Is 14-3-3 the Combination to Unlock New Pathways to Improve Metabolic Homeostasis and ß-Cell Function?

Since their discovery nearly five decades ago, molecular scaffolds belonging to the 14-3-3 protein family have been recognized as pleiotropic regulators of diverse cellular and physiological functions. With their ability to bind to proteins harboring specific serine and threonine phosphorylation motifs, 14-3-3 proteins can interact with and influence the function of docking proteins, enzymes, transcription factors, and transporters that have essential roles in metabolism and glucose homeostasis. Here, we will discuss the regulatory functions of 14-3-3 proteins that will be of great interest to the fields of metabolism, pancreatic ß-cell biology, and diabetes. We first describe how 14-3-3 proteins play a central role in glucose and lipid homeostasis by modulating key pathways of glucose uptake, glycolysis, oxidative phosphorylation, and adipogenesis. This is followed by a discussion of the contributions of 14-3-3 proteins to calcium-dependent exocytosis and how this relates to insulin secretion from ß-cells. As 14-3-3 proteins are major modulators of apoptosis and cell cycle progression, we will explore if 14-3-3 proteins represent a viable target for promoting ß-cell regeneration and discuss the feasibility of targeting 14-3-3 proteins to treat metabolic diseases such as diabetes. ARTICLE HIGHLIGHTS: 14-3-3 proteins are ubiquitously expressed scaffolds with multiple roles in glucose homeostasis and metabolism. 14-3-3ζ regulates adipogenesis via distinct mechanisms and is required for postnatal adiposity and adipocyte function. 14-3-3ζ controls glucose-stimulated insulin secretion from pancreatic ß-cells by regulating mitochondrial function and ATP synthesis as well as facilitating cross talk between ß-cells and α-cells.

The Application of High-Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes.

During the past decades, unprecedented progress in technologies has revolutionized traditional research methodologies. Among these, advances in high-throughput drug screening approaches have permitted the rapid identification of potential therapeutic agents from drug libraries that contain thousands or millions of molecules. Moreover, high-throughput-based therapeutic target discovery strategies can comprehensively interrogate relationships between biomolecules (e.g., gene, RNA, and protein) and diseases and significantly increase the authors' knowledge of disease mechanisms. Diabetes is a chronic disease primarily characterized by the incapacity of the body to maintain normoglycemia. The prevalence of diabetes in modern society has become a severe public health issue that threatens the well-being of millions of patients. Although a number of pharmacological treatments are available, there is no permanent cure for diabetes, and discovering novel therapeutic targets and agents continues to be an urgent need. The present review discusses the technical details of high-throughput screening approaches in drug discovery, followed by introducing the applications of such approaches to diabetes research. This review aims to provide an example of the applicability of high-throughput technologies in facilitating different aspects of disease research.

A high-throughput screening approach to discover potential colorectal cancer chemotherapeutics: Repurposing drugs to disrupt 14-3-3 protein-BAD interactions.

Inducing apoptosis in different types of cancer cells is an effective therapeutic strategy. However, the success of existing chemotherapeutics can be compromised by tumor cell resistance and systemic off-target effects. Therefore, the discovery of pro-apoptotic compounds with minimal systemic side-effects is crucial. 14-3-3 proteins are molecular scaffolds that serve as important regulators of cell survival. Our previous study demonstrated that 14-3-3ζ can sequester BAD, a pro-apoptotic member of the BCL-2 protein family, in the cytoplasm and prevent its translocation to mitochondria to inhibit the induction of apoptosis. Despite being a critical mechanism of cell survival, it is unclear whether disrupting 14-3-3 protein:BAD interactions could be harnessed as a chemotherapeutic approach. Herein, we established a BRET-based high-throughput drug screening approach (Z'-score= 0.52) capable of identifying molecules that can disrupt 14-3-3ζ:BAD interactions. An FDA-approved drug library containing 1971 compounds was used for screening, and the capacity of identified hits to induce cell death was examined in NIH3T3-fibroblasts and colorectal cancer cell lines, HT-29 and Caco-2. Our in vitro results suggest that terfenadine, penfluridol, and lomitapide could be potentially repurposed for treating colorectal cancer. Moreover, our screening method demonstrates the feasibility of identifying pro-apoptotic agents that can be applied towards conditions where aberrant cell growth or function are key determinants of disease pathogenesis.

14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic ß cells.

While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in ß cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in ß cell function, we generated ß cell-specific 14-3-3ζ-KO mice. Although no effects on ß cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the ß cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in ß cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan-14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of ß cell function and provides a deeper understanding of how insulin secretion is controlled in ß cells.

Metabolic Contributions of Wnt Signaling: More Than Controlling Flight.

The canonical Wnt signaling pathway is ubiquitous throughout the body and influences a diverse array of physiological processes. Following the initial discovery of the Wnt signaling pathway during wing development in Drosophila melanogaster, it is now widely appreciated that active Wnt signaling in mammals is necessary for the development and growth of various tissues involved in whole-body metabolism, such as brain, liver, pancreas, muscle, and adipose. Moreover, elegant gain- and loss-of-function studies have dissected the tissue-specific roles of various downstream effector molecules in the regulation of energy homeostasis. This review attempts to highlight and summarize the contributions of the Wnt signaling pathway and its downstream effectors on whole-body metabolism and their influence on the development of metabolic diseases, such as diabetes and obesity. A better understanding of the Wnt signaling pathway in these tissues may aid in guiding the development of future therapeutics to treat metabolic diseases.

Adipose depot-specific upregulation of Ucp1 or mitochondrial oxidative complex proteins are early consequences of genetic insulin reduction in mice.

Hyperinsulinemia plays a causal role in adipose tissue expansion. Mice with reduced insulin have increased energy expenditure, but the mechanisms remained unclear. Here we investigated the effects of genetically reducing insulin production on uncoupling and oxidative mitochondrial proteins in liver, skeletal muscle, white adipose tissue (WAT), and brown adipose tissue (BAT). Male Ins1+/+ or Ins1+/- littermates were fed either a low-fat diet (LFD) or a high-fat diet (HFD) for 4 wk, starting at 8 wk of age. Replicating our previous observations, HFD increased fasting hyperinsulinemia, and Ins1+/- mice had significantly lower circulating insulin compared with Ins1+/+ littermates. Fasting glucose and body weight were not different between genotypes. We did not observe robust significant differences in liver or skeletal muscle. In mesenteric WAT, Ins1+/- mice had reduced Ndufb8 and Sdhb, while Ucp1 was increased in the context of HFD. HFD alone had a dramatic inhibitory effect on Pparg abundance. In inguinal WAT, Ins1+/- mice exhibited significant increases in oxidative complex proteins, independent of diet, without affecting Ucp1, Pparg, or Prdm16:Pparg association. In BAT, lowered insulin increased Sdhb protein levels that had been reduced by HFD. Ucp1 protein, Prdm16:Pparg association, and Sirt3 abundance were all increased in the absence of diet-induced hyperinsulinemia. Our data show that reducing insulin upregulates oxidative proteins in inguinal WAT without affecting Ucp1, whereas in mesenteric WAT and BAT, reducing insulin upregulates Ucp1 in the context of HFD. Preventing hyperinsulinemia has early depot-specific effects on adipose tissue metabolism and helps explain the increased energy expenditure previously reported in Ins1+/- mice.

14-3-3ζ mediates an alternative, non-thermogenic mechanism in male mice to reduce heat loss and improve cold tolerance.

OBJECTIVE: Adaptive thermogenesis, which is partly mediated by sympathetic input on brown adipose tissue (BAT), is a mechanism of heat production that confers protection against prolonged cold exposure. Various endogenous stimuli, for example, norepinephrine and FGF-21, can also promote the conversion of inguinal white adipocytes to beige adipocytes, which may represent a secondary cell type that contributes to adaptive thermogenesis. We previously identified an essential role of the molecular scaffold 14-3-3ζ in adipogenesis, but one of the earliest, identified functions of 14-3-3ζ is its regulatory effects on the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine. Herein, we examined whether 14-3-3ζ could influence adaptive thermogenesis via actions on BAT activation or the beiging of white adipocytes. METHODS: Transgenic mice over-expressing a TAP-tagged human 14-3-3ζ molecule or heterozygous mice without one allele of Ywhaz, the gene encoding 14-3-3ζ, were used to explore the contribution of 14-3-3ζ to acute (3 h) and prolonged (3 days) cold (4 °C) exposure. Metabolic caging experiments, PET-CT imaging, and laser Doppler imaging were used to determine the effect of 14-3-3ζ over-expression on thermogenic and vasoconstrictive mechanisms in response to cold. RESULTS: Transgenic over-expression of 14-3-3ζ (TAP) in male mice significantly improved tolerance to acute and prolonged cold. In response to cold, body temperatures in TAP mice did not decrease to the same extent when compared to wildtype (WT) mice, and this was associated with increased UCP1 expression in beige inguinal white tissue (iWAT) and BAT. Of note was the paradoxical finding that cold-induced changes in body temperatures of TAP mice were associated with significantly decreased energy expenditure. The marked improvements in tolerance to prolonged cold were not due to changes in sensitivity to ß-adrenergic stimulation or BAT or iWAT oxidative metabolism; instead, over-expression of 14-3-3ζ significantly decreased thermal conductance and heat loss in mice via increased peripheral vasoconstriction. CONCLUSIONS: Despite being associated with elevations in cold-induced UCP1 expression in brown or beige adipocytes, these findings suggest that 14-3-3ζ regulates an alternative, non-thermogenic mechanism via vasoconstriction to minimize heat loss during cold exposure.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function.

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are critical lipolytic enzymes, and their phosphorylation generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. Although we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on ATGL and HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Here we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and free fatty acid (FFA) release. Unexpectedly, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of peroxisome proliferator-activated receptor (PPAR)γ2 protein and expression of mature adipocyte genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated preadipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.

Can 14-3-3 proteins serve as therapeutic targets for the treatment of metabolic diseases?

Since their initial characterization as abundant brain proteins more than 5 decades ago, a resurgence into understanding the cellular functions of 14-3-3 proteins has emerged. While one of the earliest functions attributed to this eukaryotic scaffold protein family was the activation of enzymes involved in catecholamine and serotonin biosynthesis, 14-3-3 proteins have since been implicated in the regulation of several cellular processes including cell-cycle control, apoptosis, and metabolism. Moreover, increasing lines of evidence demonstrate links between changes in 14-3-3 protein function and the pathogenesis of chronic diseases. As a result, this has raised the question of whether 14-3-3 proteins represent viable targets for pharmacological intervention against diseases such as obesity, diabetes and cancer. In addition to providing an overview of the 14-3-3 protein family, we will discuss their connections to metabolism and metabolic diseases. We will also elaborate on the potential of targeting 14-3-3 proteins, as well as components of their interactomes, for developing novel therapies for treating metabolic diseases, including diabetes and obesity.

Scaffold Proteins: From Coordinating Signaling Pathways to Metabolic Regulation.

Among their pleiotropic functions, scaffold proteins are required for the accurate coordination of signaling pathways. It has only been within the past 10 years that their roles in glucose homeostasis and metabolism have emerged. It is well appreciated that changes in the expression or function of signaling effectors, such as receptors or kinases, can influence the development of chronic diseases such as diabetes and obesity. However, little is known regarding whether scaffolds have similar roles in the pathogenesis of metabolic diseases. In general, scaffolds are often underappreciated in the context of metabolism or metabolic diseases. In the present review, we discuss various scaffold proteins and their involvement in signaling pathways related to metabolism and metabolic diseases. The aims of the present review were to highlight the importance of scaffold proteins and to raise awareness of their physiological contributions. A thorough understanding of how scaffolds influence metabolism could aid in the discovery of novel therapeutic approaches to treat chronic conditions, such as diabetes, obesity, and cardiovascular disease, for which the incidence of all continue to increase at alarming rates.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis (i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.

Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain.

Excess circulating insulin is associated with obesity in humans and in animal models. However, the physiologic causality of hyperinsulinemia in adult obesity has rightfully been questioned because of the absence of clear evidence that weight loss can be induced by acutely reversing diet-induced hyperinsulinemia. Herein, we describe the consequences of inducible, partial insulin gene deletion in a mouse model in which animals have already been made obese by consuming a high-fat diet. A modest reduction in insulin production/secretion was sufficient to cause significant weight loss within 5 wk, with a specific effect on visceral adipose tissue. This result was associated with a reduction in the protein abundance of the lipodystrophy gene polymerase I and transcript release factor ( Ptrf; Cavin) in gonadal adipose tissue. RNAseq analysis showed that reduced insulin and weight loss also associated with a signature of reduced innate immunity. This study demonstrates that changes in circulating insulin that are too fine to adversely affect glucose homeostasis nonetheless exert control over adiposity.-Page, M. M., Skovsø, S., Cen, H., Chiu, A. P., Dionne, D. A., Hutchinson, D. F., Lim, G. E., Szabat, M., Flibotte, S., Sinha, S., Nislow, C., Rodrigues, B., Johnson, J. D. Reducing insulin via conditional partial gene ablation in adults reverses diet-induced weight gain.

Reduced Circulating Insulin Enhances Insulin Sensitivity in Old Mice and Extends Lifespan.

The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk of age-related disease in mammals. We compared Ins2+/- mice to Ins2+/+ littermate controls, on a genetically stable Ins1 null background. Proteomic and transcriptomic analyses of livers from 25-week-old mice suggested potential for healthier aging and altered insulin sensitivity in Ins2+/- mice. Halving Ins2 lowered circulating insulin by 25%-34% in aged female mice, without altering Igf1 or circulating Igf1. Remarkably, decreased insulin led to lower fasting glucose and improved insulin sensitivity in aged mice. Moreover, lowered insulin caused significant lifespan extension, observed across two diverse diets. Our study indicates that elevated insulin contributes to age-dependent insulin resistance and that limiting basal insulin levels can extend lifespan.

A causal role for hyperinsulinemia in obesity.

Insulin modulates the biochemical pathways controlling lipid uptake, lipolysis and lipogenesis at multiple levels. Elevated insulin levels are associated with obesity, and conversely, dietary and pharmacological manipulations that reduce insulin have occasionally been reported to cause weight loss. However, the causal role of insulin hypersecretion in the development of mammalian obesity remained controversial in the absence of direct loss-of-function experiments. Here, we discuss theoretical considerations around the causal role of excess insulin for obesity, as well as recent studies employing mice that are genetically incapable of the rapid and sustained hyperinsulinemia that normally accompanies a high-fat diet. We also discuss new evidence demonstrating that modest reductions in circulating insulin prevent weight gain, with sustained effects that can persist after insulin levels normalize. Importantly, evidence from long-term studies reveals that a modest reduction in circulating insulin is not associated with impaired glucose homeostasis, meaning that body weight and lipid homeostasis are actually more sensitive to small changes in circulating insulin than glucose homeostasis in these models. Collectively, the evidence from new studies on genetic loss-of-function models forces a re-evaluation of current paradigms related to obesity, insulin resistance and diabetes. The potential for translation of these findings to humans is briefly discussed.

14-3-3ζ: A numbers game in adipocyte function?

Molecular scaffolds are often viewed as passive signaling molecules that facilitate protein-protein interactions. However, new evidence gained from the use of loss-of-function or gain-of-function models is dispelling this notion. Our own recent discovery of 14-3-3ζ as an essential regulator of adipogenesis highlights the complex roles of this member of the 14-3-3 protein family. Depletion of the 14-3-3ζ isoform affected parallel pathways that drive adipocyte development, including pathways controlling the stability of key adipogenic transcription factors and cell cycle progression. Going beyond adipocyte differentiation, this study opens new avenues of research in the context of metabolism, as 14-3-3ζ binds to a variety of well-established metabolic proteins that harbor its canonical phosphorylation binding motifs. This suggests that 14-3-3ζ may contribute to key metabolic signaling pathways, such as those that facilitate glucose uptake and fatty acid metabolism. Herein, we discuss these novel areas of research, which will undoubtedly shed light onto novel roles of 14-3-3ζ, and perhaps its related family members, on glucose homeostasis.

Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro ß-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal ß-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms.

Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells.

OBJECTIVE: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. METHODS: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. RESULTS: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. CONCLUSIONS: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

14-3-3ζ coordinates adipogenesis of visceral fat.

The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27(Kip1). Indeed, concomitant knockdown of p27(Kip1) or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27(Kip1) abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BACKGROUND: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. METHODS: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. RESULTS: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. CONCLUSIONS: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.