Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.     

Incorporation of glucose into lipid of perirenal and subcutaneous adipocytes of rats and sheep: influence of insulin

Adipocytes were prepared by collagenase digestion of perirenal and subcutaneous fat from rats and sheep and were incubated in vitro with various concentrations of glucose and insulin. The lipogenic rate of perirenal and subcutaneous adipocytes of rats showed a quadratic response to glucose concentration. The addition of 10 nM insulin increased lipogenesis, especially at low lipogenic rates. At constant glucose concentrations, insulin concentrations up to 50 nM stimulated lipogenesis a similar amount in adipocytes from both depots. The rate of lipogenesis increased relative to cell volume in perirenal adipocytes only. The lipogenic rate of perirenal and subcutaneous adipocytes of sheep showed a positive linear response to glucose concentration, but insulin did not affect the rate of lipogenesis in adipocytes from either depot. In both rats and sheep, the rate of lipogenesis was higher in the perirenal adipocytes. It was concluded that insulin is unlikely to be the agent responsible for the differential growth rates of subcutaneous and perirenal fat depots in rats or sheep.     

Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro

1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.     

Insulin resistance in human adipocytes occurs downstream of IRS1 after surgical cell isolation but at the level of phosphorylation of IRS1 in type 2 diabetes

Insulin resistance is a cardinal feature of type 2 diabetes and also a consequence of trauma such as surgery. Directly after surgery and cell isolation, adipocytes were insulin resistant, but this was reversed after overnight incubation in 10% CO(2) at 37 degrees C. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)1 was insulin sensitive, but protein kinase B (PKB) and downstream metabolic effects exhibited insulin resistance that was reversed by overnight incubation. MAP-kinases ERK1/2 and p38 were strongly phosphorylated after surgery, but was dephosphorylated during reversal of insulin resistance. Phosphorylation of MAP-kinase was not caused by collagenase treatment during cell isolation and was present also in tissue pieces that were not subjected to cell isolation procedures. The insulin resistance directly after surgery and cell isolation was different from insulin resistance of type 2 diabetes; adipocytes from patients with type 2 diabetes remained insulin resistant after overnight incubation. IRS1, PKB, and downstream metabolic effects, but not insulin-stimulated tyrosine phosphorylation of insulin receptor, exhibited insulin resistance. These findings suggest a new approach in the study of surgery-induced insulin resistance and indicate that human adipocytes should recover after surgical procedures for analysis of insulin signalling. Moreover, we pinpoint the signalling dysregulation in type 2 diabetes to be the insulin-stimulated phosphorylation of IRS1 in human adipocytes.     

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Insulinotropin PACAP potentiates insulin-stimulated glucose uptake in 3T3 L1 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized in pancreatic nerve fibers and islets and potently augments glucose-induced insulin secretion. The present study explored a possible extra-pancreatic action of PACAP. The specific PACAP receptor (PAC1 receptor) was expressed in the rat fat tissue and 3T3-LI adipocytes. PACAP-38 (10 nM) significantly enhanced insulin-induced 2-deoxyglucose uptake by 3T3-L1 adipocytes. Insulin-stimulated phosphatidylinositol 3-kinase activity was further increased by PACAP-38, whereas the tyrosine-phosphorylation of insulin receptor beta-subunit and insulin receptor substrate-1 was unaltered by PACAP-38. These results reveal that PACAP-38 enhances insulin-induced glucose uptake, an effect probably mediated by insulin-stimulated phosphatidyl-inositol 3-kinase, and that PACAP potentiates not only insulin secretion, but also insulin action in adipocytes.     

Docosahexaenoic acid attenuates macrophage-induced inflammation and improves insulin sensitivity in adipocytes-specific differential effects between LC n-3 PUFA

ObjectiveAdipose tissue inflammation with immune cell recruitment plays a key role in obesity-induced insulin resistance (IR). Long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory potential; however, their individual effects on adipose IR are ill defined. We hypothesized that EPA and DHA may differentially affect macrophage-induced IR in adipocytes.MethodsJ774.2 macrophages pretreated with EPA or DHA (50 μM for 5 days) were stimulated with lipopolysaccharide (LPS, 100 ng/ml for 30 min–48 h). Cytokine secretion profiles and activation status of macrophages were assessed by enzyme-linked immunosorbent assay and flow cytometry. Pretreated macrophages were seeded onto transwell inserts and placed over 3T3-L1 adipocytes for 24–72 h; effects on adipocyte–macrophage cytokine cross-talk and insulin-stimulated ³H-glucose transport into adipocytes were monitored.ResultsDHA had more potent anti-inflammatory effects relative to EPA, with marked attenuation of LPS-induced nuclear factor (NF)κB activation and tumor necrosis factor (TNF)α secretion in macrophages. DHA specifically enhanced anti-inflammatory interleukin (IL)-10 secretion and reduced the expression of proinflammatory M1 (F4/80⁺/CD11⁺) macrophages. Co-culture of DHA-enriched macrophages with adipocytes attenuated IL-6 and TNFα secretion while enhancing IL-10 secretion. Conditioned media (CM) from DHA-enriched macrophages attenuated adipocyte NFκB activation. Adipocytes co-cultured with DHA-enriched macrophages maintained insulin sensitivity with enhanced insulin-stimulated ³H-glucose transport, GLUT4 translocation and preservation of insulin-receptor substrate-1 expression compared to co-culture with untreated macrophages. We confirmed that IL-10 expressed by DHA-enriched macrophages attenuates the CM-induced proinflammatory IR phenotype in adipocytes.ConclusionsWe demonstrate an attenuated proinflammatory phenotype of DHA-pretreated macrophages, which when co-cultured with adipocytes partially preserved insulin sensitivity.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

p38 MAPK alpha mediates cytokine-induced IL-6 and MMP-3 expression in human cardiac fibroblasts

Pre-clinical studies suggest that the p38 MAPK signaling pathway plays a detrimental role in cardiac remodeling, but its role in cardiac fibroblast (CF) function is not well defined. We aimed to identify the p38 MAPK subtypes expressed by human CF, study their activation in response to proinflammatory cytokines, and determine which subtypes were important for expression of specific cytokines and matrix metalloproteinases (MMPs).Quantitative real-time RT-PCR analysis of mRNA levels in human CF cultured from multiple patients revealed a consistent pattern of expression with p38α being most abundant, followed by p38γ, then p38δ and only low expression of p38β (3% of p38α mRNA levels). Immunoblotting confirmed marked protein expression of p38α, γ and δ, with little or no expression of p38β. Phospho-ELISA and combined immunoprecipitation/immunoblotting techniques demonstrated that the proinflammatory cytokines IL-1α and TNFα selectively activated p38α and p38γ, but not p38δ. Selective p38α siRNA gene silencing reduced IL-1α-induced IL-6 and MMP-3 mRNA expression and protein secretion, without affecting IL-1α-induced IL-1β and MMP-9 mRNA expression.In conclusion, human CF express the α, γ and δ subtypes of p38 MAPK, and the α subtype is important for IL-1α-induced IL-6 and MMP-3 expression in this cell type.     

Viability criteria of islets of Langerhans isolated and purified using Ficoll

Rejection of islet allografts is generally explained by immunologic problems, due to both cellular and antibody mechanisms. But another great problem is in the isolation of intact and viable islets of Langerhans: it is necessary to use a good method of pancreas distention, to determine the optimal concentration of collagenase for digestion, to select an effective technique for purifying the islets. This study correlates the morphology of isolated pancreatic islets of rats and dogs with secretion of insulin. The islets are incubated in a perifusion system and are tested during four periods; the glucose concentrations of the perifusion fluid are: 5.5 mM during the initial 70 min. period, 16.5 mM during the second 60 min. period, 5.5 mM during the third 60 min. period and 16.5 during the fourth 50 min. period. This "double glucose stimulation" is a good test of islet viability. The intact, viable isolated islets showed a significant increase of insulin secretion during the two 16.5 mM glucose periods. Damaged islets with some little morphologic alterations after showed a good insulin release during the first glucose stimulation, but a very poor insulin response to glucose during the second stimulation period.     

Proinflammatory adipocytokines induce TIMP-1 expression in 3T3-L1 adipocytes

Tissue inhibitor of metalloproteinase (TIMP)-1 is an adipocyte-secreted protein upregulated in obesity which promotes adipose tissue development. Furthermore, the proinflammatory adipocytokines tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-6 induce insulin resistance, and plasma concentrations are increased during weight gain. In the current study, the impact of TNFalpha and IL-6 on TIMP-1 mRNA and protein expression was determined in 3T3-L1 adipocytes. Interestingly, TNFalpha and IL-6 induced TIMP-1 protein secretion more than 3- and 2-fold, respectively. Furthermore, TIMP-1 mRNA was upregulated in a time- and dose-dependent fashion. Inhibitor experiments suggested that nuclear factor kappaB and p 44/42 mitogen-activated protein kinase are involved in both, basal and adipocytokine-induced TIMP-1 expression. Moreover, the thiazolidinedione troglitazone partly reversed TNFalpha- but not IL-6-induced TIMP-1 synthesis. Taken together, we demonstrate that TIMP-1 expression is selectively upregulated in fat cells by proinflammatory adipocytokines and might play a role in maintaining adipose tissue mass in obesity.     

Rosiglitazone regulates IL-6-stimulated lipolysis in porcine adipocytes

Interleukin (IL)-6, a proinflammatory cytokine, stimulates adipocyte lipolysis and induces insulin resistance in obese and diabetic subjects. However, the effects of the anti-diabetic drug rosiglitazone on IL-6-stimulated lipolysis and the underlying molecular mechanism are largely unknown. In this study, we demonstrated that rosiglitazone suppressed IL-6-stimulated lipolysis in differentiated porcine adipocytes by inactivation of extracellular signal-related kinase (ERK). Meanwhile, rosiglitazone enhanced the lipolysis response of adipocytes to isoprenaline. In addition, rosiglitazone significantly reversed IL-6-induced down-regulation of several genes such as perilipin A, peroxisome proliferators activated receptor gamma (PPARγ), and fatty acid synthetase, as well as the up-regulation of IL-6 mRNA. However, mRNA expression of PPARγ coactivator-1 alpha (PCG-1α) was enhanced by rosiglitazone in IL-6-stimulated adipocytes. These results indicate that rosiglitazone suppresses IL-6-stimulated lipolysis in porcine adipocytes through multiple molecular mechanisms.     

Interleukin-6 and interleukin-15 are selectively regulated by lipopolysaccharide and interferon-gamma in primary pig adipocytes

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.     

Effects of insulin on lipolysis and lipogenesis in hamster white adipocytes with high sensitivity to hormones

A modified procedure for preparation of hamster adipocytes by collagenase digestion under carefully controlled conditions has been developed. The adipocytes were 4- to 8-fold more sensitive to catecholamine stimulation of lipolysis than cells prepared by a commonly used method (Hittelman, K.J., Wu, C.F. and Butcher, R.W. (1973) Biochim. Biophys. Acta 304, 188-196) and also more sensitive to the anti-lipolytic action of insulin. The effects of insulin on lipogenesis, measured as [3H]glucose conversion to cell lipids, and on catecholamine-stimulated lipolysis were compared under identical conditions with the same cell batch. Isoprenaline-stimulated lipolysis was found to be half-maximally inhibited by an insulin concentration 8-fold lower than that stimulating lipogenesis to a corresponding extent (half-maximal effects at insulin concentrations of 40 vs. 300 pM). A similar difference was found when cells had been stimulated with adrenaline instead of isoprenaline.     

Plasma PTX3 protein levels inversely correlate with insulin secretion and obesity, whereas visceral adipose tissue PTX3 gene expression is increased in obesity

Plasma acutephase protein pentraxin 3 (PTX3) concentration is dysregulated in human obesity and metabolic syndrome. Here, we explore its relationship with insulin secretion and sensitivity, obesity markers, and adipose tissue PTX3 gene expression. Plasma PTX3 protein levels were analyzed in a cohort composed of 27 lean [body mass index (BMI) ≤ 25 kg/m(2)] and 48 overweight (BMI 25-30 kg/m(2)) men (cohort 1). In this cohort, plasma PTX3 was negatively correlated with fasting triglyceride levels and insulin secretion after intravenous and oral glucose administration. Plasma PTX3 protein and PTX3 gene expression in visceral (VAT) and subcutaneous (SAT) whole adipose tissue and adipocyte and stromovascular fractions were analyzed in cohort 2, which was composed of 19 lean, 28 overweight, and 15 obese subjects (BMI >30 kg/m(2)). An inverse association with body weight and waist/hip ratio was observed in cohort 2. In VAT depots, PTX3 mRNA levels were higher in subjects with BMI >25 kg/m(2) than in lean subjects, positively correlated with IL-1β mRNA levels, and higher in the adipocyte than stromovascular fraction. Human preadipocyte SGBS cell line was used to study PTX3 production in response to factors that obesity entails. In SGBS adipocytes, PTX3 gene expression was enhanced by IL-1β and TNFα but not IL-6 or insulin. In conclusion, the negative correlation between PTX3 and glucose-stimulated insulin secretion suggests a role for PTX3 in metabolic control. PTX3 gene expression is upregulated in VAT depots in obesity, despite lower plasma PTX3 protein, and by some proinflammatory cytokines in cultured adipocytes.     

Short-term regulation of adiponectin secretion in rat adipocytes

Adiponectin belongs to the group of biologically active substances secreted by adipocytes and referred to as adipokines. Disturbances in its secretion and/or action are thought to be involved in the pathogenesis of some metabolic diseases. However, regulation of adiponectin secretion is poorly elucidated. In the present study, short-term regulation of adiponectin secretion in primary rat adipocytes was investigated. Isolated rat adipocytes were incubated in Krebs-Ringer buffer containing 5 mM glucose and insulin alone or in the combination with epinephrine, dibutyryl-cAMP, adenosine A(1) receptor antagonist (DPCPX), palmitate, 2-bromopalmitate or inhibitor of mitochondrial electron transport (rotenone). Adipocyte exposure for 2 h to insulin (1-100 nM) significantly increased secretion of adiponectin compared with secretion observed without insulin. Furthermore, secretion of adiponectin from adipocytes incubated with glucose and insulin was reduced by 1 and 2 microM epinephrine, but not by 0.25 and 0.5 microM epinephrine. Under similar conditions, 1 and 2 mM dibutyryl-cAMP substantially diminished secretion of adiponectin, whereas 0.5 mM dibutyryl-cAMP was ineffective. Secretion of adiponectin was found to be effectively decreased by DPCPX. Moreover, adipocyte exposure to rotenone also resulted in a substantial diminution of secretory response of adipocytes incubated for 2 h with glucose and insulin. It was also demonstrated that palmitate and 2-bromopalmitate (0.06-0.5 mM) failed to affect secretion of leptin. The obtained results indicated that in short-term regulation of adiponectin secretion, insulin and epinephrine exert the opposite effects. These effects appeared as early as after 2 h of exposure. Moreover, deprivation of energy or blockade of adenosine action substantially decreased secretion of adiponectin.     

Inhibition of pancreatic islet monoamine oxidase by adrenergic antagonists and ethanol.

Although the alpha-adrenergic antagonist phentolamine potentiates glucose-stimulated insulin secretion of intact animals, it either does not alter, or it inhibits in vitro insulin secretion. This may be because in the higher concentration used in in vitro studies, phentolamine exerts a second pharmacological effect that counterbalances its primary effect of blocking monoamine action. We recently demonstrated that pancreatic islets contain substantial amounts of monoamine oxidase (MAO), and that MAO inhibitors such as iproniazid and tranylcypromine can alter insulin secretion. In the present study, we determined if other drugs that affect insulin secretion, alter the MAO activity of homogenates of rabbit pancreatic islets (collagenase technique) or liver. Phentolamine, phenoxybenzamine and propranolol (10 muM and 100 muM) inhibit islet and hepatic MAO. Haloperidol (10muM) inhibits hepatic but not islet MAO, while haloperidol (10muM) does not inhibit MAO in either tissue. Ethanol (270 to 2.7mM) inhibits islet MAO. Hepatic MAO is inhibited by high (270 to 180mM) but not by low (27 to 2.7mM) concentrations of ethanol. Collagenase digestion does not increase the sensitivity of islet and liver MAO to inhibition by phentolamine or ethanol. In the absence of added monoamines, phentolamine and phenoxybenzamine do not alter basal or glucose-stimulated insulin secretion from rabbit pancreas. Preincubation of rabbit pancreas with the serotonin precursor 5-hydroxytryptophan (5-HTP) increases the beta cell serotonin content and inhibits glucose-stimulated insulin secretion. Alpha adrenergic antagonists not only fail to block, but actually potentiate the serotonin inhibition of insulin secretion. We conclude that inhibition of islet MAO may cause an increase in islet monoamine content and these monoamines may alter in vitro insulin secretion. One mechanism through which adrenergic antagonists and ethanol modify in vitro insulin secretion may be by inhibiting pancreatic islet MAO.