Abstract
In this work, our aim was to determine whether l-arginine (a known insulinotropic amino acid) can promote a shift of β-cell intermediary metabolism favoring glutathione (GSH) and glutathione disulfide (GSSG) antioxidant responses, stimulus–secretion coupling and functional integrity. Clonal BRIN-BD11 β-cells and mouse islets were cultured for 24 h at various l-arginine concentrations (0–1.15 mmol/l) in the absence or presence of a proinflammatory cytokine cocktail (interleukin 1β, tumour necrosis factor α and interferon γ). Cells were assessed for viability, insulin secretion, GSH, GSSG, glutamate, nitric oxide (NO), superoxide, urea, lactate and for the consumption of glucose and glutamine. Protein levels of NO synthase-2, AMP-activated protein kinase (AMPK) and the heat shock protein 72 (HSP72) were also evaluated. We found that l-arginine at 1.15 mmol/l attenuated the loss of β-cell viability observed in the presence of proinflammatory cytokines. l-Arginine increased total cellular GSH and glutamate levels but reduced the GSSG/GSH ratio and glutamate release. The amino acid stimulated glucose consumption in the presence of cytokines while also stimulating AMPK phosphorylation and HSP72 expression. Proinflammatory cytokines reduced, by at least 50%, chronic (24 h) insulin secretion, an effect partially attenuated by l-arginine. Acute insulin secretion was robustly stimulated by l-arginine but this effect was abolished in the presence of cytokines. We conclude that l-arginine can stimulate β-cell insulin secretion, antioxidant and protective responses, enabling increased functional integrity of β-cells and islets in the presence of proinflammatory cytokines. Glucose consumption and intermediary metabolism were increased by l-arginine. These results highlight the importance of l-arginine availability for β-cells during inflammatory challenge.
Introduction
Obesity, insulin resistance and type 2 diabetes (T2DM) are known to induce a proinflammatory state that, together with the adverse effects of hyperglycemia and hyperlipidemia, leads to the progressive dysfunction and demise of pancreatic β-cells (Newsholme et al. 2010). Among the factors involved in the reduction of β-cell viability is the local inflammatory process in the islets of Langerhans. This is induced by islet dendritic cell activation and infiltration by T-lymphocytes and macrophages, leading to microenvironmental changes, resulting in β-cell injury and death (Masters et al. 2010).
Destruction of β-cells during islet inflammation can be mediated by direct contact with activated macrophages and by exposure to soluble mediators secreted by dendritic cells, macrophages and T-lymphocytes, including cytokines, oxygen free radicals and nitric oxide (NO; Eizirik & Mandrup-Poulsen 2001). Indeed, overproduction of NO by β-cells themselves is one of the most significant mechanisms leading to β-cell dysfunction and death (Eizirik & Mandrup-Poulsen 2001). The production of NO is promoted by inflammatory cytokines which activate the transcription of β-cell inducible isoform of NO synthase (iNOS, encoded by the NOS-2 gene), an enzyme whose expression is nuclear factor κB (NFκB)-driven and which uses l-arginine as substrate (Eizirik & Mandrup-Poulsen 2001). While excessive NO production within β-cells may trigger oxidative/nitrosative stress leading to cell death, NO also serves as an important β-cell stimulus–secretion coupling factor (Smukler et al. 2002, Newsholme et al. 2005, 2007a,b, 2010, Krause & de Bittencourt 2008). l-Arginine is recognized as one of the most powerful insulin secretagogues (Palmer et al. 1976) and while it can contribute to promotion of exocytosis via transport-associated membrane depolarization, there are possibly many other mechanisms by which it positively modulates insulin secretion. At specific concentrations, evidence suggests that NO is a physiological regulator of insulin secretion in β-cells (Spinas 1999, Smukler et al. 2002). The presence of an NFκB-dependent iNOS while beneficial at low concentration/activity, predisposes β-cells to development of oxidative/nitrosative stress at higher levels, because NO may inhibit metabolism, mitochondrial activity and generation of stimulus–secretion coupling factors (Krause & de Bittencourt 2008).
Macrophages can release, into the inflammatory islet microenvironment, the enzyme arginase (Murphy & Newsholme 1998), which splits l-arginine into urea and l-ornithine, thus avoiding its conversion into NO and favoring resolution of inflammation (Zhai et al. 2009). Interestingly, the depletion of l-arginine itself is sufficient to inhibit T-cell proliferation by the downregulation of the ζ-chain, the main signal transduction component of the T-cell receptor complex (Bronte & Zanovello 2005). It has also been demonstrated that β-cells possess a cytokine-inducible arginase activity (Stickings et al. 2002), which may account for reduction in NO synthesis under appropriate conditions. Although release of arginase by infiltrating macrophages may promote the resolution of inflammation, restriction of arginine availability in the islet microenvironment may be detrimental for β-cell metabolism, antioxidant defenses and insulin secretion (Newsholme et al. 2010). Interestingly, decreased plasma and intracellular concentrations of l-arginine have been reported in patients with type 2 diabetes (Pieper & Dondlinger 1997). l-Arginine also increased β-cell neogenesis and antioxidant defenses in rats treated with alloxan and aided recovery of endothelium-dependent relaxation in patients with type 2 diabetes (Pieper & Dondlinger 1997, Vasilijevic et al. 2007). Administration of l-arginine has also been reported to reduce adiposity in obese–diabetic humans, genetic and diet-induced obese rats as well as finishing pigs (McKnight et al. 2010).
To further investigate the importance of l-arginine for β-cell function, we determined changes in clonal β-cell insulin secretion, metabolism, redox status and integrity in vitro in response to manipulation of l-arginine concentration in tissue culture in the absence or presence of proinflammatory cytokines. Key experimental findings were subsequently further explored in heterogeneous (mixed endocrine cell) mouse islet incubations.
Materials and Methods
Culture of BRIN-BD11 pancreatic β-cells, proinflammatory cocktail challenge and measurement of insulin secretion at different l-arginine concentrations
The clonal rat insulin-secreting β-cell line BRIN-BD11 was chosen because its metabolic, signaling and secretory responses to glucose, amino acids and other stimuli have been extensively characterized (McClenaghan et al. 1996, McClenaghan & Flatt 1999, Brennan et al. 2003). BRIN-BD11 cells were maintained in culture overnight as described previously (Kiely et al. 2007). After washing with PBS, cells were incubated in fresh (l-arginine free) RPMI-1640 medium, supplemented with 11.1 mmol/l d-glucose, 2 mmol/l l-glutamine and different concentrations of l-arginine (0, 0.1, 0.25 and 1.15 mmol/l). Cells were incubated in the presence or absence of a non-lethal (with respect to cells cultured in normal culture medium) proinflammatory cytokine cocktail (interleukin 1β (IL1β) 0.3125 U/ml, tumour necrosis factor α (TNFα) 31.25 U/ml and interferon γ (IFNγ) 15.625 U/ml; Kiely et al. 2007). IL1β has been implicated in macrophage associated β-cell dysfunction typical of type 2 diabetes (Masters et al. 2010). The l-arginine concentration range tested was chosen to include absence (0 mM), the physiological range in human blood (0.1–0.25 mmol/l) and the normal concentration found in the RPMI-1640 culture medium (1.15 mmol/l l-arginine). After 24 h, an aliquot of the medium was removed for insulin assay or centrifuged at 16 000 g for 10 min at 4 °C for the later determination of metabolites. In addition, for the set of experiments designed to test the effect of 24 h culture in various concentrations of l-arginine in the absence or presence of proinflammatory cytokines on subsequent acutely stimulated insulin secretion, cells were cultured for 24 h as above, preincubated for 40 min at 1.1 mmol/l glucose and then acutely stimulated for 20 min in the presence of 16.7 mM glucose+10 mM alanine. The latter combination has been shown to evoke a robust and reproducible insulin secretion response (Brennan et al. 2002, Kiely et al. 2007). Aliquots of incubation medium were taken for analysis of insulin using the Mercodia Ultrasensitive Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden).
Islet isolation and culture; viability and chronic (24 h) insulin secretion
Pancreatic islets were isolated from wild-type C57 black mice. Each pancreas was excised and inflated with a Liberase TL grade solution (Roche 1815032; 8 mg/ml) and chopped into small pieces. Digestion was initiated during sample incubation at 37 °C for 3 min with constant shaking. The digest was washed with 0.1% BSA Krebs solution (5.6 mM glucose) and the islets were sedimented by gentle centrifugation (500 g for 10 min at 4 °C) with Histopaque 1077 (Sigma–Aldrich). Islets were resuspended in Krebs buffer containing 0.1% BSA, individually picked and cultured for 24 h with RPMI-1640 culture medium (as above), in the presence or absence of a non-lethal proinflammatory cytokine cocktail (IL1β 0.3125 U/ml, TNFα 31.25 U/ml and IFNγ 15.625 U/ml; Kiely et al. 2007) at 0 or 1.15 mmol/l of l-arginine. Chronic (24 h) insulin release was determined using the Mercodia Ultrasensitive Mouse Insulin ELISA kit.
Cell viability measurements
The neutral red uptake assay provides a quantitative estimation of the number of viable cells based on their ability to incorporate and bind the dye neutral red in lysosomes. BRIN-BD11 cells were exposed to different concentrations of l-arginine, with or without the proinflammatory cytokine cocktail as described earlier. After 2 h incubation in presence of neutral red (100 μg/ml), cells were washed with PBS followed by disruption with acid ethanol (alcohol/glacial acetic acid, 50:1v/v). Aliquots of the resulting solution were transferred to 96-well plates and absorbance at 540 nm was recorded using a microplate spectrophotometer (Molecular Devices SpectraMax Plus 384, Sunnyvale, CA, USA). This procedure was highly reproducible and comparible with other cytotoxicity tests (tetrazolium salts, enzyme release, or DNA content; Repetto et al. 2008). To determine cell membrane integrity (an indirect measurement of cell viability), a lactate dehydrogenase (LDH) release assay (Biovision, Dublin, Ireland) was used and the results expressed as percentage of islet LDH released.
There was good correlation between cell membrane integrity, mitochondrial function and level of apoptosis and necrosis in a clonal β-cell line exposed to proinflammatory cytokines (Kiely et al. 2007). Islet β-cells contain a relatively low content of LDH but this is clearly measurable using the Biovision kit (Mountain View, CA, USA).
Enzymatic determination of metabolites
Urea and nitrite production, glucose and glutamine consumption and production of glutamate and lactate plus intracellular glutamate concentration were determined as described previously (Brennan et al. 2002, Kiely et al. 2007).
Measurement of glutathione and glutathione disulfide content
BRIN-BD11 cells were seeded into six-well plates (1×106 cells/well), allowed to adhere overnight and then washed with PBS after which they were incubated for 24 h in fresh media as described earlier. Cells were then rinsed twice with PBS and disrupted in 200 μl of 5% (w/v) metaphosphoric acid on ice. After centrifugation (14 000 g, 5 min at room temperature), cell lysates were spectrophotometrically (415 nm) assayed in a temperature-controlled microplate reader (Molecular Devices SpectraMax Plus 384) by a modification of the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)/glutathione disulfide (GSSG) reductase recycling method, using the N-ethylmaleimide conjugating technique for GSSG sample preparation (Krause et al. 2007). Samples (10 μl) were assayed in 105 μl final volume in 96-well polystyrene plates at 37 °C in the presence of 10 mM DTNB, 0.17 mM β-NADPH (dissolved in 0.5% (w/v) NaHCO3 as a stabilizing agent) and 0.5 U/ml GSSG reductase (EC 1.6.4.2).
Superoxide production in BRIN-BD11 cells
Dihydroethidium (DHE) is widely used as a probe to measure superoxide (O2•). DHE is cell permeable and reacts with (O2•) to form ethidium, which in turn intercalates with DNA, providing nuclear fluorescence at an excitation wavelength of 525 nm and an emission wavelength of 590 nm (Benov et al. 1998). BRIN-BD11 cells were incubated (1×104 cells/100 μl per well) in a 96-well black plate (Corning, Inc. Costar 3603, Dublin, Ireland) allowed to adhere overnight. Cells were then washed with PBS and incubated in various conditions for 24 h as described earlier. Subsequently DHE was added to the culture medium for 30 min to allow for the reaction between the dye and the superoxide to reach completion. The fluorescence was then read (ex=525/em=590). The results were expressed as average of fluorescence intensity/milligram of protein per 24 h.
Preparation of protein extracts from BRIN-BD11 cells
BRIN-BD11 cells were lysed in 150 μl of RIPA lysis buffer (MSC, Dublin, Ireland) containing protease inhibitors. Cell lysates were transferred to fresh ice-cold microcentrifuge tubes and placed on a shaker at 4 °C for 15 min. The cells were then centrifuged at 14 000 g for 15 min at 4 °C. The supernatant fraction was transferred to a fresh tube and stored at −20 °C. Cellular protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA; kit no. 23225), which utilizes a modification of the biuret reaction.
Western blot analysis
Cells were seeded into six-well plates (1.5×106 cells/well), allowed to adhere overnight and then washed with PBS after which they were incubated in fresh media for 24 h, in various conditions as described earlier. Subsequently cells were lysed and equal amounts of BRIN-BD11 cell protein extracts were prepared and subjected to 10% SDS–PAGE then electrophoretically transferred to a nitrocellulose membrane. The membranes were blocked in 5% (milk protein or BSA) and probed with polyclonal antibodies anti-NOS-2 and heat shock protein 72 (HSP72; Sigma–Aldrich); anti-AMP-activated protein kinase (AMPK), phosphorylated AMPK (AMPK-P; Cell Signalling Technologies, Denvers, MA, USA). The blots were washed and visualized with a HRP-based Supersignal West Pico chemiluminescent substrate (Pierce). Results of digitalized images were expressed as means±s.d. using anti-GAPDH antibodies for GAPDH detection (Cell Signalling Technologies) as an expression control.
Statistical analysis
Following confirmation of a normal distribution through the use of repeated Shapiro–Wilks tests, paired and unpaired two-tailed Student's t-tests were used where appropriate to evaluate the statistical significance of differences between the group means and ANOVA was used for multiple comparisons. Data are presented as means±s.d. and differences were considered significant at a P value of <0.05.
Results
Effect of l-arginine on BRIN-BD11 cell viability in the absence or presence of a proinflammatory cytokine cocktail
The effects of 24 h culture in the presence of various concentrations of l-arginine on the viability of BRIN-BD11 cells in the absence or presence of proinflammatory cytokines was investigated using the neutral red assay. Consistent with the importance of l-arginine for the maintenance of β-cell function, viability of BRIN-BD11 cells decreased from a normalized value of 100–61% in the absence of the amino acid (Table 1). Loss of viability was more dramatic when the proinflammatory cytokine cocktail was added, reducing viability to 40% of control in the absence of l-arginine. While ‘viability’ measurements invariably determine loss of cell function, e.g. loss of plasma membrane integrity by LDH release, or reduced lysosomal function via binding of neutral red, or activation of enzymes associated with cell apoptosis, key cell structures may remain intact at the time of assay. Thus, BRIN-BD11 cells were still able to metabolize glutamine at high rates and maintain their GSSG/glutathione (GSH) ratio, even when neutral red binding capacity was severely reduced (see below).
Pancreatic β-cell viability (%, by neutral red assay). Effect of 24 h culture in the presence of various concentrations of l-arginine on the viability of BRIN-BD11 cells in the absence or presence of proinflammatory cytokines. Results are presented as percentage of control values. Data are means±s.d. of three separate preparations
| Cytokine cocktail | ||
|---|---|---|
| − | + | |
| Condition | ||
| 1.15 mM l-arginine | 100±8.7†,‡ | 96.3±8.1†,‡,§ |
| 0.25 mM l-arginine | 94±3*,† | 56.3±10.9†,∥ |
| 0.1 mM l-arginine | 89±4.6*,† | 56.1±20.9†,∥ |
| No l-arginine | 61±4.6* | 42.1±4.5 |
*P<0.05 versus cytokine treatment at the same l-arginine concentration; †P<0.05 vs 0 mmol/l l-arginine; ‡P<0.05 vs 0.1 mmol/l l-arginine; §P<0.05 vs 0.25 mmol/l l-arginine; and ∥P<0.05 vs 1.15 mmol/l l-arginine.
Effect of l-arginine on GSH levels, intracellular glutamate levels, glutamate release and glutamine consumption in BRIN-BD11 cells in the absence or presence of a proinflammatory cytokine cocktail
The effects of 24 h culture in the presence of various concentrations of l-arginine on GSH levels, cellular redox state, glutamate levels, glutamate release and glutamine consumption of BRIN-BD11 cells in the absence or presence of proinflammatory cytokines was investigated. Since glutamine (and the immediate product of its metabolism, glutamate) is important precursors for β-cell GSH synthesis (Brennan et al. 2003), both reduced and oxidized forms of GSH were determined as was the GSSG/GSH ratio in addition to intracellular glutamate levels, glutamate release plus glutamine consumption. The intracellular levels of reduced GSH increased substantially in the presence of l-arginine (Table 2) from 0.13±0.01 μmol/mg (0 mmol/l l-arginine) to 0.92±0.14 μmol/mg (1.15 mmol/l l-arginine). This greater than sixfold increase evoked by l-arginine was concentration-dependent (Table 2). Levels of GSH disulfide were also increased but to a much lesser extent. Intracellular glutamate did not change when cells were exposed to physiologic or high concentrations of l-arginine (Table 3). However, in the total absence of l-arginine, intracellular glutamate concentration was decreased by ∼50% indicating that a minimum concentration of l-arginine was essential for the maintenance of β-cell glutamate levels (Table 3). Glutamate release was increased approximately fourfold by cytokine exposure, but amounts of glutamate released were lowered when the l-arginine concentration was increased. With respect to glutamine consumption, an approximate fourfold increase (from 3.52±0.87 to 15.05±3.05 μmol/mg) was observed with all cytokine incubations. This was unchanged by manipulation of l-arginine concentration (Table 3).
Glutathione metabolism (mmol/mg protein) and redox state (GSSG/GSH (AU)) over 24 h incubation. Pancreatic β-cell glutathione metabolism. Effect of 24 h culture in the presence of various concentrations of l-arginine on glutathione levels and cellular redox state of BRIN-BD11 cells in the absence or presence of proinflammatory cytokines. Data are means±s.d. of three separate preparations
| Experimental condition | Total glutathione | GSSG | GSH | GSSG/GSH | ||||
|---|---|---|---|---|---|---|---|---|
| Cytokine cocktail | − | + | − | + | − | + | − | + |
| 1.15 mM l-arginine | 1.45±0.19†,‡,§ | 1.7±0.18†,‡,§ | 0.26±0.029†,‡,§ | 0.36±0.06†,‡,§ | 0.91±0.14†,‡ | 0.96±0.18†,‡ | 0.28±0.02†,‡,§ | 0.39±0.13†,‡ |
| 0.25 mM l-arginine | 1.21±0.15†,‡,∥ | 1.18±0.07†,‡,∥ | 0.19±0.03†,‡,∥ | 0.22±0.021†,‡,∥ | 0.828±0.08†,‡ | 0.73±0.09†,‡ | 0.23±0.02†,‡ | 0.31±0.06†,‡ |
| 0.1 mM l-arginine | 0.49±0.07§,∥ | 0.5±0.05§,∥ | 0.107±0.02§,∥ | 0.12±0.008§,∥ | 0.28±0.02†,§,∥ | 0.24±0.05†,§,∥ | 0.37±0.07†,‡,∥ | 0.37±0.07†,‡,∥ |
| No l-arginine | 0.44±0.02 | 0.44±0.03 | 0.15±0.01 | 0.15±0.02 | 0.13±0.007 | 0.13±0.034 | 1.23±0.35 | 1.19±0.21 |
*P<0.05 versus cytokine treatment at the same l-arginine concentration; †P<0.05 vs 0 mmol/l l-arginine; ‡P<0.05 vs 0.1 mmol/l l-arginine; §P<0.05 vs 0.25 mmol/l l-arginine; and ∥P<0.05 vs 1.15 mmol/l l-arginine.
Pancreatic β-cell metabolites consumption/production (mmol/mg protein) over 24 h incubation. Effect of 24 h culture in the presence of various concentrations of l-arginine on glucose and glutamine consumption, lactate production, intracellular and extracellular glutamate from BRIN-BD11 cells in the absence or presence of proinflammatory cytokines. Data are means±s.d. of three separate preparations
| Experimental condition | Glucose consumption | Lactate production | Glutamine consumption | Extracellular glutamate | Intracellular glutamate | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Cytokine cocktail | − | + | − | + | − | + | − | + | − | + |
| 1.15 mM l-arginine | 27.6±2*,†,‡,§ | 120±18†,‡ | 31±3*,†,‡,§ | 140±32†,‡ | 3.82±0.6* | 14.07±4.08 | 2.46±0.4*,† | 13.05±2.25† | 12.8±0.7† | 13.18±0.9† |
| 0.25 mM l-arginine | 19.01±4*,†,∥ | 123±40.6†,‡ | 19±1.3*,†,‡,∥ | 106±24†,∥ | 3.16±0.71* | 15.6±6.7 | 2.9±0.28*,† | 16.48±3.42† | 13.3±0.4† | 13.12±0.4† |
| 0.1 mM l-arginine | 19.4±2*,†,∥ | 74.71±11§,∥ | 16.8±1*,†,‡,∥ | 99.92±16†,∥ | 3.52±0.8* | 15.04±3.05 | 2.96±0.46*,† | 16.99±3.42† | 12.6±0.2† | 13.31±0.19† |
| No l-arginine | 15.57±2.35* | 71.15±8.5 | 10.24±1.77* | 20.83±4.6 | 5.62±1.7* | 17.97±3.01 | 4.15±0.4* | 21.88±3.03 | 6.42±0.5 | 5.9±0.6 |
*P<0.05 versus cytokine treatment at the same l-arginine concentration; †P<0.05 vs 0 mmol/l l-arginine; ‡P<0.05 vs 0.1 mmol/l l-arginine; §P<0.05 vs 0.25 mmol/l l-arginine; and ∥P<0.05 vs 1.5 mmol/l l-arginine.
Effect of l-arginine on chronic (24 h) or acute (20 min) insulin secretion, glucose consumption and lactate production by BRIN-BD11 cells in the absence or presence of a proinflammatory cytokine cocktail
The effects of 24 h culture in the presence of various concentrations of l-arginine on insulin secretion, glucose consumption and lactate production from BRIN-BD11 cells in the absence or presence of proinflammatory cytokines was investigated. As expected, l-arginine stimulated chronic (24 h) tissue culture insulin release (by 28%) from cells cultured at 1.15 mM compared with 0 mM l-arginine (928 μg/mg protein compared with 727 μg/mg protein, Fig. 1A). In the presence of proinflammatory cytokines, insulin secretion was blunted by at least 50% in all incubations (Fig. 1A). Glucose consumption and lactate production were augmented by proinflammatory cytokine challenge (Table 3). Interestingly, glucose consumption and lactate production were further increased at higher l-arginine concentrations. It is also important to note that l-arginine increased glucose consumption and lactate production in the absence of cytokines, indicating l-arginine is able to alter β-cell glycolytic metabolism to pyruvate, so promoting lactate production and pyruvate oxidation. In a separate experiment BRIN-BD11 cells were cultured for 24 h in various concentrations of l-arginine in the absence or presence of proinflammatory cytokines and then subsequently stimulated for 20 min with a potent insulinotropic nutrient mixture (glucose+alanine, Fig. 1B–E). Cells cultured for 24 h in the absence of l-arginine were associated with reduced GSH levels, an elevated GSSG/GSH ratio and reduced intracellular glutamate (a key stimulus secretion coupling factor) as described earlier, which contribute to attenuation of acute glucose+alanine stimulated insulin secretion (Fig. 1E). This increase of glucose metabolism in cells incubated in the absence of cytokines was associated with enhanced insulin release from cells acutely stimulated with glucose+alanine (Fig. 1B–D). However, addition of cytokine cocktail potently attenuated nutrient-stimulated insulin secretion, an effect that has been previously reported (Kiely et al. 2007).
Effect of 24 h culture in the presence of various concentrations of l-arginine in the absence or presence of proinflammatory cytokines on chronic insulin secretion and subsequent 20 min acute nutrient stimulation of insulin secretion by BRIN-BD11 cells. (A) Chronic insulin secretion. 24 h preincubation in 1.15 mmol/l l-arginine (B); 24 h preincubation in 0.25 mmol/l l-arginine (C); 24 h preincubation in 0.10 mmol/l l-arginine (D); and 24 h preincubation in 0 mmol/l l-arginine (E). Cytokines were added to the 24 h culture where indicated. Data are means±s.d. of three separate preparations. Significance: *P<0.05 versus cytokine treatment at the same l-arginine concentration; ΔP<0.05 vs 0 mmol/l l-arginine; ¥P<0.05 versus respective basal; ‡P<0.05 vs 0.1 mmol/l l-arginine; †P<0.05 vs 0.25 mmol/l l-arginine; and ψP<0.05 vs 1.15 mmol/l l-arginine.
Citation: Journal of Endocrinology 211, 1; 10.1530/JOE-11-0236
Effect of l-arginine on NOS-2 expression, production of nitrite, urea and superoxide by BRIN-BD11 cells in the absence or presence of a proinflammatory cytokine cocktail
The effects of 24 h culture in the presence of various concentrations of l-arginine on NOS-2 expression, nitrite, urea production and superoxide generation in BRIN-BD11 cells in the absence or presence of proinflammatory cytokines was investigated. Since proinflammatory cytokines are likely to induce oxidative stress by the generation of oxygen- and nitrogen-based free radicals, iNOS expression, NO and superoxide production were examined. Cytokine addition resulted in a large increase in iNOS protein levels (Fig. 2). This increment was dependent on extracellular l-arginine and showed clear concentration-dependency. iNOS was not detectable in the absence of the cytokine cocktail. As a consequence greater amounts of NO were detected (by the production of nitrite) in proportion to the extracellular l-arginine concentration (Fig. 2C). Urea levels were determined in the absence or presence of cytokines at all l-arginine concentrations (Fig. 2D), but while cytokines increased urea production this was not altered by l-arginine concentration. l-arginine availability (with the exception of zero concentration) was inversely proportional to superoxide production (Fig. 2E) perhaps as a result of the effect of the lack of l-arginine availability on the uncoupled iNOS phenomenon (Wu & Meininger 2009).
Effect of 24 h culture in the presence of various concentrations of l-arginine on NOS-2 expression, nitrite, urea production and superoxide generation in BRIN-BD11 cells in the absence or presence of proinflammatory cytokines. NOS-2 expression (A and B) is shown relative to GAPDH as control. Parts of the same representative gel are mounted and shown for each immunoblot. Results are shown for (C) NO production evaluated by measuring nitrite, (D) urea production and (E) superoxide production. Data are means±s.d. of three separate preparations. Significance: *P<0.05 versus cytokine treatment at the same l-arginine concentration; ΔP<0.05 vs 0 mmol/l l-arginine; ‡P<0.05 vs 0.1 mmol/l l-arginine; †P<0.05 vs 0.25 mmol/l l-arginine; and ψP<0.05 vs 1.15 mmol/l l-arginine.
Citation: Journal of Endocrinology 211, 1; 10.1530/JOE-11-0236
Effect of l-arginine on the phosphorylation state and protein levels of AMPK and protein levels of HSP72 in the absence or presence of a proinflammatory cytokine cocktail
Since β-cell metabolism is dependent on regulation by the activity of key enzymes such as AMPK (Kiely et al. 2007, Newsholme et al. 2007b), protein levels of AMPK and its AMP-P were determined following 24 h incubation in various concentrations of l-arginine in the absence or presence of proinflammatory cytokines. l-Arginine evoked concentration-dependent activation of AMPK (as determined by phosphorylation status) that was further enhanced by the addition of the cytokine cocktail, at the higher concentrations of the amino acid (Fig. 3). HSP72, a protein involved in stress adaptation in all cell types (Krause & Rodrigues-Krause 2011), was also investigated. l-Arginine increased HSP72 levels in a dose-dependent manner but, with the exception of the 1.15 mM l-arginine condition, cytokine exposure did not significantly change HSP72 content (Fig. 3).
Effect of 24 h culture in the presence of various concentrations of l-arginine on the expression of AMPK and its AMPK-P and also the levels of HSP72 in BRIN-BD11 cells in the absence or presence of proinflammatory cytokines. Results are expressed as mean±s.d. of at least three separate preparations in duplicate using GAPDH as an expression control. Parts of the same representative gel are mounted and shown for each immunoblot. Data are means±s.d. Significance: *P<0.05 versus cytokine treatment at the same l-arginine concentration; ΔP<0.05 vs 0 mmol/l l-arginine; ‡P<0.05 vs 0.1 mmol/l l-arginine; †P<0.05 vs 0.25 mmol/l l-arginine; and ψP<0.05 vs 1.15 mmol/l l-arginine.
Citation: Journal of Endocrinology 211, 1; 10.1530/JOE-11-0236
Effect of l-arginine on LDH release and chronic (24 h) insulin secretion from mouse islets in the absence or presence of a proinflammatory cytokine cocktail
To confirm validity of observations using BRIN-BD11 cells reported above, experiments were made using mouse islets as primary cell model. The effect of 24 h culture in the presence of either 0 or 1.15 mmol/l l-arginine on cell viability and chronic insulin secretion of mouse islets after 24 h incubation in the absence or presence of proinflammatory cytokines were investigated. Islets incubated in media lacking l-arginine were associated with a significant increase in LDH release compared with islets incubated in the presence of 1.15 mM l-arginine (Fig. 4A). This finding, together with the results on islet function (chronic insulin secretion) confirmed that l-arginine is essential for viability of primary islet cells as well as clonal β-cells. Chronic (24 h) insulin release from cultured islets was also significantly attenuated (by ∼70%) in the absence of l-arginine, an effect that was also evident in the presence of inhibitory cytokine cocktail irrespective of the absence or presence of l-arginine (Fig. 4B).
Effect of 24 h culture in the presence of either 0 or 1.15 mM l-arginine on membrane integrity as assessed by LDH release (A) and chronic (24 h) insulin secretion (B) of mouse islets in the absence or presence of proinflammatory cytokines. Cellular LDH release results are presented as percentage of cellular LDH released over 24 h of incubation according to the manufacturer's instructions (Biovision, Dublin, Ireland) following incubation in the presence of either 0 or 1.15 mmol/l. Insulin secretion results are expressed as nanograms insulin released/islet for insulin secretion. Results are expressed as mean±s.d. of three separate preparations. Significance: *P<0.05 for control versus cytokine treatment at the same l-arginine concentration and ΔP<0.05 vs 1.15 mmol/l l-arginine.
Citation: Journal of Endocrinology 211, 1; 10.1530/JOE-11-0236
Discussion
l-Arginine is synthesized from glutamine, glutamate and proline via the intestinal-renal axis in humans and most other mammals (Wu et al. 2009). It is essential for metabolism and function of multiple body organs, with decreased plasma and cellular levels reported in type 2 diabetic subjects (Pieper & Dondlinger 1997). Since the amino acid is the precursor of NO that serves as a key cell signaling molecule, restriction in the availability of l-arginine is likely to contribute to derangements in the secretion and action of insulin (Newsholme et al. 2010).
While excessive NO production can trigger oxidative/nitrosative stress and is undoubtedly a key contributor to β-cell death (Palmer et al. 1976, Newsholme et al. 2005, 2007a,b, 2010, Michalska et al. 2010), evidence now suggests that lesser amounts of NO, produced by the NFκB-regulated iNOS, serve as an important coupling factor in insulin-secreting cells (Palmer et al. 1976, Newsholme et al. 2005, 2007a,b, 2010, Michalska et al. 2010). We have demonstrated herein that l-arginine is an important stimulator of β-cell glucose consumption and intermediary metabolism. Such actions lead to increased insulin secretion, enhanced antioxidant and protective responses with greater functional integrity when challenged with proinflammatory cytokines. Given that insulin-secreting cells have very low expression levels of antioxidant enzymes such as catalase and GSH peroxidase (Lenzen et al. 1996, Tiedge et al. 1997), β-cells are particularly prone to chemical stress in the diabetogenic or inflammatory environment typical of type 1 and possibly type 2 diabetes (Rocie et al. 1997, Santini et al. 1997, Kiely et al. 2007, Newsholme et al. 2007a, Michalska et al. 2010). The novel finding that l-arginine increased glucose consumption and lactate production in pancreatic β-cells, both in the presence or absence of cytokines, indicates that l-arginine is able to promote β-cell glycolytic flux in a concentration-dependent manner. Because glycolysis is essential to produce ATP (for stimulus–secretion coupling) and other energy-dependent process such as protein and GSH synthesis, this cellular response may be essential during inflammation of the pancreatic islet. The molecular mechanisms of the effect of l-arginine on glycolysis remains unknown but may be mediated via interaction at key regulatory points such as GLUT2, glucokinase, or phosphofructokinase 1. As T2DM patients are associated with lower levels of plasma l-arginine then the reduced availability of this amino acid could, at least in part, explain the reduced insulin secretory response during metabolic challenge in vivo (Salehi et al. 2010).
These positive actions of l-arginine on viability, antioxidant status and insulin secretion are likely to reflect, in large part, the importance of GSH and the GSH reductase/peroxidase systems as the main line of antioxidant defense in β-cells. To maintain GSH, β-cells may either regenerate it from GSSG via a GSSG reductase catalyzed reaction or synthesize it, de novo, through the concerted action of γ glutamylcysteine synthetase and GSH synthetase, which are ATP-consuming enzymes (see Fig. 5 for metabolic scheme). Regeneration of GSH from GSSG, which utilizes NADPH as a cofactor but does not require ATP, is metabolically less expensive than the de novo synthesis from the constituent amino acids. However, pentose phosphate shunt activity is relatively low in β-cells (Droge 2002), which is exacerbated by the high flux of glucose directed toward ATP production (Krause & de Bittencourt 2008). Therefore, β-cell NADPH can be obtained from the cytosolic malic enzyme, capable of converting malate to pyruvate with the concomitant production of NADPH from NADP+ (MacDonald 1995). De novo GSH synthesis, on the other hand, is completely dependent on the supply of glutamate, not only because this amino acid is a constituent of the GSH molecule, but is also an amino acid donor in the synthesis of serine, which subsequently can be converted to glycine, via a reaction requiring tetrahydrofolate. In fact, previous reports from our laboratories have highlighted the importance of glutamine and alanine (which give rise to glutamate) for GSH generation, insulin secretion and protection against proinflammatory cytokines (Brennan et al. 2002, 2003, Cunningham et al. 2005).
Scheme depicting major pathways relating to l-arginine metabolism in pancreatic β-cells. Entry of l-arginine into β-cells occurs via cationic transporters (CAT) in the plasma membrane and can be enhanced by cytokines. Once inside the cell l-arginine can be metabolized by iNOS to produce nitric oxide and citrulline and/or by arginase producing ornithine and urea. Ornithine can be converted subsequently in a sequence of reactions to pyrroline-5-carboxylate (P5C), glutamate semialdehyde and finally to glutamate. The glutamate produced is used for the synthesis of glutathione to enhance cell protection. Under normal secretagogue-induced insulin release, both NO and GSH are obligatory intermediates. Accordingly, β-cells have an intricate iNOS-centered machinery to produce NO, which potentiates insulin secretion physiologically. At the same time, β-cells utilize glutamate-derived GSH to maintain redox status needed to allow secretion and to avoid a possible NO-mediated cytotoxicity. l-Arginine is an established insulin secretagogue that depolarizes the cell membrane by virtue of its positive charge, leading to influx and elevation of intracellular Ca2+. On the other hand, l-arginine increases β-cell metabolism leading to increments in glucose consumption, NADPH and lactate. Lack of l-arginine will result in decreased metabolism and reduced glutathione production, leading to a fragile state against stress factors such as inflammation and free radicals. Low l-arginine availability would also result in decreased ATP production, blunting the insulin secretion. Enzymes/reactions: 1, iNOS (inducible nitric oxide synthase); 2, arginase; 3, ornithine aminotransferase; 4, pyrroline-5-carboxylate dehydrogenase; 5, aspartate-aminotransferase (a: mitochondrial and b: cytosolic); 6, α-ketoglutarate dehydrogenase; 7, succinate thiokinase; 8, succinate dehydrogenase; 9, fumarase; 10a, malate dehydrogenase; 10b, cytosolic malate dehydrogenase; 11, citrate synthase; 12, aconitase; 13, isocitrate dehydrogenase; 14, argininosuccinate synthase; 15, argininosuccinate lyase; 16, γ-glutamylcysteine synthetase; 17, glutathione synthetase; 18, glutathione peroxidase; 19, glutathione reductase; 20, malic enzyme; 21, lactate dehydrogenase; 22, glucokinase; 23, pentose phosphate pathway; 24, pyruvate carboxylase; 25, pyruvate dehydrogenase; 26, GLUT2; 27, glutaminase.
Citation: Journal of Endocrinology 211, 1; 10.1530/JOE-11-0236
The observation reported herein, that l-arginine is able to increase β-cell GSH synthesis, regardless of the presence or absence of proinflammatory cytokines, sheds light on an as yet unappreciated facet of β-cell metabolism, namely that l-arginine could be a precursor of GSH via glutamate generation (as l-arginine enhanced glutamate production and decreased glutamate release). This scenario is illustrated in a metabolic scheme depicted in Fig. 5. Increasing l-arginine availability allows for an increase in metabolic flux that produces glutamate from l-arginine by coupling production of l-ornithine to l-glutamate formation via pyrroline-5-carboxylate dehydrogenase and ornithine aminotransferase as described previously (Malaisse et al. 1989). This latter pathway may be important when l-glutamine or l-alanine availability is compromised. Inclusion of 0.25 mmol/l l-arginine in culture was sufficient to provide redox protection, as estimated by GSSG–GSH ratio. This suggests that l-arginine is an essential source of l-glutamate for GSH synthesis and maintenance of redox state during periods of inflammatory stress. Superoxide production evoked by proinflammatory cytokines was significantly reduced at higher l-arginine levels (Fig. 5).
The activation of the iHSP70 is sine qua non for the promotion of tissue repair, since the expression of this chaperone confers cytoprotection and also exerts anti-inflammatory effects (Krause & Rodrigues-Krause 2011). The increase in the levels of HSP72 induced by l-arginine in β-cells could be one of the key mechanisms mediating the observed protection against stress and inflammation. Thus, the higher level of HSP72 together with the increment in GSH synthesis induced by l-arginine may provide effective β-cell protection against inflammatory insult.
The stimulation of insulin release by l-arginine involves transport of the cationic amino acid into β-cells (via the amino acid transporter mCAT2A). This leads to direct membrane depolarization, activation of voltage-dependent Ca2+ channels, Ca2+ influx, elevation of intracellular Ca2+ and discharge of insulin by exocytosis (Newsholme et al. 2005). The finding that l-arginine significantly increased β-cell glucose consumption is a novel finding and suggests that l-arginine promotes diversion of glucose carbon from mitochondrial oxidation toward the formation of NADPH via the cytosolic malic enzyme, so requiring that glucose-derived-malate is transported from the mitochondrial matrix to the cytosol. Indeed, we believe that, in the presence of l-arginine, glutamate can be generated from both l-arginine and glucose (via 2-oxoglutarate formation and transamination of glutamate) and is subsequently utilized for GSH synthesis (Fig. 5). However, in an early study, oxidation of l-arginine was barely detectable in the presence or absence of glucose, with the authors concluding that the amino acid does not give rise to oxidative intermediates (Hellman et al. 1971). This is entirely in agreement with our findings, as l-arginine conversion to glutamate and GSH does not require oxidation of l-arginine carbon, thus does not directly result in stimulation of ATP synthesis. However, the l-arginine-dependent stimulation of glucose consumption may lead to elevated ATP levels. Thus, the long-accepted view that l-arginine exerts a stimulatory effect on insulin secretion in the presence of glucose, simply due to membrane depolarization induced by transport of a positively charged amino acid across the membrane must be revised. We propose that l-arginine exerts its positive effects via a combination of membrane depolarization, its metabolism to NO (and citrulline) or ornithine (and urea) and subsequent conversion to glutamate and GSH as well as an enhancement of glucose metabolism, so contributing to both stimulus–secretion coupling and antioxidant status. The fact that proinflammatory cytokines can impair insulin secretion even in the presence of high concentrations of l-arginine may indicate that non-lethal concentrations of proinflammatory cytokines shift β-cell metabolism away from energy generation and stimulus–secretion coupling and toward a catabolic state which may be related to cell defense (Kiely et al. 2007), as indicated by the GSH metabolism changes reported here.
The significance of AMPK in β-cell stimulus–secretion coupling is as yet unclear (Newsholme et al. 2007b), but l-arginine enhanced the level of AMPK-P in BRIN-BD11 cells (Fig. 3), thus favoring fatty acid oxidation. Interestingly, l-arginine supplementation in Zucker diabetic fatty rats, resulted in powerful activation of HO-3, AMPK and PGC-1α (PPARγ coactivator 1α), which would be expected to increase mitochondrial biogenesis and increase oxidative metabolism in skeletal and cardiac muscle, brain, liver and adipose tissue (Fu et al. 2005). It has been suggested that changes in AMPK activity may also contribute to β-cell functional integrity (Wang et al. 2007, Nyblom et al. 2008). Thus, activation of AMPK by l-arginine may not only promote fatty acid oxidative metabolism thereby driving ATP production, but it may also serve to counter β-cell glucolipotoxicity.
In summary, l-arginine exerts a broad spectrum of beneficial effects on clonal β-cells and isolated islets in addition to simple membrane depolarization and triggering of insulin secretion. The novel findings reported in this paper suggest an important role of l-arginine in promotion of GSH synthesis and antioxidant defense that may encourage the development of novel strategies for the protection of β-cells against chemical/immune insult and diabetes.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Technological Sector Research (TSR): strand III – Core Research Strengths Enhancement Scheme (Ireland), the Coordination for the Improvement of Higher Level- or Education- Personnel (CAPES, Brazil), and the National Council for Scientific and Technological Development (CNPq, Brazil).
Author contribution statement
M S K completed all experiments described in this manuscript. M S K and P N co wrote the manuscript. N H M, P R F, P I H and C M provided experimental advice and helped with manuscript revision. N M was responsible for the presentation style of all figures. C M and P N were responsible for grant support with respect to TSR: strand III – Core Research Strengths Enhancement Scheme (Ireland).
Acknowledgements
We thank UCD School of Biomolecular and Biomedical Science, the Department of Science, Institute of Technology Tallaght (Dublin, Ireland), TSR: strand III – Core Research Strengths Enhancement Scheme (Ireland) and the National Council for Scientific and Technological Development (CNPq), Brazil, for their support of this work.
References
Benov L, Sztejnberg L & Fridovich I 1998 Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical. Free Radical Biology and Medicine 25 826–831. doi:10.1016/S0891-5849(98)00163-4.
Brennan L, Shine A, Hewage C, Malthouse JP, Brindle KM, McClenaghan N, Flatt PR & Newsholme P 2002 A nuclear magnetic resonance-based demonstration of substantial oxidative l-alanine metabolism and l-alanine-enhanced glucose metabolism in a clonal pancreatic beta-cell line: metabolism of l-alanine is important to the regulation of insulin secretion. Diabetes 51 1714–1721. doi:10.2337/diabetes.51.6.1714.
Brennan L, Corless M, Hewage C, Malthouse JP, McClenaghan NH, Flatt PR & Newsholme P 2003 13C NMR analysis reveals a link between l-glutamine metabolism, d-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line. Diabetologia 46 1512–1521. doi:10.1007/s00125-003-1184-7.
Bronte V & Zanovello P 2005 Regulation of immune responses by l-arginine metabolism. Nature Reviews. Immunology 5 641–654. doi:10.1038/nri1668.
Cunningham GA, McClenaghan NH, Flatt PR & Newsholme P 2005 l-Alanine induces changes in metabolic and signal transduction gene expression in a clonal rat pancreatic beta-cell line and protects from pro-inflammatory cytokine-induced apoptosis. Clinical Science 109 447–455. doi:10.1042/CS20050149.
Droge W 2002 Free radicals in the physiological control of cell function. Physiological Reviews 82 47–95. doi:10.1152/physrev.00018.2001.
Eizirik DL & Mandrup-Poulsen T 2001 A choice of death-the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia 44 2115–2133. doi:10.1007/s001250100021.
Fu WJ, Haynes TE, Kohli R, Hu J, Shi W, Spencer TE, Carroll RJ, Meininger CJ & Wu G 2005 Dietary l-arginine supplementation reduces fat mass in Zucker diabetic fatty rats. Journal of Nutrition 135 714–721..
Hellman B, Sehlin J & Taljedal IB 1971 Effects of glucose and other modifiers of insulin release on the oxidative metabolism of amino acids in micro-dissected pancreatic islets. Biochemical Journal 123 513–521..
Kiely A, McClenaghan NH, Flatt PR & Newsholme P 2007 Pro-inflammatory cytokines increase glucose, alanine and triacylglycerol utilization but inhibit insulin secretion in a clonal pancreatic beta-cell line. Journal of Endocrinology 195 113–123. doi:10.1677/JOE-07-0306.
Krause MS & de Bittencourt PI Jr 2008 Type 1 diabetes: can exercise impair the autoimmune event? The l-arginine/glutamine coupling hypothesis. Cell Biochemistry and Function 26 406–433. doi:10.1002/cbf.1470.
Krause M & Rodrigues-Krause JD 2011 Extracellular heat shock proteins (eHSP70) in exercise: Possible targets outside the immune system and their role for neurodegenerative disorders treatment. Medical Hypotheses 76 286–290. doi:10.1016/j.mehy.2010.10.025.
Krause MS, Oliveira LP Jr, Silveira EM, Vianna DR, Rossato JS, Almeida BS, Rodrigues MF, Fernandes AJ, Costa JA & Curi R et al. 2007 MRP1/GS-X pump ATPase expression: is this the explanation for the cytoprotection of the heart against oxidative stress-induced redox imbalance in comparison to skeletal muscle cells? Cell Biochemistry and Function 25 23–32. doi:10.1002/cbf.1343.
Lenzen S, Drinkgern J & Tiedge M 1996 Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radical Biology and Medicine 20 463–466. doi:10.1016/0891-5849(96)02051-5.
MacDonald MJ 1995 Feasibility of a mitochondrial pyruvate malate shuttle in pancreatic islets. Further implication of cytosolic NADPH in insulin secretion. Journal of Biological Chemistry 270 20051–20058. doi:10.1074/jbc.270.34.20051.
Malaisse WJ, Blachier F, Mourtada A, Camara J, Albor A, Valverde I & Sener A 1989 Stimulus-secretion coupling of arginine-induced insulin release. Metabolism of l-arginine and l-ornithine in pancreatic islets. Biochimica et Biophysica Acta 1013 133–143. doi:10.1016/0167-4889(89)90041-4.
Masters SL, Dunne A, Subramanian SL, Hull RL, Tannahill GM, Sharp FA, Becker C, Franchi L, Yoshihara E & Chen Z et al. 2010 Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nature Immunology 11 897–904. doi:10.1038/ni.1935.
McClenaghan NH & Flatt PR 1999 Engineering cultured insulin-secreting pancreatic B-cell lines. Journal of Molecular Medicine 77 235–243. doi:10.1007/s001090050344.
McClenaghan NH, Barnett CR, Ah-Sing E, Abdel-Wahab YH, O'Harte FP, Yoon TW, Swanston-Flatt SK & Flatt PR 1996 Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 45 1132–1140. doi:10.2337/diabetes.45.8.1132.
McKnight JR, Satterfield MC, Jobgen WS, Smith SB, Spencer TE, Meininger CJ, McNeal CJ & Wu G 2010 Beneficial effects of l-arginine on reducing obesity: potential mechanisms and important implications for human health. Amino Acids 39 349–357. doi:10.1007/s00726-010-0598-z.
Michalska MWG, Walther R & Newsholme P 2010 Effects of pharmacologic inhibition of NADPH oxidase and iNOS on pro-inflammatory cytokine, palmitic acid or H2O2-induced mouse islet or clonal pancreatic beta cell dysfunction. Bioscience Reports 30 445–453. doi:10.1042/BSR20090138.
Murphy C & Newsholme P 1998 Importance of glutamine metabolism in murine macrophages and human monocytes to l-arginine biosynthesis and rates of nitrite or urea production. Clinical Science 95 397–407. doi:10.1042/CS19980194.
Newsholme P, Brennan L, Rubi B & Maechler P 2005 New insights into amino acid metabolism, beta-cell function and diabetes. Clinical Science 108 185–194. doi:10.1042/CS20040290.
Newsholme P, Haber EP, Hirabara SM, Rebelato EL, Procopio J, Morgan D, Oliveira-Emilio HC, Carpinelli AR & Curi R 2007a Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity. Journal of Physiology 583 9–24. doi:10.1113/jphysiol.2007.135871.
Newsholme P, Keane D, Welters HJ & Morgan NG 2007b Life and death decisions of the pancreatic beta-cell: the role of fatty acids. Clinical Science 112 27–42. doi:10.1042/CS20060115.
Newsholme P, Homem De Bittencourt PI, Ó Hagan C, De Vito G, Murphy C & Krause MS 2010 Exercise and possible molecular mechanisms of protection from vascular disease and diabetes: the central role of ROS and nitric oxide. Clinical Science 118 341–349. doi:10.1042/CS20090433.
Nyblom HK, Sargsyan E & Bergsten P 2008 AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic beta-cells without changing triglyceride levels. Journal of Molecular Endocrinology 41 187–194. doi:10.1677/JME-08-0006.
Palmer JP, Benson JW, Walter RM & Ensinck JW 1976 Arginine-stimulated acute phase of insulin and glucagon secretion in diabetic subjects. Journal of Clinical Investigation 58 565–570. doi:10.1172/JCI108502.
Pieper GM & Dondlinger LA 1997 Plasma and vascular tissue arginine are decreased in diabetes: acute arginine supplementation restores endothelium-dependent relaxation by augmenting cGMP production. Journal of Pharmacology and Experimental Therapeutics 283 684–691..
Repetto G, del Peso A & Zurita JL 2008 Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols 3 1125–1131. doi:10.1038/nprot.2008.75.
Rocie B, Vucić M, Knezević-Cuća J, Radica A, Pavlic-Renar I, Profozić V & Metelko Z 1997 Total plasma antioxidants in first degree relatives of patients with insulin-dependent diabetes. Experimental and Clinical Endocrinology and Diabetes 105 213–217. doi:10.1055/s-0029-1211754.
Salehi M, Aulinger B, Prigeon RL & D'Alessio DA 2010 Effect of endogenous GLP-1 on insulin secretion in type 2 diabetes. Diabetes 59 1330–1337. doi:10.2337/db09-1253.
Santini SA, Marra G, Giardina B, Cotroneo P, Mordente A, Martorana GE, Manto A & Ghirlanda G 1997 Defective plasma antioxidant defenses and enhaced susceptibility to lipid peroxidation in uncomplicated IDDM. Diabetes 46 1853–1858. doi:10.2337/diabetes.46.11.1853.
Smukler SR, Tang L, Wheeler MB & Salapatek AM 2002 Exogenous nitric oxide and endogenous glucose-stimulated beta-cell nitric oxide augment insulin release. Diabetes 51 3450–3460. doi:10.2337/diabetes.51.12.3450.
Spinas GA 1999 The dual role of nitric oxide in islet beta-cells. News in Physiological Sciences 14 49–54..
Stickings P, Mistry SK, Boucher JL, Morris SM & Cunningham JM 2002 Arginase expression and modulation of IL-1beta-induced nitric oxide generation in rat and human islets of Langerhans. Nitric Oxide 7 289–296. doi:10.1016/S1089-8603(02)00122-2.
Tiedge M, Lortz S, Drinkgern J & Lenzen S 1997 Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46 1733–1742. doi:10.2337/diabetes.46.11.1733.
Vasilijevic A, Buzadzic B, Korac A, Petrovic V, Jankovic A & Korac B 2007 Beneficial effects of l-arginine nitric oxide-producing pathway in rats treated with alloxan. Journal of Physiology 584 921–933. doi:10.1113/jphysiol.2007.140277.
Wang X, Zhou L, Shao L, Qian L, Fu X, Li G, Luo T, Gu Y, Li F & Li J et al. 2007 Troglitazone acutely activates AMP-activated protein kinase and inhibits insulin secretion from beta cells. Life Sciences 81 160–165. doi:10.1016/j.lfs.2007.04.034.
Wu G & Meininger CJ 2009 Nitric oxide and vascular insulin resistance. BioFactors 35 21–27. doi:10.1002/biof.3.
Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE & Yin Y 2009 Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37 153–168. doi:10.1007/s00726-008-0210-y.
Zhai Z, Solco A, Wu L, Wurtele ES, Kohut ML, Murphy PA & Cunnick JE 2009 Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells, indicative of alternative macrophage activation. Journal of Ethnopharmacology 122 76–85. doi:10.1016/j.jep.2008.11.028.
