Glucose uptake in human and animal muscle cells in culture

Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs.     

Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.     

Glucose transporter content and glucose uptake in skeletal muscle constructs engineered in vitro

Engineered muscle may eventually be used as a treatment option for patients suffering from loss of muscle function. The metabolic and contractile function of engineered muscle has not been well described; therefore, the purpose of this experiment was to study glucose transporter content and glucose uptake in engineered skeletal muscle constructs called myooids. Glucose uptake by way of 2-deoxyglucose and GLUT-1 and GLUT-4 transporter protein content was measured in basal and insulin-stimulated myooids that were engineered from soleus muscles of female Sprague-Dawley rats. There was a significant increase in the basal 2-deoxyglucose uptake of myooids compared with adult control (fivefold), contraction-stimulated (3.4-fold), and insulin-stimulated (threefold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). In addition, there was a significant increase in the insulin-stimulated 2-deoxyglucose uptake of myooids compared with adult control soleus muscles in basal conditions (6.5-fold) and adult contraction-stimulated (4.5-fold) and insulin- stimulated (3.9-fold) soleus muscles (P = 0.0001, 0.0001, and 0.0001, respectively). There was a significant 30% increase in insulin-stimulated compared with basal 2-deoxyglucose uptake in the myooids. The myooid GLUT-1 protein content was 820% of the adult control soleus muscle, whereas the GLUT-4 protein content was 130% of the control soleus muscle. Myooid GLUT-1 protein content was 6.3-fold greater than GLUT-4 protein content, suggesting that the glucose transport of the engineered myooids is similar in several respects to that observed in both fetal and denervated skeletal muscle tissue.     

Glucose uptake and oxidation in fat cells of trained and sedentary pregnant rats

The purpose of this investigation was to examine the relationship between an exercise program and fetal development to determine whether training could influence insulin sensitivity in the pregnant rat. Prior to impregnation one group of animals was exercise trained on a Quinton shock-stimulus rodent treadmill. The exercised group was trained to run 5 days/wk, for 2.0 h/day at 31 m/min up an 8 degree incline for 8 wk before mating. Following mating the training intensity was reduced to 27 m/min up a 5 degree incline, and the exercise period decreased to 1 h/day. On day 19 of gestation, 24 h postexercise for the trained mothers, the animals were killed in the fed state and the parametrial fat pads were removed. The parametrial depot of the trained mother was smaller than the sedentary control dam. This was due to a change in cell size and did not involve alterations in cell number. Isolated adipocytes of the parametrial fat pads were used to measure the rates of 2-deoxy-D-[3H]glucose uptake and D-[1-14C]glucose oxidation to 14CO2. The results indicated that the adipocytes from the dam trained prior to and during pregnancy were significantly (P less than 0.05) more responsive to insulin than those of animals remaining sedentary during the same period. At the maximal insulin concentration tested, the fat cells from trained mothers were able to take up and metabolize approximately twice as much glucose as the sedentary control dams. However, the increase in insulin responsiveness induced by the training program did not match the changes observed in trained nonpregnant rats of prior investigations.     

Clonal aspects of plant cell proliferation and their applications to animal cells and bacteria

Abstract.   Objectives : Extensive mathematical studies have been made on cell clone development but little has been advanced in the mathematics of small clone formation and virtually no actual data of small clone size has been collected. Materials and methods : Small clone sizes in leaf marginal cells of the aquatic plant Elodea and aleurone spot sizes in the grain of Zea were counted for later statistical analyses of mean, variance and probability distribution frequencies. Results : Simple mathematical models were developed and their calculated results are comparable to data collected on actual plant clones. The parameters in these models were original cell size ( s ₀), growth rate ( T ), duration of growth ( t ) and cell division inequality ( i ). Conclusions : Given T and t , the critical parameter is s ₀. Plant tissue is ideal material to collect data on clone development because growth rate is uniform across a tissue and cells remain in place, so clone size can be measured, unlike microbes and animal cells that have neither feature. In the light of the results, traditional methods for calculating cell cycle duration and mutation rate are questioned. The applications of these plant features to studies on animal cell populations are discussed.     

In vitro inhibition of cholesterol uptake in human and animal arteries by 7-ketocholesterol

Human and pig coronary arteries and rabbit aortas were perfused with pulsatile pressure in a modified Lindbergh apparatus with blood plasma obtained from the same species. Uptake of cholesterol by the arterial wall was measured using [³H]-cholesterol as tracer. Percent distribution of synthesized lipid fractions was determined by thin-layer chromatography and liquid scintillation counting. Inhibition of cholesterol uptake by the arterial wall was studied by the addition of 7-ketocholesterol (concentrations of from 0.05 to 1 μmoles/ml in the perfusate). The addition of 7-ketocholesterol to the perfusate reduced cholesterol uptake by the vessel by an average of 90%. At concentrations of from 0.1 to 1 μmoles/ml of perfusate, 7-ketocholesterol inhibition remained unchanged. Inhibition was reduced at concentrations of ketocholesterol of 0.05 μmoles/ml. Inhibition was present in all species, and was not due to oxidation of cholesterol to 7-ketocholesterol in the perfusate. The results suggest inhibition of cholesterol uptake in the arterial wall by a competitive process.     

Metabolism of exogenous polyisoprenoids by animal cells cultured in vitro

The content and type of dolichols in chicken embryo fibroblasts was estimated and the presence of enzymic activities of various dolichyl phosphate sugar transferases was documented. Chicken embryo fibroblasts effectively take up various polyprenoids from the culture medium and catalyse both formation of polyprenyl fatty acid esters and their hydrolysis. With fully unsatured polyprenols the hydrogenation of the terminal alpha-isoprene residue was observed. In baby hamster kidney cells the formation of mannolipids was enhanced by exogenous polyprenols.     

Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures

To evaluate the extent to which single-cell glucose uptake rates determine the overall specific growth rate of a culture, dilute chemostat cultures of Escherichia coli BL21 were grown in defined medium under glucose limitation. The glucose uptake dynamics of the cell population was examined at the single-cell level using the fluorescent glucose analog, 2-NBDG. Between dilution rates of 0.12 h(-1) and 0.40 h(-1), mean cellular protein content and steady-state, extracellular glucose concentrations increased with increasing dilution rate. However, the distribution of 2-NBDG uptake rates in the population remained constant over the range of dilution rates studied. This indicates that the growth of cells in continuous culture is not limited by the maximum rate of uptake of glucose but by the availability of glucose for transport. The work represents an example of how quantitative flow cytometry can be applied to gain detailed insight into microbial growth physiology.     

Stimulation of differentiation and proliferation of haemopoietic cells in vitro

A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co-cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non-attaching population. In the (co-cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non-attaching cells was seen in the (co-cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co-cultures) for at least 14 days. In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono-nuclear cells. In the (co-cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co-cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation. By feeding the (co-cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFU_S were detectable, but variable numbers of agar colony forming cells (not classical CFU_C) were present at least for ten weeks.     

Glucose reduces PDGF production and cell proliferation of cultured vascular endothelial cells

The effect of glucose on PDGF production and cell proliferation was studied on cultured bovine aortic endothelial cells. PDGF levels were measured using an enzyme-linked immunosorbent assay technique newly developed in our laboratory. The cell proliferation rate was determine on the basis of 3H-thymidine incorporation into cellular DNA. PDGF levels in culture medium were below the detection limit of the assay. However, PDGF levels were measurable in cultured endothelial cells at confluence. Both PDGF production and thymidine incorporation were significantly reduced in the endothelial cells cultured with high concentrations of glucose. These results suggest that reduced PDGF production and cell proliferation may be involved in altered vascular endothelial function in diabetics.     

Alpha-fetoprotein enhances the proliferation of human hepatoma cells in vitro

Although the biological functions of alpha-fetoprotein ( AFP ) have been extensively studied, little is known about its effect on tumor cell growth. Our previous work has found that human AFP significantly stimulates the growth of mouse hepatoma cells in vitro. The purpose of the present study is to observe the effect of AFP on the proliferation of human hepatoma cells in vitro. Using a MTT- microculture tetrazolium assay, we found that the proliferation of human hepatoma cells was enhanced by in vitro treatment of AFP. However, the same concentrations of AFP had no effect on HL - 60 human leukemia cell proliferation, indicating that the human hepatoma cell proliferation - promoting role of AFP was not simply due to non-specific addition of exogenous protein and the proliferation enhancement of AFP showed certain tumor cell specificity. On the other hand, the growth stimulation of AFP could be diminished by rabbit anti - human AFP antibody. The anti- AFP antibody alone suppressed the growth of BEL - 7404 human hepatoma cells, not affecting HL - 60 cell proliferation. BEL - 7404 cell proliferation was not inhibited by normal rabbit immunoglobulins to demonstrate the specificity of anti-AFP effect. Taken together, it is concluded that AFP enhances the proliferation of human hepatoma cells in vitro, and this effect is seemingly mediated by an AFP/receptor autocrine pathway.     

Glucose uptake and lactate production in cells exposed to CoCl(2) and in cells overexpressing the Glut-1 glucose transporter

Glut-1-mediated glucose transport is augmented in response to a variety of conditions and stimuli. In this study we examined the metabolic fate of glucose in cells in which glucose transport is stimulated by exposure to CoCl(2), an agent that stimulates the expression of a set of hypoxia-responsive genes including several glycolytic enzymes and the Glut-1 glucose transporter. Similarly, we determined the metabolic fate of glucose in stably transfected cells overexpressing Glut-1. Exposure of Clone 9 liver cell line, 3T3-L1 fibroblasts, and C(2)C(12) myoblasts to CoCl(2) resulted in an increase glucose uptake and in the activity of glucose phosphorylation ("hexokinase") and lactate dehydrogenase. In cells treated with CoCl(2), the net increase in glucose taken up was accounted for by its near-complete conversion to lactate. Cells stably transfected to overexpress Glut-1 also exhibited enhanced net uptake of glucose with the near-complete conversion of the increased glucose taken up to lactate; however, the effect in these cells was observed in the absence of any change in the activity of two glycolytic enzymes examined. These findings suggest that in cells in which glucose transport is rate-limiting for glucose metabolism, enhancement of the glucose entry step per se results in a near-complete conversion of the extra glucose to lactate.     

Glucose uptake and phosphorylation in Pseudomonas fluorescens

Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase (d-glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [(14)C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [(14)C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5'-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens. The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens. A general unifying scheme for glucose utilization in the aerobic fluorescent pseudomonads is suggested for the purpose of clarifying glucose uptake in these bacteria.     

Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells

The flavanone hesperetin is known to decrease basal glucose uptake, although the inhibitory mechanism is largely unknown. Here, we used MDA‐MB‐231 breast cancer cells to investigate the molecular pathways affected by hesperetin. The results indicate that the suppression of glucose uptake is caused by the down‐regulation of glucose transporter 1 (GLUT1). Hesperetin was also found to inhibit insulin‐induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4). In addition, the phosphorylation of the insulin receptor‐beta subunit (IR‐beta) and Akt was suppressed. Hesperetin also decreased cellular proliferation, which is likely due to the inhibition of glucose uptake. Cancer cells are highly dependent on glucose and hesperetin may, therefore, have potential application as an anticancer agent. Copyright © 2012 John Wiley & Sons, Ltd.