1, and 2, k being the sex ratio among scored individuals. Good agreement was found between the observed rates of homosexual copulation and those expected assuming random contacts between pairs of individuals totally indiscriminate with respect to sex. The genetic properties of the trait have been investigated by means of a diallel analysis of six highly inbred lines derived from the same population and their F1 crosses. Significant general and specific combining ability effects were detected. When noninbred females were used for testing, the rate of homosexual copulation is expected to be higher for inbred than for noninbred males. This prediction, implying the existence of inbreeding depression for the trait, also was confirmed by the data.

The response to artificial selection from new mutations in Drosophila melanogaster.

Twenty generations of divergent selection for abdominal bristle number were carried out starting from a completely homozygous population of Drosophila melanogaster. All lines were selected with the same proportion (20%) but at two different numbers of selected parents of each sex (5 or 25). A significant response to selection was detected in eight lines (out of 40) and, in most cases, it could be wholly attributed to a single mutation of relatively large effect (0.5-2 phenotypic standard deviations). The ratio of new mutational variance to environmental variance was estimated to be (0.33 +/- 0.11) X 10(-3). The distribution of mutant effects was asymmetrical, both with respect to bristle number (85% of it was negative) and to fitness (most detected bristle mutations were lethal or semilethal). Moreover, this distribution was leptokurtic, due to the presence of major genes. Gene action on bristles ranged from additive to completely recessive, no epistatic interactions being found. In agreement with theory, larger responses in each direction were achieved by those lines selected at greater effective population sizes. Furthermore, the observed divergence between lines selected in opposite directions was proportional to their effective size, as predicted for mutations of large effect.

Accumulation of lethals in highly selected lines of Drosophila melanogaster.

Four synthetic lines of D. melanogaster selected for low sternopleural bristle number for 50 generations were screened for lethals on chromosome III when their mean score equalled 2.5. Each line originated from a cross between line M (previously selected for the same trait during 130 generations) and a different unselected cage population. Line M was already known to carry a recessive lethal on chromosome III affecting the selected trait, such that the bristle score of the lethal heterozygote was lower than that of the viable homozygote. Tests revealed 18 lethals, 15 of these present in at least two lines. Each line carried from 10 to 16 lethals. All lines carried groups of lethals present on the same chromosome, and at least six lethals in each line were included in such an association with a frequency of 0.18 or higher. It appears that the lethal affecting bristle score in line M has protected a segment of chromosome III from natural selection and that the remaining 14 lethals have accumulated later in that line.