Alpha-glycerophosphate Dehydrogenase Activity Research Articles

ДИНАМИКА ПАТОМОРФОЛОГИЧЕСКИХ ИЗМЕНЕНИЙ В ИШЕМИЗИРОВАННОМ СПИННОМ МОЗГЕ КРЫС ПОСЛЕ ЛЕЧЕНИЯ РЕКОМБИНАНТНЫМ ЭРИТРОПОЭТИНОМ (ЭКСПЕРИМЕНТАЛЬНОЕ ИССЛЕДОВАНИЕ)

Aim. To study how experimental autoimmune liver injury in mother influences morphofunctional features of monocyte development in the bone marrow of progeny. Materials and methods. The subjects of study were female Wistar rats with experimental liver injury and their progeny on days 30 and 60 of postnatal development divided into two groups: control and experimental. Chronic autoimmune liver injury was modeled using BCG vaccine via subcutaneous immunization with periodic intraperitoneal administration of liver antigen each three days. Results. The study showed that absolute cell count at all stages of development in the experimental group of animals decreased in comparison with the control group. Besides, the decrease was observed in PAS positive cell number, acid phosphatase activity and level of alpha-glycerophosphate dehydrogenase in monocyte series of the bone marrow and in peripheral blood monocytes of the groups of animals under observation. Conclusion. Mothers with experimental autoimmune hepatobiliary disorder give birth to progeny with disturbance expressed in functional immaturity of monocytes.

Free fatty acid inhibition of alpha-glycerophosphate dehydrogenase activity in rat brain.

Free fatty acid inhibition of alpha-glycerophosphate dehydrogenase activity in rat brain.

Different fibre populations distinguished by their calcium transient characteristics in enzymatically dissociated murine flexor digitorum brevis and soleus muscles

Enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres from mouse were used to compare the kinetics of electrically elicited Ca2+ transients of slow and fast skeletal muscle fibres, using the fast Ca2+ dye MagFluo4-AM, at 20-22 degrees C. For FDB two Ca2+ transient morphologies, types I (MT-I, 11 fibres, 19%) and II (MT-II, 47 fibres, 81%), were found, the kinetic parameters (amplitude, rise time, half width, decay time, and time constants of decay) being statistically different. For soleus (n = 20) only MT-I was found, with characteristics similar to MT-I from FDB. Correlations with histochemically determined mATPase, reduced nicotinamide adenine dinucleotide diaphorase and alpha-glycerophosphate dehydrogenase activities, as well as immunostaining and myosin heavy chain electrophoretic analysis of both muscles suggest that signals classified as MT-I may correspond to slow type I and fast IIA fibres while those classified as MT-II may correspond to fast IIX/D fibres. The results point to the importance of Ca2+ signaling for characterization of muscle fibres, but also to its possible role in determining fibre function.

Specific Metabolic Properties of Rat Oculorotatory Extraocular Muscles Can Be Linked to Their Low Force Requirements

To gain insight into the metabolic pathways of oculorotatory extraocular muscle (EOM) fiber types at the cellular level to explain their high fatigue resistance, rapid contraction, and low force output. In consecutive sections of adult rat EOMs, the cross-sectional area (CSA) was calculated, and the activities of succinate-dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPDH) were measured by quantitative histochemistry of different fiber types classified by the myofibrillar adenosine triphosphatase (mATPase) staining pattern. In the orbital regions, type 1 (fast) fibers were present, showing small CSA, medium SDH, and low GPDH activity. The type 2 (slow) fibers exhibited extremely small CSA and low SDH and GPDH activity. In the global region, fast types 3 to 5 fibers were found, forming a continuum with an inverse correlation between CSA and enzyme activity. SDH and GPDH activity showed an unusual positive relationship in contrast to the skeletal muscle fiber types. The type 6 fibers were slow forming a close and clearly separated group with medium CSA and extremely low SDH and low GPDH activity. Muscle fibers in adult rat EOMs show unique metabolic properties not seen in other skeletal muscles, covering their extraordinary functional demands. It can be speculated that the EOMs embedded within the orbit material do not need to perform with high force, and therefore they could develop extensive systems that ensure both fatigue resistance (many mitochondria) and fast contraction with high mATPase activity (a well-developed sarcotubular system).

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.

運動性筋損傷から回復後に見られる極細筋線維の機能特性

Markedly smaller myofibers including branched myofibers (SF) were contained in regenerated skeletal muscles. However, its functional properties are not yet sufficiently understood. The present study examined the metabolic, contractile properties and hypertrophy of SF included in rat skeletal plantaris muscles regenerated from eccentric contraction-induced muscle injury. Succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activity of SF were respectively similar to that of other normal size myofibers (NF) within regenerated muscles. The shortening velocity of skinned fibers prepared from branched myofibers isolated from regenerated muscles was slightly slower than that of control muscles. In addition, the extent of glycogen depletion in SF was similar to that of NF after exhaustive running. Therefore, SF were innervated by motoneurons and recruited during the contractile activity of the skeletal muscle. No matter when the regenerated muscle was loaded by synergistic ablation, the continued existence of SF was observed. Therefore, the ability of hypertrophy in SF may be distinct from that in NF. The physiological properties of SF were similar to that of normal myofiber, except for the ability to regulate hypertrophy.

Detection of individual variation in enzyme activity in natural populations of Drosophila melanogaster.

The method allows the determination of the activity level of enzymes in a single fly and assessing the genetic composition of the given individual at these enzyme loci. Three isofemale lines were constructed which were monomorphic at several enzyme loci. Samples were prepared in two different ways: (i) individual samples--individuals were homogenised separately; (ii) collective samples--a common homogenate was prepared from several individuals. Oregon-R strain was also used to prepare a standard homogenate. The activities of alcohol dehydrogenase (ADH), alpha-glycerophosphate dehydrogenase (alpha GPDH), isocitrate dehydrogenase (IDH), and 6-phosphogluconate dehydrogenase (6PGDH), were measured in each sample on starch gel after the proteins were separated by electrophoresis. Enzyme activities were assessed by the optical density of the bands. Gel and position weights were estimated on the basis of the statistical analyses of the activities measured in the standard samples. Gel weights were then used to account for the activity differences among the gels while position weights were applied to correct for the general tendencies in the activities observed within the gels. The gel and position weighted activities of individual and collective samples were compared in the isofemale lines. The individual samples had approximately two times as much variation as the collective samples for all four enzymes. The electrophoretic method is sensitive enough to study the structure of the phenotypic variation in enzyme activity in the natural populations. The total variation among the standard samples was close to the within subline component of variation obtained for the collective samples (measurement error). This shows that the standard samples can be used to estimate the size of the measurement error.

Exercise Performance and Skeletal Muscles in Patients With Advanced Chagas Disease

This study was designed to evaluate the peripheral muscle metabolic and structural characteristics in patients with advanced Chagas disease (ChD), and whether they were related with exercise performance. Cross-sectional study. Outpatient cardiomyopathy clinic of a university hospital. We studied 11 stage II patients, 8 stage III patients, and 11 healthy volunteers. All patients underwent exercise testing and peripheral muscle biopsies. The muscles were also studied in control subjects. Muscle biopsy specimens were analyzed for histochemical characteristics. In six patients, the muscle was studied ultrastructurally. The data demonstrate more glycolytic and less oxidative capacity of the peripheral muscle in patients with advanced ChD (increased proportion of type IIb fibers, increased proportion of fibers with low nicotinamide adenine dinucleotide diaphorase activity, high proportion of darkly stained fibers for alpha-glycerophosphate dehydrogenase activity, and lower levels of citrate synthase). Many capillaries in patients with ChD had an abnormal aspect: they were either closed or showed a thicker wall. The ultrastructural study also showed fiber atrophy and abnormal capillaries even in patients with normal functional capacity. Some muscle characteristics (enzyme activity, mean cross-sectional area of the fiber, and capillarity) related with exercise parameters (anaerobic threshold, and peak oxygen pulse). These findings indicate that patients with advanced ChD have decreased oxidative capacity and a shift to anaerobic metabolism in the skeletal muscle. They also suggest that muscular abnormalities are related to oxygen delivery, which is probably reduced in part by the abnormal muscle microvasculature. Those changes could affect oxygen extraction, and therefore exercise tolerance in these patients.

Metabolic Modulation of Muscle Fiber Properties Unrelated to Mechanical Stimuli

The effects of chronically increasing (creatine-fed) or decreasing (beta-guanidinopropionic acid [beta-GPA]-fed) high-energy phosphates for up to 8 weeks on daily voluntary activity levels, swimming endurance capacity, electromyogram (EMG) activity, and the morphological and metabolic properties of single fibers in the soleus and extensor digitorum longus (EDL) muscles in young rats were determined. High-energy phosphate, voluntary activity, and soleus-integrated EMG levels were lower in beta-GPA-fed rats than in control rats. Endurance capacity was higher at a relatively low intensity of swimming and lower at a relatively high intensity in beta-GPA-fed rats than in control rats. Muscle mass and fiber size were smaller, and the percentage of slow fibers was higher in the soleus and EDL of beta-GPA-fed rats than in control rats. Succinate dehydrogenase activity was higher in both the fast and slow fibers of the EDL of beta-GPA-fed rats than in control rats. Thus, a reduction in high-energy phosphates transformed some fast fibers toward a slow phenotype. Creatine supplementation had minimal effects: The only significant change was an increase in alpha-glycerophosphate dehydrogenase activity in the fast fibers of the EDL. These results indicate that the metabolic environment of a muscle fiber can influence the prominence of a given muscle fiber independent of the activity level of muscle.

The effect of high-intensity exhaustive exercise studied in isolated mitochondria from human skeletal muscle.

Six young men performed five 1-min bicycle exercise bouts to exhaustion. Muscle lactate increased to congruent with 114 mmol x kg(-1) dwt and pH decreased to congruent with 6.6. Mitochondria were prepared from a needle biopsy sample taken from m. vastus lateralis immediately after the last exercise bout. No significant effect of exhaustion on the proton permeability and amount of cytochromes c and aa3 in isolated mitochondria was detected. The activities of the following enzymes and systems were not altered either: citrate synthase, succinate dehydrogenase, cytochrome oxidase, succinate + glutamate respiration, malate + glutamate respiration, the respiratory chain, and the reactions involved in ATP synthesis. Thus, the mitochondria did not appear globally altered upon exhaustion. However, the following NAD-linked activities were significantly lowered: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase and fatty acid beta-oxidation. The activities of alpha-glycerophosphate dehydrogenase and exo-NADH oxidase, enzymes that might catalyze the oxidation of sarcoplasmic NADH, were increased. These changes may be due to the action of reactive oxygen species, protons and Ca2+. Transient opening of the permeability transition pore may also be involved. Some effects may have been reversed during isolation of the mitochondria and the changes in mitochondrial function in situ upon exhaustion may have been more extensive than observed.

Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains.

Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sectional area (CSA), capillary and nuclear densities] features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform and, furthermore, it allowed a semiquantitative delineation of fast hybrid fibres according to the predominant MyHC isoform expressed. The phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. Slow fibres had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPD activity (glycolytic metabolism) and glycogen content, the smallest CSA, the greatest capillary and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IID/X fibres, and type IIA fibres had intermediate properties. Hybrid IIAD/X fibres had mean values intermediate to those of their respective pure phenotypes. Discrimination of fibres according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafibre interdependencies suggest that, even when controlled by different mechanisms, myofibres of control horses exhibit a high degree of co-ordination in their physiological, biochemical and anatomical features.

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes.

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.

Decrease and recovery of N-acetylaspartate/creatine in rat brain remote from focal injury.

Magnetic resonance spectroscopy (MRS) studies on traumatic brain injury (TBI) have shown that the neuronal metabolite N-acetylaspartate (NAA) may be reduced in regions of brain remote from sites of focal injury. Such reductions have generally been attributed to diffuse axonal injury (DAI) or neuron death. The aim of the present study was to investigate the contribution of metabolic depression, in the absence of DAI or cell death, to remote NAA reduction after TBI. The right sensorimotor cortices of adult rats were injured by weight drop. Two and six days later, tissue slices from the ipsilateral occipital cortex, or from the same region in uninjured rats, were superfused and examined by 1H-MRS. The occipital cortex has been shown to have negligible DAI or cell death but marked transient metabolic depression in this model of TBI. Two days after injury, the ratio of the NAA peak height to the total creatine peak height (NAA/TCr) was 14% lower than in control samples. Six days after injury, NAA/TCr recovered to within 7% of the control value. The time course of NAA/TCr decrease and recovery was similar to the time courses of widespread depression and recovery of 2-deoxyglucose uptake and mitochondrial alpha-glycerophosphate dehydrogenase activity measured previously in this model of TBI. Together, these results suggest that at least one component of remote NAA depression after TBI may be associated with a widespread and reversible metabolic depression that is unrelated to either DAI or cell death.

Alpha-glycerophosphate dehydrogenase activity in flight muscles of triatomine bugs Panstrongylus megistus and Triatoma sordida

The alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity in flight muscles of Panstrongylus megistus and Triatoma sordida, vectors of Chagas disease in Brazil, was studied. Both species showed higher enzymatic activities in fliers than in non-fliers insects. T. sordida exhibited a higher proportion of flier insects than P. megistus. A possible role of alpha-GPDH on triatomines flight is discussed.

Interrelationships of myofibrillar ATPase activity and metabolic properties of myosin heavy chain-based fibre types in rat skeletal muscle.

Myofibrillar ATPase (mATPase), succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities and cross-sectional area (CSA) were measured in fibres of rat medial gastrocnemius muscle using quantitative histochemistry. The same fibres were typed immunohistochemically using monoclonal antibodies specific to selected myosin heavy chain (MHC) isoforms. The values of mATPase, SDH, GPD and CSA formed a continuum, but significant differences in mean values were observed among fibre types of presumed homogeneous MHC content. Type I fibres had the lowest mATPase activity, followed in rank order by type IIA < type IID/X < type IIB. Type IIA fibres had the highest SDH activity, followed in rank order by type IID/X > type I > type IIB. The mean GPD activity was consistently ranked according to fibre type such that type IIB > type IID/X > type IIA > type I. Type IIA fibres were the smallest, type IIB fibres were the largest and types I and IID/X were of intermediate size. Significant interrelationships between mATPase, SDH, GPD and CSA values were found on a fibre-to-fibre basis. Consequently, discrimination of fibres according to their MHC content was possible on the basis of their mATPase, SDH, GPD and CSA profiles. These intrafibre interrelationships suggest that the MHC isoform is associated with phenotypic differences in contractile, metabolic and size properties of muscle fibre types.

Proliferation and differentiation of unilocular fat cells in the bone marrow.

Fat cells contribute not only to systemic lipid metabolism, but also to osteogenesis and hemopoiesis in the bone marrow. The present study represents the first attempt to culture mature unilocular fat cells of the bone marrow. Two methods devised in our laboratory were employed: one is the "ceiling culture method" that utilizes the floating property of the cells; the other, a three-dimensional collagen gel matrix culture that captures unilocular fat cells in the gel matrix. Using these methods, the proliferation of unilocular fat cells from the bovine metacarpal bone marrow was demonstrated. First, we confirmed the proliferative ability of unilocular fat cells derived from the bone marrow, using autoradiography to study 3H-thymidine incorporation into the nuclei. The unilocular fat cells de-differentiated into fibroblast-like fat cells and then proceeded to proliferate. When they underwent a contact inhibition of growth, re-differentiation from fibroblast-like fat cells into unilocular fat cells occurred at a high rate. A specific enzymatic marker of the fat cell, alpha-glycerophosphate dehydrogenase activity related to lipogenesis, was then demonstrated in the cultured fat cells. We examined the functional reactivity of the fat cells by treatment with insulin and cyclic-AMP, and both lipogenesis and lipolysis were also confirmed in them. We concluded that unilocular fat cells from the bone marrow de-differentiated, proliferated and re-differentiated in culture. The present results may help to clarify the various functions of fat cells in the bone marrow.

Metabolic properties of the sensory neurons in the rat dorsal root ganglion.

The dorsal root ganglion (DRG) neurons were classified in the rat on the basis of their metabolic enzyme properties as determined by quantitative analysis in histochemical staining. In particular, nicotinamide adenine dinucleotide-diaphorase (NADH-d) and alpha-glycerophosphate dehydrogenase (alpha-GPD) activities were examined on two serial sections from the same neurons in the lumbar (L4) DRG. The DRG neurons were classified into three groups based on the soma diameter distribution; small, intermediate and large size DRG neurons. The NADH-d activity showed a unimodal distribution in all size groups, while the alpha-GPD activity clearly showed a bimodal distribution in the intermediate and large size neurons, but not in the small size neurons.

Effects of muscle length on the response to unloading.

Muscle mass, distribution of fiber types, fiber cross-sectional areas (CSA) and selected enzyme activities were determined in rats hindlimb-suspended free of immobilization (Susp-Free), suspended with the ankle dorsiflexed (Susp-DF, soleus stretched) or plantarflexed (Susp-PF, soleus shortened) for 10 days and compared to cage-control (Con) rats. Reduction of muscle weight associated with suspension was prevented in Susp-DF rats. The mean CSAs of slow fibers were Con = Susp-DF > Susp > PF > Susp-Free and of fast and intermediate fiber tended to be Susp-DF > Con > Susp-PF = Susp-Free. Mean activities of succinate dehydrogenase (SDH), alpha-glycerophosphate dehydrogenase (GPD) and myofibrillar adenosine triphosphatase (mATPase) in slow and fast fibers were similar in Con and Susp-Free rats. Mean SDH activity in slow fibers was higher in Susp-DF and Susp-PF than in Con and Susp-Free. No significant differences in SDH activities of fast fibers were observed among groups. GPD activity was higher in slow fibers of Susp-DF and Susp-PF compared to Con. The mATPase activity was higher in slow fibers of Susp-DF compared to Con and Susp-Free rats and lower in fast fibers of Susp-DF compared to Con rats. Thus, when compared to control, the patterns of adaptation were more similar in the Susp-DF and Susp-PF than in the Susp-Free. Although these results are consistent with previous studies demonstrating that the load placed on a muscle can affect protein metabolism, the direction and magnitude of the adaptive responses observed in the present study were closely associated with the chronically imposed changes in muscle length, i.e. fixed at either a shortened or a lengthened position.

Effects of hypophysectomy on soleus muscle fibers and spinal motoneurons in rats.

The fiber type distribution of the soleus muscle in male and female rats was investigated 4 weeks after hypophysectomy. Oxidative enzyme activity of the soleus motoneurons in the spinal cord was also examined by enzyme histochemical assay. In male rats, the total number of fibers in the soleus muscle was not changed after hypophysectomy, but the percentage of intermediate (INT) fibers (with intermediate adenosine triphosphatase activity following alkaline preincubation, and high succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activities) was increased. All types of fibers in the soleus muscle of hypophysectomized rats showed high adenosine triphosphatase activity following acid preincubation. Oxidative enzyme activity of the motoneurons innervating the soleus muscle was not changed after hypophysectomy. Similar results were obtained in female rats. It is suggested that the increased percentage of INT fibers in the rat soleus muscle after hypophysectomy is due to a lack or reduced levels of growth hormones, and that the metabolic capacities of the muscle fibers and of the innervating motoneurons are affected independently by hypophysectomy.

Cellular structure and function of rat fat cells in the primary culture.

A primary culture of undigested fat tissue fragments was used to obtain fat cells in vitro. On day 2 of culture, immature fat cells, which are fibroblast-like fat cells containing fine lipid droplets, appeared, surrounding the fat tissue fragments, and began to proliferate extensively. Afterwards, these fibroblast-like fat cells grew to become multilocular fat cells containing larger intracytoplasmic lipid droplets, and differentiated further into unilocular fat cells containing a single large intracytoplasmic lipid droplet. Treatment with dibutyryl-cAMP, which is a second messenger of the lipolytic factor, caused the cultured fat cells to retract, and the intracytoplasmic lipid droplets of those fat cells became finely granulated and decreased along with an increase of hormone-sensitive lipase activities. Conversely, administration of insulin caused the lipid droplets in the fat cells to increase and become larger along with an increase of alpha-glycerophosphate dehydrogenase activities. These findings indicate the occurrence of lipolysis and lipogenesis of fat cells in vitro. Immuno-cytochemistry revealed that vimentin surrounded intracytoplasmic lipid droplets, and became distinct with an increase of lipid droplets through lipogenesis in the fat cells. Vimentin seems to be correlated to the behavior of lipid droplets in the fat cells. Fat cells in this study showed the appropriate cellular structures and functions in response to stimulation of lipolysis and lipogenesis under culture conditions. It is expected that in vitro culture of fat cells will facilitate cell biological elucidation of obesity in the future.