Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.     

Ethanolamine Glycerophospholipid Formation by Decarboxylation of Serine Glycerophospholipids in Myelinating Organ Cultures of Cerebellum

Abstract: Serine decarboxylation as a source of glycer-ophospholipid ethanolamine is known to occur in mammals. However, early investigators failed to demonstrate the pathway in brain. In the present study serine is shown to be decarboxylated to glycerophospholipid ethanolamine in myelinating organ cultures of rat cerebellum up to 32 days in vitro. The pattern of incorporation of l -[3-¹⁴C]serine into culture phospholipids strongly suggests a precursor-product relationship between serine glycero-phospholipids (SGP) and ethanolamine glycerophospho-lipids (EGP), with serine label appearing in the ethanolamine moiety of EGP. The time course of labelling was similar for both acid-stable and acid-labile EGP In contrast DL-[l-¹⁴C]serine failed to label EGP significantly due to the loss of serine carbon C₁ on decarboxylation. Through the systematic hydrolysis of phospholipids from cerebellar cultures incubated with l -[3-¹⁴C], it was clear that in SGP, acid-stable EGP, and acid-labile EGP >70% of radiolabel resides in the base moiety of each of these molecular species. It is proposed that serine decarboxylation as a source of EGP ethanolamine may be important in the early stages of brain development.     

The effects of pH, buffers, and fatty acid concentration on the incorporation of radioactive arachidonate into endogenous neuronal nuclear lipids

Using neuronal nuclei (N1) isolated from cerebral cortices of 15-day-old rabbits the incorporation of [3H]arachidonate into N1 lipids was followed in vitro. Arachidonate was principally incorporated into triacylglycerol and phosphatidylinositol. When low concentrations (32 mM) of Tris-HC1 (pH 7.4) were used, rates of total arachidonate incorporation were small and phosphatidylinositol received the bulk (greater than 84%) of the arachidonate. When the concentration of Tris-HC1 (pH 7.4) or, in certain cases, the concentration of arachidonate was increased, there was a rise in total arachidonate incorporation into N1, with an increasing proportion of radioactivity entering triacylglycerol until it was the predominantly labelled lipid. Using other buffers (phosphate, imidazole, HEPES, pH 7.4), the shift from phosphatidylinositol to triacylglycerol as principal labelled lipid, with buffer concentration, was not as marked as with Tris-HC1 (pH 7.4). When the buffer concentration was maintained at 107 mM and the pH was lowered to 6.5, the three amine-containing buffers showed a sizeable decline in arachidonate incorporation into N1 lipids and a corresponding decrease in triacylglycerol labelling. The proportion of the total radioactivity in N1 phosphatidylinositol rose as the pH declined. Of the buffers used, Tris-HC1 showed the greatest changes over the pH range. Based upon pK values for the amine buffers, it is suggested that an increased proportion of the protonated amine may be inhibitory to arachidonate incorporation in N1. Studies of acyl-CoA synthetase in N1 indicated this enzyme as the site of the inhibition.     

Insulin administration in the mild hyperglycaemia changes expression of proinflammatory adhesion molecules on human aortic endothelial cells

An overexpression of cell adhesion molecules (CAMs) on the surface of endothelial cells is one of the first steps in a high glucose-mediated endothelial dysfunction in diabetic patients. The effect of insulin administration in the condition of elevated glucose concentration on the E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) expression on human aortic endothelial cells (HAEC) was investigated. Cells were cultured for 4 h in a medium supplemented with homocysteine (7 pM) and different concentration of glucose (5.5, 8.0, 12.0 and 16.5 mM respectively) with or without insulin (1 mlU/mL) addition. Expression of CAMs was analysed by flow-cytometry using monoclonal antibodies. Controls were CAMs expression in the medium with a corresponding glucose concentration. Obtained results show that short-term exposure of HAECs to moderate high glucose concentrations results in increased expression of E-selectin (2-fold), VCAM-1 (3-fold) and ICAM-1 (47%). At the same time, HAEC grown with 12 mM glucose expressed lesser E-selectin and, more ICAM-1 (for 64%) and VCAM-1 (41%) molecules. 16.5 mM glucose decreased expression of all investigated adhesion molecules. Addition of insulin was not changed expression of CAMs in a medium with 5.5 mM glucose. In conditions of elevated glucose concentration (12 mM), addition of insulin significantly dropped E-selectin (27%) and increased VCAM-1 (23%) expression. In conclusion, moderate elevated glucose concentration increased expression of cell adhesion molecules on HAEC. Insulin administration in the mild hyperglycaemia reduces an expression of the proinflammatory adhesion molecule E-selectin which could contribute in deceleration of macrovascular complications development in diabetic patients.     

Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.     

Calmodulin-like protein and the phospholipids of Mycobacterium smegmatis

When Mycobacterium smegmatis TMC1546 was grown at different concentrations of glucose supplemented to a synthetic medium already containing 2% v/v glycerol, the following changes were observed. Amount of calmodulin-like protein (CAMLP), total and individual phospholipids (PLs) namely phosphatidylethanolamine, cardiolipin, phosphatidylglycerol and phosphatidylinositol mannosides and total lipids and growth increased up to 5% w/v but decreased at higher concentrations of glucose (7.5% w/v and above). Cyclic AMP content of the whole cells decreased continuously with increase in glucose concentration in the medium. Incorporation of 32Pi into total phospholipids was inhibited by two calmodulin antagonists trifluoperazine and phenothiazine (50% at 40 microM) and the calcium-specific chelator ethylene glycol bis (beta-aminoethyl ether) N,N,N',N'-tetraacetate (EGTA) 35% at 2 mM. Total lipids, CAMLP and growth of this organism are also modulated in a similar way in response to the glucose concentration in the growth medium. Taking these observations together it is suggested that CAMLP has some effect on the metabolism of PLs.     

Uptake studies in adipocytes isolated from rainbow trout (Salmo gairdnerii). A comparison with adipocytes from rat and cat

Adipocytes were isolated from mesenteric adipose tissue of rainbow trout (Salmo gairdnerii) by incubation of tissue slices at 20 degrees C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37 degrees C). Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.     

Decreased insulin secretory response of pancreatic islets during culture in the presence of low glucose is associated with diminished 45Ca2+ net uptake, NADPH/NADP+ and GSH/GSSG ratios

In isolated rat pancreatic islets maintained at a physiologic glucose concentration (5.6 mM) the effect of glucose on parameters which are known to be involved in the insulin secretion coupling such as NADPH, reduced glutathione (GSH), 86Rb+ efflux, and 45Ca++ net uptake were investigated. The insulinotropic effect of 16.7 mM glucose was decreased with the period of culturing during the first 14 days being significant after 2 days though in control experiments both protein content and ATP levels per islet were not affected and insulin content was only slightly decreased. Both NADPH and GSH decreased with time of culture. 86Rb+ efflux which is decreased by enhancing the glucose concentration from 3 to 5.6 mM in freshly isolated islets was not affected by culturing whatsoever, even not after 14 days of culture when there was no longer any insulin responsiveness to glucose. The 45Ca++ net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2) that more likely it is the result of a diminished 45Ca++ net uptake as a consequence of the inability of islet cells to maintain proper NADPH and GSH levels, and (3) that potassium (86Rb+) efflux may not be related to changes of NADPH and GSH.     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Effects of 2-deoxy-d-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 10⁷ cells/ml. ³¹P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by ¹³C- and ¹H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-¹³C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.     

The Effect of Glucose Concentration on Insulin-Induced 3T3-L1 Adipose Cell Differentiation

We examined the effect of glucose concentration on insulin-induced 3T3-L1 adipose cell differentiation. Oil Red O staining of neutral lipid, cellular triglyceride mass, and glycerol phosphate dehydrogenase (GPDH) activity, were greater in 3T3-L1 cells cultured at 5 mM vs. 25 mM glucose. GPDH activity was 2- to 4-fold higher at 5 mM vs. 25 mM glucose over a range of insulin concentrations (0. 1 to 100 nM). Insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was 1. 7-fold greater, and insulinstimulated phosphoinositide 3-kinase association with IRS-1 was 2. 3-fold higher, at 5 mM vs. 25 mM glucose. These effects of glucose were not caused by alterations in IRS-1 mass or cell-surface insulin binding. In preadipose cells at 5 mM glucose, expression of the leukocyte antigen-related (LAR) protein tyrosine phosphatase (negative regulator of insulin signaling) was 63% of the level at 25 mM glucose. Our data demonstrate that glucose concentration affects insulin-induced 3T3-L1 adipose cell differentiation as well as differentiation-directed insulin signaling pathways. Alterations in LAR expression potentially may be involved in modulating these responses.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Glycerolipid formation and PGE2 and PGF2α production of rat renal papillae in vitro, the effects of urea

The glycerolipid production by rat renal papillary slices varied inversely with the urea concentration (0a–1660 mM) whether the production was measured as labelling of the glycerol backbone from glucose or as incorporation of labelled arachidonic acid and palmitic acid. The rate of phospholipid formation was most dependent on medium urea concentrations in the range between 0 and 1100 mM. The production of prostaglandins PGE₂ and PGF_2α, measured radioimmunologically or by an isotope derivative method was in the same range inversely related to the production of glycerolipids and chain elongations. The effect of urea on prostaglandin formation is probably indirectly caused by the inhibition of the phospholipid formation and chain elongation, since the effect was abolished by 1% defatted albumin in the medium. The data suggest that the level of free arachidonic acid within the cells is controlled to an important extent by glycerolipid formation and chain elongation.     

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.     

Spermatogenic cell differentiation in vitro

Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l0⁷ cells/25 cm²) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis.     

Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

Characterizations of Phospholipids from Paramecium tetraurelia Cells and Cilia*,†

Phospholipids from Paramecium tetraurelia strains 51s and d,95 cultures and isolated cilia were characterized. The following classes of phospholipids were identified in whole cell lipids: the 1-alkyl-2–acylglyceryl and the 1,2–diacylglyceryl forms of phosphonylethanolamine, phosphorylethanolamine. and phosphorylcholine; cardiolipin: ceramide aminoethylphosphonate and 5 other sphingolipids: phosphatidylserine; phosphatidylinositol; and lyso derivatives of the major glycerophospholipids. Cilia lipids were rich in ether lipids, phosphonolipids. and sphingolipids. Net lipid biosynthesis did not occur, as determined by the weight of lipids extracted from culture medium compared with the weight of lipids extracted from culture medium and ciliates after 7 days of growth. Total lipids/cell decreased with culture age. changes in the neutral lipid fraction accounting for the major decrease. Phospholipid class distributions changed with culture age—the glyceryl phosphorylethanolamine and choline content of cells decreased, while the glyceryl ohosphonylethanolamine content remained relatively constant: hence, the ratio of phosphonolipids to total phospholipids increased. All fatty acids observed in total lipids from cells and cilia were also present in the glycerophospholipids. Total lipids from cilia contained a greater percent of polyunsaturated fatty acid than those of whole cells. Whole cells and cilia glyceryl phosphonolipids contained up to 93% eicosatetraenoic plus eicosapentaenoic fatty acids. The enrichment of phosphonolipids in cilia accounted for most of the polyunsaturated fatty acid enrichment observed in cilia total lipids. The fatty acid composition of all major whole cell glycerophospholipid classes changed dramatically with culture age, while only small changes occurred in cilia glycerophospholipid fatty acids.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

Effects of a surfactant-associated protein and calcium ions on the structure and surface activity of lung surfactant lipids

Previous studies have demonstrated that lung-specific proteins are associated with surfactant lipids, particularly the highly surface active subfraction known as tubular myelin. We have isolated a surfactant-associated protein complex with molecular weight components of 36 000, 32 000, and 28 000 and reassembled it with protein-free lung surfactant lipids prepared as small unilamellar liposomes. The effects of divalent cations on the structure and surface activity of this protein-lipid mixture were investigated by following (1) the state of lipid dispersion by changes in turbidity and by electron microscopy and (2) the ability of the surfactant lipids to form a surface film from an aqueous subphase at 37 degrees C. The protein complex markedly increased the rate of Ca2+-induced surfactant-lipid aggregation. Electron microscopy demonstrated transformation of the small unilamellar liposomes (median diameter 440 A) into large aggregates. The threshold Ca2+ concentration required for rapid lipid aggregation was reduced from 13 to 0.5 mM by the protein complex. This protein-facilitated lipid aggregation did not occur if Mg2+ was the only divalent cation present. Similarly, 5 mM Ca2+ but not 5 mM Mg2+ improved the ability of the protein-lipid mixture to form a surface film at 37 degrees C. Extensive aggregation of the surfactant lipids without protein by 20 mM Ca2+ or 20 mM Mg2+ did not promote rapid surface film formation. These results add to the growing evidence that specific Ca2+-protein-lipid interactions are important in determining both the structure and function of extracellular lung surfactant fractions.