Objective: A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM).
Methods: LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro, using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice.
A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25–8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5–10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5–15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176.
Results: LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist.
A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25–15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: −49.12 mg/dL [−78.14, −20.12] and −43.15 mg/dL [−73.06, −13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: −1.75 kg [−3.38, −0.12], −5.09 kg [−6.72, −3.46] and −4.61 kg [−6.21, −3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: −2.62 kg [−3.79, −1.45] and −2.07 kg [−3.25, −0.88], respectively.
The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity.
Conclusions: Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity.[Hide abstract]
Objective: The liver performs a central role in regulating energy homeostasis by increasing glucose output during fasting. Recent studies on Argonaute2 (Ago2), a key RNA-binding protein mediating the microRNA pathway, have illustrated its role in adaptive mechanisms according to changes in metabolic demand. Here we sought to characterize the functional role of Ago2 in the liver in the maintenance of systemic glucose homeostasis.
Methods: We first analyzed Ago2 expression in mouse primary hepatocyte cultures after modulating extracellular glucose concentrations and in the presence of activators or inhibitors of glucokinase activity. We then characterized a conditional loss-of-function mouse model of Ago2 in liver for alterations in systemic energy metabolism.
Results: Here we show that Ago2 expression in liver is directly correlated to extracellular glucose concentrations and that modulating glucokinase activity is adequate to affect hepatic Ago2 levels. Conditional deletion of Ago2 in liver resulted in decreased fasting glucose levels in addition to reducing hepatic glucose production. Moreover, loss of Ago2 promoted hepatic expression of AMP-activated protein kinase α1 (AMPKα1) by de-repressing its targeting by miR-148a, an abundant microRNA in the liver. Deletion of Ago2 from hyperglycemic, obese, and insulin-resistant Lepob/ob mice reduced both random and fasted blood glucose levels and body weight and improved insulin sensitivity.
Conclusions: These data illustrate a central role for Ago2 in the adaptive response of the liver to fasting. Ago2 mediates the suppression of AMPKα1 by miR-148a, thereby identifying a regulatory link between non-coding RNAs and a key stress regulator in the hepatocyte.[Hide abstract]
Objective: Non-alcoholic fatty liver disease (NAFLD) risk begins in utero in offspring of obese mothers. A critical unmet need in this field is to understand the pathways and biomarkers underlying fetal hepatic lipotoxicity and whether maternal dietary intervention during pregnancy is an effective countermeasure.
Methods: We utilized a well-established non-human primate model of chronic, maternal, Western-style diet induced obesity (OB-WSD) compared with mothers on a healthy control diet (CON) or a subset of OB-WSD mothers switched to the CON diet (diet reversal; OB-DR) prior to and for the duration of the next pregnancy. Fetuses were studied in the early 3rd trimester.
Results: Fetuses from OB-WSD mothers had higher circulating triglycerides (TGs) and lower arterial oxygenation suggesting hypoxemia, compared with fetuses from CON and OB-DR mothers. Hepatic TG content, oxidative stress (TBARs), and de novo lipogenic genes were increased in fetuses from OB-WSD compared with CON mothers. Fetuses from OB-DR mothers had lower lipogenic gene expression and TBARs yet persistently higher TGs. Metabolomic profiling of fetal liver and serum (umbilical artery) revealed distinct separation of CON and OB-WSD groups, and an intermediate phenotype in fetuses from OB-DR mothers. Pathway analysis identified decreased tricarboxylic acid cycle intermediates, increased amino acid (AA) metabolism and byproducts, and increased gluconeogenesis, suggesting an increased reliance on AA metabolism to meet energy needs in the liver of fetuses from OB-WSD mothers. Components in collagen synthesis, including serum protein 5-hydroxylysine and hepatic lysine and proline, were positively correlated with hepatic TGs and TBARs, suggesting early signs of fibrosis in livers from the OB-WSD group. Importantly, hepatic gluconeogenic and arginine related intermediates and serum levels of lactate, pyruvate, several AAs, and nucleotide intermediates were normalized in the OB-DR group. However, hepatic levels of CDP-choline and total ceramide levels remained high in fetuses from OB-DR mothers.
Conclusions: Our data provide new metabolic evidence that, in addition to fetal hepatic steatosis, maternal WSD creates fetal hypoxemia and increases utilization of AAs for energy production and early activation of gluconeogenic pathways in the fetal liver. When combined with hyperlipidemia and limited antioxidant activity, the fetus suffers from hepatic oxidative stress and altered intracellular metabolism which can be improved with maternal diet intervention. Our data reinforce the concept that multiple “first hits” occur in the fetus prior to development of obesity and demonstrate new biomarkers with potential clinical implications for monitoring NAFLD risk in offspring.[Hide abstract]
Objective: Although debated, metabolic health characterizes 10–25% of obese individuals and reduces risk of developing life-threatening co-morbidities. Adipose tissue is a recognized endocrine organ important for the maintenance of whole-body metabolic health. Adipocyte transcriptional signatures of healthy and unhealthy obesity are largely unknown.
Methods: Here, we used a small cohort of highly characterized obese individuals discordant for metabolic health, characterized their adipocytes transcriptional signatures, and cross-referenced them to mouse phenotypic and human GWAs databases.
Results and Conclusions: Our study showed that glucose intolerance and insulin resistance co-operate to remodel adipocyte transcriptome. We also identified the Nuclear Export Mediator Factor (NEMF) and the Ectoderm-Neural Cortex 1 (ENC1) as novel potential targets in the management of metabolic health in human obesity.
Objectives: Brown adipose tissue (BAT) and BAT-like adipose tissues, referred to as ‘beige’ adipose tissues uncouple respiration from ATP synthesis via uncoupling protein one (UCP-1). There is a sexual dimorphism with respect to beige and BAT tissues; pre-menopausal women have more BAT and are more sensitive to BAT activation than men or postmenopausal women. We hypothesized selective activation of adipose tissue estrogen receptor alpha (ERα) induces beiging of WAT through induction of lipolysis mediated by adipose tissue triglyceride lipase (ATGL).
Methods: 3T3-L1 and primary adipocytes were treated with the selective ERα agonist pyrazole triol (PPT), and selection deletion of ERα (using siRNA) was used to determine if selective ERα activation, or inhibition, influences the adipose tissue expression of genes associated with beiging. In a second series of experiments, ERα was selectively added back to adipose tissue of mice lacking total body ERα (ERKO) to determine if add back of ERα changed the morphology of adipose tissue to resemble beige tissues. Additionally, WT and ERKO mice were exposed to cold and FDG labeled glucose uptake was measured to determine the ability of cold to induce UCP-1 in ERKO mice. To begin to mechanistically probe how activation of ERα facilitates beiging, we tested the influence of PPT to activate the lipolytic pathway through ATGL. Finally, since ERα exerts its effects both at the genomic and non-genomic level depending on its cellular location, we determined in vivo if beiging occurs in mice expressing ERα only at the plasma membrane (MOER mice) or only at nucleus (NOER mice).
Results: Selective ERα activation by PPT increased markers of beiging in vitro in 3T3-L1 and primary adipocytes, whereas, knockdown of ERα with siRNA reduced the ability of PPT to induce beiging in vitro. ERα add back to the adipose tissue of ERKO mice resulted in multilocular adipose tissue resembling a beige phenotype. Following cold exposure, FDG labeled glucose in BAT tissues of ERKO mice was reduced when compared to weight-matched controls. Glycerol release and ATGL expression were increased after PPT treatment, while pre-treatment with the ATGL inhibitor prevented PPT's ability to increase UCP-1 expression. Finally, MOER mice were more sensitive to beiging of adipose tissues when compared to NOER mice.
Conclusion: Our results demonstrate for the first time that selective-activation of ERα in adipocytes increases markers of beiging and this is likely through induction of AMPK and ATGL-mediated lipolysis providing FFAs as a fuel to activate UCP-1.[Hide abstract]
There is emerging evidence to support an important role for the transient receptor potential vanilloid type 1 (TRPV1) sensory innervation in glucose homeostasis. However, it remains unknown whether the glucoregulatory action of these afferent neurons is sex-biased and whether it is pancreatic β-cell-mediated.
Objective: We investigated in male and female mice whether denervation of whole-body or pancreas-projecting TRPV1 sensory neurons regulates adult functional β-cell mass and alters systemic glucose homeostasis.
Methods: We used a combination of pharmacological and surgical approaches to ablate whole-body or pancreatic TRPV1 sensory neurons and assessed islet β-cell function and mass, aspects of glucose and insulin homeostasis, and energy expenditure.
Results: Capsaicin-induced chemodenervation of whole-body TRPV1 sensory neurons improved glucose clearance and enhanced glucose-stimulated insulin secretion without alterations in β-cell proliferation and mass, systemic insulin sensitivity, body composition, and energy expenditure. Similarly, denervation of intrapancreatic TRPV1 afferents by pancreas intraductal injection of capsaicin or surgical removal of the dorsal root ganglia projecting into the pancreas lowered post-absorptive glucose levels and increased insulin release upon glucose stimulation. The beneficial effects of TRPV1 sensory denervation on glucose tolerance and β-cell function were observed in male but not female mice.
Conclusion: Collectively, these findings suggest that TRPV1 neurons regulate glucose homeostasis, at least partly, through direct modulation of glucose-induced insulin secretion and that this regulation operates in a sex-dependent manner.[Hide abstract]
Objective: Studies using the estrogen receptor alpha (ERα) knock-out (αERKO) mice have demonstrated that ERα plays a crucial role in various estrogen-mediated metabolic regulations. ERα is a ligand dependent transcription regulator and its activity is regulated by estrogenic compounds. ERα consists of two transcriptional activation domains, AF-1 and AF-2. The activities of these domains are regulated through different mechanisms; however, the specific physiological role in metabolic regulation by these domains is still unclear.
Methods: We utilized an ERα AF-2 mutant knock-in mouse (AF2ERKI) to evaluate the physiological functionality of ERα transactivation domains. Due to the estrogen insensitive AF-2 mutation, the phenotypes of AF2ERKI mice are seemingly identical to the global αERKO including obesity in the females. Distinct from the αERKO, the AF-1 function of AF2ERKI mice can be activated by tamoxifen (Tam). Ovariectomized (OVX) AF2ERKI and WT females were treated with Tam and fed a high-fat diet (HFD) for 10 weeks. Additionally, indirect calorimetric analysis was performed using metabolic chambers with food intake and locomotor activity recorded for Tam-treated AF2ERKI and αERKO females.
Results: Obesity in HFD-fed AF2ERKI females was prevented by Tam treatment; particularly, inguinal fat accumulation was strongly blocked by Tam treatment. Alterations in fat metabolism genes, however, were not found in either inguinal fat nor visceral fat to be Tam-regulated, even though fat accumulation was strongly reduced by Tam treatment. Indirect calorimetric analysis revealed that without alteration of food intake and locomotor activity Tam treatment increased energy expenditure in AF2ERKI but not αERKO females.
Conclusion: These results suggest that the activation of ERα AF-1 prevents fat accumulation. The prevention of obesity through AF-1 is mediated by induction of energy expenditure rather than ERα AF-1 functionality of lipid metabolism gene regulation in fat tissues.[Hide abstract]
Objective: Melanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2.
Methods: To target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII.
Results: Mc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females.
Conclusion: Our data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure.[Hide abstract]
Objective: The blood–brain barrier (BBB) regulates the entry of substrates and peptides into the brain. Ghrelin is mainly produced in the stomach but exerts its actions in the central nervous system (CNS) by crossing the BBB. Once present in the CNS, ghrelin can act in the hypothalamus to regulate food intake, in the hippocampus to regulate neurogenesis, and in the olfactory bulb to regulate food-seeking behavior. The goal of this study was to determine whether the primary signaling receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), mediates the transport of ghrelin from blood to brain.
Methods: We utilized the sensitive and quantitative multiple-time regression analysis technique to determine the transport rate of mouse and human acyl ghrelin (AG) and desacyl ghrelin (DAG) in wildtype and Ghsr null mice. We also measured the regional distribution of these ghrelin peptides throughout the brain. Lastly, we characterized the transport characteristics of human DAG by measuring the stability in serum and brain, saturability of transport, and the complete transfer across the brain endothelial cell.
Results: We found the transport rate across the BBB of both forms of ghrelin, AG, and DAG, were not affected by the loss of GHSR. We did find differences in the transport rate between the two isoforms, with DAG being faster than AG; this was dependent on the species of ghrelin, human being faster than mouse. Lastly, based on the ubiquitous properties of ghrelin throughout the CNS, we looked at regional distribution of ghrelin uptake and found the highest levels of uptake in the olfactory bulb.
Conclusion: The data presented here suggest that ghrelin transport can occur independently of the GHSR, and ghrelin uptake varies regionally throughout the brain. These findings better our understanding of the gut-brain communication and may lead to new understandings of ghrelin physiology.[Hide abstract]
Objective: Imprinted genes are crucial for the growth and development of fetal and juvenile mammals. Altered imprinted gene dosage causes a variety of human disorders, with growth and development during these crucial early stages strongly linked with future metabolic health in adulthood. Neuronatin (Nnat) is a paternally expressed imprinted gene found in neuroendocrine systems and white adipose tissue and is regulated by the diet and leptin. Neuronatin expression is downregulated in obese children and has been associated with stochastic obesity in C57BL/6 mice. However, our recent studies of Nnat null mice on this genetic background failed to display any body weight or feeding phenotypes but revealed a defect in glucose-stimulated insulin secretion due to the ability of neuronatin to potentiate signal peptidase cleavage of preproinsulin. Nnat deficiency in beta cells therefore caused a lack of appropriate storage and secretion of mature insulin.
Methods: To further explore the potential role of Nnat in the regulation of body weight and adiposity, we studied classical imprinting-related phenotypes such as placental, fetal, and postnatal growth trajectory patterns that may impact upon subsequent adult metabolic phenotypes.
Results: Here we find that, in contrast to the lack of any body weight or feeding phenotypes on the C57BL/6J background, deletion of Nnat in mice on 129S2/Sv background causes a postnatal growth restriction with reduced adipose tissue accumulation, followed by catch up growth after weaning. This was in the absence of any effect on fetal growth or placental development. In adult 129S2/Sv mice, Nnat deletion was associated with hyperphagia, reduced energy expenditure, and partial leptin resistance. Lack of neuronatin also potentiated obesity caused by either aging or high fat diet feeding.
Conclusion: The imprinted gene Nnat plays a key role in postnatal growth, adult energy homeostasis, and the pathogenesis of obesity via catch up growth effects, but this role is dependent upon genetic background.[Hide abstract]
Objective: Hypothalamic Pro-opiomelanocortin (POMC) and Neuropeptide Y/Agouti-Related Peptide (NPY/AgRP) neurons are critical nodes of a circuit within the brain that sense key metabolic cues as well as regulate metabolism. Importantly, these neurons retain an innate ability to rapidly reorganize synaptic inputs and electrophysiological properties in response to metabolic state. While the cellular properties of these neurons have been investigated in the context of obesity, much less is known about the effects of exercise training.
Methods: In order to further investigate this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY/AgRP neurons for patch-clamp electrophysiology experiments.
Results: Using whole-cell patch-clamp electrophysiology, we found exercise depolarized and increased firing rate of arcuate POMC neurons. The increased excitability of POMC neurons was concomitant with increased excitatory inputs to these neurons. In agreement with recent work suggesting leptin plays an important role in the synaptic (re)organization of POMC neurons, POMC neurons which express leptin receptors were more sensitive to exercise-induced changes in biophysical properties. Opposite to effects observed in POMC neurons, NPY neurons were shunted toward inhibition following exercise.
Conclusions: Together, these data support a rapid reorganization of synaptic inputs and biophysical properties in response to exercise, which may facilitate adaptations to altered energy balance and glucose metabolism.[Hide abstract]
Objective: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN.
Methods: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis.
Results: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure.
Conclusions: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.[Hide abstract]
Objective: Obesity-associated metabolic dysfunctions are linked to dysregulated production of adipokines. Accumulating evidence suggests a role for fat-derived extracellular vesicles (EVs) in obesity-metabolic disturbances. Since EVs convey numerous proteins we aimed to evaluate their contribution in adipokine secretion.
Methods: Plasma collected from metabolic syndrome patients were used to isolate EV subtypes, namely microvesicles (MVs) and exosomes (EXOs). Numerous soluble factor concentrations were measured successively on total, MV- and EXO-depleted plasma by multiplexed immunoassays.
Results: Circulating MVs and EXOs were significantly increased with BMI, supporting a role of EVs as metabolic relays in obesity. Obesity was associated with dysregulated soluble factor production. Sequential depletion of plasma MVs and EXOs did not modify plasma levels of these molecules, with the exception of Macrophage Migration Inhibitory Factor (MIF). Half of plasma MIF circulated within MVs, and this MV secretory pathway was conserved over different MIF-producing cells. Although MV-associated MIF triggered rapid ERK1/2 activation in macrophages, these functional MV-MIF effects specifically relied on MIF tautomerase activity.
Conclusion: Our results emphasize the importance of reconsidering MIF-metabolic actions with regard to its MV-associated form and opening new EV-based strategies for therapeutic MIF approaches.[Hide abstract]
Objectives: The deficit of Glyoxalase I (Glo1) and the subsequent increase in methylglyoxal (MG) has been reported to be one the five mechanisms by which hyperglycemia causes diabetic late complications. Aldo-keto reductases (AKR) have been shown to metabolize MG; however, the relative contribution of this superfamily to the detoxification of MG in vivo, particularly within the diabetic state, remains unknown.
Methods: CRISPR/Cas9-mediated genome editing was used to generate a Glo1 knock-out (Glo1−/−) mouse line. Streptozotocin was then applied to investigate metabolic changes under hyperglycemic conditions.
Results: Glo1−/− mice were viable and showed no elevated MG or MG-H1 levels under hyperglycemic conditions. It was subsequently found that the enzymatic efficiency of various oxidoreductases in the liver and kidney towards MG were increased in the Glo1−/− mice. The functional relevance of this was supported by the altered distribution of alternative detoxification products. Furthermore, it was shown that MG-dependent AKR activity is a potentially clinical relevant pathway in human patients suffering from diabetes.
Conclusions: These data suggest that in the absence of GLO1, AKR can effectively compensate to prevent the accumulation of MG. The combination of metabolic, enzymatic, and genetic factors, therefore, may provide a better means of identifying patients who are at risk for the development of late complications caused by elevated levels of MG.[Hide abstract]
Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells; however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development.
Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors: Oct4, Sox2, Klf4, cMyc. We performed pluripotency characterization and directed the differentiation of control and IRKO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm), and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IRKO iPSCs.
Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3, and cMyc were upregulated, while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold, respectively, in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk and an increase in the total mTor and Erk proteins in IRKO iPSCs in the basal unstimulated state. Stimulation with leukemia inhibitory factor (LIF) showed a ∼33% decrease of phospho-ERK in IRKO iPSCs. On the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparγ, Fabp4, C/ebpα, and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1, and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network including several key proteins involved in diverse aspects of growth and embryonic development.
Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1, and LIF stimulation in mouse iPSCs to reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.[Hide abstract]