Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release

Ragnar Henningsson1, Per Alm2, Erik Lindström1, and Ingmar Lundquist1

Institute of Physiological Sciences, 1 Departments of Pharmacology and 2 Pathology, University of Lund, Lund, Sweden


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.

chronic nitric oxide synthase blockade; nitric oxide and carbon monoxide; insulin and glucagon secretion; isolated islets; cyclic nucleotides


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is today considered as a ubiquitous messenger molecule in many different organ systems (11, 28). It is also produced in the islets of Langerhans (16, 41, 47, 50) and might serve as a physiological regulator of islet hormone secretion (2-5, 7, 16, 17, 21, 26, 27, 31, 39-41, 47-50, 52). NO is a short-lived free radical gas that results from oxidation of L-arginine into L-citrulline, a reaction that is catalyzed by nitric oxide synthase (NOS; see Ref. 28). There are two main forms of NOS: a Ca2+/calmodulin-dependent constitutive (cNOS) and a Ca2+/calmodulin-independent inducible (iNOS) form (28). Furthermore, there are at least two isoforms of the constitutive enzyme, i.e., endothelial cNOS and neuronal cNOS (ncNOS; see Refs. 11 and 28). The different forms of NOS are inhibited by L-arginine analogs (53). The simplest of these compounds, NG-nitro-L-arginine, is reportedly an inhibitor of NOS and is widely used as its methyl ester analog NG-nitro-L-arginine methyl ester (L-NAME; see Ref. 53). Also, nonarginine analogs such as nitrated indazoles, e.g., 7-nitroindazole, have recently been shown to be potent NOS inhibitors (10, 53).

We and others have shown that both cNOS and iNOS occur in the islets of Langerhans (5, 16, 19, 41, 39, 47, 49, 50). There are increasing amounts of data suggesting that NO derived from iNOS is implicated in the autoimmune destruction and dysfunction of beta -cells in insulin-dependent diabetes mellitus (16, 17, 19) and that different cytokines induce the expression of iNOS (16, 17, 19). However, the role of islet cNOS, which produces NO in a pulsatile way and in much lower absolute amounts than iNOS, is more controversial (2-3, 7, 17, 21, 26, 27, 31, 40, 41, 47-50, 52), especially since the earliest studies suggested that NO was either a stimulator (31, 50), an inhibitor (41), or had no effect (27) on insulin secretion. In a number of studies concerning short-term effects of blockade and stimulation of cNOS, we have shown that NO derived from cNOS inhibits insulin secretion induced by glucose, L-arginine, and cholinergic stimulation, whereas it increases glucagon secretion (2-4, 26, 39, 41, 44, 47-49). In the present study, the aim was focused on the possible effects of a long-term NOS blockade on insulin and glucagon secretion by giving mice the NOS blocker L-NAME in their drinking water for 4-6 wk. A few earlier studies did not reveal any effects of such a treatment on in vivo insulin secretion (43, 55), and the results thus suggested that NO did not have any appreciable effects on islet hormone secretion. However, the direct effects of chronic L-NAME treatment on islet NOS activity and NO production have never been investigated. Only general indirect indexes of NOS blockade (e.g., increase in blood pressure and aortic wall cGMP content) have been used (8, 43, 55). Interestingly, recent investigations on long-term L-NAME treatment did not reveal any inhibition of NO synthesis in the intestine or the liver because of a compensatory increase in iNOS activity (37). Moreover, because we very recently have discovered (24, 25) that the islets of Langerhans express the constitutive isoform of the CO-producing enzyme heme oxygenase (HO)-2 and because our data suggested (24) that the CO produced could suppress islet NOS activity and stimulate both insulin and glucagon release, we were interested to see whether long-term blockade of NOS would affect islet CO production and thus, in turn, the secretion of insulin and glucagon. The effects of chronic NOS blockade were compared both in freely fed and 24-h-fasted animals, since preliminary data in our laboratory suggested that the islet NO-producing system might be activated by fasting and thus could be an important factor behind the diabetic-like condition displayed in starvation (51). To get a more complete biochemical pattern of islet NOS activity in the present experimental situation, we have now adapted our original assay of total NOS activity in isolated islets (47) to include determination of both cNOS and iNOS activity. Complementary data were obtained by immunoblotting for both neuronal (n)cNOS and iNOS. Hence, the present investigation was conducted to study in detail the effects of a long-term NOS blockade on insulin and glucagon secretion in relation to expression and activity of mouse islet cNOS and iNOS.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals. Female mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 25-30 g were used in all studies. Freely fed animals were fed a standard pellet diet (B&K) and tap water ad libitum before and during all experiments. In the fasting experiments, food was withdrawn for 24 h before the experiment started. These mice had free access to water during this period. The animal experiments were approved by the local animal welfare committee.

Chronic NOS blockade. The mice were given 50 mg · kg-1 · day-1 L-NAME in the drinking water for 4-6 wk. This dose has been proved to cause NOS blockade in rats (43). Control mice were given drinking water only. The L-NAME-drinking mice displayed a normal weight gain, and their body weights at the time of islet isolation or the performance of in vivo experiments were similar to the controls.

Drugs and chemicals. Collagenase (CLS 4) was obtained from Worthington Biochemicals (Freehold, NJ). BSA was from ICN Biochemicals (High Wycombe, UK). 7-Nitroindazole was obtained from Calbiochem (La Jolla, CA). L-NAME, forskolin, and all other chemicals were from Sigma Chemical (St. Louis, MO). The RIA kits for insulin and glucagon determination were obtained from Diagnostika (Falkenberg, Sweden) and Euro-Diagnostica (Malmö, Sweden).

Immunocytochemistry. Pancreatic glands from freely fed L-NAME-drinking mice were dissected and divided in pieces and were processed further for immunocytochemistry of ncNOS and iNOS as previously described in detail (5, 24). In control experiments, iNOS immunoreactivity was only observed in animals injected with endotoxin (lipopolysaccharide) in, e.g., macrophages in lung and liver, in which no ncNOS immunoreactivity could be visualized. The immunocytochemical results were also tested by observers not knowing original data.

In vivo experiments. D-Glucose was dissolved in 0.9% NaCl (saline). Forskolin was dissolved in DMSO and then diluted in saline. Glucose (3.3 mmol/kg) and forskolin (7.3 µmol/kg) were injected intravenously (volume load: 5 µl/g mouse) into a tail vein. Controls received either saline (glucose) or vehicle (forskolin). Blood sampling was performed as described previously (44). The concentrations of insulin and glucagon in plasma were determined by RIA (1, 22, 42). Plasma glucose concentrations were determined enzymatically (14).

Hormone secretion in vitro. Preparation of isolated pancreatic islets from mouse was performed by retrograde injection of a collagenase solution via the bile-pancreatic duct (20). Islets were then isolated and hand picked under a stereomicroscope at room temperature. The islets were then preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer (29), pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% BSA, and 1 mmol/l glucose. Each incubation vial contained 10 islets in 1.0 ml buffer solution and was gassed with 95% O2-5% CO2 to obtain constant pH and oxygenation. After preincubation for 30 min, the buffer was changed to a medium containing 1, 7, or 20 mmol/l glucose and the different test agents, and the islets were then incubated for 60 min. All incubations were performed at 37°C in an incubation box (30 cycles/min). Immediately after incubation, aliquots of the medium were removed and frozen for subsequent assays of insulin and glucagon (1, 22, 42).

Assay of islet NOS. Freshly isolated islets were thoroughly washed and collected in ice-cold buffer (200 islets in 840 µl buffer) containing 20 mmol/l HEPES, 0.5 mmol/l EDTA, and 1 mmol/l DL-dithiothreitol (DTT), pH 7.2, and were immediately frozen at -20°C. For comparative purposes, small pieces of mouse cerebellum were also removed and frozen in the same DTT-containing buffer as described above. On the day of assay, the islets and the specimens of cerebellum were sonicated on ice, and the buffer solution containing the islet homogenate was reconstituted to contain, in addition to the above-mentioned compounds, also 0.2 mmol/l L-arginine, 0.45 mmol/l CaCl2, 2 mmol/l NADPH, and 25 units calmodulin in a total volume of 1 ml. To determine iNOS activity, both Ca2+ and calmodulin were omitted. The buffer composition was essentially the same as previously described for assay of NOS in brain tissue using radiolabeled L-arginine (13). The crude homogenate was then incubated at 37°C under constant air bubbling (1.0 ml/min) for 3 h. Aliquots of the incubated homogenate (200 µl) were then passed through a 1-ml Amprep CBA cation-exchange column for HPLC analysis of the L-citrulline formed, according to Carlberg (15). Because L-citrulline is created in equimolar concentrations to NO and because L-citrulline is stable whereas NO is not, L-citrulline is the preferred parameter when measuring NO production. The method has been described in detail earlier (15, 47). cNOS activity was calculated as total NO production minus Ca2+/calmodulin-independent NO production. Protein was determined by the method of Bradford (12) on samples from the original homogenate.

Western blot analysis. Approximately 200 islets were hand picked in Hanks' buffer under a stereomicroscope and were sonicated on ice (3 × 10 s). The protein content was determined according to Bradford (12). Homogenate samples representing 20 µg of total protein from islet tissue were then processed as previously described (24). Immunoblotting with rabbit anti-mouse ncNOS (N-7155), iNOS (N-7782; 1:2,000; Sigma), and HO-2 and HO-1 (1:2,000) antibodies (StressGen Biotechnol, Victoria, Canada) was performed for 16 h at room temperature. Antibody binding to ncNOS, iNOS, HO-2, and HO-1 was detected using 0.25 mmol/l CDP-Star (Boehringer Mannheim, Mannheim, Germany) and the signal enhancer Nitro Block II (Tropix, Bedford, MA) for 5 min at room temperature. The chemiluminescence signal was visualized by exposing the membranes to Dupont Cronex X-ray films for 1-5 min.

Measurement of islet cAMP and cGMP. The methodology has been described previously (40). Incubation of isolated islets in the presence of the phosphodiesterase inhibitor IBMX (0.2 mmol/l) was stopped by removal of the buffer and the addition of 0.5 ml of ice-cold 10% TCA followed by immediate freezing in a -70°C ethanol bath. Before assay, 0.5 ml of H2O was added, and the samples were sonicated for 3 × 5 s followed by centrifugation at 1,100 g for 15 min. The supernatants were collected and extracted with 4 × 2 ml of water-saturated diethyl ether. The aqueous phase was removed and freeze-dried using a Lyovac GT 2 freeze-drier. The residue was then dissolved in 450 µl of 50 mmol/l sodium acetate buffer (pH 6.2). The amounts of cAMP and cGMP were quantified with a 125I-labeled cAMP and 125I-labeled cGMP RIA kit (RIANEN; Du Pont, Boston, MA). [3H]cGMP was added to the TCA islet homogenate to determine the recovery of cAMP and cGMP during the ether extraction. The mean recovery was 85%.

Measurement of islet HO activity. CO production was determined with a sensitive gas chromatographic method essentially as previously described (24, 25). Islets were isolated and hand picked under a stereomicroscope at room temperature and then, unless otherwise stated, thoroughly washed and collected in ice-cold phosphate buffer (0.1 mol/l, pH 7.4, ~300 islets in 200 µl buffer). Thereafter, the islets were immediately frozen at -20°C. On the day of assay, the islets were sonicated on ice, and methemalbumin (30 µl), beta -NADPH [100 µl; 4 mg dissolved in 1 ml phosphate buffer (0.1 mol/l)], and Hb (2 mg) were added together with phosphate buffer up to a final volume of 1 ml. Methemalbumin solution was prepared by dissolving 25 mg hemin, 82.5 mg NaCl, and 12.1 mg Tris base in 5 ml of 0.1 mol/l NaOH followed by the addition of 5 ml albumin solution (20 g/l) and 5 ml distilled water. The homogenate was then incubated in a water bath at 37°C under protection from light. Aliquots (330 µl) were taken after 6 min of incubation, which was terminated by placing the tubes on ice. The samples were then injected in reaction tubes containing ferricyanide-citric acid (100 µl). Nitrogen was used as a carrier gas and to purge the reaction vessels for 4 min before the samples were injected in them. After a reaction time of 4 min, the liberated CO was brought to a nickel catalyst, mixed with H2, and then brought further as methane to the detector. CO (99.9%) was used as a standard. The amount of CO produced was calculated from the area under the curve. Protein was determined according to the method of Bradford (12) on samples from the original homogenate.

Statistics. Statistical significance between sets of data was assessed using unpaired Student's t-test or, where applicable, ANOVA followed by Tukey-Kramer's multiple comparisons test. Results are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Effect of chronic L-NAME treatment on total NO production (total NOS activity) from pancreatic islets and cerebellum. The total NO production was determined in the presence of Ca2+ and calmodulin, and the results obtained reflect the total NO production of both cNOS (Ca2+/calmodulin-dependent) activity and iNOS (Ca2+/calmodulin-independent) activity. The total production of NO in mouse islets from freely fed and 24-h-fasted mice and from cerebellum of freely fed mice is shown in Fig. 1A. In freely fed control mice, total islet NOS activity was approx 25 pmol NO · min-1 · mg protein-1, whereas islets from freely fed NAME-drinking mice showed a considerably (more than doubled) higher total NO production (52 pmol NO · min-1 · mg protein-1; Fig. 1A). Moreover, fasting the control mice for 24 h was found to markedly increase islet NO production (37 pmol NO · min-1 · mg protein-1) compared with islets of the freely fed controls. In islets from L-NAME-drinking fasted mice, the production of NO was not further increased compared with L-NAME-drinking freely fed mice (47 vs. 52 pmol NO · min-1 · mg protein-1). NO production was, however, still significantly greater in islets from fasted NAME-drinking mice (47 pmol NO · min-1 · mg protein-1) than in fasted control mice (37 pmol NO · min-1 · mg protein-1). In comparison, also in cerebellar specimens of freely fed mice, NO production was considerably increased after L-NAME drinking (Fig. 1A).


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Fig. 1.   A: Total nitric oxide synthase (NOS) activity in islets isolated from freely fed and 24-h-fasted control mice (open bars) or mice treated for 4-6 wk with NG-nitro-L-arginine methyl ester (L-NAME) in their drinking water (filled bars; freely fed and fasted) and cerebellum specimens taken from freely fed control mice (open bars) or L-NAME-treated mice (filled bars). Nitric oxide (NO) production was measured as L-citrulline formation (pmol · min-1 · mg protein-1). Values are means ± SE for 4-6 batches of islets or 4 specimens of cerebellum taken from 2-3 mice in each group. Asterisks indicate probability level of random difference for freely fed and fasted L-NAME drinking vs. freely fed and fasted control mice: *P < 0.05, **P < 0.01 and ***P < 0.001. Crosses indicate probability level of random difference for islets of freely fed control mice vs. islets of fasted control mice: xxx P < 0.001. B and C: islet total NOS activity seen in A separated into constitutive NOS (cNOS)- and inducible NOS (iNOS)-derived NO production in islets from freely fed control mice (open bars; B) or in islets from fasted control mice (open bars; C) as well as in islets from mice treated with L-NAME in their drinking water (filled bars; freely fed in B, fasted in C). D and E: Western blots of islets taken from freely fed mice. Lanes 1 and 2, incubation with ncNOS antibody; lanes 3 and 4, incubation with iNOS antibody; lanes 1 and 3, 20 µg of islet protein from control mice; lanes 2 and 4, 20 µg of islet protein from L-NAME-treated mice. Molecular masses (kDa) are indicated on left in D and E.

Ca2+/calmodulin-dependent (cNOS activity) and Ca2+/calmodulin-independent (iNOS activity) NO production in islets from L-NAME drinking mice and control mice. iNOS activity was estimated as NO formation measured in the absence of Ca2+ and calmodulin (Fig. 1, B and C). In islets from freely fed L-NAME-drinking mice, iNOS activity attained a high level (40 ± 2.1 pmol NO · min-1 · mg protein-1), whereas very low iNOS activity (1.1 ± 1.1 pmol NO · min-1 · mg protein-1; not different from 0) was demonstrated in freely fed control mice (Fig. 1B). In comparison, islets of fasted control mice expressed an overt iNOS activity in their islets (15 ± 1.3 pmol NO · min-1 · mg protein-1), and in islets of fasted L-NAME-drinking mice the iNOS activity was even higher (26 ± 3.0 pmol NO · min-1 · mg protein-1; P < 0.02; Fig. 1C).

In islets of freely fed L-NAME-drinking mice, cNOS activity was significantly lower (P < 0.002) than in freely fed controls (12 vs. 24 pmol NO · min-1 · mg protein-1; Fig. 1B). In contrast, no significant difference in cNOS activity could be recorded in islets of fasted controls and fasted L-NAME-drinking mice (18 pmol NO · min-1 · mg protein-1 for both; Fig. 1C), with the levels being no different from the level in freely fed controls (compare Fig. 1B).

Western blot analysis. ncNOS expression could be detected in islets from both control and L-NAME-drinking freely fed mice. However, the expression was clearly stronger in the control group (Fig. 1D). In comparison, the opposite pattern could be detected for iNOS expression, i.e., no iNOS expression in control islets but strong iNOS expression in islets after L-NAME drinking (Fig. 1E). Moreover, iNOS expression could also be clearly detected in islets of 24-h-fasted control mice (data not shown).

Immunocytochemical findings. In L-NAME-drinking mice, most islet cells exhibited significant iNOS immunofluorescence (Fig. 2B), which contrasted against the exocrine tissue. By comparison, in control mice, the islet cells displayed a very weak tissue fluorescence comparable to that in surrounding exocrine tissue (Fig. 2A). In control mice, most islet cells expressed ncNOS immunofluorescence (Fig. 2C), which was clearly reduced in intensity in L-NAME-drinking mice (Fig. 2D).


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Fig. 2.   Effect of long-term L-NAME drinking on islet iNOS and neuronal (n)cNOS immunocytochemistry. A: islet of freely fed control mice displaying almost no immunofluorescence. B: islet of a freely fed L-NAME-drinking mouse showing clear iNOS immunofluorescence. C: islet of a freely fed control mouse expressing ncNOS immunofluorescence. D: islet of a freely fed L-NAME-drinking mouse exhibiting significant reduction in ncNOS immunofluorescence compared with C. Left, a ncNOS positive cell body, probably of neuronal nature, close to the islet. Bars = 100 µm.

Effects of chronic L-NAME treatment on basal plasma levels of insulin, glucagon, and glucose in freely fed and 24-h-fasted mice. L-NAME-drinking mice did not show significant differences in basal plasma levels of insulin or glucagon compared with controls, irrespective of whether they were freely fed or fasted for 24 h (data not shown). Basal plasma glucose levels, however, were increased in fasted L-NAME-drinking mice (controls 5.72 ± 0.29 vs. L-NAME drinking 8.83 ± 0.32; P < 0.001) but not in L-NAME-drinking freely fed mice (controls 11.80 ± 0.44 vs. L-NAME drinking 11.90 ± 0.74).

Effect of chronic L-NAME treatment on insulin and glucagon release from isolated islets in the presence of low and high glucose concentrations in freely fed and 24-h-fasted animals. Figure 3A shows that insulin secretion at a high glucose concentration (20 mmol/l) was sixfold greater compared with a low glucose concentration (1 mmol/l) in both freely fed controls and L-NAME-drinking mice. Furthermore, there was no apparent difference compared with controls after L-NAME drinking, either at low or high glucose concentrations. However, insulin secretion was significantly greater at a high concentration of glucose in islets of fasted L-NAME-drinking mice compared with islets of fasted control mice (Fig. 3C). This difference was not seen at the low concentration of glucose. In comparison, glucagon secretion was higher both at low and high glucose concentrations in the L-NAME drinking group in both freely fed (Fig. 3B) and 24-h-fasted animals (Fig. 3D).


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Fig. 3.   Effect of long-term L-NAME drinking on hormone secretion from isolated islets. Secretion of insulin (A) and glucagon (B) in the presence of 1 mmol/l glucose (G1) or 20 mmol/l glucose (G20) from islets isolated from freely fed control mice (open bars in A and B), L-NAME-drinking mice (filled bars in A and B), fasted controls (open bars in C and D), and fasted L-NAME-drinking mice (filled bars in C and D). Means ± SE are shown for 8-12 batches of islets in each category. Incubation time was 60 min. Results are from 4 separate experiments performed on different days. Asterisks indicate probability level of random difference for L-NAME-drinking group vs. control group; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Effects of chronic L-NAME treatment on the in vivo dynamics of insulin, glucagon, and glucose in plasma after an acute glucose injection in freely fed and 24-h-fasted animals. No significant differences were observed between freely fed L-NAME-drinking mice and controls with regard to the insulin and glucagon response after an intravenous glucose load (Fig. 4, A and B). There was, however, a very slight improvement of the glucose tolerance curve in the L-NAME group (Fig. 4C). In fasted control mice (Fig. 4, D-F) the acute insulin response after glucose injection was markedly impaired compared with the freely fed animals, but it was significantly increased in fasted L-NAME-drinking mice compared with fasted controls (Fig. 4D). This was accompanied by a greatly improved glucose tolerance curve (Fig. 4F). There was no apparent difference in the glucagon response between fasted controls and fasted L-NAME-drinking mice (Fig. 4E).


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Fig. 4.   Effect of long-term L-NAME drinking on the plasma concentrations of insulin (A and D), glucagon (B and E), and glucose (C and F) at 2, 6, 15, and 30 min after an iv injection of glucose (3.3 mmol/kg). The effects on freely fed mice are shown in A-C and the effects on fasted mice are in D-F. , L-NAME-drinking mice; open circle , control mice. Values are means ± SE for 8-16 animals in each group. Asterisks indicate probability level of random difference vs. control: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Short-term in vitro effects of two different NOS inhibitors (L-NAME and 7-nitroindazole) in the presence of 20 mmol/l glucose on hormone release and NO production from isolated islets taken from control or L-NAME-drinking mice. To assess the effects of the two chemically different NOS inhibitors L-NAME and 7-nitroindazole on islet cNOS and iNOS activities, we measured the NO production from control islets incubated at 20 mM glucose in the presence of these inhibitors. Figure 5A shows that islet cNOS activity was dose dependently inhibited by 1 and 5 mM L-NAME and that 7-nitroindazole (1 mM) was a potent cNOS inhibitor. Moreover, 7-nitroindazole markedly inhibited both cNOS and iNOS, whereas L-NAME was a good inhibitor of cNOS but displayed practically no effect on iNOS activity during the present experimental conditions (Fig. 5A). In addition, it should be noted that islet incubation with 20 mM glucose itself apparently stimulated iNOS activity (compare with Fig. 1B showing no iNOS activity in freshly isolated nonincubated islets).


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Fig. 5.   A: effect of the NOS inhibitors L-NAME (1.0 and 5.0 mmol/l) and 7-nitroindazole (1.0 mmol/l) on islet cNOS and iNOS activities in control islets. B and C: secretions of insulin (B) and glucagon (C) from isolated islets taken from control mice (open bars) or long-term L-NAME-drinking mice (filled bars). D and E: effect on islet hormone production from fasted control mice (open bars) and fasted L-NAME-drinking mice (filled bars). Glucose concentration was 20.0 mmol/l. In A, means ± SE for 3-4 pools of islets taken from 2-3 mice in each group are shown. Incubation time was 60 min. Asterisks denote effects of the NOS inhibitors vs. control: P < 0.05, *** P < 0.001. In B-E, means ± SE are shown for 8-10 batches of islets in each category. Incubation time was 60 min. The results are from 8 separate experiments performed on different days. Asterisks denote probability level of random difference for L-NAME drinking vs. control: * P < 0.05, ** P < 0.01, and *** P < 0.001. Crosses denote effects of L-NAME or 7-nitroindazole (7-NI) in control islets (× P < 0.05, ×× P < 0.01, ××× P < 0.001). Stars denote difference between islets from L-NAME-drinking mice in the absence or presence of L-NAME or 7-NI: * P < 0.05 and ** P < 0.01.

Addition of L-NAME to the incubation medium (60-min incubation) increased insulin secretion from isolated islets taken from both L-NAME-drinking and control freely fed mice in the presence of 20.0 mmol/l glucose (Fig. 5B). This effect was much more pronounced in islets from L-NAME-drinking mice than in the control islets. In the presence of 5 mmol/l L-NAME, the secretion of insulin was almost doubled [9.64 ± 1.21 (L-NAME drinking) vs. 5.33 ± 0.79 (control) ng insulin · islet-1 · h-1]. Addition of 7-nitroindazole also markedly increased insulin secretion from islets of both controls and L-NAME-drinking mice (Fig. 5B). Figure 5C shows that the highly increased glucagon secretion in the L-NAME-drinking group was inhibited by the addition of 5 mmol/l L-NAME or 1 mmol/l 7-nitroindazole to the incubation medium. Also in fasted animals, L-NAME and 7-nitroindazole potentiated insulin secretion from islets of both controls and L-NAME-drinking mice (Fig. 5D). However, only at 5 mmol/l of L-NAME was a slightly greater secretion of insulin seen from islets of fasted L-NAME-drinking mice compared with the effects of L-NAME in fasted controls (Fig. 5D). In contrast, at this high glucose concentration, glucagon secretion in fasted control animals was not affected by the addition of the NOS inhibitors (Fig. 5E). However, in islets of fasted L-NAME-drinking animals, the high rate of glucagon release in islets could still be significantly inhibited by both L-NAME and 7-nitroindazole (Fig. 5E).

Influence of chronic L-NAME treatment on islet cAMP and cGMP content. Islets from freely fed L-NAME-drinking mice incubated in the presence of 1 mmol/l glucose for 1 h showed a fourfold greater content of cAMP than control islets (Table 1). Incubation of islets at a high concentration of glucose (20 mmol/l) evoked a further increase in cAMP levels in control islets but not in islets of L-NAME-treated mice. In fact, at this high concentration of glucose, the cAMP content was somewhat lower (-30%) in islets of L-NAME-drinking mice. cGMP levels were significantly greater (+60%) in islets of L-NAME-drinking mice than in islets of control mice after incubation at a low (1 mmol/l) concentration of glucose (Table 1). At a high concentration of glucose (20 mmol/l), islet cGMP content was increased to the same level in both controls and L-NAME-drinking mice.

                              
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Table 1.   Content of cAMP and cGMP in isolated islets taken from freely fed controls or L-NAME-drinking mice and incubated in the presence of low (1 mmol/l) or high (16.7 mmol/l) concentrations of glucose

Effect of chronic L-NAME treatment on insulin and glucagon release stimulated by the adenylate cyclase activator forskolin in freely fed and 24-h-fasted animals. In view of the increased islet cAMP levels after L-NAME drinking, the effect of the adenylate cyclase activator forskolin was tested. Figure 6A shows that the in vivo insulin response to forskolin was of similar magnitude in freely fed L-NAME-drinking and control animals. In fasted animals, however, the insulin response was markedly increased in the L-NAME-drinking group. In comparison, there was no effect by L-NAME drinking on the glucagon response to forskolin in either freely fed or fasted animals (Fig. 6B).


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Fig. 6.   Influence of the adenylate cyclase activator forskolin on islet hormone secretion in vivo and in vitro. A and B: effect of long-term L-NAME drinking on the plasma concentrations of insulin (A) and glucagon (B) at 2 min after an iv injection of forskolin (7.3 µmol/kg). The effects on both freely fed and fasted mice are shown. Filled bars, L-NAME-drinking mice; open bars, control mice. Values are means ± SE for 8 animals in each group. Asterisks indicate probability level of random difference vs. control: ** P < 0.01. C-F: effects of long-term L-NAME drinking on hormone secretion from isolated islets. Secretion of insulin (C) and glucagon (D) in the presence of 7 mmol/l glucose (G7) or 7.0 mmol/l glucose + 20 µmol/l forskolin (G7 + F) from islets isolated either from control mice (open bars) or from L-NAME-drinking mice (filled bars). In E and F, the secretory responses from islets of fasted control mice (open bars) and fasted L-NAME-treated mice (filled bars) are shown. Means ± SE are shown for 6-10 batches of islets in each group. Incubation time was 60 min. The results were obtained from 4 separate experiments. Asterisks indicate probability level of random difference for control group vs. L-NAME-drinking group: * P < 0.05 and *** P < 0.001. Crosses indicate probability level of random difference for controls (G7) vs. control + forskolin (G7 + F): × P < 0.05, ×× P < 0.01, and ××× P < 0.001.

Figure 6, C and E, shows that, in isolated islets, the insulin secretory response to forskolin in the presence of 7 mmol/l glucose was increased over basal levels, being twofold greater than incubated islets isolated from L-NAME-drinking freely fed and fasted mice compared with islets of freely fed and fasted control mice. Similarly, the forskolin-stimulated glucagon release was also greatly increased after L-NAME treatment (Fig. 6, D and F). There was no difference between controls and L-NAME-drinking mice with regard to insulin secretion from islets incubated at 7 mM glucose in the absence of forskolin (Fig. 6, A and C). In contrast, glucagon release was slightly increased after L-NAME drinking (Fig. 6, B and D).

Effect of chronic L-NAME treatment on CO production and protein expression of the CO-producing isoenzymes HO-2 and HO-1 in pancreatic islets. To elucidate a possible contribution by the newly discovered HO-2-CO pathway having a stimulatory effect on insulin secretory mechanisms (24, 25) in the restoration of glucose-stimulated insulin release from islets of L-NAME-drinking mice, we measured CO production and expression of HO isoenzymes. In islets from freely fed L-NAME-drinking mice, the production of CO was fivefold higher compared with control mice (Fig. 7A). Immunoblotting revealed that this increase was accompanied by an increased expression of the constitutive HO-2 protein (Fig. 7B). In contrast, no HO-1 expression could be seen in islet tissue of either freely fed mice or controls after L-NAME drinking (Fig. 7B).


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Fig. 7.   Effect of long-term L-NAME drinking on islet heme oxygenase (HO). A: HO activity in isolated islets from freely fed control and L-NAME-drinking mice. Data are expressed as CO formation (pmol · mg protein-1 · min-1). Values are means ± SE for 3-5 pools of islets taken from 3-4 mice in each group. Asterisks denote probability level of random difference vs. controls: *** P < 0.001. B: Western blots of islets isolated from freely fed mice. Lanes 1 and 2, incubation with HO-2 antibody; lanes 3 and 4, incubation with HO-1 antibody; lanes 1 and 3, 20 µg of islet protein from islets of control mice; lanes 2 and 4, 20 µg of islet protein from islets of L-NAME-drinking mice. Molecular masses are indicated on left.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunoblots, immunocytochemistry, and biochemistry demonstrated the presence of a constitutive NOS isoform (ncNOS) in the islets of both control and L-NAME-drinking mice. The expression of cNOS, however, was markedly decreased after L-NAME drinking. This was in accordance with the biochemical findings of a decreased islet cNOS activity. An accidental but interesting immunocytochemical observation in this context was that the ncNOS immunofluorescence exhibited by a cell body of probably neuronal nature situated close to the islet shown in Fig. 2D (L-NAME-drinking mice) was not at all reduced by L-NAME treatment, being suggestive of a different reaction pattern to chronic L-NAME treatment in peripheral nitrinergic neurons compared with the islet endocrine cells.

No iNOS was found in freshly isolated islets of freely fed control mice, but there was a strong expression of iNOS in the islets of L-NAME-treated mice that contributed to an increase in total islet NO production. The compensatory increase in iNOS activity after chronic L-NAME drinking suggests the occurrence of a regulatory NO-producing system within the islets of Langerhans trying to assure adequate production of NO by inducing iNOS expression when cNOS activity is suppressed and thus maintain the function of NO as a negative feedback modulator of glucose-stimulated insulin release (24, 40, 47-49). One possible mechanism responsible for the compensatory induction of islet iNOS could be the decrease in cNOS-derived NO production after L-NAME drinking. It should be noted that we found no evidence of insulitis or macrophage invasion in the islets of our L-NAME-drinking mice, and thus the measured NOS activities in the present study do not involve cytokine stimulation and are to be referred to as islet endocrine cells. This is also emphasized by our immunocytochemical data.

In control mice we found, unexpectedly, that a fasting period of 24 h evoked an increase in islet total NO production due to a marked induction of iNOS expression. Because fasting is known to greatly reduce glucose-stimulated insulin release (32) and because NO is a negative modulator of the insulin response to glucose (24, 40, 47-49), the present data suggested to us that the increased total NO production in islets from fasted animals might be an important mechanism in fasting-induced impairment of glucose-stimulated insulin release (32) and thus also an essential factor in the induction of the diabetic-like condition known to occur in starvation, such as increased lipolysis and glycolysis, increased gluconeogenesis, increased peripheral insulin resistance, and impaired insulin response to glucose (51).

We now expected that chronic treatment with L-NAME would inhibit starvation-induced NO production and thus improve this "diabetic" condition in fasted mice. In fact, the starvation diabetes was modestly improved, at least with regard to glucose-stimulated insulin release and glucose tolerance, but not in the way we had anticipated, since, similar to what happened in freely fed mice, chronic L-NAME drinking induced a further increase in islet NO production (due to iNOS). This improvement might be explained, at least partially, by a previous observation (30) that 8-bromo-cGMP (8-BrcGMP) potentiated glucose-stimulated insulin release in islets from starved but not freely fed rats.

NO is known to increase soluble guanylate cyclase activity and cGMP production (28, 33). In accordance with this, islet cGMP levels, probably due to increased iNOS activity, were increased after long-term L-NAME treatment when islets were incubated in the presence of a low glucose concentration. In contrast, at a high glucose concentration, which by itself increases islet NO production (Fig. 5A and Ref. 24), no appreciable difference was seen. It is not inconceivable that the NO-evoked negative feedback effect on glucose-stimulated insulin release (24, 40, 47) might be mediated by the cGMP system. However, we have also recently shown that cGMP might be involved as a possible short-term modulator of CO-stimulated insulin and glucagon secretion (24, 25). Such a cGMP-mediated direct stimulatory mechanism is apparently not operating for NO with regard to glucose-stimulated insulin release. However, the enhanced NO production in islets of L-NAME-drinking mice to increase islet cAMP levels after incubation in low glucose is probably attributed to the cGMP-inhibited cAMP-phosphodiesterase (56). Hence, this cGMP effect upregulating the cAMP system might contribute to the enhanced insulin and glucagon secretion at low glucose in islets from L-NAME-drinking mice. At high glucose, however, the further increase in NO production might adversely affect the cAMP production, as previously described (18).

Insulin secretion after chronic L-NAME drinking. Insulin secretion is regulated by a complex chain of events exerted in response to different metabolic, nervous, and hormonal factors (59). Glucose, the major insulin secretagogue, is known to initiate insulin release through closure of ATP-sensitive K+ channels, with subsequent depolarization and opening of voltage-dependent Ca2+ channels leading to increased intracellular Ca2+ and subsequent exocytosis of insulin. This secretory process is regulated by different intracellular modulatory systems, among which the importance of cNOS-derived NO as a negative or positive modulator has been discussed (26, 27, 47, 49, 50).

Chronic NOS inhibition in the rat has been reported to have no effects on in vivo insulin secretion and, furthermore, no influence on glucose tolerance or insulin resistance (8, 43, 55). Our observation that glucose-stimulated insulin secretion was unaffected after L-NAME drinking might at first glance favor the assumption that the secretion of insulin is independent of NO production (27, 43) more than our previous suggestion that NO is an important negative modulator of glucose-induced insulin release (24, 40, 47-49). However, when studying the effects of in vitro addition of two chemically different NOS inhibitors, L-NAME and 7-nitroindazole (10, 53), on glucose-stimulated insulin secretion, we found that insulin release from islets of both controls and L-NAME-drinking mice was greatly increased in the presence of the NOS inhibitors. Moreover, we could directly show here by biochemical assay of islet cNOS and iNOS activities that the increase of glucose-stimulated insulin release in the presence of L-NAME or 7-nitroindazole was accompanied by a parallel decrease in cNOS activity. Interestingly, such a parallel decrease was not found with regard to iNOS, thus suggesting that iNOS-derived NO might be less efficient than cNOS-derived NO in suppressing insulin release, at least during short-term glucose stimulation. Indeed, the present data suggest that L-NAME is a potent inhibitor of islet cNOS, whereas 7-nitroindazole is a slightly more efficient inhibitor against iNOS than against cNOS. Moreover, the results also underline the fact emphasized in a recent review (53) that different NOS inhibitors display different inhibitory effects against cNOS and iNOS dependent on the type of tissue investigated.

We have repeatedly found that NO is a negative modulator of insulin secretion induced by glucose, L-arginine, and cholinergic stimulation (2, 4, 26, 39, 40, 47-49). The mechanisms of the inhibitory effects of NO on nutrient-induced insulin secretion are still unclear but most probably are exerted through the formation of S-nitrosothiols (54), thereby impairing important regulatory thiol groups, which have long been known to be essential for glucose-induced insulin secretion (6, 23). Hence, any possible stimulating effect of NO on insulin release exerted via the cGMP system in the beta -cell is likely to be strongly counteracted and overshadowed by the formation of S-nitrosothiols, a reaction pattern that we have suggested to negatively modulate stimulus-secretion coupling of glucose-induced insulin secretion. It should be noted that the inhibitory effect on glucose-stimulated insulin release exerted by excessive production of NO through cytokine-stimulated iNOS activity (16, 17, 19) is currently ascribed to a reduction of oxidative metabolism of glucose resulting in reduced levels of ATP. This has been explained by NO reacting with iron sulfur centers in certain enzymes, such as the tricarboxylic acid cycle enzyme aconitase (57). Such a mechanism cannot be excluded in the present context. However, with regard to our present results, we favor the assumption that NO exerts its inhibitory action at more distal steps in the stimulus-secretion coupling, since we have recently shown (26, 48, 49) that this inhibitory effect could be abolished by adding NOS inhibitors to islets exposed to high K+ (30 mM) and diazoxide (to keep the ATP-sensitive K+ channels open). The precise nature of these presumably thiol-dependent enzymes and regulatory proteins that are attacked by NO, remains, however, to be elucidated.

With regard to the fasted animals, we found that, although the NO production was markedly increased already in fasted control islets, it was further increased in the islets of fasting L-NAME-drinking mice. Similar to the islets of freely fed L-NAME-drinking animals, this increase in NO production was derived from iNOS. However, in contrast to islets from freely fed mice, the cNOS activity in islets from L-NAME-drinking fasted animals did not decrease, suggesting that the negative feedback of NO on cNOS activity was impaired in these islets. Furthermore, in spite of the increased NO production in the islets of L-NAME-drinking fasted mice compared with islets from fasted controls, we found an increased insulin response to glucose in these islets both in vitro and in vivo, suggesting that fasted islets are less susceptible to S-nitrosylation. Such a reaction pattern could lend support to previous data (30) discussed above, showing that 8-BrcGMP is a positive modulator of glucose-stimulated insulin release from starved but not freely fed rats. However, a compensatory increase in the cAMP system and the HO-CO activity is probably more important (see below). In the in vivo situation, the enhanced insulin response to glucose was accompanied by a marked improvement of the glucose tolerance curve, most probably due to both the increase in plasma insulin levels and an NO-dependent augmentation of insulin-stimulated glucose uptake in peripheral tissues (46). It should be noted that, when an NOS inhibitor (L-NAME or 7-nitroindazole) was added to the islet incubation medium in the fasting series of experiments, insulin secretion was potentiated almost equally from the islets of control and L-NAME-drinking animals, largely reflecting the decreased difference in NO production between the two groups seen after fasting compared with that between the two groups of freely fed animals. Addition of the highest concentration of L-NAME (5 mmol/l) could still induce a slight increase in insulin secretion from fasting L-NAME-drinking mice, probably because the NO production was somewhat greater in islets of these mice also after fasting. It is tempting to speculate that the increased iNOS-derived NO production in islets of fasted animals might be an important factor behind the impairment of glucose-stimulated insulin secretion in starvation.

Glucagon secretion after chronic L-NAME drinking. The physiological mechanisms behind the secretion of glucagon are not so well understood. It has been known for a long time that the glucagon-producing cells, being located in the periphery of the islets, are richly innervated by both adrenergic and cholinergic nerves (34, 38, 58), that adrenergic as well as cholinergic stimulation induces an increase in glucagon secretion (38, 45, 58), and that Ca2+ and the cAMP system are important intracellular messengers in mediating stimulus-secretion coupling of glucagon release (45). We originally proposed (39) that NO should be looked upon as a novel physiological stimulator of glucagon secretion, and we have now demonstrated that the glucagon-producing alpha -cells indeed harbor the cNOS enzyme (5). Moreover, we have previously shown that glucagon secretion induced by L-arginine or by cholinergic stimulation is inhibited by different types of NOS inhibitors both in vivo and in vitro (2-4, 26, 47, 49), thus suggesting that NO is a positive modulator of glucagon release. Furthermore, we have recently found that addition of NO gas to isolated islets stimulates glucagon secretion (24). In the present paper, we now show that islets taken from L-NAME-drinking mice display a markedly increased NO production in parallel with an increased glucagon secretion at both low and high glucose and after stimulating the cAMP system by forskolin. The greatest increase in glucagon release, relative to control islets, was found at 1 mM glucose in islets from fasted animals, thus being in accordance with the notion that glucose is a potent inhibitor of glucagon release. Interestingly, and somewhat unexpectedly, islets from L-NAME-drinking mice displayed a marked increase in glucagon secretion relative to controls, even when incubated at high (20 mM) glucose. This was especially notable in islets from fasted L-NAME-drinking animals, where also insulin, a well-known inhibitor of glucagon release (36), was markedly enhanced. These findings might be explained, at least partly, by our recent observation (24) that glucose itself is a potent stimulator of islet NO production. In addition, NO-stimulated glucagon release seems to be less efficiently inhibited by insulin. Our observation that NO might be a very potent stimulator of glucagon release also in the presence of 20 mM glucose is shown, although indirectly, in islets from fasted L-NAME-drinking mice. The greatly increased glucagon release from these islets was markedly reduced after in vitro addition of the NOS inhibitors L-NAME and 7-nitroindazole. The mechanism of action of NO to stimulate glucagon release is presently unclear. However, much evidence suggests that the cGMP-protein kinase G system constitutes an important transduction pathway (cf. Table 1).

Possible islet compensatory mechanisms against the increased NO production during chronic L-NAME treatment. The present results showed that chronic L-NAME treatment of mice resulted in a marked increase in islet NO production. The inhibitory action of NO on glucose-stimulated insulin release (16, 39, 40, 47), however, had been clearly compensated for during the long-term L-NAME treatment, since the insulin response to glucose was found to be essentially normal both in vitro and in vivo in freely fed mice and even increased in fasted mice. As previously discussed, this could partly be explained by iNOS-derived NO being less efficient than cNOS-derived NO as an inhibitor of glucose-stimulated insulin release. However, evidently, the islet adenylate cyclase-cAMP secretory pathway contributed to the compensatory mechanisms, since islets taken from L-NAME-drinking mice displayed a marked increase in the sensitivity and activity of the cAMP system, as documented by a great increase in islet cAMP content at low glucose and a greatly enhanced insulin response to the adenylate cyclase activator forskolin. Furthermore, the present results also suggested that a marked increase in the newly discovered (24, 25) HO-2-CO pathway for insulin release is another compensatory mechanism operating to counteract the increased NO production. We have very recently shown (24) that CO can promote insulin release, both by directly stimulating the guanylate cyclase-cGMP system and by indirectly inhibiting islet NOS activity. Moreover, additional CO-induced mechanisms to stimulate insulin release cannot be excluded. Hence, the very high production of CO in islets from L-NAME-drinking mice is most probably an important compensatory mechanism to potentiate glucose-stimulated insulin release in islets displaying increased NO formation. It is notable that the compensatory CO production was conveyed by HO-2 and not by HO-1, which previously has been found to be induced in various situations that are toxic to the islets (57). With regard to glucagon release, our data showed that chronic L-NAME treatment resulted in a pronounced hypersecretion of glucagon compared with controls in the in vitro situation both at low and high glucose and after forskolin stimulation. The glucagon hypersecretion was most probably due to the increased formation of NO in the islets, since it was virtually abolished by the addition of the NOS inhibitors L-NAME or 7-nitroindazole. Furthermore, the NO-stimulated hypersecretion of glucagon seen in isolated islets was apparently counteracted in vivo, since both basal and stimulated glucagon secretion was essentially normal in these mice, apart from an increased glucagon response to forskolin after fasting. The present data do not tell the nature of the mechanisms that inhibit the NO-stimulated increase in glucagon secretion in the vivo situation. Further studies will hopefully reveal whether in situ neural and/or endocrine mechanisms are involved.

Our accumulated data show that chronic L-NAME treatment results in an induction of islet iNOS activity and an increase in total NO production and emphasize the necessity of measuring both cNOS and iNOS activities in the islets, and certainly also in other tissues (35), when investigating the effects of both chronic and acute effects of different NOS inhibitors. Fasting, which enhanced the total NO production also in islets from control mice by inducing the expression of the iNOS protein, only slightly increased the NO production after L-NAME drinking and did not increase it above the values achieved in freely fed L-NAME-drinking mice. It is not inconceivable that the increase in islet NO production in starvation is an important factor contributing to the impaired glucose-stimulated insulin release in the fasting state. This effect is, however, improved by L-NAME drinking, since in this situation the cAMP system and the increase in HO activity compensate for this defect. The observed effects of both L-NAME drinking and fasting as well as of in vitro addition of NOS inhibitors on insulin and glucagon secretion support our idea of cNOS-derived NO as a physiological negative modulator and feedback inhibitor of glucose-stimulated insulin secretion and further strengthen our hypothesis of NO being an important positive modulator of glucagon secretion. Important compensatory mechanisms counteracting and reversing the NO-induced suppression of glucose-stimulated insulin release include an enhanced sensitivity and capacity of the islet cAMP-protein kinase A system and the newly discovered HO-2-CO pathway.


    ACKNOWLEDGEMENTS

The skillful technical assistance of Britt-Marie Nilsson, Maj-Britt Johansson, and Lillemor Thuresson is gratefully acknowledged.


    FOOTNOTES

This study was supported by the Swedish Medical Research Council (14X-4286 and 11205), the Swedish Diabetes Association, and the foundations of Magnus Bergvall, Crafoord, Anna-Lisa and Sven-Erik Lundgren, Albert Påhlsson, Åke Wiberg, and Thelma Zoega.

Address for reprint requests and other correspondence: R. Henningsson, Dept. of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden (E-mail: Ragnar.Henningsson{at}farm.lu.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 29 September 1999; accepted in final form 23 February 2000.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 279(1):E95-E107
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