Billy W. Geer

Dietary ethanol and lipid synthesis in Drosophila melanogaster

When cultured on a defined diet, ethanol was an efficient substrate for lipid synthesis in wild-type Drosophila melanogaster larvae. At certain dietary levels both ethanol and sucrose could displace the other as a lipid substrate. In wild-type larvae more than 90% of the flux from ethanol to lipid was metabolized via the alcohol dehydrogenase (ADH) system. The ADH and aldehyde dehydrogenase activities of ADH were modulated in tandem by dietary ethanol, suggesting that ADH provided substrate for lipogenesis by degrading ethanol to acetaldehyde and then to acetic acid. The tissue activity of catalase was suppressed by dietary ethanol, implying that catalase was not a major factor in ethanol metabolism in larvae. The activities of lipogenic enzymes, sn-glycerol-3-phosphate dehydrogenase, fatty acid synthetase (FAS), and ADH, together with the triacylglycerol (TG) content of wild-type larvae increased in proportion to the dietary ethanol concentration to 4.5% (v/v). Dietary ethanol inhibited FAS and repressed the accumulation of TG in ADH-deficient larvae, suggesting that the levels of these factors may be subject to a complex feedback control.

Regulation of alcohol dehydrogenase in Drosophila melanogaster by dietary alcohol and carbohydrate

Abstract When fed to Drosphila melanogaster Canton-S wild-type larvae cultured axenically on Sangs modified medium C with 0.5% (w/v) sucrose, 2.5% (v/v) ethanol induced an alcohol dehydrogenase (ADH) activity at least 2.5-fold higher than the activity in larvae fed an unsupplemented 0.5% sucrose diet. Ethanol induced only a 2-fold change in ADH cross reacting material (CRM), suggesting that there was also a post-translational modification of ADH. The addition of dietary sucrose to the diet (at 5%, w/v) exerted similar, but lesser effects on ADH activity and CRM. Increasing dietary sucrose to 5% in the presence of 2.5% ethanol decreased ADH activity and CRM to levels below those attained in the 0.5% sucrose-2.5% ethanol diet. Upon examination, dietary ethanol was found to decrease the relative concentration of ADH-1 and increase the concentrations of ADH-3 and ADH-5 in larvae; sucrose had a similar but lesser effect on the isozyme pattern. Because dietary ethanol increased both the total NAD content and the NADH NAD + ratio, the diet-induced changes in isozyme pattern were proposed to be linked to the relative amounts of NADH, NAD+ and ADH enzyme in larval tissues. Larval ADH activity is modulated by both dietary alcohol and carbohydrate.

Relationship of the oxidative pentose shunt pathway to lipid synthesis in Drosophila melanogaster

The tissue activities of the oxidative pentose shunt enzymes, glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44), in the larvae of Drosophila melanogaster are not dependent on the amount of flux through the oxidative pentose shunt pathway. An oxidative pentose shunt deficiency effects about a 40% reduction in the NADPH concentration in early third instar larvae, resulting in a six-fold difference in the NADPH/NADP+ ratio between wild-type and pentose-shunt-deficient larvae. The capacity of pentose-shunt-deficient larvae to synthesize triglyceride in response to a high concentration of dietary sucrose is only 73% of the wild-type level. Environmental temperature influences on the fatty acid composition of larvae are not altered by an oxidative pentose shunt deficiency.

Effects of dietary sucrose and environmental temperature on fatty acid synthesis in Drosophila melanogaster

Abstract The proportion of dietary sucrose devoted to lipid synthesis in the larvae of Drosophila melanogaster increased when the concentration was raised from 9 mM to 145 mM. A high dietary sucrose concentration increased the microsomal 9-desaturase activity and altered the specificity for de novo fatty acid synthesis termination to shorter chain lengths (14 and 16-C). Both neutral and phospholipid fatty acid contents were modified by dietary sucrose. A low environmental temperature favored the synthesis of monounsaturated fatty acids but had little effect on the length of fatty acids formed by de novo synthesis. Dietary sucrose minimized the utilization of dietary long chain fatty acids for lipid synthesis and diminished dietary fatty acid effects on fatty acid desaturation and chain elongation.

Genetic variation in the dietary sucrose modulation of enzyme activities in Drosophila melanogaster

Genetic variation in the modulating effect of dietary sucrose was assessed in Drosophila melanogaster by examining 27 chromosome substitution lines coisogenic for the X and second chromosomes and possessing different third isogenic chromosomes derived from natural populations. An increase in the concentration of sucrose from 0·1% to 5% in modified Sangs medium C significantly altered the activities of 11 of 15 enzyme activities in third instar larvae, indicating that dietary sucrose modulates many, but not all, of the enzymes of D. melanogaster . A high sucrose diet promoted high activities of enzymes associated with lipid and glycogen synthesis and low activities of enzymes of the glycolytic and Krebs cycle pathways, reflecting the physiological requirements of the animal. Analyses of variance revealed significant genetic variation in the degrees to which sucrose modulated several enzyme activities. Analysis of correlations revealed some relationships between enzymes in the genetic effects on the modulation process. These observations suggest that adaptive evolutionary change may depend in part on the selection of enzyme activity modifiers that are distributed throughout the genome.

The effect of dietary ethanol on the composition of lipids of Drosophila melanogaster larvae

At a moderate concentration (2.5%, v/v) dietary ethanol reduced the chain length of total fatty acids (FA) and increased the desaturation of short-chain FA in Drosophila melanogaster larvae with a functional alcohol dehydrogenase (ADH). The changes in length in total FA were postulated to be due to the modulation of the termination specificity of fatty acid synthetase. Because the ethanol-stimulated reduction in the length of unsaturated FA was blocked by linoleic acid, it was thought to reflect the properties of FA 9-desaturase. Although the ethanol-stimulated reduction in chain length of unsaturated FA was also observed in ADH-null larvae, ethanol promoted an increase in the length of total FA of the mutant larvae. Thus, the ethanolstimulated change in FA length was ADH dependent but the ethanol effect on FA desaturation was not. Ethanol also stimulated a decrease in the relative amount of phosphatidylcholine and an increase in phosphatidylethanolamine. Because similar ethanol-induced changes have been found in membrane lipids of other animals, ethanol may alter the properties of membranes in larvae. It is proposed that ethanol tolerance in D. melanogaster may be dependent on genes that specify lipids that are resistant to the detrimental effects of ethanol.

Alcohol Dehydrogenase and Alcohol Tolerance in Drosophila melanogaster

Drosophila melanogaster has often been used as a model system for investigations of the genetic factors that underlie ethanol tolerance. The species not only is very tolerant to environmental ethanol (McKenzie and Parsons, 1972; David et al., 1976), but it is able to use ethanol as a food source (McKechnie and Geer, 1984; Geer et al., 1985). Judgement of the degree of ethanol tolerance in D. melanogaster is complicated by the complex nature of the trait, and by the diversity of the tests that have been used to measure tolerance. Ethanol tolerance is a composite of the abilities to grow and to survive in the presence of ethanol, and, as shown in this study, to utilize dietary ethanol as a foodstuff. The conditions for the tolerance test and the diagnostic traits in previous studies have varied. Many tolerance tests have been performed by adding ethanol to the medium (Gibson, 1970; McDonald et al., 1977; Cavener and Clegg, 1978), but different life stages have been tested. Ethanol tolerance in adult D. melanogaster has been equated to the concentration of alcohol in sealed tubes that kills 50% of the individuals in a treatment of fixed duration (David et al., 1978, 1984).

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth‐limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol‐3‐phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol‐free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.

The involvement of catalase in alcohol metabolism in Drosophila melanogaster larvae.

The involvement of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in the metabolism of alcohols was investigated by comparing Drosophila melanogaster larvae in which catalase was inhibited by dietary 3-amino-1,2,4-triazole (3AT) to larvae fed a diet without 3AT. 3AT inhibited up to 80% of the catalase activity with concordant small increases in the in vitro activities of sn-glycerol-3-phosphate dehydrogenase, fumarase, and malic enzyme, but with a 16% reduction in the in vivo incorporation of label from [14C]glucose into lipid. When the catalase activity was inhibited to different degrees in ADH-null larvae, there was a simple linear correlation between the catalase activity and flux from [14C]ethanol into lipid. By feeding alcohols simultaneously with 3AT, ethanol and methanol were shown to react efficiently with catalase in wild-type larvae at moderately low dietary concentrations. Drosophila catalase did not react with other longer chain alcohols. Catalase apparently represents a minor pathway for ethanol degradation in D. melanogaster larvae, but it may be an important route for methanol elimination from D. melanogaster larvae.

The epistasis of Adh and Gpdh allozymes and variation in the ethanol tolerance of Drosophila melanogaster larvae

The role of epistatic interaction of allozymes in the determination of variation in larval ethanol tolerance of Drosophila melanogaster was examined. Isofemale lines from the Tahbilk Winery were made homozygous for different common alleles of alcohol dehydrogenase ( Adh ) and sn-glycerol-3-phosphate dehydrogenase ( Gpdh ). When fed 6% ethanol, all the lines had reduced survival and, in the survivors, reduced body weight and lengthened development time. A strong positive correlation between tolerance and development time suggested that alleles responsible for slowing development on ethanol also increased ethanol tolerance. Analysis of larval ethanol tolerance over four generations showed that larvae of the Adh ff Gpdh ff , and Adh ss Gpdh ss allelic combinations were more tolerant than larvae with the other combinations. However, these genotypes were not associated with the slowing of development nor the weight loss on ethanol. Hence, larvae with certain combinations of Adh and Gpdh allozymes may have a greater capacity to metabolize ethanol and be more tolerant to its toxic effects.