Pulmonary arterial hypertension - progress in understanding the disease and prioritizing strategies for drug development.

Pulmonary arterial hypertension (PAH), at one time a largely overlooked disease, is now the subject of intense study in many academic and biotech groups. The availability of new treatments has increased awareness of the condition. This in turn has driven a change in the demographics of PAH, with an increase in the mean age at diagnosis. The diagnosis of PAH in more elderly patients has highlighted the need for careful phenotyping of patients and for further studies to understand how best to manage pulmonary hypertension associated with, for example, left heart disease. The breadth and depth of expertise focused on unravelling the molecular pathology of PAH has yielded novel insights, including the role of growth factors, inflammation and metabolic remodelling. The description of the genetic architecture of PAH is accelerating in parallel, with novel variants, such as those reported in potassium two-pore domain channel subfamily K member 3 (KCNK3), adding to the list of more established mutations in genes associated with bone morphogenetic protein receptor type 2 (BMPR2) signalling. These insights have supported a paradigm shift in treatment strategies away from simply addressing the imbalance of vasoactive mediators observed in PAH towards tackling more directly the structural remodelling of the pulmonary vasculature. Here, we summarize the changing clinical and molecular landscape of PAH. We highlight novel drug therapies that are in various stages of clinical development, targeting for example cell proliferation, metabolic, inflammatory/immune and BMPR2 dysfunction, and the challenges around developing these treatments. We argue that advances in the treatment of PAH will come through deep molecular phenotyping with the integration of clinical, genomic, transcriptomic, proteomic and metabolomic information in large populations of patients through international collaboration. This approach provides the best opportunity for identifying key signalling pathways, both as potential drug targets and as biomarkers for patient selection. The expectation is that together these will enable the prioritization of potential therapies in development and the evolution of personalized medicine for PAH.

Degradation of islet amyloid polypeptide by neprilysin.

AIMS/HYPOTHESIS: A progressive loss of pancreatic beta cell function, a decrease in beta cell mass and accumulation of islet amyloid is characteristic of type 2 diabetes mellitus. The main constituent of islet amyloid is islet amyloid polypeptide (IAPP). In this study, we examined the ability of the peptidase neprilysin to cleave IAPP and prevent human IAPP-induced pancreatic beta cell toxicity. METHODS: Neprilysin and a catalytically compromised neprilysin mutant were tested for their ability to inhibit human IAPP fibrillisation and human IAPP-induced pancreatic beta cell cytotoxicity. Degradation of human IAPP by neprilysin was followed by HPLC, and the degradation products were identified by MS. RESULTS: Neprilysin prevented IAPP fibrillisation by cleaving IAPP at Arg(11)-Leu(12), Leu(12)-Ala(13), Asn(14)-Phe(15), Phe(15)-Leu(16), Asn(22)-Phe(23) and Ala(25)-Ile(26). It also appears to prevent human IAPP fibrillisation through a non-catalytic interaction. Neprilysin protected against beta cell cytotoxicity induced by exogenously added or endogenously produced human IAPP. CONCLUSIONS/INTERPRETATION: The data presented support a potential therapeutic role for neprilysin in preventing type 2 diabetes mellitus. This study supports the hypothesis that extracellular human IAPP contributes to human IAPP-induced beta cell cytotoxicity. Whether human IAPP exerts its cytotoxic effect through a totally extracellular mechanism or through a cellular reuptake mechanism is unclear at this time.

Iron deficiency in pulmonary arterial hypertension: a potential therapeutic target.

Iron deficiency is known to be common and detrimental in chronic left heart failure, where parenteral iron treatment has been shown to improve exercise capacity, New York Heart Association functional class and patient wellbeing. There is now increasing interest in the role of iron in the natural history of pulmonary arterial hypertension (PAH). Iron availability influences the pulmonary vasoconstrictor response to hypoxia and accumulating evidence indicates that iron deficiency is prevalent in idiopathic and heritable forms of PAH, iron status being related to exercise capacity, symptoms and poorer survival in patients with idiopathic PAH (IPAH). Potential mechanisms behind iron deficiency in IPAH include inhibition of dietary iron uptake by the master iron regulator hepcidin. High hepcidin levels underlie the anaemia of chronic disease. Possible stimuli of the observed high levels of hepcidin in IPAH include dysfunctional bone morphogenetic protein receptor type II signalling and inflammation. Iron status may influence outcomes through modulation of the pulmonary circulation as well as myocardial and skeletal muscle function. Two parallel studies, from our centre (Hammersmith Hospital, London, UK) and others in the UK and Amsterdam (the Netherlands), investigating the safety and potential benefit of iron supplementation in patients with PAH are currently under way.

Arsenic: toxicity, oxidative stress and human disease.

Arsenic (As) is a toxic metalloid element that is present in air, water and soil. Inorganic arsenic tends to be more toxic than organic arsenic. Examples of methylated organic arsenicals include monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)]. Reactive oxygen species (ROS)-mediated oxidative damage is a common denominator in arsenic pathogenesis. In addition, arsenic induces morphological changes in the integrity of mitochondria. Cascade mechanisms of free radical formation derived from the superoxide radical, combined with glutathione-depleting agents, increase the sensitivity of cells to arsenic toxicity. When both humans and animals are exposed to arsenic, they experience an increased formation of ROS/RNS, including peroxyl radicals (ROO•), the superoxide radical, singlet oxygen, hydroxyl radical (OH•) via the Fenton reaction, hydrogen peroxide, the dimethylarsenic radical, the dimethylarsenic peroxyl radical and/or oxidant-induced DNA damage. Arsenic induces the formation of oxidized lipids which in turn generate several bioactive molecules (ROS, peroxides and isoprostanes), of which aldehydes [malondialdehyde (MDA) and 4-hydroxy-nonenal (HNE)] are the major end products. This review discusses aspects of chronic and acute exposures of arsenic in the etiology of cancer, cardiovascular disease (hypertension and atherosclerosis), neurological disorders, gastrointestinal disturbances, liver disease and renal disease, reproductive health effects, dermal changes and other health disorders. The role of antioxidant defence systems against arsenic toxicity is also discussed. Consideration is given to the role of vitamin C (ascorbic acid), vitamin E (α-tocopherol), curcumin, glutathione and antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase in their protective roles against arsenic-induced oxidative stress.

Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass.

AIMS/HYPOTHESIS: Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition. METHODS: Wistar rats (2 and 6 months old) received a 72 h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry. RESULTS: A 72 h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets. CONCLUSIONS/INTERPRETATION: In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.

The influence of viral infection on a plankton ecosystem undergoing nutrient enrichment.

It is increasingly recognised that viruses are a significant active component of oceanic plankton ecosystems. They play an important role in biogeochemical cycles as well as being implicated in observed patterns of species abundance and diversity. The influence of viral infection in plankton ecosystems is not fully understood. Here we use a number of well-founded mathematical models to investigate the interplay of the ecological and epidemiological interactions of plankton and viruses in the sea. Of particular interest is the role of nutrient on the population dynamics. Nutrient forcing has been suggested as a means of absorbing excess anthropogenic atmospheric carbon dioxide by stimulating increased phytoplankton primary productivity. Here we show that enriching nutrient levels in the sea may decrease the amount of infected phytoplankton species thereby additionally enhancing the efficiency of the biological pump, a means by which carbon is transferred from the atmosphere to the deep ocean.

The Lévy flight paradigm: random search patterns and mechanisms.

Over recent years there has been an accumulation of evidence from a variety of experimental, theoretical, and field studies that many organisms use a movement strategy approximated by Lévy flights when they are searching for resources. Lévy flights are random movements that can maximize the efficiency of resource searches in uncertain environments. This is a highly significant finding because it suggests that Lévy flights provide a rigorous mathematical basis for separating out evolved, innate behaviors from environmental influences. We discuss recent developments in random-search theory, as well as the many different experimental and data collection initiatives that have investigated search strategies. Methods for trajectory construction and robust data analysis procedures are presented. The key to prediction and understanding does, however, lie in the elucidation of mechanisms underlying the observed patterns. We discuss candidate neurological, olfactory, and learning mechanisms for the emergence of Lévy flight patterns in some organisms, and note that convergence of behaviors along such different evolutionary pathways is not surprising given the energetic efficiencies that Lévy flight movement patterns confer.

Variational data assimilation with epidemic models.

Mathematical modelling is playing an increasing role in developing an understanding of the dynamics of communicable disease and assisting the construction and implementation of intervention strategies. The threat of novel emergent pathogens in human and animal hosts implies the requirement for methods that can robustly estimate epidemiological parameters and provide forecasts. Here, a technique called variational data assimilation is introduced as a means of optimally melding dynamic epidemic models with epidemiological observations and data to provide forecasts and parameter estimates. Using data from a simulated epidemic process the method is used to estimate the start time of an epidemic, to provide a forecast of future epidemic behaviour and estimate the basic reproductive ratio. A feature of the method is that it uses a basic continuous-time SIR model, which is often the first point of departure for epidemiological modelling during the early stages of an outbreak. The method is illustrated by application to data gathered during an outbreak of influenza in a school environment.

Contact rate calculation for a basic epidemic model.

Mass-action epidemic models are the foundation of the majority of studies of disease dynamics in human and animal populations. Here, a kinetic model of mobile susceptible and infective individuals in a two-dimensional domain is introduced, and an examination of the contact process results in a mass-action-like term for the generation of new infectives. The conditions under which density dependent and frequency dependent transmission terms emerge are clarified. Moreover, this model suggests that epidemics in large mobile spatially distributed populations can be well described by homogeneously mixing mass-action models. The analysis generates an analytic formula for the contact rate (beta) and the basic reproductive ratio (R0) of an infectious pathogen, which contains a mixture of demographic and epidemiological parameters. The analytic results are compared with a simulation and are shown to give good agreement. The simulation permits the exploration of more realistic movement strategies and their consequent effect on epidemic dynamics.

The effect of superspreading on epidemic outbreak size distributions.

Recently, evidence has been presented to suggest that there are significant heterogeneities in the transmission of communicable diseases. Here, a stochastic simulation model of an epidemic process that allows for these heterogeneities is used to demonstrate the potentially considerable effect that heterogeneity of transmission will have on epidemic outbreak size distributions. Our simulation results agree well with approximations gained from the theory of branching processes. Outbreak size distributions have previously been used to infer basic epidemiological parameters. We show that if superspreading does occur then such distributions must be interpreted with care. The simulation results are discussed in relation to measles epidemics in isolated populations and in predominantly urban scenarios. The effect of three different disease control policies on outbreak size distributions are shown for varying levels of heterogeneity and disease control effort.

The balance between proinsulin biosynthesis and insulin secretion: where can imbalance lead?

Insulin is stored in pancreatic beta-cells in beta-granules. Whenever insulin is secreted in response to a nutrient secretagogue, there is a complementary increase in proinsulin biosynthesis to replenish intracellular insulin stores. This specific nutrient regulation of proinsulin biosynthesis is predominately regulated at the translational level. Recently, a highly conserved cis-element in the 5'-untranslated region (UTR) of preproinsulin mRNA, named ppIGE, has been identified that is required for specific translational regulation of proinsulin biosynthesis. This ppIGE is also found in the 5'-UTR of certain other translationally regulated beta-granule protein mRNAs, including the proinsulin processing endopeptidases, PC1/3 and PC2. This provides a mechanism whereby proinsulin processing is adaptable to changes in proinsulin biosynthesis. However, relatively few beta-granules undergo secretion, with most remaining in the storage pool for approximately 5 days. Aged beta-granules are retired by intracellular degradation mechanisms, either via crinophagy and/or autophagy, as another long-term means of maintaining beta-granule stores at optimal levels. When a disconnection between insulin production and secretion arises, as may occur in type 2 diabetes, autophagy further increases to maintain beta-granule numbers. However, if this increased autophagy becomes chronic, autophagia-mediated cell death occurs that could then contribute to beta-cell loss in type 2 diabetes.

Exendin 4 controls insulin production in rat islet beta cells predominantly by potentiation of glucose-stimulated proinsulin biosynthesis at the translational level.

AIMS/HYPOTHESIS: Ideally, a therapeutic insulin secretagogue should coordinately increase insulin production and insulin secretion to maintain islet beta cell secretory capacity. We compared the incretin mimetic exendin 4 and the sulfonylurea glibenclamide (known as glyburide in the USA and Canada) for their effects in upholding a balance between (pro)insulin biosynthesis and insulin secretion in pancreatic islets. METHODS: Isolated rat islets were incubated for 1 or 16 h over a range of glucose concentrations (2.8-16.7 mmol/l) with or without exendin 4 (10 nmol/l) or glibenclamide (1 micromol/l). Islets were then analysed for preproinsulin mRNA expression by RNase protection and quantitative real-time RT-PCR assays. Proinsulin biosynthesis was analysed by metabolic pulse-radiolabelling, immunoprecipitation and PAGE. Insulin secretion and insulin content were analysed by radioimmunoassay. RESULTS: Neither exendin 4 nor glibenclamide affected islet preproinsulin mRNA expression. However, exendin 4 significantly increased glucose-induced proinsulin biosynthesis at the translational level within 1 h, in marked contrast to glibenclamide, which inhibited proinsulin biosynthesis, especially at basal and intermediate glucose concentrations. Exendin 4 potentiated insulin secretion in a glucose-dependent manner, whereas glibenclamide stimulated insulin secretion independently of glucose. Exendin 4 better maintained rat islet insulin content compared with glibenclamide, which depleted intracellular stores of insulin in islet beta cells by 40% within 16 h. CONCLUSIONS/INTERPRETATION: Exendin 4 maintains insulin stores and beta cell secretory capacity primarily by translational control of proinsulin biosynthesis in parallel to insulin secretion. Glibenclamide does not regulate insulin production in coordination with stimulated insulin secretion, and consequently depletes islet insulin stores, compromising secretory capacity. Thus, at the level of the beta cell, incretin mimetics have an advantage over sulfonylureas for treatment of type 2 diabetes.

Free radicals, metals and antioxidants in oxidative stress-induced cancer.

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular, ROS activation of AP-1 (activator protein) and NF-kappaB (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu, Zn-SOD, Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of carcinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappaB, AP-1 are also reviewed.

Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.

AIMS/HYPOTHESIS: The gut hormone glucagon-like peptide-1 (GLP-1) decreases beta cell apoptosis in a protein kinase B (PKB)-dependent fashion, and increases islet cell mass and function in vivo. In contrast, cytokines induce beta cell apoptosis, leading to decreased islet mass and type 1 diabetes. In the present study we used rat INS-1E beta cells and primary rat islet cells to examine the potential role of PKB as a mediator of the effect of GLP-1 on cytokine-induced apoptosis. METHODS: Cell viability was determined by MTT assay, and apoptosis and necrosis by Hoechst 33342-propidium iodide staining. Immunoblot analysis was used to detect changes in protein expression, including active (phosphorylated) and total PKB, phosphorylated and total glycogen synthase kinase-3beta, activated caspase-3 and inducible nitric oxide synthase. Reactive oxygen species were determined by 1,7-dichlorofluorescein (DCF) analysis, and mutant forms of PKB were introduced into cells using adenoviral vectors. RESULTS: Incubation of INS-1E cells with cytokines (IL-1beta, TNF-alpha and interferon-gamma; 10-50 ng/ml) for 18 h significantly decreased cell viability (by 44%, p<0.001), cell proliferation (by 80%, p<0.001), and activation of PKB (by 67%, p<0.001). Pre-treatment with exendin-4 (10(-7) mol/l), a long-acting GLP-1 receptor agonist, partially protected the cells against cytokine-induced toxicity (p<0.01) in association with a reduction in cytokine-induced inhibition of PKB phosphorylation (p<0.05). Exendin-4 pre-treatment did not change cell proliferation. Cytokine treatment increased apoptosis (by 156%, p<0.05) and necrosis (from undetectable to 2.6% of cells). These increases were both reduced by pre-treatment with exendin-4 (p<0.05-0.01). Furthermore, cytokine-induced apoptosis and necrosis were significantly increased in cells infected with kinase-dead PKB (p<0.05), and the protective effect of exendin-4 on both parameters was fully abolished in these cells. Similar changes were observed in primary islet cells. In parallel with these changes, exendin-4 decreased the cytokine-induced activation of caspase-3 (by 46%, p<0.05), and decreased levels of inducible nitric oxide synthase (by 71%, p<0.05) and reactive oxygen species (by 27%, p<0.05). CONCLUSIONS/INTERPRETATION: The results of our study indicate that GLP-1 plays a protective role against cytokine-induced apoptosis and necrosis in beta cells through a PKB-dependent signalling pathway.

Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic beta-cells.

Several proteins play a role in the mechanism of insulin exocytosis. However, these 'exocytotic proteins' have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet beta-cells as experimental tools. Syncollin is not normally expressed in beta-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of beta-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5 microM). Consistent with specific localization of syncollin to beta-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in beta-cells has highlighted the importance of intralumenal beta-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a 'fluorescent tag' to track beta-granule transport and exocytosis in real time.

Glucagon-like peptide-1 prevents beta cell glucolipotoxicity.

AIMS/HYPOTHESIS: We have provided evidence that glucagon-like peptide-1, a potential therapeutic agent in the treatment of diabetes, activates phosphatidylinositol-3 kinase/protein kinase B signalling in the pancreatic beta cell. Since this pathway promotes cell survival in a variety of systems, we tested whether glucagon-like peptide-1 protects beta cells against cell death induced by elevated glucose and/or non-esterified fatty acids. METHODS: Human islets and INS832/13 cells were cultured at glucose concentrations of 5 or 25 mmol/l in the presence or absence of palmitate. Apoptosis was evaluated by monitoring DNA fragmentation and chromatin condensation. Wild-type and protein kinase B mutants were overexpressed in INS832/13 cells using adenoviruses. Nuclear factor-kappa B DNA binding was assayed by electrophoretic mobility shift assay. RESULTS: In human pancreatic beta cells and INS832/13 cells, glucagon-like peptide-1 prevented beta cell apoptosis induced by elevated concentrations of (i) glucose (glucotoxicity), (ii) palmitate (lipotoxicity) and (iii) both glucose and palmitate (glucolipotoxicity). Overexpression of a dominant-negative protein kinase B suppressed the anti-apoptotic action of glucagon-like peptide-1 in INS832/13 cells, whereas a constitutively active protein kinase B prevented beta cell apoptosis induced by elevated glucose and palmitate. Glucagon-like peptide-1 enhanced nuclear factor-kappa B DNA binding activity and stimulated the expression of inhibitor of apoptosis protein-2 and Bcl-2, two anti-apoptotic genes under the control of nuclear factor-kappa B. Inhibition of nuclear factor-kappa B by BAY 11-7082 abolished the prevention of glucolipotoxicity by glucagon-like peptide-1. CONCLUSIONS/INTERPRETATION: The results demonstrate a potent protective effect of glucagon-like peptide-1 on beta cell gluco-, lipo- and glucolipotoxicity. This effect is mediated via protein kinase B activation and possibly its downstream target nuclear factor-kappa B.

Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1).

It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.

Regulation of the production and secretion of insulin

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