Increased neuronal nitric oxide synthase dimerisation is involved in rat and human pancreatic beta cell hyperactivity in obesity
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Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans.
Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy.
Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules.
Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.
KeywordsBeta cell Insulin secretion NO synthase Obesity Type 2 diabetes
N ω-nitro-D-arginine methyl ester
N ω-nitro-l-arginine methyl ester
Neuronal nitric oxide synthase
Protein inhibitor of neuronal NOS
Voltage-dependent anion channel
Type 2 diabetes is a heterogeneous disease due to a combination of genetic and environmental factors that contribute to insulin sensitivity and pancreatic beta cell function. Obesity and associated insulin resistance are major risk factors for the development of type 2 diabetes. During the long-lasting silent phase that occurs before the appearance of the clinical signs of diabetes, pancreatic beta cells compensate for insulin resistance by increased insulin secretion.
The physiological mechanisms underlying pancreatic beta cell compensation have been widely studied using the Zucker fa/fa rat , a relevant model that displays hyperphagia, obesity, insulin resistance and hyperinsulinaemia, resulting from a mutation in the leptin receptor . The fa/fa rats compensate for insulin resistance partially through an increase in beta cell mass . In addition, a primary defect in the beta cells also occurs, as neither in vivo starvation nor in vitro culture at low glucose concentration is able to decrease glucose-stimulated insulin secretion . Furthermore, an innate hyperactivity of pancreatic beta cell response to feeding has been proposed to occur at least in some obese humans .
Early studies have shown that islets from fa/fa rats exhibit an increased sensitivity to glucose . An increase in glucose utilisation and oxidation, resulting from increased metabolic fluxes through pyruvate carboxylase and the malate–pyruvate and citrate–pyruvate shuttles, has been shown in islets from fa/fa rats . More recently, insulin hypersecretion in these animals has been proposed to result from an enhanced glucose-responsive fatty acid esterification/lipolysis process in beta cells . In contrast, there have not been any studies in humans, until now, concerning the mechanism involved in beta cell hyperactivity.
To further investigate the mechanisms involved in beta cell hyperactivity in Zucker fa/fa rats and obese individuals, we explored the function of neuronal nitric oxide (NO) synthase (nNOS), an enzyme responsible for the production of NO . We have recently demonstrated that a neuronal isoform of NOS, produced in rat pancreatic beta cells, is able to control insulin secretion via both inhibitory and stimulatory effects . Interestingly, we found nNOS mainly associated with the insulin secretory granules, but also with mitochondria and nuclei in beta cells. We also showed that the monomer/dimer equilibrium of the enzyme is able to influence the kinetics of glucose-induced insulin secretion and NO sensitivity .
Changes in nNOS activity/function could be involved in the pathogenesis of type 2 diabetes, as abnormally increased NO production has been shown to be involved in beta cell dysfunction in Goto-Kakizaki rats . Using the Zucker fa/fa rat model and human pancreatic islets from obese individuals, we investigated whether nNOS changes could, at least partly, account for pancreatic beta cell hyper-responsiveness in obesity and/or insulin-resistant states.
Male Zucker fa/fa rats and their control littermates, fa/+ rats, were purchased from Harlan (Indianapolis, IN, USA). Tail blood samplings were taken before they were killed at 7 to 9 weeks of age. All experiments were conducted in accordance with the Principles of Laboratory Care.
Human islets were isolated at the University of Geneva Cell Isolation and Transplantation Centre from human pancreases harvested from brain-dead, heart-beating, multi-organ donors. The islets originated from two populations: lean individuals (BMI < 25 kg/m2) and obese donors (BMI > 30 kg/m2). We obtained islets from three lean individuals (two men, one woman, mean age 59 ± 5.6 years, mean BMI 24 ± 0.55 kg/m2) and from two obese women (mean age 54.5 ± 8.5 years; mean BMI 30.7 ± 0.3 kg/m2).
Isolation of rat and human islets
Rat islets were isolated using digestion with collagenase P (Roche, Basel, Switzerland). Islets were handpicked under a stereomicroscope after separation with a Ficoll density gradient.
Human islets were isolated in Geneva as previously described by Bucher et al.  following a protocol approved by the Institutional Ethical Committee of Geneva University Hospital. They were cultured at 37°C in CMRL medium containing 5.6 mmol/l glucose and 10% fetal calf serum for 3–4 days before being sent to Montpellier for the next stage of the experiment.
Total RNA from human islets was extracted with RNA Now Reagent (Biogentex, League City, TX, USA). First-strand cDNA was obtained from 5 μg total RNA using Superscript II RNAse H− Reverse Transcriptase (Invitrogen, Paisley, UK). Quantitative real-time RT-PCR was performed with FastStart DNA Master SYBR Green I mix and a LightCycler (Roche).
Human and freshly isolated rat islets were homogenised in 20 mmol/l Tris lysis buffer pH 7.4, containing 150 mmol/l NaCl, 1% Triton X-100 (vol./vol.), 0.1% SDS (vol./vol.) and a cocktail of protease inhibitors (Roche).
Proteins (50 μg) were separated on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Incubation with anti-nNOS (BD Biosciences, Franklin Lakes, NJ, USA), anti-phospho-Ser847-nNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-α-tubulin (as an internal control; Sigma, St Louis, MO, USA) antibodies was performed overnight and immunoreactivity was revealed by chemiluminescence. Signals were acquired and quantified by Bio 1D image analysis software (Vilber Lourmat, Marne-la-Vallée, France). Each experiment shown is representative of three independent western blots.
Low-temperature 6% SDS-PAGE at 10°C without boiling proteins (50 μg) was used to study nNOS dimerisation . In the case of INS-1 cell extracts, the cells were first incubated in the presence of 5 mmol/l glucose ± 5 mmol/l N ω-nitro-l-arginine methyl ester (L-NAME) and 10 μmol/l miconazole for 1 h. After incubation, the cells were homogenised in lysis buffer as described above.
Cells isolated from pancreatic islets were obtained after dissociation with 0.025% trypsin/0.27 mmol/l EDTA (Sigma) and seeded on a poly-l-lysine (Sigma) coated Lab-Tek System. After 3 days of culture, they were immunostained overnight with anti-nNOS (Euro-Diagnostica, Malmö, Sweden) and anti-insulin (Sigma) antibodies. Mitochondria were labelled with the mitochondrion-selective dye MitoTracker DeepRed 633 (Invitrogen). The negative control was performed by incubating the cells with only the secondary antibodies. Fluorescence was observed with the Bio-Rad MRC 1024 confocal microscope (Montpellier Rio Imaging, Montpellier, France). We used the ImageJ software (JACoP plugins) to calculate the Pearson coefficient with a Costes’ automatic threshold.
Freshly isolated islets were fixed using 2.5% paraformaldehyde (wt/vol.) and 0.1% glutaraldehyde (vol./vol.) and routinely embedded in LR White. Ultrathin sections of 60 nm were immunostained with anti-nNOS (Euro-Diagnostica) and anti-insulin (Sigma) antibodies and with secondary antibodies labelled with 15 or 5 nm gold particles, respectively (British Biocell, Cardiff, UK). Sections were observed with a transmission electron microscope (Hitachi H-7100). The specificity of the immune reaction was tested by incubating the sections with only the secondary antibodies.
Isolated rat islets pooled from three different animals were blended with a Dounce homogeniser. Nuclei and unbroken cells were removed by centrifugation at 1,000 g for 10 min. Mitochondria were then isolated from the cytosolic fraction by further centrifugation for 15 min at 16,000 g. The pellet of mitochondria was resuspended in a mixture of 10 mmol/l Tris–HCl pH 7.5, 250 mmol/l sucrose and 1 mmol/l EDTA–0.1% ethanol (vol./vol.) and the suspension was subsequently centrifuged for 15 min at 16,000 g at 4°C. The purity of cytosolic and mitochondrial fractions was assessed by, respectively, anti-α-tubulin (Sigma) and anti-voltage-dependent anion channel (VDAC; Abcam, Cambridge, UK) antibodies.
Isolated islets incubation experiments
Human isolated islets were stabilised in KRB with 2.8 mmol/l glucose for 2 h at 37°C. Groups of ten islets were then incubated in KRB with 2.8, 8.3 or 16.7 mmol/l glucose for 90 min at 37°C ± 10 mmol/l l-NAME, 10 mmol/l N ω-nitro-d-arginine methyl ester (d-NAME) or 5 μmol/l miconazole. Each experimental condition was repeated ten times. Supernatant fractions were sampled and insulin release estimated by an HTRF Insulin assay (Cis Bio, Saclay, France).
Isolated perfused rat pancreas studies
Lean (∼200 g) and obese (∼300 g) age-matched Zucker rats were anaesthetised with sodium pentobarbitone (60 mg/kg i.p.). Isolated pancreases were perfused according to procedures described by Lajoix et al. . Insulin secretion values, obtained by RIA (Linco, St Charles, MO, USA), are plotted on the figures as means ± SEM but also as mean integrated data obtained by calculating the AUC during 20 min of high glucose (11 mmol/l) administration.
nNOS catalytic activity assay
nNOS catalytic activity was estimated in islet extracts by measuring the production of radiolabelled [3H]citrulline from [3H]arginine (MP Biomedicals, Irvine, CA, USA), according to the manufacturer’s recommendations (Nitric Oxide Synthase Assay Kit; Merck Biosciences, Whitehouse Station, NJ, USA).
Asymmetric dimethyl-arginine immunoassay
Asymmetric dimethyl-arginine (ADMA) concentrations were determined in proteic extracts from rat and human islets using the ADMA ELISA kit (Immundiagnostik, Bensheim, Germany) according to the manufacturer’s recommendations.
Experimental values represent the mean of n experiments and are plotted on the graphs as means ± SEM. The data were analysed using a Student’s t test.
Expression of the neuronal isoform of NOS in human islets
Evidence for beta cell hyperactivity in Zucker fa/fa rat isolated pancreas and in human islets from obese humans
Effects of nNOS blockade with L-NAME on glucose-induced insulin secretion
Alterations of nNOS catalytic activity
Increased nNOS expression in pancreatic islets
Quantitative RT-PCR (Fig. 4c) and western blotting (Fig. 4e) analysis revealed respectively a 3.6- and a sevenfold overexpression of nNos in fa/fa rats (p < 0.05; n = 3), which contrasts with the unchanged nNOS activity in these animals. However, when nNOS activity is expressed relative to nNOS protein level, the ratio achieved was six- to sevenfold lower in fa/fa vs fa/+ rats, pointing to a strong decrease in the enzyme catalytic activity. In human islets from obese individuals, nNOS mRNA (Fig. 4d) and protein (Fig. 4f) was also found to be overexpressed by, respectively, 78% and threefold compared with lean individuals (p < 0.05; n = 2 or 3). nNOS activity expressed relative to nNOS protein content was also decreased by threefold in human islets from obese individuals.
Increased nNOS dimerisation and phosphorylation in pancreatic islets
Effects of downregulation of nNOS dimerisation by miconazole on glucose-induced insulin secretion
Alterations in nNOS localisation in pancreatic beta cells
In our early studies , we showed that inhibition of nNOS by L-NAME, a potent competitive inhibitor of constitutive NOS activity, was able to convert the biphasic pattern of insulin response to glucose into a significantly greater monophasic one . Interestingly, the L-NAME effect was associated with changes in the dimer/monomer ratio, with the appearance of a higher amount of stable nNOS dimers . These observations prompted us to propose that changes in nNOS function and dimerisation could be involved in certain hyperinsulinaemic states associated with insulin resistance and obesity.
In Zucker fa/fa rats, hyperinsulinaemia that compensates for insulin resistance clearly results from an increased sensitivity of the beta cell to glucose [4, 6, 7, 8]. In addition, beta cell hyperactivity in fa/fa rats is associated with a 3.8% increased mass. Indeed, previous studies in obese rats have reported the presence of enlarged islets with an increase in beta cell mass, as well as beta cell hypertrophy [6, 18]. Beta cell hypertrophy is accompanied by an increased amount of secretory cells at 5.5–11 mmol/l glucose, increased secretion per cell at low glucose concentrations, and decreased insulin content after high glucose exposure [7, 19]. Hence, insulin hypersecretion appears mainly due to an increased recruitment and an exaggerated secretory response of fa/fa beta cells.
In our study, we bring evidence for insulin hypersecretion in islets from obese individuals, together with an increased sensitivity to glucose, as previously described in vivo . Unlike in fa/fa rats, no changes in islet size could be observed for obese humans, but we cannot exclude an increase in beta cell mass. In a recent review, obesity was suggested to be associated with a modest expansion of beta cell mass, reaching 10–30% per 10 kg weight gain . Interestingly, insulin hypersecretion that generally occurs as a compensatory mechanism for the reduced insulin sensitivity associated with obesity  has also been found in non-diabetic obese individuals in the absence of insulin resistance . As information concerning the insulin sensitivity states of our obese donors was lacking, we cannot speculate on the nature of the state, obesity or insulin resistance, that primarily triggers insulin hypersecretion. However, we can conclude that, in addition to beta cell expansion , obesity is also an actual condition of beta cell hyperactivity.
The failure of L-NAME to affect glucose-induced insulin secretion in islets issued from fa/fa rats and obese individuals provides pharmacological evidence for a defect in pancreatic beta cell nNOS functional activity. A decrease in nNOS oxidative catalytic activity has previously been reported in the stomach fundus and the hypothalamus of fa/fa rats , whereas no data are available concerning possible changes in nNOS in obese humans. In our study, we found no change in nNOS catalytic activity in islet protein extracts. However, owing to a drastic increase in nNOS protein level, the overall enzyme activity appeared drastically reduced. nNOS overexpression could be due to an enhanced activity of transcription factors, as nuclear factor kappa-B (NFκB) and cAMP response element (CRE) are reportedly involved in nNOS induction [24, 25, 26]. In addition, the dimeric conformation of nNOS has been shown to protect the protein from proteolysis by the ubiquitin–proteasome pathway . However, despite a sevenfold increase in nNOS protein level, we found only a 2.7-fold increase in phospho-Ser847 in fa/fa rats, which plays probably only a minor role in the relative reduction of nNOS activity.
Of main interest is the strong increase in the amount of SDS-resistant nNOS dimers we found in fa/fa rats and obese individuals. The ability of L-NAME to induce stable nNOS dimers is shared by N ω-methyl-l-arginine (l-MMA) and ADMA, two endogenous inhibitors of nNOS  derived from in vivo proteolysis of methylated arginine residues on various proteins. In this respect, a positive correlation has been described between plasma concentrations of ADMA and insulin resistance , as well as obesity . Importantly, we observed increased ADMA levels within islets from fa/fa rats and obese individuals. Indeed, methylarginines are transported and concentrated into the cell in competition with arginine by using the cationic amino acid transporters . Our results suggest that ADMA participates in the marked increment in nNOS catalytic inactive dimers we found, which together with our functional data obtained with miconazole and L-NAME, argue that the effect of nNOS on insulin secretion results from a mechanism independent of its classic enzymatic function.
Concerning nNOS subcellular localisation, we observed a greater association of nNOS with insulin secretory granules in beta cells from fa/fa rats and obese humans. Indeed, we have previously shown by electron microscopy that nNOS was present at the level of insulin granules in rat beta cells , where the enzyme interacts with its endogenous protein inhibitor, protein inhibitor of neuronal NOS (PIN) . PIN is a highly conserved protein that blocks the enzyme dimerisation and subsequent NO production . However, when nNOS is stabilised in the dimeric conformation by l-NAME, l-MMA or ADMA, PIN is unable to prevent nNOS dimerisation , which could explain the high number of dimers we observed in islets from fa/fa rats and obese humans.
It must be emphasised that PIN is also the light chain of cytoplasmic and flagellar dyneins and myosin V, two cytoskeletal components involved in flagellar movements and the traffic of intracellular organelles . In pancreatic beta cell, myosin Va has been involved in the transport of dense-core secretory vesicles  and granule recruitment during late-phase secretion . As we have recently characterised a ternary complex between nNOS, PIN and myosin V in INS-1 cells (data not shown), we hypothesise that a greater association of nNOS dimers with PIN and myosin Va could be involved in insulin hypersecretion in fa/fa rats and obese individuals through enhanced intracellular granule trafficking. Such a possibility is strongly supported by the increased association of nNOS with insulin granules we found in fa/fa rats and obese humans that could favour the simultaneous association of the inactive nNOS dimers with PIN/myosin V and insulin granules. This is consistent with the observation that downregulation of nNOS dimers by miconazole brought insulin secretion in fa/fa rats and obese humans back to levels close to those observed in, respectively, fa/+ rats and lean individuals.
In addition to insulin secretory granules, nNOS is also located in beta cell mitochondria , which are likely to be affected by decreased NO production and to participate in beta cell hyperactivity. We observed modifications in mitochondrial morphology in fa/fa beta cells, as previously shown in islets from the Zucker diabetic fatty rat . In this respect, NO has been shown to act as a key messenger activating mitochondrial biogenesis in diverse cell types  and mitochondrial density has been found lowered in tissues of nNOS knockout animals . NO has also been reported to inhibit mitochondrial respiratory chain complexes I, II and III, and nanomolar concentrations of NO are able to reversibly inhibit cytochrome c oxidase . Therefore, it is tempting to suggest that the decrease in mitochondrial nNOS abundance that appears to occur from our immunofluorescence and subcellular fractionation studies could also be implicated in beta cell hyperactivity.
In conclusion, our data obtained in two models of obesity, the Zucker fa/fa rat and islets from obese humans, provide evidence for a number of nNOS changes likely to participate in hypersecretion of insulin. A major one is the increased formation of resistant nNOS dimers, which could be related to high intracellular amounts of ADMA. In addition, a greater association of nNOS dimers with PIN and myosin Va at the level of insulin secretory granules could significantly increase insulin granule migration and release, and account for, at least partly, insulin hypersecretion in fa/fa rats and obese humans. Our study could be of pathophysiological relevance as islets from obese organ donors are now used for transplantation in patients with uncontrolled type 1 diabetes .
Human islets were obtained thanks to grant no. 31-2008-416 from the Juvenile Diabetes Research Foundation. A. D. Lajoix was also supported by a grant from the Fondation pour la Recherche Médicale (FRM). We thank M. Manteghetti, S. Dietz, and C. Aknin for their technical contribution.
All authors contributed to the conception, design or analysis and interpretation of data, to drafting and revising the manuscript, and to approval of the final version of the manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.