Selective induction of inducible nitric oxide synthase in pancreatic islet of rat after an intravenous glucose or intralipid challenge

Nutrition 22 (2006) 652– 660 www.elsevier.com/locate/nut

Basic nutritional investigation

Selective induction of inducible nitric oxide synthase in pancreatic islet of rat after an intravenous glucose or intralipid challenge Mats Ekelund, M.D., Ph.D.a, Saleem S. Qader, M.D., Ph.D.a, Javier Jimenez-Feltstrom, Ph.D.b, and Albert Salehi, Ph.D.b,* a

b

Department of Surgery, Division of Diabetes, Metabolism and Endocrinology, University of Lund, Lund, Sweden Department of Experimental Medical Sciences, Division of Diabetes, Metabolism and Endocrinology, University of Lund, Lund, Sweden Manuscript received September 27, 2005; accepted December 23, 2005.

Abstract:

Objective: Constant exposure of pancreatic islets to high levels of glucose or free fatty acids can lead to irreversible ␤-cell dysfunction, a process referred to as glucotoxicity or lipotoxicity, respectively. In this context a role for nitric oxide generated by pancreatic islet has been suggested. The present investigation examined whether the route of glucose administration, i.e., given orally (OG) or infused intravenously (IVG), could have any effect on the expression and activity of inducible nitric oxide synthase (iNOS) in pancreatic islets. Methods: Rats were infused with glucose (50%) or Intralipid intravenously for 24 h or given glucose orally. A freely fed control group (FF) was also included. At 24 h rats were killed and blood samples were drawn for analysis of plasma insulin, glucagon, and glucose. Pancreatic islets were harvested from each animal and investigated for the occurrence of iNOS by the use of confocal microscopy, western blot, and high-performance liquid chromatographic analysis. The effect of intravenously infused glucose was then compared with the effect of an intravenous infusion of Intralipid (IL). Results: Plasma insulin levels were markedly decreased after 24 h of infusion of glucose (IVG group) or Intralipid (IL group) compared with the FF or OG group. Plasma glucagon and glucose levels were markedly increased in the IVG group, whereas both parameters were decreased in the IL group. No significant differences in plasma insulin, glucagon, or glucose were found between the OG and FF groups. Immunocytochemical (confocal microscopy), western blot, and biochemical (high-performance liquid chromatographic) analyses showed that a sustained increase in plasma level of glucose or free fatty acids by an intravenous infusion of either nutrient for 24 h resulted in a marked expression and activity of iNOS in pancreatic islets. No sign of iNOS expression could, however, be detected in the islets of FF control or OG rats. Conclusion: The data suggest that impaired ␤-cell function found after 24 h of an intravenous infusion of glucose or Intralipid might be mediated, at least in part, by the induction of iNOS in pancreatic islets. This may subsequently result in an exclusive production of nitric oxide, which is deleterious for ␤-cells. © 2006 Elsevier Inc. All rights reserved.

Keywords:

Pancreatic islets; Insulin secretion; Inducible nitric oxide synthase; Glucose challenge

Introduction It is well known that the gastrointestinal tract harbors hormones that contribute to the control of insulin secretion [1–3].

This study was supported by grants from the Swedish Research Council (K2006-04X), NOVO Nordic, the Albert Påhlsson, the Swedish Diabetes Foundation, and the Crafoord Foundation. * Corresponding author. Tel.: ⫹46-46222-7586; fax: ⫹46-46222-7763. E-mail address: [email protected] (A. Salehi). 0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2006.01.006

The route of glucose administration is important for ␤-cell function and insulin secretion. It has been shown that more insulin is secreted in response to an oral glucose challenge than in response to an equivalent intravenous (IV) glucose challenge [1]. Thus, an oral glucose load is metabolized faster than an equivalent IV glucose load. It is also known that sustained increased plasma glucose (hyperglycemia) or free fatty acid (FFA) levels (hyperlipidemia) negatively affect pancreatic ␤-cell response to glucose challenge, a process that has been referred to as glucotoxicity or lipotoxicity, respectively [4 – 6].

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

The nature of hyperglycemia-induced ␤-cell dysfunction is as yet undetermined, but hyperglycemia has been reported to increase superoxide production and uncoupling protein-2 expression in the pancreatic islets, which subsequently inhibits insulin secretion [7]. Previous studies have clearly demonstrated that an increase in intracellular generation of nitric oxide (NO) contributes to the development of several pathophysiologic conditions [5,6,8 –11]. An exaggerated NO production in the pancreatic islet by inducible NO synthase (iNOS) is implicated in the pathogenesis of ␤-cell dysfunction [5,6,10,11]. Inducible NOS has been implicated in different defense mechanisms of the body, where it is induced by a variety of inflammatory agents and cytokines [12,13]. We recently presented evidence for a “non-cytokine”– induced expression of iNOS in pancreatic islets during in vitro incubation of islets at high glucose concentration [5,6,14]. Overnight culturing of islets in the presence of a high level of FFA also increased NO generation by islets [15]. The data suggested that some signal or metabolite derived from glucose or FFA metabolism could be involved in the induction of iNOS. Hyperglycemic and hyperlipidemic subjects have been reported to display a markedly impaired insulin response to IV glucose [16]. In this context, it should be mentioned that hyperglycemia and hyperlipidemia are prominent characteristics of type-2 diabetes [4]. This is especially interesting because a combination of Intralipid and glucose infusion in total parenteral nutrition (TPN) is frequently used in patients undergoing intensive care [17]. We previously reported that long-term (⬎8 d) TPN with a solution containing slightly high amounts of glucose, Intralipid, and amino acids causes expression of iNOS in pancreatic islets of TPN-treated animals [5,6,18]. However, the time course for the induction of iNOS is not known. Further, it has been shown that fasting, preoperative IV glucose infusion, and surgical trauma are associated with postoperative insulin resistance and high blood glucose levels that can be avoided with preoperative carbohydrate feeding [19,20]. Based on these findings, we hypothesized that iNOS might be expressed rapidly during IV glucose infusion and therefore might contribute to the defect in insulin release by pancreatic islets [19,20]. The Intralipid and glucose solutions used in this study were developed for rats, with components similar to those used in clinical practice, to establish an animal model where the effect of IV infusion of glucose or Intralipid could be compared with oral intake of food or glucose. The present study investigated the effect of short-term (24 h) infusion of glucose or Intralipid on the expression of iNOS in pancreatic islets. Freely fed controls or a group given glucose orally were also included for comparison.

653

Materials and methods Animals Male Sprague-Dawley rats (B&K, Sollentuna, Sweden) weighing 200 to 225 g were used in all experiments. The rats intended for IV nutrient administration were intraperitoneally anesthetized with 5% chloral hydrate before operation. The neck of the rat was shaved and the operative field was washed with iodine solution. The operation was performed under sterile conditions. A catheter (silicon-rubber) was inserted into the right external jugular vein. The catheter was delivered to the skull subcutaneously and connected to a swivel via a protective coil attached to the skin of skull. The rats serving as freely fed controls or those orally fed glucose underwent the same operative procedure, including catheter insertion, without any infusion. At the end of experiment (24 h later), the catheter was flushed with low-molecular-weight heparin before aspiration of blood from all animals. A detailed description of the methodology has previously been reported [5,6,18,21]. Treatment The rats were assigned to one of four groups: freely fed (FF), oral glucose (OG), IV glucose (IVG), or IV Intralipid (IL). Rats in the IVG group received a continuous IV infusion (2 mL/h) of a 50% glucose solution (Sweden) for 24 h. Rats in the IL group received a continuous IV infusion (2 mL/h) of Intralipid (Fresenius-Kabi, Uppsala, Sweden). The amount of solutions given (glucose and Intralipid) equaled or slightly exceeded what is given during TPN to rats. Rats in the OG group were provided with batches of 6 mL of glucose (50%) each third hour for 24 h, thus ensuring that they had continuous access to glucose. Rats ingested all the solution provided. Rats serving as FF controls had free access to a standard pellet diet (B&K) and tap water ad libitum. All animals were housed in metabolic cages with constant temperature (22°C) and 12-h light/dark cycles. The local animal welfare committee (Lund, Sweden) approved the experimental protocols. Chemicals Bovine serum albumin was purchased from ICN Biochemicals (High Wycombe, UK). Radioimmunoassay kits for insulin and glucagon determinations were obtained from Diagnostika (Falkenberg, Sweden) and Euro-Diagnostika (Malmö, Sweden). All other drugs were obtained from Sigma Chemicals (St. Louis, MO, USA). Isolation of pancreatic islets Preparation of isolated pancreatic islets from rat was performed by retrograde injection of a collagenase solution

654

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

through the bile-pancreatic duct [22]. Islets were then collected under a stereomicroscope at room temperature. Immunocytochemistry Freshly isolated islets were fixed with 4% formaldehyde and permeabilized with 5% Triton X-100, and unspecific sites were blocked with 5% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA, USA). neuronal constitutive NOS (ncNOS) and iNOS were detected with corresponding rabbit-raised primary antibodies (BD Transduction Lab, San Diego, CA, USA) in combination with Cy2-conjugated anti-rabbit immunoglobulin G (Jackson Immunoresearch Laboratories). For staining of insulin, islets were incubated with a guinea pig–raised antiinsulin antibody (Euro-Diagnostika) followed by a Cy5conjugated anti-guinea pig antibody (Jackson Immunoresearch Laboratories). Fluorescence was visualized with a Zeiss LSM510 confocal microscope by sequentially scanning at (excitation/emission) 488/505–530 nm (Cy2) and 633/⬎650 nm (Cy5), as previously reported [23]. Western blot analysis Approximately 250 islets (n ⫽ 5 per group) were collected in Hanks’ buffer (100 ␮L) and sonicated on ice (3 ⫻ 10 s). Homogenate samples representing 20 ␮g of total protein from islet tissue were then run on 10% sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrotransfer (10 – 15 V, 60 min; semi-dry transfer cell, Bio-Rad, Richmond, CA, USA). Membranes were blocked in 9 mmol/L of Tris-HCl (pH 7.4) containing 5% non-fat milk powder for 40 min at 37°C. Immunoblotting with rabbit anti-mouse iNOS (N-7782) or ncNOS (N-7155; 1:2000; Sigma) was performed for 16 h at room temperature. The membrane was washed twice and then incubated with alkaline-phosphatase– conjugated goat anti-rabbit immunoglobulin G (1:10 000; Sigma) for 90 min. Antibody binding to ncNOS and iNOS was detected by using 0.25 mmol/L of CDP-Star (Tropix, Bedford, MA, USA) for 5 min at room temperature. The chemiluminiscence signal was visualized by exposing the membranes to Dupont Cronex X- ray films (Dupont, Wilmington, DE, USA) for 1 to 5 min. An appropriate standard, i.e., molecular mass marker, was run in all analyses. Band intensities were quantified by densitometry (Bio-Rad GS-710 Densitometer). Measurement of NOS activities After isolation, islets were thoroughly washed and collected in ice-cold buffer (200 ␮L) containing HEPES (20.0 mmol/L), ethylenediaminetetra-acetic acid (0.50 mmol/L) and D,L-dithiothreitol (1.0 mmol/L), pH 7.2, and stored at ⫺20°C for subsequent NOS analysis [8,13,22]. In brief, after sonication on ice, the buffer solution containing the

islet homogenate was supplemented to contain also CaCl2 (0.45 mmol/L), calmodulin (25 U/mL), nicotinamide adenine dinucleotide phosphate (reduced; 2.0 mmol/L), and L-arginine (0.2 mmol/L) in a total volume of 450 ␮L. For the assay of iNOS, calmodulin and CaCl2 were omitted from the buffer, as previously described [12]. The homogenate was then incubated at 37°C under constant air bubbling (1.0 mL/min) for 3 h. Aliquots of the incubated medium (200 ␮L) were mixed with an equal volume of o-phtaldialdehyde reagent solution in a glass vial and then passed through an 1-mL Amprep CBA cation-exchange column for high-performance liquid chromatographic analysis. The amount of L-citrulline formed (NO and L-citrulline were produced in equimolar concentrations) was then measured in a Hitachi F1000 fluorescence spectrophotometer (Merck, Darmstadt, Germany), as previously described [9,14]. Protein Protein was determined according to the method of Bradford [24]. Statistics Results are expressed as mean ⫾ SEM for the indicated number of observation or illustrated by an observation representative of a result obtained from different experiments. Probability levels of random differences were determined by analysis of variance followed by Tukey-Kramer multiple comparisons test. Results Plasma levels of insulin, glucagon, and glucose after glucose or Intralipid infusion First, we studied the influence of short-term (24 h) glucose or Intralipid infusion on plasma levels of insulin, glucagon, and glucose. As shown in Figure 1A, no differences in plasma concentration of insulin or glucagon could be found in FF versus OG animals. Animals in the IVG or IL group showed a modest decrease in plasma insulin. Plasma glucagon in the IVG group was moderately higher than that in the FF control or OG group (Fig. 1B). Regarding the IL group, plasma level of glucagon was markedly lower than levels in the other groups (Fig.1B). Plasma glucose level was increased only in the IVG group, whereas levels in the IL group were slightly lower than those in the FF group (Fig. 1C). Confocal microscopy (immunocytochemical) and western blot findings The next series of experiments was designed to study the influence of short-term glucose or Intralipid infusion

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

655

islet cells isolated from OG and IL rats showed a great immunoreactivity for iNOS. Double immunolabeling of these islets showed that most iNOS-immunoreactive cells also expressed insulin immunoreactivity (Fig. 2). Expression of iNOS after IV infusion of glucose or Intralipid was further confirmed by western blot analysis, which visually demonstrated a marked expression of iNOS protein in the islets (Fig. 3). Moreover, islet ncNOS expression was not significantly modulated by the different treatments except for IL infusion, which resulted in the decrease of ncNOS protein as evaluated by densitometry analysis; 16.4 ⫾ 1.5 units (n ⫽ 5) for IL, versus 31.1 ⫾ 3.5 for FF, 25.3 ⫾ 2.5 for OG, and 27.3 ⫾ 3.1 for IVG (P ⬍ 0.05, IL versus other groups; Fig. 3B). Influence of different routes of nutrient administration on islet NOS activities. As seen in Figure 4, IV infusion of glucose or intralipid for 24 h brought about a strong upregulation of islet iNOS activity (IVG and IL groups). No iNOS activity could be detected in islets isolated from the FF and OG groups. However, because of a marked increase in iNOS activity, total NOS activity was clearly increased in the islets of the IVG and IL groups (Fig. 4). No apparent differences in ncNOS activity were found in islets from FF the group compared with the OG and IVG groups (Fig. 4), whereas ncNOS activity in the islets of IL was slightly decreased (P ⬍ 0.05; Fig. 4). Discussion

Fig. 1. Plasma insulin (A), glucagon (B), and glucose (C) in FF control rats, OG (50% glucose) rats, IVG rats, and IL rats after 24 h of treatment. Values are means ⫾SEM from 9–12 animals in each group. Stars denote probability level of random difference for islets from FF versus total parenteral nutrition–treated rats. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001. FF, freely fed; IL, intravenous Intralipid; IVG, intravenous glucose; NS, not significant; OG, oral glucose.

on iNOS expression in pancreatic islets. As seen in Figure 2 no iNOS immunoreactivity was observed in pancreatic islets isolated from FF or OG rats, whereas most

Glucose tolerance tests, orally or intravenously, performed in hyperglycemic or hyperlipidemic subjects have shown a decreased glucose tolerance level [4,25]. The insulin response relative to plasma glucose concentration is markedly lower in hyperglycemic or hyperlipidemic subjects. A previous report claimed that glucose intolerance in such individuals could be due to an increased glucagon level in plasma [19,20]. This could in turn affect the liver and thus increase glycogen breakdown, leading to the increased plasma glucose level seen in these subjects. An inhibitory effect of increased NO production due to an increased activity of cNOS (pathophysiologic condition) or after induction of iNOS (inflammatory condition) on pancreatic ␤-cell function and insulin secretion has been highlighted by reports concerning ␤-cell dysfunction as a consequence of increased NOS isoenzyme activities [8 –10, 26]. NO is now widely accepted as a mediator of ␤-cell dysfunction and apoptosis [5,6,9,18,27,28]. Due to the extremely low levels of NO metabolizing enzymes such as catalase and glutathione peroxidase, pancreatic ␤-cells are susceptible to high levels of intracellularly produced NO [29]. An increased generation of NO may interact with vital sites in the ␤-cell such as Kreb’s cycle enzyme aconitase [30], ion channels [31], or other enzymes

656

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

Fig. 2. Immunostaining and confocal micrographs of formaldehyde-fixed islets from freely fed rats (A–C), orally fed glucose (50%) rats (D–F), intravenous glucose (50%) treated rats (G–I), and Intralipid-infused rats (J–L) for 24 h showing the distribution of insulin (A, D, G, J), inducible nitric oxide synthase (B, E, H, K), and the overlay (C, F, I, L). In endocrine pancreas of freely fed control or orally fed glucose rats, no immunoreactivity for inducible nitric oxide synthase could be detected (B and E), whereas that in endocrine pancreas from intravenous glucose or Intralipid-infused protein ions could be clearly visualized (H and K).

important for normal ␤-cell function [5,6,18,32]. It has been demonstrated in several reports that inhibition of NOS isoenzyme (iNOS and ncNOS) activities by specific inhibitors is always accompanied by increased glucose-stimulated insulin secretion in vitro and in vivo [5,6,9,14].

Effect of IV glucose infusion on iNOS expression in pancreatic islets In a very recent investigation, we found evidence for the expression of iNOS by pancreatic islets where no inflam-

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

657

24 h stimulated the expression and activity of iNOS called our attention to a possible involvement of increased production of NO in the pathogenesis of glucotoxicity and lipo-

Fig. 3. Representative western blots of islets isolated from FF control rats (lane 1), OG (50% glucose rats (lane 2), IVG (50% glucose rats (lane 3), and IL rats (lane 4) and incubated with iNOS or ncNOS antibodies. Blots were performed with 20 ␮g of islet protein on each lane. Arrow indicates molecular weights of 130 kDa (iNOS) and 150 kDa (ncNOS). FF, freely fed; IL, intravenous Intralipid; IVG, intravenous glucose; iNOS, inducible nitric oxide synthase; ncNOS, neuronal constitutive nitric oxide synthase; OG, oral glucose.

matory agents were present. We observed that glucose at high concentrations (⬎12 mmol/L) induced expression of iNOS in incubated pancreatic islets from normal healthy mice and rats when islets were incubated for 60 to 90 min [5,6,14]. In line with our previous in vitro results are the present in vivo data, which further provide evidence that a “hyperglycemia” episode of 24 h can induce the expression of iNOS in pancreatic islets. Interestingly, in accordance with our data regarding induction of iNOS by glucose is a recent study showing that perfusion of rat heart with 33.3 mmol/L of glucose for 2 h results in the induction of iNOS gene expression concomitant with increased NO production [33]. The present study also demonstrates that the route of glucose administration and plasma level of sugar are critical factors concerning the observed effect of glucose on the expression of iNOS in the ␤-cell. The expression pattern of iNOS illustrated by confocal microscopic images from rat receiving IV glucose or Intralipid were in perfect agreement with immunoblots of iNOS protein and biochemically recorded iNOS activity in the islets. This further confirmed our previous hypothesis that ␤-cell dysfunction is associated with route of glucose administration and induction of iNOS may at least in part play a contributing role in such scenario. Prominent characteristics of the metabolic syndrome and diabetes are abnormalities in plasma glucose and plasma FFA (hyperglycemia and hyperlipidemia, respectively). The relative importance of glucotoxicity versus lipotoxicity in inducing ␤-cell dysfunction and apoptosis remains controversial. In a recent study, Robertson et al. [4] proposed that hyperlipidemia-evoked lipotoxicity alone is not sufficient to cause any harmful effect on pancreatic ␤-cell function when no sign of glucotoxicity is present. In contrast, our finding that glucose or Intralipid infusion for a period as short as

Fig. 4. Total NOS, iNOS, and cNOS activities measured as L-citrulline formation (pmol/min per mg protein) in islets isolated from FF control rats, OG (50% glucose) rats, IVG (50% glucose) rats, and IL rats. Mean ⫾ SEM for 9 –12 rats in each group is shown. Asterisks denote probability levels of random differences for islets from FF or OG versus IVG or IL rats. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001. FF, freely fed; IL, intravenous Intralipid; IVG, intravenous glucose; NO, nitric oxide; cNOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; OG, oral glucose.

658

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

toxicity. Our hypothesis is further strengthened by previous studies of cultured pancreatic islets or HIT-T15 cells (a hamster insulin-secreting cell line) reporting a progressive decrease in glucose-stimulated insulin secretion and insulin content on 24 to 48 h of exposure to high glucose of FFA [34,35]. Data obtained thus far in rodent investigations have indicated that the ␤-cell is extremely sensitive to sustained increases in circulating glucose and/or FFA concentrations. Several cellular abnormalities resulting from exposure to high glucose or FFA have been observed in pancreatic ␤-cells, including altered gene expression, increased expression of uncoupling protein-2, increased flux of glucose through the hexosamine biosynthesis pathway, activation of serine/threonine kinases such as protein kinase-C and microtubule-associated protein kinase and enhanced activation of transcriptional factors such as peroxisome proliferator-activated receptor-␥ and nuclear factor-␬B [36 –39]. The precise metabolic signal that results in the induction of iNOS in the ␤-cells is not clear but might likely involve the transcriptional factors peroxisome proliferator-activated receptor-␥, nuclear factor-␬B, or microtubule-associated protein kinase, which may then alter ␤-cell gene expression (negatively modulating ␤-cell replication capacity and/or even inducing ␤-cell death). In contrast to the present finding is a previous report showing that long-term (24 h) hyperglycemia has a beneficial effect on insulin secretion [36]. A reason for this discrepancy might be that the animals in that investigation had free access to food during the period of IV infusion of glucose [36]. Ingested food stimulates the release of gastrointestinal hormones (incretins) such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) that have been shown to be important for optimal function of pancreatic ␤-cells [6,40 – 43]. Conversely, GLP-1 deficiency and/or GLP-1 resistance have been implicated in the pathogenesis of ␤-cell dysfunction in type-2 diabetic patients [43]. An orally administrated glucose load has a comparably greater effect on insulin secretion than an IV glucose load [1]. Thus, the plasma insulin response after glucose ingestion is much greater than that after IV glucose, despite equivalent increases in plasma glucose concentration. This potentiating effect of oral glucose administration is reportedly related to the release of GLP-1 from endocrine cells in gastrointestinal tissues [40 – 42]. It is worth mentioning that GLP-1 not only potentiates glucose-stimulated insulin secretion and suppresses glucagon release but also upregulates genes that control the glucose-sensing machinery, stimulate ␤-cell neogenesis, differentiation and proliferation, and inhibit apoptosis [40,42]. Expression of iNOS in the IVG and IL groups but not in the OG and FF groups can be explained by the loss of IVG or IL to stimulate the release of GLP-1 and GIP, whose secretion is dependent on the orally ingested carbohydrate (GLP-1) or FFA (GIP) [40,42]. It should also be emphasized that the present results are in good agreement with our previous observations that activators of the cyclic adenosine

monophosphate/protein kinase-A pathway, i.e., GLP-1, phosphodiesterase inhibitor, isobuthylmethyl xantine, or pituitary adenylate cyclase activator, suppress the expression and activity of iNOS in isolated islets, thus implicating the importance of the cyclic adenosine monophosphate/protein kinase-A system as a negative modulator of iNOS expression [5,6]. The slight decrease in ncNOS expression and activity observed in the islets of IL-infused rats suggests interactive mechanisms between FFA and islet ncNOS, in which a signal generated by FFA might exert an inhibitory effect on ncNOS expression and activity. Apart from insulin, glucagon also plays a central role in the maintenance of a normal blood glucose level [44,45]. Although insulin stimulates glucose uptakes by peripheral tissues and suppresses endogenous glucose production by the liver, glucogon exerts an opposite effect [44,45]. Thus, increased plasma levels of glucagon are concomitantly followed by an increased plasma glucose level [45]. Numerous studies have indicated that the effectiveness of increased plasma glucose concentrations in suppressing glucagon release may be dependent on a permissive effect of insulin [40,41,44,45]. A disturbed insulin secretory response in ␤- cells due to induction of iNOS might be the cause of the exaggerated glucagon secretion seen in the IVG group. It should be mentioned that NO has a dual action on insulin and glucagon secretions, i.e., inhibiting insulin release but positively modulating glucagon release [5,6,9,46]. The detailed mechanism of action of NO in stimulating glucagon release is unclear. However, we previously showed that hydroxylamine (an intracellular NO donor) markedly stimulates glucagon secretion from isolated pancreatic islets [47]. Regarding plasma glucose levels, most studies thus far have demonstrated that plasma glucose and glucose uptake by peripheral tissues over a period of 4 to 12 h after ingestion of an oral glucose load is normal or even slightly decreased [1,19]. Changes in plasma glucagon levels are closely associated with similar changes in plasma glucose concentrations, which are amply illustrated in the present study. Effect of IV lipid infusion on iNOS expression in pancreatic islets Our finding of a decreased plasma insulin level in response to lipid infusion for 24 h is in good accordance with previous investigations on isolated pancreatic islets that showed that long-term exposure of islets to FFA (24 to 72 h) is accompanied by morphologic changes in ␤-cells associated, with a marked decrease in insulin secretory response to glucose [48], and a recent study has suggested a clear relation between the high production of NO (intracellularly) and chromatin clumping [49]. The present study indicates that this defect might, at least in part, be explained by the induction of iNOS in pancreatic islet cells. Expression of iNOS represents a sustained and exaggerated source of NO generation in the islet cells [5,6]. Although induction of

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

iNOS could not account entirely for alterations in ␤-cell survival, i.e., induction of apoptosis, it might negatively modulate the secretory function of ␤-cells. In this respect, it should be mentioned that long-term (24 to 72 h) exposure of ␤-cells to FFA results in a marked production of reactive oxygen species such as superoxide anion (O2⫺) [7,49]. Combination of NO and O2⫺ results in the formation of peroxynitrite, which is a powerful oxidant and cytotoxic molecule. The increase in NO, O2⫺, and peroxynitrite concentrations are positively correlated to mitochondria and DNA damage in the ␤-cell [27,50]. It is well known that an increased plasma FFA obtained by IV infusion of lipids results in a decreased plasma level of glucagon in humans and rats [51]. Conversely, lowering of plasma FFA with the antilipolytic agent, nicotinic acid, raises plasma glucagon levels in humans and rats [49]. Glucagon stimulates lipolysis in adipocytes and decreases hepatic triacylglycerol synthesis, resulting in increased plasma FFA [16,51]. Thus, an increased plasma FFA concentration during IV lipid infusion (present study) may curtail an exaggerated glucagon release and thereby suppress glucose production through decreased glycogenesis and gluconeogenesis in the liver (Fig. 1). Further, IV treatment with nutrients is regularly seen in clinical situations, preferably pre- and postoperatively [16,19]. If IV glucose induces increased NO production in human ␤-cells, this may well explain some of the adverse events seen during IV treatment with nutrients. Conclusion From the present data we propose that a sustained hyperglycemic episode, as caused by an IV infusion of glucose for 24 h, is capable of inducing iNOS expression in pancreatic islet cells. From a clinical point of view, our finding might be one explanation to the disturbed glucose homeostasis seen postoperatively in patients treated with IVG and fasting. Regarding iNOS expression in islets, a comparable result was observed when Intralipid was infused for the same period. The mediator of action of glucose or FFA on the induction of iNOS is presently unknown. Studies are in progress to explore the origin and detailed mechanisms of such mediators. Acknowledgment The technical assistance of Britt-Marie Nilsson is gratefully acknowledged. References [1] Salehi A, Chen D, Håkanson R, Nordin G, Lundquist I. Gastrectomy induces impaired insulin and glucagon secretion: evidence for a gastro-insular axis in mice. J Physiol 1999;514(pt2):579 –91.

659

[2] Sudo T, Ishiyama K, Takemoto M, Kawamura M, Umemura H, Shiraha S. Pancreatic endocrine function after total gastrectomy and truncal vagotomy. Am J Surg 1982;144:539 – 44. [3] Vigili de Kreutzenberg S, Lisato G, Riccio A, Giunta F, Bonato R, Petolillo M. Metabolic control during total parenteral nutrition: use of an artificial endocrine pancreas. Metabolism 1988;37:510 –3. [4] Robertson RP, Harmon J, Tran PO, Poitout V. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes 2004;53(suppl 1):S119 –24. [5] Salehi A, Ekelund M, Henningsson R, Lundquist I. Total parenteral nutrition modulates hormone release by stimulating expression and activity of inducible nitricoxide synthase in rat pancreatic islets. Endocrine 2001;16:97–104. [6] Salehi A, Ekelund M, Lundquist I. Total parenteral nutrition-stimulated activity ofinducible nitric oxide synthase in rat pancreatic islets is suppressed by glucagon-like peptide-1. Horm Metab Res 2003;35:48 –54. [7] Krauss S, Zhang CY, Scorrano L, Dalgaard LT, St-Pierre J, Grev ST, Lowell BB. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction. J Clin Invest 2003;112:1831– 42. [8] Corbett JA, McDaniel ML. Does nitric oxide mediate autoimmune destruction of beta-cells? Possible therapeutic interventions in IDDM. Diabetes 1992;41:897–903. [9] Salehi A, Carlberg M, Henningson R, Lundquist I. Islet constitutive nitric oxidesynthase: biochemical determination and regulatory function. Am J Physiol 1996;270(6 pt 1):C1634 – 41. [10] Delaney CA, Eizirik DL. Intracellular targets for nitric oxide toxicity to pancreatic beta-cells. Braz J Med Biol Res 1996;29:569 –79. [11] Delaney CA, Tyrberg B, Bouwens L, Vaghef H, Hellman B, Eizirik DL. Sensitivity of human pancreatic islets to peroxynitrite-induced cell dysfunction and death. FEBS Lett 1996;394:300 – 6. [12] Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001;357(pt 3):593– 615. [13] Forstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn Schmiedebergs Arch Pharmacol 1995;352:351– 64. [14] Henningsson R, Salehi A, Lundquist I. Role of nitric oxide synthase isoforms in glucose-stimulated insulin release. Am J Physiol Cell Physiol 2002;283:C296 –304. [15] Shimabukuro M, Ohneda M, Lee Y, Unger RH. Role of nitric oxide in obesity-induced beta cell disease. J Clin Invest 1997;100:290 –5. [16] Knapke CM, Owens JP, Mirtallo JM. Management of glucose abnormalities in patients receiving total parenteral nutrition. Clin Pharm 1989;8:136 – 44. [17] Quirk J. Malnutrition in critically ill patients in intensive care units. Br J Nurs 2000;9:537– 41. [18] Salehi zA, Fan BG, Ekelund M, Nordin G, Lundquist I. TPN-evoked dysfunction of islet lysosomal activity mediates impairment of glucose-stimulated insulin release. Am J Physiol Endocrinol Metab 2001;281:E171–9. [19] Ljungqvist O, Soreide E. Preoperative fasting. Br J Surg 2003;90: 400 – 6. [20] Ljungqvist O, Thorell A, Gutniak M, Haggmark T, Efendic S. Glucose infusion instead of preoperative fasting reduces postoperative insulin resistance. J Am Coll Surg 1994;178:329 –36. [21] Fan BG, Salehi A, Sternby B, Axelson J, Lundquist I, AndrenSandberg A, Ekelund M. Total parenteral nutrition influences both endocrine and exocrine function of rat pancreas. Pancreas 1997;15: 147–53. [22] Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP. An improved method for isolation of mouse pancreatic islets. Transplantation 1985; 40:437– 8. [23] Qader SS, Ekelund M, Andersson R, Obermuller S, Salehi A. Acute pancreatitis, expression of inducible nitric oxide synthase and defective insulin secretion. Cell Tissue Res 2003;313:271–9.

660

M. Ekelund et al. / Nutrition 22 (2006) 652– 660

[24] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. [25] Unger RH. Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications. Diabetes 1995;44:863– 70. [26] Eizirik DL, Flodstrom M, Karlsen AE, Welsh N. The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells. Diabetologia 1996;394:875–90. [27] Eizirik DL, Delaney CA, Green MH, Cunningham JM, Thorpe JR, Pipeleers DG, et al. Nitric oxide donors decrease the function and survival of human pancreatic islets. Mol Cell Endocrinol 1996;118: 71– 83. [28] de-Mello MA, Flodstrom M, Eizirik DL. Ebselen and cytokineinduced nitric oxide synthase expression in insulin-producing cells. Biochem Pharmacol 1996;52:1703–9. [29] Ammon HP, Mark M. Thiols and pancreatic beta-cell function: a review. Cell Biochem Funct 1985;3:157–71. [30] Welsh N, Sandler S. Interleukin-1 beta induces nitric oxide production and inhibits the activity of aconitase without decreasing glucose oxidation rates in isolated mouse pancreatic islets. Biochem Biophys Res Commun 1992;182:333– 40. [31] Tsuura Y, Ishida H, Hayashi S, Sakamoto K, Horie M, Seino Y. Nitric oxide opens ATP-sensitive K⫹channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells. J Gen Physiol 1994;104:1079 –98. [32] Tsuura Y, Ishida H, Shinomura T, Nishimura M, Seino Y. Endogenous nitric oxide inhibits glucose-induced insulin secretion by suppression of phosphofructokinase activity in pancreatic islets. Biochem Biophys Res Commun 1998;252:34 – 8. [33] Ceriello A, Quagliaro L, D’Amico M, Di Filippo C, Marfella R, Nappo F, et al. Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat. Diabetes 2002;51:1076 – 82. [34] Kajimoto Y, Kaneto H. Role of oxidative stress in pancreatic beta-cell dysfunction. Ann N Y Acad Sci 2004;1011:168 –76. [35] Harmon JS, Gleason CE, Tanaka Y, Poitout V, Robertson RP. Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats. Diabetes 2001;50:2481– 6 [36] N’Guyen JM, Magnan C, Laury MC, Thibault C, Leveteau J, Gilbert M, et al. involvement of autonomic nervous system in the in vivo memory to glucose of pancreatic ␤cell in rats. Clin Invest 1994;94: 1456 – 62. [37] Busch AK, Cordery D, Denyer GS, Biden TJ. Expression profiling of palmitate- and oleate-regulated genes provides novel insights into the effects of chronic lipid exposure on pancreatic beta-cell function. Diabetes 2002;51:977– 87. [38] Kim WH, Lee JW, Suh YH, Hong SH, Choi JS, Lim JH, et al. Exposure to chronic high glucose induces {beta}-cell apoptosis

[39]

[40]

[41]

[42]

[43]

[44] [45]

[46]

[47]

[48]

[49]

[50]

[51]

through decreased interaction of glucokinase with mitochondria: downregulation of glucokinase in pancreatic {beta}-cells. Diabetes 2005;54:2602–11. Wang X, Li H, De Leo D, Guo W, Koshkin V, Fantus IG, et al. Gene and protein kinase expression profiling of reactive oxygen speciesassociated lipotoxicity in the pancreatic beta-cell line M1N6. Diabetes 2004;53:129 – 40. Hansotia T, Drucker DJ GIP and GLP-1 as incretin hormones: lessons from single and double incretin receptor knockout mice Regul Pept 2005;128:125–34. Holst JJ, Orskov C. The incretin approach for diabetes treatment: modulation of islet hormone release by GLP-1 agonism. Diabetes 2004;53(suppl 3):S197–204. Herbach N, Goeke B, Schneider M, Hermanns W, Wolf E, Wank R. Overexpression of a dominant negative GIP receptor in transgenic mice results in disturbed postnatal pancreatic islet and beta-cell development. Regul Pept 2005;125:103–17. Preitner F, Ibberson M, Franklin I, Binnert C, Pende M, Gjinovci A, et al. Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors. J Clin Invest 2004;113:635– 45. Jiang G, Zhang BB. Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 2003;284:E671– 8. Shah P, Vella A, Basu A, Schwenk WF, Rizza RA. Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab. 2000; 85:4053–9. Henningsson R, Alm P, Lindstrom E, Lundquist I. Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release. Am J Physiol Endocrinol Metab: 2000;279:E95–107. Salehi A, Parandeh F, Lundquist I. Signal transduction in islet hormone release: interaction of nitric oxide with basal and nutrientinduced hormone responses. Cell Signal 1998;10:645–51. Lupi R, Dotta F, Marselli L, Del Guerra S, Masini M, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated.Diabetes 2002;51:1437– 42. Koshkin V, Wang X, Scherer PE, Chan CB, Wheeler MB, Mitochondrial functional state in clonal pancreatic beta-cells exposed to free fatty acids. J Biol Chem 2003;278:19709 –15. Heitmeier MR, Arnush M, Scarim AL, Corbett JA. Pancreatic betacell damage mediated by beta-cell production of interleukin-1. A novel mechanism for virus-induced diabetes. J Biol Chem 2001;276: 11151– 8. Gerich JE, Langlois M, Schneider V, Karam JH, Noacco C. Effects of alternations of plasma free fatty acid levels on pancreatic glucagon secretion in man. J Clin Invest 1974;53:1284 –9.