Cytotoxicity and cytoprotective effects of citrus flavonoids on insulin-secreting cells BRIN-BD11: beneficial synergic effects.

Flavonoids, in general, have potent antioxidant activity and they can be used in treating chronic diseases involving oxidative stress, such as diabetes mellitus. The purpose of this study was to evaluate the cytotoxicity and cytoprotective effects of citrus flavonoids on the functionality of BRIN-BD11 cells. The assessment of cytotoxic and cytoprotective flavonoid tested was performed using the MTT reduction assay. The flavonoids did not show cytotoxic effects in any of the tested concentrations (5-20 µM) and also negative insulinotropic effects were not observed. To cytoprotective assay, the IC50 of H2O2 in treatment of 2 h (acute oxidative stress) was measured (350 µM). Moreover, under acute oxidative stress, the isolated flavonoids (10 µM) had no cytoprotective effects. Besides an antioxidant role of the flavonoids was only observed when using in association. Thus future experiments are needed, varying the experimental condition, to better evaluate the possible mechanisms of action of these flavonoids.

Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line.

AIMS/HYPOTHESIS: Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). METHODS: Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. RESULTS: Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. CONCLUSIONS/INTERPRETATION: NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

Molecular mechanisms of glutamine action.

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions.

Changes of fatty acid composition in incubated rat pancreatic islets.

OBJECTIVE: The hypothesis that changes in fatty acId composition of pancreatic islets occur during incubation was investigated. METHODS: The content and composition of fatty acIds (FA) from rat pancreatic islets and culture medium after incubation for 1 and 3 hours in the absence or in the presence of 5.6, 8.3, or 16.7 mM glucose were determined by HPLC analysis. RESULTS: The FA content of pancreatic islets was reduced after 1 hour incubation in the absence of glucose. However, the total FA content was restored by incubating in the presence of 5.6 mM glucose and exceeded by incubating in the presence of 8.3 mM or 16.7 mM glucose. Saturated FA contributed a substantially greater proportion of the total FA increase in comparison to unsaturated FA, being palmitic and stearic acIds the most important. The total lipId content of pancreatic islets was not increased if the period of incubation in the presence of glucose was extended to 3 hours. A substantial amount of FA was found in the medium after 1 hour incubation in the absence of glucose: 141 ng per 80 islets for saturated and 75 ng per 80 islets for unsaturated. The release of FA from islets is increased in the presence of glucose. CONCLUSION: The release of FA from islets is a novel finding and may be related to modulation of B-cell function.

Transfer of arachidonic acid from lymphocytes to macrophages.

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mphi) in co-culture, and the subsequent functional impact on Mphi phagocytosis were investigated. The rate of incorporation of [1-14C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158 +/- 8 nmol/10(10) LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [14C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/10(10) cells in untreated-LY and 636 nmol/10(10) cells in TG-LY. [14C]AA was transferred from LY to co-cultured Mphi in substantial amounts (8.7 nmol for untreated and 15 nmol per 10(10) for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mphi co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 microM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mphi. TG treatment abolished the AA-induced inhibition of phagocytosis in Mphi co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.

Glutamine and glutamate as vital metabolites.

Glucose is widely accepted as the primary nutrient for the maintenance and promotion of cell function. This metabolite leads to production of ATP, NADPH and precursors for the synthesis of macromolecules such as nucleic acids and phospholipids. We propose that, in addition to glucose, the 5-carbon amino acids glutamine and glutamate should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine/glutamate are many, i.e., they are substrates for protein synthesis, anabolic precursors for muscle growth, they regulate acid-base balance in the kidney, they are substrates for ureagenesis in the liver and for hepatic and renal gluconeogenesis, they act as an oxidative fuel for the intestine and cells of the immune system, provide inter-organ nitrogen transport, and act as precursors of neurotransmitter synthesis, of nucleotide and nucleic acid synthesis and of glutathione production. Many of these functions are interrelated with glucose metabolism. The specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells are discussed in the context of glucose requirements and cell function.

Glutamine and glutamate as vital metabolites

Glucose is widely accepted as the primary nutrient for the maintenance and promotion of cell function. This metabolite leads to production of ATP, NADPH and precursors for the synthesis of macromolecules such as nucleic acids and phospholipids. We propose that, in addition to glucose, the 5-carbon amino acids glutamine and glutamate should be considered to be equally important for maintenance and promotion of cell function. The functions of glutamine/glutamate are many, i.e., they are substrates for protein synthesis, anabolic precursors for muscle growth, they regulate acid-base balance in the kidney, they are substrates for ureagenesis in the liver and for hepatic and renal gluconeogenesis, they act as an oxidative fuel for the intestine and cells of the immune system, provide inter-organ nitrogen transport, and act as precursors of neurotransmitter synthesis, of nucleotide and nucleic acid synthesis and of glutathione production. Many of these functions are interrelated with glucose metabolism. The specialized aspects of glutamine/glutamate metabolism of different glutamine-utilizing cells are discussed in the context of glucose requirements and cell function

Transfer of arachidonic acid from lymphocytes to macrophages

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mö) in co-culture, and the subsequent functional impact on Mö phagocytosis were investigated. The rate of incorporation of [1-14C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158 ± 8 nmol/1010 LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [14C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/1010 cells in untreated-LY and 636 nmol/1010 cells in TG-LY. [14C]AA was transferred from LY to co-cultured Mö in substantial amounts (8.7 nmol for untreated and 15 nmol per 1010 for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mö co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 ìM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mö. TG treatment abolished the AA-induced inhibition of phagocytosis in Mö co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion.

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.

Metabolic response of macrophages to injury promoted by the activated complement system.

In nucleated cells, the swelling promoted by a complement system (CS) attack is not enough to promote cell death, because unlike erythrocytes these cells are able to eliminate cytolytic complement channels from the plasma membrane, by processes that include endocytosis. Several studies have demonstrated that the resistance of nucleated cells to the injury promoted by the CS is related to the cellular metabolism. Despite this, to the present day, no study has clearly related cell survival capacity to injury by the CS to its energetic metabolic status. In macrophages, the challenge imposed by the CS provoked an increase in the total amount of glucose incorporated into fatty acids, including phospholipids and cholesterol; substrates for membrane synthesis. The inhibition of cholesterol synthesis promoted an increase of the cell death rate. These data support the importance of cholesterol metabolism for macrophage resistance to necrosis induced by the activated complement system.

Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils.

Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of glutamine utilization and its importance in such cells have raised the question as to the source of this glutamine, which, according to current evidence, appears to be muscle.

Effects of adrenaline on glucose and glutamine metabolism and superoxide production by rat neutrophils.

Despite the large body of information on the role of corticosteroids in regulating lymphocyte and phagocyte function, the role of the hormone adrenaline in immunoregulation is an under-investigated topic. The present study has addressed the effects of adrenaline on the rates of utilization and oxidation of glucose and glutamine, the phagocytic capacity and the rate of superoxide production by rat neutrophils. Incubation of rat neutrophils in the presence of 50 microM adrenaline caused a marked elevation in glucose metabolism, an effect that could be blocked by propranolol. Adrenaline caused a partial inhibition of glutamine utilization by neutrophils, an effect that was also blocked by propranolol. These effects of adrenaline could be mimicked by 100 microM dibutyryl cAMP. Phosphate-dependent glutaminase activity was significantly elevated in neutrophils incubated in the presence of 50 microM adrenaline or 100 microM dibutyryl cAMP for 1 h, whereas glutamine oxidation was significantly depressed (P<0.05) under these conditions. The elevation in enzyme activity was only partially blocked by propranolol. The phagocytic activity of rat neutrophils was not altered by adrenaline in the presence of either glucose or glutamine. The rate of phorbol 12-myristate 13-acetate-induced superoxide production in the presence of glucose was potently reduced by the addition of 5 nM or 50 microM adrenaline. This effect could be mimicked by dibutyryl cAMP. However, when rat neutrophils were incubated in the presence of glutamine plus adrenaline (5 nM or 50 microM), the rate of superoxide production was only marginally reduced. These findings support the proposition that adrenaline may deviate the flux of glucose from the NADPH-producing pentose phosphate pathway, thus reducing substrate availability for the superoxide-generating NADPH oxidase. However, glutamine metabolism may still give rise to substantial quantities of NADPH from the glutaminolysis pathway. We postulate that glutamine metabolism may thus provide a protective mechanism against the inhibitory effect of adrenaline on superoxide production by neutrophils.

Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils

Eric Newsholme's laboratory was the first to show glutamine utilization by lymphocytes and macrophages. Recently, we have found that neutrophils also utilize glutamine. This amino acid has been shown to play a role in lymphocyte proliferation, cytokine production by lymphocytes and macrophages and phagocytosis and superoxide production by macrophages and neutrophils. Knowledge of the metabolic fate of glutamine in these cells is important for the understanding of the role and function of this amino acid in the maintenance of the proliferative, phagocytic and secretory capacities of these cells. Glutamine and glucose are poorly oxidized by these cells and might produce important precursors for DNA, RNA, protein and lipid synthesis. The high rate of glutamine utilization and its importance in such cells have raised the question as to the source of this glutamine, which, according to current evidence, appears to be muscle

Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats.

Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced by adrenodemedullation in the lymphoid organs (MLN) (28%), thymus (40%) and spleen (42%)) and gastrocnemius muscle (67%). G6PDh activity was enhanced in the MLN (69%) and reduced in the spleen (28%) and soleus muscle (75%). CS activity was reduced in all tissues (MLN (75%), spleen (71%), gastrocnemius (61%) and soleus (43%)), except in the thymus which displayed an increment of 56%. Cu/Zn-SOD activity was increased in the MLN (126%), thymus (223%), spleen (80%) and gastrocnemius muscle (360%) and was reduced in the soleus muscle (31%). Mn-SOD activity was decreased in the MLN (67%) and spleen (26%) and increased in the thymus (142%), whereas catalase activity was reduced in the MLN (76%), thymus (54%) and soleus muscle (47%). It is particularly noteworthy that in ADM rats the activity of glutathione peroxidase was not detectable by the method used. These data are consistent with the possibility that epinephrine might play a role in the oxidative stress of the lymphoid organs. Whether this fact represents an important mechanism for the establishment of impaired immune function during stress remains to be elucidated.

Changes in the TBARs content and superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of adrenodemedullated rats

Thiobarbituric acid reactant substances (TBARs) content, and the activities of glucose-6-phosphate dehydrogenase (G6PDh), citrate synthase (CS), Cu/Zn- and Mn-superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) were measured in the lymphoid organs (thymus, spleen, and mesenteric lymph nodes (MLN)) and skeletal muscles (gastrocnemius and soleus) of adrenodemedullated (ADM) rats. The results were compared with those obtained for sham-operated rats. TBARs content was reduced by adrenodemedullation in the lymphoid organs (MLN (28 percent), thymus (40 percent) and spleen (42 percent)) and gastrocnemius muscle (67 percent). G6PDh activity was enhanced in the MLN (69 percent) and reduced in the spleen (28 percent) and soleus muscle (75 percent). CS activity was reduced in all tissues (MLN (75 percent), spleen (71 percent), gastrocnemius (61 percent) and soleus (43 percent)), except in the thymus which displayed an increment of 56 percent. Cu/Zn-SOD activity was increased in the MLN (126 percent), thymus (223 percent), spleen (80 percent) and gastrocnemius muscle (360 percent) and was reduced in the soleus muscle (31 percent). Mn-SOD activity was decreased in the MLN (67 percent) and spleen (26 percent) and increased in the thymus (142 percent), whereas catalase activity was reduced in the MLN (76 percent), thymus (54 percent) and soleus muscle (47 percent). It is particularly noteworthy that in ADM rats the activity of glutathione peroxidase was not detectable by the method used. These data are consistent with the possibility that epinephrine might play a role in the oxidative stress of the lymphoid organs. Whether this fact represents an important mechanism for the establishment of impaired immune function during stress remains to be elucidated

Effect of adrenaline and phorbol myristate acetate or bacterial lipopolysaccharide on stimulation of pathways of macrophage glucose, glutamine and O2 metabolism. Evidence for cyclic AMP-dependent protein kinase mediated inhibition of glucose-6-phosphate dehydrogenase and activation of NADP+-dependent 'malic' enzyme.

Adrenaline has recently been shown to stimulate both glucose metabolism and H2O2 release by macrophages but the activity of the key pentose phosphate pathway enzyme, glucose-6-phosphate dehydrogenase (which generates the NADPH crucial for the reduction of molecular oxygen), was reduced under these conditions [Costa Rosa, Safi, Cury and Curi (1992) Biochem. Pharmacol. 44, 2235-2241]. We report here that adrenaline activates another NADPH-producing enzyme, NADP(+)-dependent 'malic' enzyme, while also inhibiting glucose-6-phosphate dehydrogenase, via cyclic AMP-dependent protein kinase (PKA) activation. Regulation of glucose-6-phosphate dehydrogenase activity by PKA has not been reported elsewhere. The sparing of some glucose from pentose phosphate pathway consumption may be important in the provision of glycerol 3-phosphate which in the macrophage may be required for new phospholipid synthesis. Glutamine oxidation was also stimulated by adrenaline thus providing increased substrate (malate) for NADP(+)-dependent 'malic' enzyme and therefore shifting some of the burden of NADPH production from glucose to glutamine metabolism. We also report a novel synergistic effect of adrenaline and some bacterial products and/or gamma-interferon in stimulating secretory and metabolic pathways in macrophages which may be a part of a larger network of signals that lead to enhanced macrophage activity.

Propionate modifies lipid biosynthesis in rat peritoneal macrophages.

1. This study examines the effect of propionate, normally produced in the gut, on lipid metabolism of resident macrophage. This cell is very abundant in the epithelial lining of the gut. 2. The activity of propionyl-CoA synthetase in macrophages was shown to be 0.39 nmol/min per mg protein, so this cell presents the ability to use propionate. Propionate at concentrations varying from 0.5 to 5 mM did not affect the activities of carnitine acetyltransferase, ATP-citrate lyase, acetoacetyl-CoA thiolase and 3-oxoacid-CoA transferase. 3. Thus this short chain fatty acid did not alter the capacity for transferring acetyl-CoA from mitochondria to cytosol and for ketone bodies formation and oxidation. However, propionate (40 mM) inhibited the incorporation of [1-14C]-palmitate into phospholipids, cholesterol, cholesterol ester and triacylglycerol and the incorporation of [3-14C]-pyruvate into phospholipids. 4. These findings suggest that fibre-rich diet by generating propionate may regulate macrophage lipid metabolism.

Maximal activities of key enzymes of glutaminolysis, glycolysis, Krebs cycle and pentose-phosphate pathway of several tissues in mature and aged rats.

1. The maximum activities of some key enzymes, which provide a quantitative indices of flux through several important pathways have been measured in brain, liver, muscle, white and brown adipose tissue and lymphocytes of mature and aged rats. 2. The results were expressed as mumol/min per g fresh weight and nmol/min per mg protein. 3. On the both basis, as compared to mature rats, hexokinase activity is decreased in brown adipose tissue and increased in soleus muscle. 4. Glucose-6-phosphate dehydrogenase activity is decreased in most tissues and increased in brain. 5. Citrate synthase activity, which provides a qualitative index of the Krebs cycle, is decreased in white adipose tissues and lymphocytes. 6. Glutaminase activity is decreased in brain, white and brown adipose tissues but is increased in lymphocytes.