Fine structure and metabolism of multiply innervated fast muscle fibres in teleost fish

Summary Both the fast and slow muscle fibres of advanced teleost fish are multiply innervated. The fraction of slow-fibre volume occupied by mitochondria is 31.3%, 25.5% and 24.6%, respectively, for the myotomal muscles of brook trout (Salvelinus fontinalis), crucian carp (Carassius carassius), and plaice (Pleuronectes platessa), respectively. The corresponding figures for the fast muscles of these species are 9.3%, 4.6% and 2.0%, respectively. Cytochrome-oxidase and citrate-synthetase activities in the fast muscles of 9 species of teleost range from 0.20–0.93 moles substrate utilised, g wet weight muscle^-1 min^-1 (at 15° C) or around 4–17% of that of the corresponding slow fibres. Ultrastructural analyses reveal a marked heterogeneity within the fast-fibre population. For example, the fraction of fibres with <1% or >10% mitochondria is 0,4,42% and 36, 12 and 0%, respectively, for trout, carp and plaice. In general, small fibres (<500 m²) have the highest and large fibres (>1,500 m²) the lowest mitochondrial densities. The complexity of mitochondrial cristae is reduced in fast compared to slow fibres.Hexokinase activities range from 0.4–2.5 in slow and from 0.08–0.7 moles, g wet weight^-1 min^-1 in fast muscles, indicating a wide variation in their capacity for aerobic glucose utilisation. Phosphofructokinase activities are 1.2 to 3.6 times higher in fast than slow muscles indicating a greater glycolytic potential. Lactate dehydrogenase activities are not correlated with either the predicted anaerobic scopes for activity or the anoxic tolerances of the species studied. The results indicate a considerable variation in the aerobic capacities and principal fuels supporting activity among the fast muscles of different species. Brook trout and crucian carp are known to recruit fast fibres at low swimming speeds. For these species the aerobic potential of the fast muscle is probably sufficient to meet the energy requirements of slow swimming.     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

Quantitative changes in the lysosomal vacuolar system of rat hepatocytes during short-term starvation

Summary Ultrastructural morphometric analysis was used to study time-dependent variations in macro and microautophagy in rat hepatocytes. Except during periods of shortterm starvation for up to 24 h, animals were kept under standardized conditions of food intake.In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level.Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises.The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.     

The effect of starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius carassius)

Summary The effect of 16 weeks total starvation on the ultrastructure of the red and white myotomal muscles of the crucian carp (Carassius Carassius) has been investigated. In the white fibres the amount of myofibrillar material fell from 89.6% to 70.7% of the total fibre volume whilst in the red fibres the fall was from 72.2% to 70.3%. The sarcoplasmic reticulum appeared to have become swollen during starvation in both fibre types. In the white fibres the terminal cisternae of some triads seem to have fused. The volume of the red fibres occupied by mitochondria was reduced from 16.2 % to 5.9 %. The concentration of mitochondria in the white fibres was too low to detect any quantitative changes. A marked reduction in the amount of euchromatin material was observed in most white fibre nuclei and many red fibre nuclei. Many of the ultrastructural changes noted in the present study can be correlated with biochemical changes known to occur in the red and white myotomal muscles of fish during starvation. This work was supported by a grant from the Natural Environmental Research Council.     

Quantitative studies on the distribution of myofilaments in intrafusal muscle fibres

Summary Muscle spindles contain two types of intrafusal muscle fibre, nuclear bag fibres and nuclear chain fibres. The intrafusal fibres of rabbit and guinea pig spindles have been studied using quantitative stereological techniques at the ultrastructural level. The crosssectional areas occupied by myofilaments have been measured in the polar and equatorial regions of both types of intrafusal fibre. There are considerably fewer myofilaments in the equatorial regions of both types of fibre compared with their polar regions.This work was carried out with the aid of grants from the Medical Research Council and the Science Research Council of Great Britain.     

Drinking rates and Na+ effluxes in response to temperature change in two species of marine flatfish: dab,Limanda limanda and plaice,Pleuronectes platessa

The relationship between fish mass and drinking rate in two species of flatfish, dab and plaice, weighing between 1 and 150 g was investigated. Both plaice and dab showed increased drinking rates with increasing fish mass, although — when calculated on a weight-specific basis — the increase was negligible. Fish were acclimated to winter and summer temperatures of 9 and 14°C, respectively. In winter both species were acutely transferred to 5, 14 and 21°C and in summer to 5, 9, 21 and 25°C. Drinking rates, Na⁺ efflux and body ion content were measured. Dab showed lower drinking rates than plaice (e.g. the weight-specific drinking rates of summer-and winter-acclimated dab were 0.12±0.01 and 0.06±0.006 ml·h^-1·100 g^-1, respectively, compared to that of plaice which were 0.25±0.02 ml·h^-1·100 g^-1 in summer and 0.17±0.02 ml·h^-1·100g^-1 in winter). Summer dab exhibited decreased weight-specific drinking rates at 5, 9 and 25°C, while winter dab increased drinking at 21°C. Winter plaice also showed increased drinking at 21°C and a decrease at 5°C, but in contrast summer plaice did not increase drinking at either 21 or 25°C but showed a decrease at 5 and 9°C. Winter dab and plaice showed similar Na⁺ efflux rates but summer dab showed higher efflux at all temperatures except 5°C. The data indicates that (a) the osmoregulatory function of plaice is much weaker than that of dab at higher temperatures (>20°C) and (b) mass has a greater effect on drinking and Na⁺ efflux rates than temperature; although when calculated on a weight-specific basis neither drinking nor efflux showed any variation with fish mass suggesting that these functions occur at similar intensities across the entire weight range.Abbreviations ANOVA analysis of variance - ⁵¹Cr-EDTA ⁵¹chromium ethylenediaminetetra-acetic acid - SW sea water     

The ultrastructural localization of a cholinesterase in the body muscle of plaice (Pleuronectes platessa)

Summary By means of a histochemical method adapted for electron microscopy a cholinesterase in body muscle cells of plaice (Pleuronectes platessa) has been localized to the sarcolemma. The cholinesterase activity disappeared from the sarcolemma after the muscle tissue had been incubated with a bacterial enzyme, which had earlier been shown, by biochemical methods, to be able to liberate this cholinesterase activity from plaice muscle.The provision of live plaices from Kristineberg Zoological Station, Fiskebäckskil, Sweden, is gratefully acknowledged. We are greatly indebted to Dr. Åke Bovallius, FOA, who provided the starting material for the bacterial enzyme. We would like to express our sincere thanks to Prof. Lennart Nicander for valuable discussions and for placing the resources of the Department of Anatomy and Histology, Royal Veterinary College, to our disposal.     

Changes in haematological parameters associated with capture and captivity of the marine teleost, Pleuronectes platessa L

After capture by trawling, the blood parameters of plaice (Pleuronectes platessa L.) are perturbed for up to 5 days post-capture. Whole blood values recovered from an initial stress-induced haemoconcentration within 12 hr. There is a marked hyperglycaemia following capture: blood glucose concentration increased four-fold to 87.92 +/- 10.41 mg/100 ml (N = 6) after 12 hr and remained elevated for 3-4 days before returning to normal values. Monovalent blood electrolytes (Na+, K+, Cl-) significantly increased during the initial stages post-capture (4-10 hr) but then recovered. The divalent cations (Ca2+, Mg2+) similarly increased but for a longer period (24-72 hr). Liver and muscle glycogen concentrations were very variable during the recovery period. All blood parameters achieved stable values within 5 days of capture. This study provides comprehensive haematological data on post-trawl recovery and tank-acclimation in plaice, for up to 28 days following capture.     

Quantitative changes of the C-cell population in the rat thyroid during postnatal ontogenesis

Summary The number and distribution of C-cells in the rat thyroid gland, have been investigated during postnatal ontogenesis from birth to 120 days of age. The argyrophilic and metachromatic properties of these cells were used to identify them. In the thyroid of newborn rats the C-cells do not exhibit argyrophilia and metachromasia. These reactions appear at 10 days and can be seen at all subsequent ages. The number of C-cells shows a parallel increase with age as demonstrated by the change in the proportion of C-cellsF-cellscolloidstroma during development. A marked increase in C-cells was found at 50 days of age when the proportion of C-cells rose to 27.67% from the value of 16.78% at 30 days. At 70 days a decrease was noted (20.50%) which hardly changed until 120 days of age (22.20%). The numerical increase in C-cells occurs at the expense of the follicular epithelium and stroma.The C-cells occupy elongated islet-like region in the central part of the lobe, decreasing in number towards the periphery where no C-cells are present. The long axis of the C-cells area is parallel with the longitudinal axis of the lobe. The area of C-cells is largest at the centre of the lobe, corresponding to the territory of the peak of the Gaussian curve for the numerical distribution of C-cells.     

Changes in viability, respiratory activity and morphology of the marine Vibrio sp. strain S14 during starvation of individual nutrients and subsequent recovery

Abstract Starvation for different individual nutrients revealed various morphotypes of Vibrio sp. strain S14. Carbon or multiple-nutrient starved cells formed ultramicrocells with low respiratory activity and high culturability. In contrast, cells starved for nitrogen or phosphorus formed filaments or swollen rods with large inclusion bodies of PHB. These cells exhibited a 3–4 log decrease in culturability, nevertheless many were actively respiring and direct viable counts equalled at least 80% of the original number of cells. After 2–3 days of prolonged incubation, microcolonies appeared at approximately the same number of cells as at the onset of starvation. A nutrient-induced increase in respiratory activity, after 120 h of starvation, was instantaneous for cells starved for carbon or multiple-nutrients, but cells depleted of nitrogen or phosphorus exhibited a lag period of at least 3 h.     

Metabolism during starvation/dehydration stress in two species of galagos

Two species of galagos (G. senegalensis moholi andG. garnettii) were subjected to dehydration and starvation stress in order to determine whether, as is common in other animals, these hypometabolic prosimians would lower their metabolic rate even further. Dehydration was confirmed by losses in body mass, a decrease in fecal water content and a rise in urine osmolality. At the height of dehydration, 20 to 25% reduction in body mass, 30 to 40% reduction in fecal water content and urine osmolality ranging from 1.8 to 3.5 Osmol kg⁻¹ H₂O, were recorded in some of the animals. Basal metabolic rate of 0.536 ml O₂ (g·h)⁻¹ inG. s. moholi and 0.302 ml O₂ (g·h)⁻¹ inG. garnettii were recorded, representing 50 to 42% reduction in metabolic rate, respectively, compared with mass specific values. In none of the tested animals did we observe significant reduction in basal metabolism during dehydration/starvation stress compared with the rates observed during the control period. Basal metabolism in the bushbabies seems to have reached the lowest level and no further adjustment is apparently possible as a strategy for energy saving during starvation and/or dehydration stress.     

Ultrastructure and metabolism of skeletal muscle fibres in the tench: Effects of long-term acclimation to hypoxia

Summary Tench (Tinca tinca) were acclimated to either aerated (P _{O 2} 17.6 KPa) or hypoxic (P _{O 2} 1.5 KPa) water for 6 weeks.Acclimation to hypoxia resulted in a decrease in mitochondrial volume fraction in both slow (22.9 to 15.0 %) and fast glycolytic (4.5 to 1.8 %) myotomal muscles fibres (P<0.01).Intermyofibrillar mitochondrial populations (4.4 to 1.2% slow; 0.6 to 0.04% fast fibres) were affected to a greater extent than those in the subsarcolemmal zone (18.5 to 13.8% slow; 3.9 to 1.8% fast fibres). After acclimation to hypoxia, cytochrome-oxidase activities decreased by 31 and 33 % in slow and fast fibres, respectively, but were maintained in the liver.Fibre size remained unchanged and actively differentiating fibres were observed in muscles from both groups of fish. Hypoxia resulted in a significant increase in myofibrillar volume fraction in both slow (43.1 to 56.1 %) and fast glycolytic fibres (73.1 to 82.7%) (P<0.05).Glycogen concentrations (mg/100g tissue) for liver (6616) slow muscle (1892) and fast muscle (334) were similar for fish acclimated to aerated or hypoxic water. Acclimation to hypoxia increased carnitine palmitoyl transferase activity (moles substrate utilised g·dry wt_-1 min^-1) in slow (0.42 to 1.1), fast glycolytic muscle (<0.01 to 0.15) and liver (1.1 to 3.7) indicating an enhanced capacity for fatty acid oxidation.Phosphofructokinase activities of fast glycolytic fibres were similar in fish acclimated to either aerated or hypoxic water, consistent with an unaltered capacity for anaerobic glycogenolysis. Hexokinase activities (moles substate utilised, g·dry wt^-1 min^-1) decreased in fast fibres (1.2 to 0.4) but were maintained in the slow muslce (2.1 to 2.5) and liver (4.5 to 4.8) of hypoxic fish. The activities of phosphofructokinase in slow muscle and phosphofructokinase, pyruvate kinase and lactate dehydrogenase in liver were two times higher in fish acclimated to hypoxia. An enhanced capacity for glycolysis in these tissues may reflect a reduced threshold for anaerobic metabolism during activity and/or an adaptation for acute exposure to anoxia in fish acclimated to hypoxia.Abbreviations/Glossary CO cytochrome oxidase activity - CPT carnitine palmitoyltransferase activity - HK hexokinase activity - LDH lactate dehydrogenase activity - PFK phosphofructokinase activity - PK pyruvate kinase activity - Vv volume fractions of cell components - normoxic fish acclimated to aerated water - hypoxic fish acclimated to reduced oxygen tensions - P _{O 2} partial pressure of oxygen tension A preliminary account of part of this work was presented at theXth European Meeting on Muscle and Cell Motility held at Galway, Ireland, in September 1981     

Gadd34 induces autophagy through the suppression of the mTOR pathway during starvation

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.     

Gill ventilation and O2 extraction during graded hypoxia in two ecologically distinct species of flatfish,the flounder (Platichthys flesus) and the plaice (Pleuronectes platessa)

Synopsis Gill ventilation, breathing frequency, breath volume, oxygen extraction from the ventilatory water current and oxygen uptake through the gills were measured in flounder, Platichthys flesus, and plaice, Pleuronectes platessa, at water O₂ tensions ranging from 35 to 155 mm Hg at 10° C. Ventilation volumes were similar in the two species at high water O₂ tension. Exposure to hypoxic water elicited a larger increase in ventilation in the flounder. The per cent extraction of O₂ from water decreased slightly in both species as water O₂ tension was lowered. At comparable levels of ventilation O₂ extraction was higher in flounder. At the higher levels of water O₂ tension, O₂ uptake across the gills of flounder was stable, the critical O₂ tension being between 60 and 100 mm Hg. The plaice behaved as an oxygen conformer over the entire range of O₂ tensions investigated. The superior ability of the flounder in maintaining OZ uptake across the gills during a reduction in water O₂ tension may in part explain why the species, unlike plaice, inhabits very shallow waters with large fluctuations in dissolved oxygen.     

Restoration of glycogen from lactic acid in the anaerobic swimming muscle of plaice, Pleuronectes platessa L.

The conclusion from two in vivo experiments is that a significant proportion of the lactic acid, normally formed by glycolysis from glycogen and held in the muscle cells following exhausting exercise of the anaerobic swimming muscle of the teleost fish Pleuronectes platessa L, is converted by gluconeogenesis to form glycogen in the recovering muscle.
In the first experiment a technique for measurement of [³H]glucose turnover in the plaice was developed and applied to measure turnover in resting and exhausted fish. It is concluded that insufficient glucose was moved through the circulation to account for the rate of glycogen formation observed in the recovering exhausted muscle.
In the second experiment, an intramuscular injection of [¹⁴C]lactate to exhausted fish revealed a direct uptake of [¹⁴C]lactate by the recovering muscle cells, and the incorporation of substantial proportions of lactate into the restored glycogen. Simultaneous use of [³H]-mannitol allowed measurement of the isotope distribution between extra- and intracellular spaces.     

Temperature and somitogenesis in embryos of the plaice (Pleuronectes platessa)

The rate of somitogenesis was observed in plaice embryos reared at 5 and 12°C. Somite formation occurred relatively later in development at 12 than at 5°C. At any given stage of development 5°C plaice embryos had significantly more somites than embryos reared at 12°C.     

A comparison of food capture and ingestion in juveniles of two flatfish species, Pleuronectes platessa and Limanda limanda (Teleostei: Pleuronectiformes)

Juvenile plaice, Pleuronectes platessa ( n =6, 151–197 mm SL) and dab Limanda limanda ( n =6, 119–180.5 mm SL) feeding on pieces of mussel flesh were studied by high-speed (200 fields s⁻¹) videophotography at 11) C. Prey capture cycles (involving jaw protrusion and opercular expansion) were described in both species. The capture cycle was followed by at least one cycle (similar to the capture cycle in all respects except food intake) which is believed to transport food to the oesophagus. The transport cycle (or cycles) was followed by a species-specific number of normal respiratory cycles (7 or 8 in plaice, 11 or 12 in dab) and a final single cycle when food debris was violently ejected from the opercular cavity. The respiratory cycles and ejection cycle cleansed the orobranchial/opercular cavities and gills. Differences between the species were relatively minor, but plaice captured and transported food more quickly than dab.     

Hepatocyte classes during liver atrophy due to starvation in the golden hamster

Summary The total dry masses of normal hepatocytes isolated from adult golden hamsters form a multimodal frequency distribution curve (10–11 cell classes with a period of 132 pg). During starvation the hepatocytes maintain the arrangement in classes, the number of which, however, decreases. The cell percentage of the lightest classes progressively increases, but no change occurs in the class period. A similar behaviour is shown by the aqueo-insoluble dry masses of the hepatocytes. The nuclear dry masses increase until the 4th day of starvation. Later on, they decrease. The nucleo-cytoplasmic ratio progressively increases. The total number of hepatocytes per liver and the binuclear cell percentage do not show any significant changes during starvation. The mitotic index is lower in starved animals. The kinetics of decrement in dry mass of the hepatocytes during starvation and the possibility that the hepatocyte class series are the resultant of a dynamic balance of single hepatocyte mass are discussed.This work was supported by Consiglio Nazionale delle Ricerche, Roma, Italia (Grant No. 70/01811/04).The author is deeply grateful to Prof. Enrico Puccinelli for his encouragement and advice during this work. The skilful technical assistance of Mrs. Lucia Giaccardo and Mr. Emilio Madrigali is gratefully acknowledged.