Metformin acts in the gut and induces gut-liver crosstalk.

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Estradiol Protects Female ApoE KO Mice against Western-Diet-Induced Non-Alcoholic Steatohepatitis.

The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is higher in men than in women of reproductive age, and postmenopausal women are especially susceptible to developing the disease. AIM: we evaluated if female apolipoprotein E (ApoE) KO mice were protected against Western-diet (WD)-induced NASH. METHODS: Female ovariectomized (OVX) ApoE KO mice or sham-operated (SHAM) mice were fed either a WD or a regular chow (RC) for 7 weeks. Additionally, OVX mice fed a WD were treated with either estradiol (OVX + E2) or vehicle (OVX). RESULTS: Whole-body fat, plasma glucose, and plasma insulin were increased and associated with increased glucose intolerance in OVX mice fed a WD (OVX + WD). Plasma and hepatic triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) hepatic enzymes were also increased in the plasma of OVX + WD group, which was associated with hepatic fibrosis and inflammation. Estradiol replacement in OVX mice reduced body weight, body fat, glycemia, and plasma insulin associated with reduced glucose intolerance. Treatment also reduced hepatic triglycerides, ALT, AST, hepatic fibrosis, and inflammation in OVX mice. CONCLUSIONS: These data support the hypothesis that estradiol protects OVX ApoE KO mice from NASH and glucose intolerance.

Western Diet-Fed ApoE Knockout Male Mice as an Experimental Model of Non-Alcoholic Steatohepatitis.

One of the consequences of the Western lifestyle and high-fat diet is non-alcoholic fatty liver disease (NAFLD) and its aggressive form, non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC) and is rapidly becoming the leading cause of end-stage liver disease or liver transplantation. Currently, rodent NASH models lack significant aspects of the full NASH spectrum, representing a major problem for NASH research. Therefore, this work aimed to characterize a fast rodent model with all characteristic features of NASH. Eight-week-old male ApoE KO mice were fed with Western diet (WD), high fatty diet (HFD) or normal chow (Chow) for 7 weeks. Whole-body fat was increased by ~2 times in WD mice and HFD mice and was associated with increased glucose intolerance, hepatic triglycerides, and plasma ALT and plasma AST compared with Chow mice. WD mice also showed increased galectin-3 expression compared with Chow or HFD mice and increased plasma cholesterol compared with Chow mice. WD and HFD displayed increased hepatic fibrosis and increased F4/80 expression. WD mice also displayed increased levels of plasma MCP-1. Hepatic inflammatory markers were evaluated, and WD mice showed increased levels of TNF-α, MCP-1, IL-6 and IFN-γ. Taken together, these data demonstrated that the ApoE KO mouse fed with WD is a great model for NASH research, once it presents the fundamental parameters of the disease, including hepatic steatosis, fibrosis, inflammation, and metabolic syndrome.

Melatonin improves insulin sensitivity independently of weight loss in old obese rats.

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8- and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1-fold increase both after 8- and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin-induced phosphorylation of the analyzed ISP proteins increased 1.3- and 2.3-fold after 8- and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS-1, 2) remained unchanged in all investigated tissues, except for the 2-fold increase in the total amount of IRS-1 in the periepididymal adipose tissue. Therefore, the known age-related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

Diet-induced obesity impairs AKT signalling in the retina and causes retinal degeneration.

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT(1) , eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes.

A comprehensive review on phytochemicals for fatty liver: are they potential adjuvants?

Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome and, as such, is associated with obesity. With the current and growing epidemic of obesity, NAFLD is already considered the most common liver disease in the world. Currently, there is no official treatment for the disease besides weight loss. Although there are a few synthetic drugs currently being studied, there is also an abundance of herbal products that could also be used for treatment. With the World Health Organization (WHO) traditional medicine strategy (2014-2023) in mind, this review aims to analyze the mechanisms of action of some of these herbal products, as well as evaluate toxicity and herb-drug interactions available in literature.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice.

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

Reference Gene and Protein Expression Levels in Two Different NAFLD Mouse Models.

The expression levels of some reference genes and proteins are used for data normalization and quantification. However, these levels can change in response to experimental conditions or treatments. Aim. The aim of this work was to evaluate reference gene and protein expression in models of nonalcoholic fatty liver disease, using mice fed with a high-fat diet (HFD) and mice that are genetically obese (ob/ob). Main Methods. Histological staining techniques were used to verify the morphology and quantify the amount of lipid droplets present in the liver. Real-time polymerase chain reaction and immunoblotting were employed for monitoring protein expression and gene expression levels, respectively. Key Finding. The results showed that there was a substantial increase in the amount of lipid droplets in the livers of HFD and ob/ob animals when compared to the standard diet (SD) group. There was an observed reduction in the expression of ß-actin (10%), α-tubulin (6%), GAPDH (19%), and RPL3 (15%) genes when comparing the ob/ob group to the HFD group. Additionally, the ob/ob mice displayed GAPDH protein levels that were substantially, but not significantly, reduced when compared to SD. Significance. It was concluded that there are slight differences in the expression levels of reference genes and proteins in these two NAFLD animal models, and researchers should consider these alterations when working with these models.

Persistent activation of Akt or ERK prevents the toxicity induced by saturated and polyunsaturated fatty acids in RINm5F ß-cells.

The aim of this study was to investigate whether the toxicity of saturated and polyunsaturated fatty acids (PUFA) on RINm5F cells is related to the phosphorylation state of Akt, ERK and PKC delta. The regulation of these kinases was compared in three experimental designs: (a) 4h-exposure, (b) 4h-exposure and a subsequent withdrawn of the FA for a 20 h period and (c) 24h-exposure. Saturated and PUFA were toxic to RINm5F cells even at low concentrations. Also, evidence is provided for a late (i.e. the effect only appeared hours after the treatment) and a persistent regulation (i.e. maintenance of the effect for several hours) of Akt, ERK and PKC delta phosphorylation by the FA. Late activation of PKC delta seems important for palmitate cytotoxicity. Persistent activation of the survival proteins Akt and ERK by stearate, oleate and arachidonate might play an important role to prevent the toxic effect of posterior PKC delta activation. The results shown may explain why a short-period exposure to FA is not enough to induce cytotoxicity in pancreatic beta-cells, since survival pathways are activated. Besides, when this activation is persistent, it may overcome a posterior induction of death pathways.

Uncaria tomentosa improves insulin sensitivity and inflammation in experimental NAFLD.

We investigated the effect of the crude herbal extract from Uncaria tomentosa (UT) on non-alcoholic fatty liver disease (NAFLD) in two models of obesity: high fat diet (HFD) and genetically obese (ob/ob) mice. Both obese mouse models were insulin resistant and exhibited an abundance of lipid droplets in the hepatocytes and inflammatory cell infiltration in the liver, while only the HFD group had collagen deposition in the perivascular space of the liver. UT treatment significantly reduced liver steatosis and inflammation in both obese mouse models. Furthermore, serine phosphorylation of IRS-1 was reduced by 25% in the HFD mice treated with UT. Overall, UT treated animals exhibited higher insulin sensitivity as compared to vehicle administration. In conclusion, Uncaria tomentosa extract improved glucose homeostasis and reverted NAFLD to a benign hepatic steatosis condition and these effects were associated with the attenuation of liver inflammation in obese mice.

New insights into fatty acid modulation of pancreatic beta-cell function.

Insulin resistance states as found in type 2 diabetes and obesity are frequently associated with hyperlipidemia. Both stimulatory and detrimental effects of free fatty acids (FFA) on pancreatic beta cells have long been recognized. Acute exposure of the pancreatic beta cell to both high glucose concentrations and saturated FFA results in a substantial increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose-induced insulin release but palmitate can augment insulin release in the presence of nonstimulatory concentrations of glucose. These results imply that changes in physiological plasma levels of FFA are important for regulation of beta-cell function. Although it is widely accepted that fatty acid (FA) metabolism (notably FA synthesis and/or formation of LC-acyl-CoA) is necessary for stimulation of insulin secretion, the key regulatory molecular mechanisms controlling the interplay between glucose and fatty acid metabolism and thus insulin secretion are not well understood but are now described in detail in this review. Indeed the correct control of switching between FA synthesis or oxidation may have critical implications for beta-cell function and integrity both in vivo and in vitro. LC-acyl-CoA (formed from either endogenously synthesized or exogenous FA) controls several aspects of beta-cell function including activation of certain types of PKC, modulation of ion channels, protein acylation, ceramide- and/or NO-mediated apoptosis, and binding to and activating nuclear transcriptional factors. The present review also describes the possible effects of FAs on insulin signaling. We have previously reported that acute exposure of islets to palmitate up-regulates some key components of the intracellular insulin signaling pathway in pancreatic islets. Another aspect considered in this review is the potential source of fatty acids for pancreatic islets in addition to supply in the blood. Lipids can be transferred from leukocytes (macrophages) to pancreatic islets in coculture. This latter process may provide an additional source of FAs that may play a significant role in the regulation of insulin secretion.

Dehydroepiandrosterone increases beta-cell mass and improves the glucose-induced insulin secretion by pancreatic islets from aged rats.

The effect of dehydroepiandrosterone (DHEA) on pancreatic islet function of aged rats, an animal model with impaired glucose-induced insulin secretion, was investigated. The following parameters were examined: morphological analysis of endocrine pancreata by immunohistochemistry; protein levels of insulin receptor, IRS-1, IRS-2, PI 3-kinase, Akt-1, and Akt-2; and static insulin secretion in isolated pancreatic islets. Pancreatic islets from DHEA-treated rats showed an increased beta-cell mass accompanied by increased Akt-1 protein level but reduced IR, IRS-1, and IRS-2 protein levels and enhanced glucose-stimulated insulin secretion. The present results suggest that DHEA may be a promising drug to prevent diabetes during aging.

Up-regulation of the phosphatidylinositol 3-kinase/protein kinase B pathway in the ovary of rats by chronic treatment with hCG and insulin.

Polycystic ovary syndrome (PCOS) manifests as chronic anovulation, ovarian hyperandrogenism, and follicular cysts, which are amplified by insulin as well as the inability of the hormone to stimulate glucose uptake in classic target tissues such as muscle and fat. In the present study, we evaluated the regulation of the insulin-signaling pathways by using immunoprecipitation and immunoblotting in whole extracts of ovaries from non-pregnant human chorionic gonadotropin (hCG)-treated rats, hyperinsulinemic-induced rats and hyperinsulinemic-induced rats, treated with hCG for 22 consecutive days. There were increased associations of insulin receptor substrate (IRS)-1 and IRS-2 with phosphatidylinositol (PI) 3-kinase, followed by enhanced protein kinase B (Akt) serine and threonine phosphorylation, in the ovaries of rats that were treated with hCG, either alone or with insulin. In contrast, the skeletal muscle demonstrated a reduced IRS-1/PI 3-kinase/Akt pathway in hyperinsulinemic-induced rats. These intracellular modifications were accompanied by follicular cysts, detected by optical microscopy, and increased androstenedione serum levels. In summary, our data show that chronic treatment with hCG or hCG plus insulin can induce changes in ovaries that simulate PCOS. In these situations, an increase in the insulin-induced IRS/PI 3-kinase/Akt pathway occurs in the ovary, suggesting that the activation of this pathway may have a role in the development of PCOS.

ERK3 associates with MAP2 and is involved in glucose-induced insulin secretion.

The adaptation of pancreatic islets to pregnancy includes increased beta cell proliferation, expansion of islet mass, and increased insulin synthesis and secretion. Most of these adaptations are induced by prolactin (PRL). We have previously described that in vitro PRL treatment increases ERK3 expression in isolated rat pancreatic islets. This study shows that ERK3 is also upregulated during pregnancy. Islets from pregnant rats treated with antisense oligonucleotide targeted to the PRL receptor displayed a significant reduction in ERK3 expression. Immunohistochemical double-staining showed that ERK3 expression is restricted to pancreatic beta cells. Transfection with antisense oligonucleotide targeted to ERK3 abolished the insulin secretion stimulated by glucose in rat islets and by PMA in RINm5F cells. Therefore, we examined the participation of ERK3 in the activation of a cellular target involved in secretory events, the microtubule associated protein MAP2. PMA induced ERK3 phosphorylation that was companied by an increase in ERK3/MAP2 association and MAP2 phosphorylation. These observations provide evidence that ERK3 is involved in the regulation of stimulus-secretion coupling in pancreatic beta cells.

Ouabain-induced hypertension enhances left ventricular contractility in rats.

Chronic ouabain treatment produces hypertension acting on the central nervous system and at vascular levels. However, cardiac effects in this model of hypertension are still poorly understood. Hence, the effects of hypertension induced by chronic ouabain administration ( approximately 8 microg day(-1), s.c.) for 5 weeks on the cardiac function were studied in Wistar rats. Ouabain induces hypertension but not myocardial hypertrophy. Awake ouabain-treated rats present an increment of the left ventricular systolic pressure and of the maximum positive and negative dP/dt. Isolated papillary muscles from ouabain-treated rats present an increment in isometric force, and this effect was present even when inotropic interventions (external Ca(2+) increment and increased heart rate) were performed. However, the sarcoplasmic reticulum activity and the SERCA-2 protein expression did not change. On the other hand, the activity of myosin ATPase increased without changes in myosin heavy chain protein expression. In addition, the expression of alpha(1) and alpha(2) isoforms of Na(+), K(+)-ATPase also increased in the left ventricle from ouabain-hypertensive rats. The present results showed positive inotropic and lusitropic effects in hearts from awake ouabain-treated rats, which are associated with an increment of the isometric force development and of the activity of myosin ATPase and expression of catalytic subunits of the Na(+), K(+)-ATPase.

Pancreatic beta-cells express phagocyte-like NAD(P)H oxidase.

The presence of a phagocyte-like NAD(P)H oxidase in pancreatic beta-cells was investigated. Three NAD(P)H oxidase components were found in pancreatic islets by RT-PCR: gp91(PHOX), p22(PHOX), and p47(PHOX). The components p67(PHOX) and p47(PHOX) were also demonstrated by Western blotting. Through immunohistochemistry, p47(PHOX) was mainly found in the central area of the islet, confirming the expression of this component by insulin-producing cells. Activation of NAD(P)H oxidase complex in the beta-cells was also examined by immunohistochemistry. The pancreatic islets presented slower kinetics of superoxide production than HIT-T15 cells, neutrophils, and macrophages, but they reached 66% that of the neutrophil nitroblue tetrazolium (NBT) reduction after 2 h of incubation. Glucose (5.6 mmol/l) increased NBT reduction by 75% when compared with control. The involvement of protein kinase C (PKC) in the stimulatory effect of glucose was confirmed by incubation of islets with phorbol myristate acetate (a PKC activator) and bysindoylmaleimide (GF109203X) (a PKC-specific inhibitor). Diphenylene iodonium [an NAD(P)H oxidase inhibitor] abolished the increase of NBT reduction induced by glucose, confirming the NAD(P)H oxidase activity in pancreatic islets. Because reactive oxygen species are involved in intracellular signaling, the phagocyte-like NAD(P)H oxidase activation by glucose may play an important role for beta-cell functioning.

Modulation of growth hormone signal transduction in kidneys of streptozotocin-induced diabetic animals: effect of a growth hormone receptor antagonist.

Growth hormone (GH) and IGFs have a long distinguished history in diabetes, with possible participation in the development of renal complications. The implicated effect of GH in diabetic end-stage organ damage may be mediated by growth hormone receptor (GHR) or postreceptor events in GH signal transduction. The present study investigates the effects of diabetes induced by streptozotocin (STZ) on renal GH signaling. Our results demonstrate that JAK2, insulin receptor substrate (IRS)-1, Shc, ERKs, and Akt are widely distributed in the kidney, and after GH treatment, there is a significant increase in phosphorylation of these proteins in STZ-induced diabetic rats compared with controls. Moreover, the GH-induced association of IRS-1/phosphatidylinositol 3-kinase, IRS-1/growth factor receptor bound 2 (Grb2), and Shc/Grb2 are increased in diabetic rats as well. Immunohistochemical studies show that GH-induced p-Akt and p-ERK activation is apparently more pronounced in the kidneys of diabetic rats. Administration of G120K-PEG, a GH antagonist, in diabetic mice shows inhibitory effects on diabetic renal enlargement and reverses the alterations in GH signal transduction observed in diabetic animals. The present study demonstrates a role for GH signaling in the pathogenesis of early diabetic renal changes and suggests that specific GHR blockade may present a new concept in the treatment of diabetic kidney disease.

Effect of thiopental, pentobarbital and diethyl ether on early steps of insulin action in liver and muscle of the intact rat.

A large number of experimental studies have investigated insulin signaling in rats. In these studies different anaesthetics have been used to anaesthetize rats. However, the direct effects of anaesthetics on the regulation of the early steps of insulin action are not known. In the present study, we investigated the effect of thiopental, pentobarbital and diethyl ether on the plasma glucose disappearance rate, IR, IRS-1 and IRS-2 tyrosine phosphorylation, IRSs association with PI 3-kinase, Akt and Erk phosphorylation, in liver and muscle of rats. Fasting plasma glucose levels were higher in animals anaesthetized with ether. No differences in plasma glucose disappearance rates were observed, however. Insulin-induced IR, IRS-1 and IRS-2 tyrosine phosphorylation, association of these substrates with PI 3-kinase and Akt and ERK phosphorylation were similar in the three groups of animals in both tissues. These data suggest that both thiopental and pentobarbital may be used in studies where changes in insulin signaling are being measured and where adequate general anaesthesia is required.

Novel signal transduction pathway for luteinizing hormone and its interaction with insulin: activation of Janus kinase/signal transducer and activator of transcription and phosphoinositol 3-kinase/Akt pathways.

The actions of LH are mediated through a single class of cell surface LH/human chorionic gonadotropin receptor, which is a member of the G protein-coupled receptor family. In the present study we showed that LH induced rapid tyrosine phosphorylation and activation of the Janus kinase 2 (JAK2) in rat ovary. Upon JAK2 activation, tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT-1), STAT-5b, insulin receptor substrate-1 (IRS-1), and Src homology and collagen homology (Shc) were detected. In addition, LH induced IRS-1/phosphoinositol 3-kinase and Shc /growth factor receptor-binding protein 2 (Grb2) associations and downstream AKT (protein kinase B, homologous to v-AKT) serine phosphorylation and ERK tyrosine phosphorylation, respectively. The simultaneous infusion of insulin and LH induced higher phosphorylation levels of JAK2, STAT5b, IRS-1, and AKT compared with each hormone alone in the whole ovary of normal rats. By immunohistochemistry we demonstrated that these late events take place in follicular cells and both external and internal theca. These results indicate a new signal transduction pathway for LH and show that there is positive cross-talk between the insulin and LH signaling pathways at the level of phosphoinositol 3-kinase/AKT pathway in this tissue.

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets.

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.