Muscle Mitochondrial Protein Synthesis Research Articles

Lack of Increase in Muscle Mitochondrial Protein Synthesis During the Course of Aerobic Exercise and Its Recovery in the Fasting State Irrespective of Obesity.

Acute aerobic exercise induces skeletal muscle mitochondrial gene expression, which in turn can increase muscle mitochondrial protein synthesis. In this regard, the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), is a master regulator of mitochondrial biogenesis, and thus mitochondrial protein synthesis. However, PGC-1α expression is impaired in muscle of humans with obesity in response to acute aerobic exercise. Therefore, we sought to determine whether muscle mitochondrial protein synthesis is also impaired under the same conditions in humans with obesity. To this end, we measured mitochondrial and mixed-muscle protein synthesis in skeletal muscle of untrained subjects with (body fat: 34.7 ± 2.3%) and without (body fat: 25.3 ± 3.3%) obesity in a basal period and during a continuous period that included a 45 min cycling exercise (performed at an intensity corresponding to 65% of heart rate reserve) and a 3-h post-exercise recovery. Exercise increased PGC-1α mRNA expression in muscle of subjects without obesity, but not in subjects with obesity. However, muscle mitochondrial protein synthesis did not increase in either subject group. Similarly, mixed-muscle protein synthesis did not increase in either group. Concentrations of plasma amino acids decreased post-exercise in the subjects without obesity, but not in the subjects with obesity. We conclude that neither mitochondrial nor mixed-muscle protein synthesis increase in muscle of humans during the course of a session of aerobic exercise and its recovery period in the fasting state irrespective of obesity.Trial Registration: The study has been registered within ClinicalTrials.gov (NCT01824173).

Skeletal Muscle Mitochondrial Bioenergetics in a Model of Primary Osteoarthritis after Treatment with Rapamycin and/or Metformin

Impaired mitochondrial function is associated with the loss of skeletal muscle function during aging and age-related osteoarthritis (OA). Emerging evidence suggests Rapamycin (Rap), metformin (Met) or the combination of rapamycin plus metformin (Rap+Met) can extend lifespan and preserve skeletal muscle mass and function during aging. However, the mechanisms of action are not completely understood and have not been studied in specific models of age-related disease. The goal of this study was to test to test the hypothesis that Rap, Met, or Rap+Met would improve skeletal muscle mitochondrial function in an animal model of naturally occurring, age-related OA. We chose the Dunkin Hartley guinea pig because they are an outbred strain that develops naturally occurring OA by 5 months of age with a progression and pathology that highly resembles aging humans. After 5 months of age, Dunkin Hartley guinea pigs also have an age-related decline in skeletal muscle mitochondrial protein synthesis rates. Therefore, 5-month old Dunkin Hartley guinea pigs were fed a standard diet (Con; n=8), or a diet enriched with Rap (14ppm; n=8), Met (1000ppm, n=8) or Rap+Met (14+1000ppm; n=7) for 12-weeks. Mitochondrial respiration and hydrogen peroxide (H2O2) emissions were evaluated in permeabilized muscle fibers from the soleus via high-resolution respirometry and fluorometry using either a saturating bolus or titrating doses of ADP. Michaelis-Menten kinetics was used to evaluate Vmax and ADP sensitivity. All comparisons were made to control group. In the current study, Met had no effect while Rap and Rap+Met modified skeletal muscle mitochondrial bioenergetics only when ADP was titrated but not with a saturating bolus. Specifically, Rap (P<0.05) and Rap+Met (P=0.07) increased ADP sensitivity but decreased (P<0.05) maximal complex-I (CI) linked respiration. Rap and Rap+Met also tended to decrease mitochondrial H2O2 emissions but this trend was no longer apparent when mitochondrial H2O2 emissions were expressed relative to respiration. The decrease in CI-linked respiration and H2O2 emissions was attributed to both a lower CI protein abundance and a decreased index of intrinsic mitochondrial function. This is the first inquiry into how lifespan extending treatments can influence skeletal muscle mitochondria in a model of age-related OA. Further research is needed to understand if increased mitochondrial ADP sensitivity at the cost of CI-linked maximal capacity by Rap and Rap+Met is a beneficial strategy to maintain cellular energy homeostasis during age-related OA.

Skeletal Muscle Mitochondrial Protein Synthesis and Respiration Increase With Low-Load Blood Flow Restricted as Well as High-Load Resistance Training.

Purpose: It is well established that high-load resistance exercise (HLRE) can stimulate myofibrillar accretion. Additionally, recent studies suggest that HLRE can also stimulate mitochondrial biogenesis and respiratory function. However, in several clinical situations, the use of resistance exercise with high loading may not constitute a viable approach. Low-load blood flow restricted resistance exercise (BFRRE) has emerged as a time-effective low-load alternative to stimulate myofibrillar accretion. It is unknown if BFRRE can also stimulate mitochondrial biogenesis and respiratory function. If so, BFRRE could provide a feasible strategy to stimulate muscle metabolic health.Methods: To study this, 34 healthy previously untrained individuals (24 ± 3 years) participated in BFRRE, HLRE, or non-exercise control intervention (CON) 3 times per week for 6 weeks. Skeletal muscle biopsies were collected; (1) before and after the 6-week intervention period to assess mitochondrial biogenesis and respiratory function and; (2) during recovery from single-bout exercise to assess myocellular signaling events involved in transcriptional regulation of mitochondrial biogenesis. During the 6-week intervention period, deuterium oxide (D2O) was continuously administered to the participants to label newly synthesized skeletal muscle mitochondrial proteins. Mitochondrial respiratory function was assessed in permeabilized muscle fibers with high-resolution respirometry. Mitochondrial content was assessed with a citrate synthase activity assay. Myocellular signaling was assessed with immunoblotting.Results: Mitochondrial protein synthesis rate was higher with BFRRE (1.19%/day) and HLRE (1.15%/day) compared to CON (0.92%/day) (P < 0.05) but similar between exercise groups. Mitochondrial respiratory function increased to similar degree with both exercise regimens and did not change with CON. For instance, coupled respiration supported by convergent electron flow from complex I and II increased 38% with BFRRE and 24% with HLRE (P < 0.01). Training did not alter citrate synthase activity compared to CON. BFRRE and HLRE elicited similar myocellular signaling responses.Conclusion: These results support recent findings that resistance exercise can stimulate mitochondrial biogenesis and respiratory function to support healthy skeletal muscle and whole-body metabolism. Intriquingly, BFRRE produces similar mitochondrial adaptations at a markedly lower load, which entail great clinical perspective for populations in whom exercise with high loading is untenable.

Skeletal muscle mitochondrial protein synthesis and respiration in response to the energetic stress of an ultra-endurance race.

The 2016 Colorado Trail Race (CTR) was an ultra-endurance mountain bike race in which competitors cycled for up to 24 h/day between altitudes of 1,675 and 4,025 m to complete 800 km and 21,000 m of elevation gain. In one athlete, we had the unique opportunity to characterize skeletal muscle protein synthesis and mitochondrial respiration in response to a normal activity control period (CON) and the CTR. We hypothesized that mitochondrial protein synthesis would be elevated and mitochondrial respiration would be maintained during the extreme stresses of the CTR. Titrated and bolus doses of ADP were provided to determine substrate-specific oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in permeabilized muscle fibers via high-resolution respirometry. Protein synthetic rates were determined by daily oral consumption of deuterium oxide (2H2O). The endurance athlete had OXPHOS (226 pmol·s-1·mg tissue-1) and ETS (231 pmol·s-1·mg tissue-1) capacities that rank among the highest published to date in humans. Mitochondrial (3.2-fold), cytoplasmic (2.3-fold), and myofibrillar (1.5-fold) protein synthesis rates were greater during CTR compared with CON. With titrated ADP doses, the apparent Km of ADP, OXPHOS, and ETS increased after the CTR. With provision of ADP boluses after the CTR, the addition of fatty acids (-12 and -14%) mitigated the decline in OXPHOS and ETS capacity during carbohydrate-supported respiration (-26 and -31%). In the face of extreme stresses during the CTR, elevated rates of mitochondrial protein synthesis may contribute to rapid adaptations in mitochondrial bioenergetics. NEW & NOTEWORTHY The mechanisms that maintain skeletal muscle function during extreme stresses remain incompletely understood. In the current study, greater rates of mitochondrial protein synthesis during the energetic demands of ultra-endurance exercise may contribute to rapid adaptations in mitochondrial bioenergetics. The endurance athlete herein achieved mitochondrial respiratory capacities among the highest published for humans. Greater mitochondrial protein synthesis during ultra-endurance exercise may contribute to improved mitochondrial respiration and serve as a mechanism to resist cellular energetic stresses.

Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

Eicosapentaenoic acid but not docosahexaenoic acid restores skeletal muscle mitochondrial oxidative capacity in old mice.

Mitochondrial dysfunction is often observed in aging skeletal muscle and is implicated in age-related declines in physical function. Early evidence suggests that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve mitochondrial function. Here, we show that 10 weeks of dietary eicosapentaenoic acid (EPA) supplementation partially attenuated the age-related decline in mitochondrial function in mice, but this effect was not observed with docosahexaenoic acid (DHA). The improvement in mitochondrial function with EPA occurred in the absence of any changes in mitochondrial abundance or biogenesis, which was evaluated from RNA sequencing, large-scale proteomics, and direct measurements of muscle mitochondrial protein synthesis rates. We find that EPA improves muscle protein quality, specifically by decreasing mitochondrial protein carbamylation, a post-translational modification that is driven by inflammation. These results demonstrate that EPA attenuated the age-related loss of mitochondrial function and improved mitochondrial protein quality through a mechanism that is likely linked with anti-inflammatory properties of n-3 PUFAs. Furthermore, we demonstrate that EPA and DHA exert some common biological effects (anticoagulation, anti-inflammatory, reduced FXR/RXR activation), but also exhibit many distinct biological effects, a finding that underscores the importance of evaluating the therapeutic potential of individual n-3 PUFAs.

Optimizing the measurement of mitochondrial protein synthesis in human skeletal muscle.

The measurement of mitochondrial protein synthesis after food ingestion, contractile activity, and/or disease is often used to provide insight into skeletal muscle adaptations that occur in the longer term. Studies have shown that protein ingestion stimulates mitochondrial protein synthesis in human skeletal muscle. Minor differences in the stimulation of mitochondrial protein synthesis occur after a single bout of resistance or endurance exercise. There appear to be no measurable differences in mitochondrial protein synthesis between critically ill patients and aged-matched controls. However, the mitochondrial protein synthetic response is reduced at a more advanced age. In this paper, we discuss the challenges involved in the measurement of human skeletal muscle mitochondrial protein synthesis rates based on stable isotope amino acid tracer methods. Practical guidelines are discussed to improve the reliability of the measurement of mitochondrial protein synthesis rates. The value of the measurement of mitochondrial protein synthesis after a single meal or exercise bout on the prediction of the longer term skeletal muscle mass and performance outcomes in both the healthy and disease populations requires more work, but we emphasize that the measurements need to be reliable to be of any value to the field.

High insulin combined with essential amino acids stimulates skeletal muscle mitochondrial protein synthesis while decreasing insulin sensitivity in healthy humans.

Insulin and essential amino acids (EAAs) regulate skeletal muscle protein synthesis, yet their independent effects on mitochondrial protein synthesis (MiPS) and oxidative function remain to be clearly defined. The purpose of this study was to determine the effects of high or low insulin with or without EAAs on MiPS. Thirty participants were randomized to 3 groups of 10 each with each participant studied twice. Study groups comprised (1) low and high insulin, (2) low insulin with and without EAAs, and (3) high insulin with and without EAAs. The study was conducted in an in-patient clinical research unit. Eligible participants were 18 to 45 years old, had a body mass index of <25 kg/m(2), and were free of diseases and medications that might impair mitochondrial function. Low (∼ 6 μU/mL) and high (∼ 40 μU/mL) insulin levels were maintained by iv insulin infusion during a somatostatin clamp while maintaining euglycemia (4.7-5.2 mM) and replacing GH and glucagon. The EAA infusion was 5.4% NephrAmine. l-[ring-(13)C6]Phenylalanine was infused, and muscle needle biopsies were performed. Muscle MiPS, oxidative enzymes, and plasma amino acid metabolites were measured. MiPS and oxidative enzyme activities did not differ between low and high insulin (MiPS: 0.07 ± 0.009 vs 0.07 ± 0.006%/h, P = .86) or between EAAs and saline during low insulin (MiPS: 0.05 ± 0.01 vs 0.07 ± 0.01, P = .5). During high insulin, EAAs in comparison with saline increased MiPS (0.1 ± 0.01 vs 0.06 ± 0.01, P < .05) and cytochrome c oxidase activity (P < .05) but not citrate synthase (P = .27). EAA infusion decreased (P < .05) the glucose infusion rates needed to maintain euglycemia during low (∼ 40%) and high insulin (∼ 24%). EAAs increased MiPS and oxidative enzyme activity only with high insulin concentrations.

β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg(-1)·min(-1) peak O(2) uptake, 26.7 ± 2.0 kg/m(2) body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O(2) uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [(2)H(5)]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ∼2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content.

Acute β-adrenergic stimulation does not alter mitochondrial protein synthesis or markers of mitochondrial biogenesis in adult men

Exercise-induced expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is dramatically inhibited in mice pretreated with a beta-adrenergic receptor (beta-AR) antagonist, suggesting that beta-ARs play an important role in the regulation of skeletal muscle PGC-1alpha expression, and potentially, mitochondrial biogenesis. Accordingly, we hypothesized that acute beta-AR stimulation would induce transcriptional pathways involved in skeletal muscle mitochondrial biogenesis in humans. Whole body protein turnover (WBPT), myofibrillar protein synthesis (MyPS), skeletal muscle mitochondrial protein synthesis (MiPS), and mitochondrial biogenic signaling were determined in samples of vastus lateralis obtained on two separate occasions in 10 young adult males following 1 h of continuous intravenous administration of saline (CON) or a nonspecific beta-AR agonist [isoproterenol (ISO): 12 ng.kg fat free mass(-1).min(-1)], combined with coinfusion of [1,2](13)C-leucine. beta-AR stimulation induced appreciable increases in heart rate and systolic blood pressure (both P < 0.001) but did not affect mitochondrial biogenic signaling (no change in PGC-1alpha, TFAM, NRF-1, NRF-2, COX, or NADHox expression via RT-PCR; P > 0.05). Additionally, MiPS [CON: 0.099 +/- 0.028, ISO: 0.074 +/- 0.046 (mean +/- SD); P > 0.05] and MyPS (CON: 0.059 +/- 0.008, ISO: 0.055 +/- 0.009; P > 0.05), as well as measures of WBPT were unaffected. On the basis of this investigation, we conclude that acute intravenous beta-AR stimulation does not increase mitochondrial protein synthesis or biogenesis signals in skeletal muscle.

Potential Application of Essential Amino Acid Supplementation to Treat Sarcopenia in Elderly People

Aging is universally associated with a progressive decline in both physical and metabolic function across a diverse rangeofspeciesfromnematodestohumans.Moreover,progressive reductions in skeletal muscle mass, strength, and quality (referred to as sarcopenia) are often observed with aging. An associated problem is decline in maximal aerobic capacity, and all of these factors together contribute to the physical frailty of older people. In light of the rapidly expanding aging population, sarcopenia and related disorders are emerging as a major public health problem of the 21st century. This is particularly pertinent considering the loss of independence that is associated with frailty and falls that increase the risk of fractures. Another consequence of the sarcopenia of aging is a deleterious change in body composition resulting in many metabolic alterations leading on to metabolic syndrome and increased cardiovascular disease risk. Indeed, sarcopenia has received considerable attention by both researchers and clinicians over the last two decades. Muscle changes with age include reduction in fiber numbers, motor units, and a relative loss of type II fibers with a preservation of type I fibers (1) that may result from reductions in transcript levels of myosin heavy chain isoforms IIa and IIx without any decline of isoform I as reported in older people (2). Thus, a relative increase of type I fibers occurs with age, but these fibers are lower in abundance of mitochondria (3) than those in longdistance runners who also have a relatively high proportion of type I fibers but with high abundance of mitochondria. Reductionsinskeletalmuscleproteinabundances(thebalancebetween protein synthesis and degradation) are a key contributing factor to the development of sarcopenia. Mechanistically, it has been extensively reported that skeletal muscle protein synthesis is reduced in aging adults (2, 4, 5). The reduction in skeletal muscle mitochondrial protein synthesis (6) occurs in conjunction with reducedmitochondrialcapacityforATPproduction(3).Thank

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa 3 and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c 1, and aa 3, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.

The effect of chloramphenicol, ethidium bromide and cycloheximide on mortality and mitochondrial protein synthesis of adult blowflies

The effects of cycloheximide, chloramphenicol and ethidium bromide on the blowfly Calliphora erythrocephala were studied. In the first set of experiments, toxic levels were determined by examining activity and mortality of flies after injection of various doses of each drug. In the second set of experiments, the effect of drug treatment on flight muscle mitochondrial protein synthesis was determined in relation to age by following the incorporation of radioactively labelled amino acid, [3H]leucine, into mitochondrial protein in vivo. To confirm the developmental changes in flight muscle mitochondria, mitochondrial protein content per fly was estimated from emergence to 30 days of age; the highest protein level was recorded between 6 and 10 days of age. Maximum incorporation of labelled amino acid was found in newly emerged flies, and this age group was also the most sensitive to drug treatment. By the time flies had reached 6--10 days of age, amino acid incorporation had declined to about two-thirds of the rate obtained with newly emerged flies. With 6--10-day old flies, however, the highest value for flight muscle mitochondrial protein per fly was recorded, and these flies also displayed the greatest resistance to drug treatment of any age group investigated. For example, inhibition of protein synthesis following injection of 300 micrograms/fly of chloramphenicol was only about 15% below the untreated control in 6-10-day-old flies, whereas in all other age groups investigated, inhibition ranged between 30 and 50% of the controls. At 15--20 days of age, protein synthesis decreased to a third of the newly emerged flies' rate and continued to decrease further in the 30--35-day-old group, where it was less than one sixth of the youngest age group. The effect of drug treatment on these older flies was also less than that observed with newly emerged flies, especially after chloramphenicol and ethidium bromide injections. The effect of cycloheximide however, was much the same in all age groups, with inhibition of protein synthesis being 80--90% of controls. Surprisingly, cycloheximide (1--10 micrograms/fly) had little initial effect on mortality of young flies, despite almost complete blockage in the synthesis of mitochondrial proteins at these concentrations. 95% mortality occurred only when doses of 20 micrograms/fly were given. In contrast, high doses of chloramphenicol (400 micrograms/fly) and ethidium bromide (15 micrograms/fly) caused almost total mortality a few hours after injection, although such doses never induced more than about 50% inhibition of mitochondrial protein synthesis. Each drug therefore has a different site of inhibition and induces different mortality effects. Possible explanations for these differences in mortality are discussed.