Adipogranular Cells Research Articles

Microscopic anatomy of gonadal area in the deep-sea clam Calyptogena pacifica (Bivalvia: Vesicomyidae) with emphasis on somatic cells

Somatic cells in the gonadal area of male and female deep-sea clams, Calyptogena pacifica, were examined using light and transmission electron microscopy. Acini both at the pre-spawning stage and at the stage of active spermatogenesis were observed to be simultaneously present in sections through a male gonad. Oocytes of various degrees of maturity were simultaneously present in female acini. No storage tissue and cells similar to adipogranular cells or vesicular connective tissue of other mollusks were found in the gonadal area of C. pacifica. Instead, in both males and females, numerous hemocytes surround the acini. Among hemocytes, two types of granulocytes and erythrocytes were identified. Bundles of muscle cells were also found in the interacinar space. Male intraacinar accessory cells were rather large, glycogen-rich, with lipid inclusions, and phagosomes contained spermatogenic cells. Female accessory cells had well-developed endoplasmic reticulum, but they did not form any follicles around oocytes, being in their basal part, closer to basal lamina. Such a specific pattern of gonad organization can presumably be explained by both symbiosis with sulfide-oxidizing bacteria and phylogenetic aspects that should be further studied. Some evidence for continuous gametogenesis are discussed.

The expression of estrogen receptors during the Mytilus galloprovincialis ovarian cycle.

The aim of this paper is to assess, by real-time polymerase chain reaction and in situ hybridization, the expression of estrogen receptors ER1 and ER2 during the ovarian cycle of Mytilus galloprovincialis. By considering four phases of the reproductive cycle, that is stasis and previtellogenic stage (Stage 0), early vitellogenesis (Stage I), vitellogenesis (Stage II), full-grown oocyte (Stage III), our investigation demonstrates that the two receptors are differently expressed during the phases investigated of the ovarian cycle: ER1 reaches the highest level at Stage III, whereas ER2 reaches the highest level at Stage II, with ER2 always present at higher levels than ER1. The stage-dependent receptor expression was recorded within oocytes, follicle cells, and adipogranular cells. No ER1 and ER2 messenger RNAs (mRNAs) were found within vesicular cells. It is to be noted that the ER1 and ER2 expression within the growing oocytes, the follicular, and adipogranular cells overlaps with that of the mRNA for vitellogenin in the same cells, strongly suggesting that in Mytilus, as in vertebrates studied so far, the vitellogenin expression is under the control of estrogens.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17β-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3β-HSD, 17β-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

Variability of the reproductive cycle in estuarine and coastal populations of the mussel Mytilus galloprovincialis Lmk. from the SE Bay of Biscay (Basque Country)

Mussel commercial production depends on wild populations to obtain the seed. Therefore, any new farming initiative needs deep knowledge on reproductive cycle of native mussel populations. The reproductive cycle of Mytilus galloprovincialis Lmk. from five sites, three estuarine and two coastal, along the coast of the Basque Country were assessed using gonad index and stereology over 2 consecutive years (2010 and 2011). Sex ratios, developmental stages of gonads, as well as reserve tissue cycle were analyzed. The onset of gametogenesis was observed from winter to spring, with spawning occurring from spring till summer. A resting phase followed with an increase in reserve tissues (adipogranular cells) was observed in fall. Mussels stayed ripe during winter until favorable environmental conditions were met in spring. Differences in reproductive cycles were found among populations, and also, year-to-year variability was observed within each population. In two of the studied sites asynchrony in the timing of spawning was observed between genders. These timing differences, however, did not prevent mussels at those sites from reproducing successfully. The Bidasoa estuary points to the potential of such studied sites for becoming the main source of wild mussel spats for future expected commercial mussel farming in the Basque Country. To ensure year-to-year maximum reproductive output, however, wild mussels harvest should be avoided from spring to the end of summer in this region.

Spermatogenic Cycle and Steroidogenic Control of Spermatogenesis in Mytilus galloprovincialis Collected in the Bay of Naples.

The aim of the present article was to study the spermatogenic cycle of Mytilus galloprovincialis collected in the Bay of Naples during a whole year and to acquire new insights into the mechanism of control. Knowledge of the Mytilus cycle in this geographic area is of particular interest as, to the best of our knowledge, the male gonad cycle has been hitherto unexplored. Testis organization was evaluated together with the localization of the enzymes 3β-HSD, 17β-HSD, and P450-aromatase, which are strictly connected to the synthesis of two key hormones involved in the testis activity: testosterone and 17β-estradiol. It was demonstrated that: (1) the spermatogenic cycle starts in late Summer-early Fall and continues until early Winter, when the first spawning occurs; after rapid gonad restoration, several spawning events take place until June, when the testis becomes non-active again; (2) in the testis, true Leydig and Sertoli cells are present; (3) during the reproductive period, Sertoli, Leydig, germ, and adipogranular cells (ADGs) are positive to 3β-HSD and 17β-HSD, while only germ cells are positive to P450 aromatase; by contrast, during the resting period, only ADGs are positive to 3β-HSD and 17β-HSD, and P450-aromatase is no longer recognizable. The presence of a hermaphrodite sample is also described. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1881-1894, 2017. © 2017 Wiley Periodicals, Inc.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.)

The aim of this study is to assess, by RT-PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis).

Particular features of gonadal maturation and size at first maturity in <i>Atrina maura</i> (Bivalvia: Pinnidae)

The gonadal maturation of Atrina maura was examined by means of histological analysis and quantitative criteria. Particular features not previously described for this species are reported in this study: in both males and females the undifferentiated stage is absent and there is massive gamete resorption when the seawater temperature reaches 25°C; in males, there is continuous spawning concurrent with other gonadal development stages and the adipogranular cells surrounding the acini walls decrease with testis ripeness, which suggests they play an energetic role. Atresia displayed two stages: cytoplasmic structures with oocyte degeneration and digestion by hemocytes. The oocyte diameter was larger than that reported for cultured specimens. Size at first maturity was reached at 23.3 cm in shell height (SH) (12.2 cm in shell length, SL) in females and 22.8 cm SH (12.0 cm SL) in males.

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT–PCR

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv(BAS)) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv(BAS) increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.

Histochemical and ultrastructural characterisation of mantle storage cells in the hydrothermal-vent bivalve Bathymodiolus azoricus

This study reports on two types of storage cells that are present in the mantle connective tissue of the deep-sea mussel Bathymodiolus azoricus. One type corresponds to the adipogranular cells, a kind of storage cell previously described in other bivalves. In these cells extensive regions of the cytoplasm are filled with glycogen deposits and these zones became strongly stained after histochemical (PAS) or ultrastructural detection of polysaccharides. Several lipid droplets and membrane bound granules containing homogeneous electron-dense material are also present in adipogranular cells. A second type of cell contains large lysosomes in addition to numerous lipid droplets, but lacking cytoplasmic glycogen deposits. Due to these characteristics we named them adipolysosomal cells. They can be identified in semi-thin sections stained with PAS reaction because the lysosomes are the only positively stained structures. In the connective tissue of the mantle, some cells containing many lysosomes and a few lipid droplets were also observed. These cells differ from the adipolysosomal cells mainly because they have a reduced amount of lipid reserves, and could be an initial stage in the development of adipolysosomal cells. The vesicular connective tissue cells that in other Mytilidae are specialised in glycogen storage were not detected in B. azoricus. The reserves accumulated in the two types of storage cells described in B. azoricus may be important for the survival of these hydrothermal-vent bivalves if their nutrition is affected by a temporary loss or reduction of endosymbiotic bacteria due to sulphide and/or methane shortage caused by oscillations in vent activity.

왕우럭(Tresus keenae) (Mactridae: Bivalvia)의 배우자형성과정의 미세구조

The gametogenesis of the surf clam, Tresus keenae, were investigated by SEM and TEM. Both the testis and the ovary had follicle tubes surrounded by inter-tubal tissue composed of adipogranular cells that provided storage function. In the vitellogenic oocyte, lipid droplets and lipid yolk granules were found in the vacuoles formed by the Golgi apparatus. Proteid yolk granules were formed by the endoplasmic reticulum and cortical granules in the cytoplasm during vitellogenesis. The mature sperm was primitive and resembled a jar with a cover. The sperm heads were approximately <TEX>$2.00-2.30 {\cal}um$</TEX>. The acrosomal rod projected in front of the acrosome. In addition, four large mitochondria were in the midpiece.

Ultrastructure of gametogenesis in a chemosynthetic mytilid bivalve ( Bathymodiolus childressi ) from a bathyal, methane seep environment (northern Gulf of Mexico)

The ultrastructural features of gametogenesis have been described in the methane-seep mytilid bivalve Bathymodiolus childressi Gustafson, Turner, Lutz & Vrijenhoek, 1998 collected from the Gulf of Mexico in August 1995. This is the first ultrastructural description of gametogenesis in any methane-seep bivalve. B. childressi is gonochoric, and both the testis and ovary consist of acini surrounded by inter-acinal tissue composed of adipogranular cells that serve a nutrient storage function. Oocytes develop in close association with squamous follicle cells although the follicle cells do not appear to play a primary role in yolk synthesis. During the vitellogenic phase, biosynthesis of four types of organelles occurs, including lipid droplets, yolk granules, cortical granules, and unknown inclusions, which are exocytosed as part of egg envelope biogenesis. Vitellogenesis appears to be largely autosynthetic involving the Golgi complex and rough endoplasmic reticulum. Sperm differentiation resembles that reported in other mytilid bivalves and includes the differentiation of proacrosomal granules and a flagellum during the early stages of spermatogenesis. Mature sperm have a cap-like acrosome, substantial subacrosomal material, a bullet-shaped nucleus, and four to five mitochondria in the midpiece. The general features of gametogenesis closely resemble those reported in shallow-water, seasonally reproducing mytilids. Despite the relatively stable methane seep environment and presumed continuous availability of nutrients, reproduction parallels that of littoral mytilids, suggesting some phylogenetic constraints on the capacity for variability in gametogenic processes in the family Mytilidae.

Analysis of the distribution of glycoconjugates in the digestive gland of the bivalve mollusc Mytilus galloprovincialis by conventional and lectin histochemistry

We examined the distribution and pattern of reactivity of a panel of 16 lectins in the digestive gland of the bivalve mollusc Mytilus galloprovincialis at the light-microscopic level. Various chemical treatments were applied in combination with lectins to differentiate between N- and O-linked oligosaccharides. Several control reactions were carried out, including replacement of lectins by buffer and incubation with their specific competitive inhibitors. Some lectins reacted selectively with particular cell types, thus revealing a cell-specific glycoconjugate distribution pattern which is possibly related to the metabolic role of each cell type in the digestive gland. Glycoconjugates containing glucosamine, mannose, and sulfated galactose were associated with the endolysosomal system of digestive cells. These glycoconjugates were also found in small vesicles randomly distributed in the cytoplasm of adipogranular cells in the connective tissue. However galactosamine residues appeared to be associated mainly with basophilic cells. Fucose residues did not exhibit a cell-specific distribution and appeared in small amounts homogeneously distributed throughout the digestive gland tissue. Conventional histochemical reactions for carbohydrate detection revealed moderate amounts of periodic-acid-Schiff-positive, neutral carbohydrates widely distributed in digestive and connective tissues. Among the acid carbohydrates, most cell types contained complex sulfated carbohydrates, but not carboxylated ones; this agreed well with the complete lack of sialic acid residues in all cell types studied, as observed by lectin histochemistry.

Tissue and cell distribution of copper, zinc and cadmium in the mussel, Mytilus galloprovincialis, determined by autometallography

The localization of metals in selected tissues of metal-exposed mussels was investigated by means of autometallography. Mussels collected from a Zn-polluted site were (a) depurated or, alternatively, (b) exposed to either Cu, Zn or Cd for 41 d. Mussels collected from a clean site were used as experimental reference. Autometallographically demonstrated black silver deposits (BSD), indicating the presence of metals, were observed in gills, (a) in frontal cells and haemocytes of Cu-exposed mussels, (b) in secretory postlateral and abfrontal cells and in endothelial cells of Zn-exposed mussels, and (c) in frontal, postlateral and endothelial cells but mainly in abfrontal cells and haemocytes of Cd-exposed mussels. Autometallography also revealed the presence of BSD in connective tissue brown cells underlying the mantle. Additionally, adipogranular cells of the connective tissue surrounding the gonad follicles were positively stained but no BSD was found in gonad tissue. Scarce BSD were found in the cytoplasmic granules of the stomach wall of control mussels whilst depurating and metal-exposed mussels exhibited BSD lining the apex of the stomach epithelial cells. Contrary to the results previously obtained with marine gastropod molluscs, BSD were not found in the basal lamina of digestive tubules, stomach, and ducts of mussels. Highly conspicuous BSD were observed in digestive cell lysosomes of depurating and metal-exposed mussels. Basophilic cells were always devoid of BSD. Finally, BSD were also found in nephrocyte lysosomes and mineralized concretions.

Annual cycle of expression of connective tissue polypeptide markers in the mantle of the musselMytilus galloprovincialis

We suggest that gonad development in the mantle tissue of the bivalve molluscMytilus galloprovincialis Lmk. is an example of epithelial/mesenchymal interactions (i.e. soma/germline interactions) and morphogenesis in the adult state. According to this concept, the aim of the present study was to use biochemical and immunochemical methods for identifying and characterizing the mantle cell polypeptide markers whose expression is seasonally and morphogenetically regulated. We showed for the first time thatM. galloprovincialis mantle, of both males and females, contains polypeptides (with an apparent mol. wt of 45 to 53 kDa) specific for connective tissue (“mantle connective tissue polypeptides”; MCTPs). Electrophoretic, immunoblotting and immunofluorescent experiments demonstrated that MCTPs are primarily localized in the adipogranular (ADG) cells, and their expression in the mantle is seasonally regulated. There is a positive correlation between MCTP expression and connective tissue volume in the mantle. MCTPs are overexpressed during the rest period, when the mantle consists of connective tissue mainly, whereas mature gonads contain only trace amounts of MCTPs. Moreover, there is a temporal correlation between the onset and decrease of MCTP expression and the appearance and disappearance of the ADG cells in the mantle. MCTP localization in the mantle tissue should not be associated with the ADG cells only, because positive immunofluorescence was also detected in follicle membranes (but not in germ cells) and superficial mantle epithelium. Using immunoblotting and immunofluorescence, MCTPs were found in the connective tissue of the mantle, posterior adductor muscle and visceral mass, but not gills, foot or hepatopancreas. Possible mechanisms by which MCTPs could participate in the annual processes of mantle gonad/connective tissue development and involution are discussed.

Effects of infection by the protistan parasite Marteilia relringens on the reproduction of cultured mussels Mytilus galloprovincialis in Galicia (NW Spain)

Infection by the protistan Marteilia refringens affects the digestive glands of cultured mussels in Galicia, Spain. Effects of the infection on the reproduction of the mussel were studied at 3 sites. Gonad cycle of mussels involved gametogenesis utilizing stored reserves and leading to spring spawning (conservative strategy). This was followed by various sequences of gonad restoration and spawning utilizing the energy from concurrent feeding directly (opportunistic strategy). M. refringens significantly inhibited gonad development in the periods of opportunistic reproductive strategy, proportionally to infection intensity. However, the inhib~tory effect was not significant in the period of conservatlve strategy, probably because mussels had developed storage tissue before the infection progressed. Development of storage tissue was estimated by the abundance of adipogranular (ADG) cells in the mantle. Infection by M. refringens was clearly associated with inhibition of ADG cell development. The heavier the infection, the lower the abundance of ADG cells. Differences in abundance of ADG cells among categories of mussels with different infection intensity were significant at every site. No significant difference in susceptibility to progression of infection was detected between males and females and a similar mortality rate between sexes can be assumed. Evidence suggests that stress following spawning could enhance the progression of the infection.

Lectin binding sites in the digestive gland of mussel

Mussel digestive glands were fixed in modified Baker's fixative, embedded in paraffin wax and stained with a panel of 10 peroxidase conjugated lectins. The lectin Lens culinaris agglutinin-bound specifically to basiphil cells, Datura stramonium lectin bound to adipo-granular cells, and wheatgerm agglutinin ( Tritium vulgaris), Concanavalin A and Ulex europacus agglutinin I bound to digestive cells: the remaining lectins did not bind.

Cellular biochemistry of the mantle tissue of the mussel Mytilus edulis L.

In Mytilus edulis L. gametogenesis takes place in the mantle at the expense of the connective storage tissue. One of the ways in which stored reserves are mobilized for gamete formation is by controlled autophagy. As a direct approach to the study of the cellular biochemistry of the mantle tissue, we have isolated adipogranular (ADG) cells and measured changes in the biochemical composition of the excised mantle and of high-density ADG cells. The results are discussed in relation to the seasonal metabolic changes and the activities of the lysosomal proteinases Cathepsin B and Cathepsin L.

Seasonal cycle of lysosomal enzyme activities in the mantle tissue and isolated cells from the musselMytilus edulis

InMytilus edulis L., gametogenesis takes place in the mantle at the expense of the connective storage tissue. There are two main types of storage cells: vesicular (VC) cells storing large amounts of glycogen and adipogranular (ADG) cells containing large numbers of protein granules, lipid droplets and lesser amounts of glycogen. One of the ways in which stored reserves can be mobilized for gamete formation is by controlled autophagy, in which the cellular constituents are degraded by lysosomes. Mussels were collected from the Menai Strait, North Wales, and monthly measurements made, over two years (1984–1986), of the activities of lysosomal acid hydrolases (acid phosphatase,β-N-acetyl-D-glucosaminidase andβ-glucuronidase) and Cathepsins B and L in the mantle tissue, isolated ADG cells, low-density cells and, during spawning, in the mature oocytes of female mussels. The lysosomal proteinases, Cathepsins B, L and H, were further characterised by activation with thiol compounds and inhibition with thiol blockers and by leupeptin. Because of the low activity in the mantle tissue ofM. edulis, Cathepsin H was not assayed on a seasonal basis. There was a general increase in lysosomal enzyme activity during the winter, which can be related to increased autolysis in the storage cells and to the process of maturation in the developing oocytes. The activity of Cathepsin B was highest in the ADG and low-density cells, implying an important role in proteolysis within the ADG cells. By contrast, Cathepsin L displayed the highest activity in the mature oocytes, suggesting a major function of Cathepsin L in the development and maturation of the oocytes. Two differentα-glucosidase activities were measured in the monthly assays, one with a pH optimum of 4.5 (acid) and the other at pH 7.5 (neutral). Highest activities of the acidα-glycosidase were found in the low-density cells, but there were no significant seasonal changes in the mantle tissue as a whole. Activities of the neutralα-glucosidase were low in the ADG cells and mature oocytes, but showed high activities in the mantle tissue, with marked seasonal changes that corresponded to the mobilization of glycogen reserves in the VC cells.

Adipogranular cells from the mantle tissue of Mytilus edulis L. II. Seasonal changes in the distribution of dispersed cells in a preformed Percoll density gradient

Seasonal changes in the number of adipogranular (ADG) cells in mantle tissue dispersions of Mytilus edulis L. showed a decline in cell numbers during gametogenesis (October–March) as the storage tissue was replaced by gonadal cells. The data for cell counts agreed well with previously described changes in the mantle volume fraction occupied by ADG cells. The distribution of dispersed cells in preformed Percoll density gradients showed two distinct peaks at high and low density, respectively. Adipogranular cells undergo controlled autolysis in which protein and glycogen reserves are mobilized for gamete formation (Bayne et al., 1982). Early in the seasonal cycle (October–December) the ADG cells exhibited a distinct peak at high density with very few low-density ADG cells. The number of high-density ADG cells then declined (January–April), being particularly rapid during the later stages of gametogenesis. The fact that, under the light microscope, few recognizable ADG cells appeared in the low-density fraction suggests that autolysis once initiated is rapid and complete. Ultrastructural studies showed that the low-density fraction included many ADG cells in the final stages of regression. Other cells observed in this fraction were macrophages and, during early summer, new amoebocytic cells.

Adipogranular cells from the mantle tissue of Mytilus edulis L. I. Isolation, purification and biochemical characteristics of dispersed cells

High-density adipogranular (ADG) cells were isolated from the mantle tissue of female mussels Mytilus edulis L. by enzymic dispersion and centrifugation through Percoll of density 1.08 g/ml. The cells were viable as judged by microscopic examination, exclusion of Trypan Blue, nonleakage of intracellular glutamate-oxaloacetate transaminase (GOT) and malate dehydrogenase (MDH), and utilization of exogenous [2- 3H] and [U- 14C] glucose. In dual-labelled experiments, the levels of [ 3H]glucose and [ 14C]glucose in cellular glycogen were equivalent to the utilization of 80 nmol glucose/h/g wet wt and 120 nmol glucose/h/g wet wt, respectively, at an external glucose concentration of 0.5 mM. The rate of 14CO 2 production was very low (18 nmol glucose/h/g wet wt), representing 6% of the total glucose utilization as measured by detritiation of [2- 3H] glucose. Recovery of 3H 2O from [2- 3H] glucose was linear with time over a 3-h incubation and showed saturation kinetics for glucose utilization, with an apparent K m of 0.56 ± 0.09 mM and a V max of 632 ± 48 nmol glucose/h/g wet wt.