Neuronal Insulin Signaling Research Articles

Two weeks of exercise alters neuronal extracellular vesicle insulin signaling proteins and pro-BDNF in older adults with prediabetes.

Adults with prediabetes are at risk for Alzheimer's Disease and Related Dementia (ADRD). While exercise may lower ADRD risk, the exact mechanism is unclear. We tested the hypothesis that short-term exercise would raise neuronal insulin signaling and pro-BDNF in neuronal extracellular vesicles (nEVs) in prediabetes. Twenty-one older adults (18F, 60.0 ± 8.6 yrs.; BMI: 33.5 ± 1.1 kg/m2) with prediabetes (ADA criteria; 75 g OGTT) were randomized to 12 supervised work-matched continuous (n = 13, 70% HRpeak) or interval (n = 8, 90% HRpeak and 50% HRpeak for 3 min each) sessions over 2-wks for 60 min/d. Aerobic fitness (VO2peak) and body weight were assessed. After an overnight fast, whole-body glucose tolerance (total area under the curve, tAUC) and insulin sensitivity (SIis) were determined from a 120 min 75 g OGTT. nEVs were acquired from 0 and 60 min time-points of the OGTT, and levels of insulin signaling proteins (i.e., p-IRS-1, total-/p-Akt, pERK1/2, pJNK1/2, and pp38) and pro-BNDF were measured. OGTT stimulatory effects were calculated from protein differences (i.e., OGTT 60-0 min). Adults were collapsed into a single group as exercise intensity did not affect nEV outcomes. Exercise raised VO2peak (+1.4 ± 2.0 mL/kg/min, p = 0.008) and insulin sensitivity (p = 0.01) as well as decreased weight (-0.4 ± 0.9 kg, p = 0.04) and whole-body glucose tAUC120min (p = 0.02). Training lowered 0-min pro-BDNF (704.1 ± 1019.0 vs. 414.5 ± 533.5, p = 0.04) and increased OGTT-stimulated tAkt (-51.8 ± 147.2 vs. 95 ± 204.5 a.u., p = 0.01), which was paralleled by reduced pAkt/tAkt at 60 min of the OGTT (1.3 ± 0.2 vs. 1.2 ± 0.1 a.u., p = 0.04). Thus, 2 weeks of exercise altered neuronal insulin signaling responses to glucose ingestion and lowered pro-BNDF among adults with prediabetes, thereby potentially lowering ADRD risk.

Insulin Signaling in Neurons of the Tuberal Area of the Hypothalamus of Rats During Aging

Insulin Signaling in Neurons of the Tuberal Area of the Hypothalamus of Rats During Aging

Loss of Insulin Signaling in Microglia Impairs Cellular Uptake of Aβ and Neuroinflammatory Response Exacerbating Alzheimer-like Neuropathology.

Insulin receptors are present on cells throughout the body, including the brain. Dysregulation of insulin signaling in neurons and astrocytes has been implicated in altered mood, cognition, and the pathogenesis of Alzheimers disease (AD). To define the role of insulin signaling in microglia, the primary phagocytes in brain critical for maintenance and damage repair, we created mice with an inducible microglia-specific insulin receptor knockout (MG-IRKO). RiboTag profiling of microglial mRNAs revealed that loss of insulin signaling results in alterations of gene expression in pathways related to innate immunity and cellular metabolism. In vitro, loss of insulin signaling in microglia results in metabolic reprograming with an increase in glycolysis and impaired uptake of Aβ. In vivo, MG-IRKO mice exhibit alterations in mood and social behavior, and when crossed with the 5xFAD mouse model of AD, the resultant mice exhibit increased levels of Aβ; plaque and elevated neuroinflammation. Thus, insulin signaling in microglia plays a key role in microglial cellular metabolism, neuroinflammation and the ability of the cells to take up Aβ; such that reduced insulin signaling in microglia alters mood and social behavior and accelerates AD pathogenesis. Together these data indicate key roles of insulin action in microglia and the potential of targeting insulin signaling in microglia in treatment of AD.

1-Deoxynojirimycin attenuates pathological markers of Alzheimer's disease in the invitro model of neuronal insulin resistance.

Insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM), has emerged as a pathological feature in Alzheimer's disease (AD). Given the shared role of insulin resistance in T2DM and AD, repurposing peripheral insulin sensitizers is a promising strategy to preserve neuronal insulin sensitivity and prevent AD. 1-Deoxynojirimycin (DNJ), a bioactive iminosugar, exhibited insulin-sensitizing effects in metabolic tissues and was detected in brain tissue post-oral intake. However, its impact on brain and neuronal insulin signaling has not been described. Here, we investigated the effect of DNJ treatment on insulin signaling and AD markers in insulin-resistant human SK-N-SH neuroblastoma, a cellular model of neuronal insulin resistance. Our findings show that DNJ increased the expression of insulin signaling genes and the phosphorylation status of key molecules implicated in insulin resistance (Y1146-pIRβ, S473-pAKT, S9-GSK3B) while also elevating the expression of glucose transporters Glut3 and Glut4, resulting in higher glucose uptake upon insulin stimuli. DNJ appeared to mitigate the insulin resistance-driven increase in phosphorylated tau and Aβ1-42 levels by promoting insulin-induced phosphorylation of GSK3B (a major tau kinase) and enhancing mRNA expression of the insulin-degrading enzyme (IDE) pivotal for insulin and Aβ clearance. Overall, our study unveils probable mechanisms underlying the potential benefits of DNJ for AD, wherein DNJ attenuates tau and amyloid pathologies by reversing neuronal insulin resistance. This provides a scientific basis for expanding the use of DNJ-containing products for neuroprotective purposes and prompts further research into compounds with similar mechanisms of action.

WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling.

Certain areas of the brain involved in episodic memory and behavior, such as the hippocampus, express high levels of insulin receptors and glucose transporter-4 (GLUT4) and are responsive to insulin. Insulin and neuronal glucose metabolism improve cognitive functions and regulate mood in humans. Insulin-dependent GLUT4 trafficking has been extensively studied in muscle and adipose tissue, but little work has demonstrated either how it is controlled in insulin-responsive brain regions or its mechanistic connection to cognitive functions. In this study, we demonstrate that inhibition of WNK (With-No-lysine (K)) kinases improves learning and memory in mice. Neuronal inhibition of WNK enhances in vivo hippocampal glucose uptake. Inhibition of WNK enhances insulin signaling output and insulin-dependent GLUT4 trafficking to the plasma membrane in mice primary neuronal cultures and hippocampal slices. Therefore, we propose that the extent of neuronal WNK kinase activity has an important influence on learning, memory and anxiety-related behaviors, in part, by modulation of neuronal insulin signaling.

Insulin signaling accelerates the anterograde movement of Rab4 vesicles in axons through Klp98A/KIF16B recruitment via Vps34-PI3Kinase.

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis.

Age-Related Dynamics of Insulin Signaling in Neurons of the Rat Hypothalamic Tuberal Nuclei

Age-Related Dynamics of Insulin Signaling in Neurons of the Rat Hypothalamic Tuberal Nuclei

SAT047 Regulation Of The Insulin Receptor And The Insulin-like Growth Factor 1 Receptor By miR-322-5p, A miR-16 Family Member, In Hypothalamic Neuronal Models

Abstract Disclosure: W. He: None. N. Loganathan: None. K.W. Mak: None. E. McIlwraith: None. D.D. Belsham: None. Insulin signals through the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R) in hypothalamic neurons to control food intake and peripheral metabolism. A contributor to obesity, and subsequent comorbidities such type 2 diabetes and heart disease, is the development of cellular insulin resistance in hypothalamic neurons. However, the molecular changes to neuronal insulin signaling remain to be fully elucidated. MicroRNAs (miRNAs) inhibit translation of specific mRNAs; thus, this study aimed to understand the involvement of miRNAs in the regulation of insulin signaling and resistance in hypothalamic neurons. To profile miRNAs expressed in hypothalamic neurons, RNA from the whole hypothalami of 14-week-old CD1 male mice (n = 4), as well as the immortalized hypothalamic neuronal cell lines mHypoE-46 (n = 3) and mHypoA-59 (n = 3), each co-expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP), were assessed using the Affymetrix GeneChip miRNA 4.0 Array. Notably, miR-16 family members, including miR-16-5p, miR-15b-5p, and miR-322-5p, were among the most highly expressed miRNAs in each case. In the mHypoA-59 neurons, overexpression of miR-322-5p for 24 hours decreased the mRNA levels of Igf1r (-26.1%; p = 0.0007; n = 4) and the protein level of INSR-beta (-31.8%; p = 0.0023). These results suggest that the miR-16 family plays an inhibitory role in insulin signaling in hypothalamic neurons. To study the involvement of miRNAs in hyperinsulinemia-induced neuronal insulin resistance, the mHypoE-46 neurons were treated with 100 nM insulin for 24 hours to induce cellular insulin resistance, as characterized by decreased INSR-beta protein (-96.3%; p < 0.0001; n = 4) and a resulting decrease in insulin-induced phosphorylation of protein kinase B (AKT) (-67.6%; p = 0.01; n = 4). GeneChip miRNA array analysis showed that insulin overexposure disrupted the expression of 48 miRNAs (p < 0.05, n = 3), including the upregulation of miR-18a-3p, mir-322, miR-494-3p, and miR-671-3p. Upon RT-qPCR validation (n = 3), we established that prolonged (24 hours), but not acute (1-6 hours), insulin exposure upregulated miR-18a-3p (+62%; p = 0.0104), miR-671-3p (+111%; p = 0.0079), and miR-1983 (+97%; p = 0.0181). miR-671-3p, miR-494-3p, and miR-18a-3p have been shown to decrease phosphatase and tensin homolog (PTEN) levels, which can promote neuronal insulin resistance based on bioinformatic analysis. Overall, these results suggest hyperinsulinemia disrupts the expression of specific miRNAs in hypothalamic neurons to promote cellular insulin resistance. Knowledge derived from these studies will provide insight into hypothalamus-derived miRNAs that could be targeted for miRNA-based diagnostics and therapeutics for early central insulin resistance in humans.(Supported by the CRC, CIHR, NSERC, EndoSoc, and BBDC) Presentation: Saturday, June 17, 2023

PP1γ regulates neuronal insulin signaling and aggravates insulin resistance leading to AD-like phenotypes

BackgroundPP1γ is one of the isoforms of catalytic subunit of a Ser/Thr phosphatase PP1. The role of PP1γ in cellular regulation is largely unknown. The present study investigated the role of PP1γ in regulating neuronal insulin signaling and insulin resistance in neuronal cells. PP1 was inhibited in mouse neuroblastoma cells (N2a) and human neuroblastoma cells (SH-SY5Y). The expression of PP1α and PP1γ was determined in insulin resistant N2a, SH-SY5Y cells and in high-fat-diet-fed-diabetic mice whole-brain-lysates. PP1α and PP1γ were silenced by siRNA in N2a and SH-SY5Y cells and effect was tested on AKT isoforms, AS160 and GSK3 isoforms using western immunoblot, GLUT4 translocation by confocal microscopy and glucose uptake by fluorescence-based assay.ResultsResults showed that, in one hand PP1γ, and not PP1α, regulates neuronal insulin signaling and insulin resistance by regulating phosphorylation of AKT2 via AKT2-AS160-GLUT4 axis. On the other hand, PP1γ regulates phosphorylation of GSK3β via AKT2 while phosphorylation of GSK3α via MLK3. Imbalance in this regulation results into AD-like phenotype.ConclusionPP1γ acts as a linker, regulating two pathophysiological conditions, neuronal insulin resistance and AD.9kZE1HTKkUH8PMrXCf3pWdVideo

Higher untrained fitness exerts a neuroprotection in Independence to caloric restriction or exercise in high-fat diet-induced obesity

We investigated whether weight maintenance following short-term caloric restriction or exercise exerted neuroprotective effects on obesity induced by a high-fat diet. We also sought to identify whether the neuroprotective effects of higher untrained fitness persisted in the obese condition, both with and without caloric restriction or exercise. Male Wistar rats were fed with either a normal diet (ND) or a high-fat diet (HFD) for 12 weeks. At week 12, untrained fitness and blood metabolic parameters were measured. The ND-fed rats continuously received a ND for 16 additional weeks. HFD-fed rats were randomly assigned to 5 groups as of the followings: 1) an additional 16 weeks of HFD without intervention, 2) 10-week weight maintenance following 6-week short-term caloric restriction, 3) long-term caloric restriction (16 weeks), 4) 10-week weight maintenance following 6 weeks of HFD plus short-term exercise, and 5) HFD plus long-term exercise (16 weeks). Untrained fitness, blood metabolic parameters, and behavioral tests were then determined. Thereafter, the rats were euthanized for molecular studies. Our results demonstrated that long-term caloric restriction had the greatest systemic metabolic benefit among all interventions. Long-term caloric restriction and exercise equally attenuated HFD-induced cognitive impairment by improving synaptic function, blood-brain barrier integrity, mitochondrial health, and neurogenesis, and reducing oxidative stress, neuroinflammation, apoptosis, and Alzheimer's-related pathology. Weight maintenance following short-term caloric restriction showed no benefit to neurogenesis. Weight maintenance following short-term exercise exerted no benefit on synaptic function, neuronal insulin signaling and metabolism, autophagy, and neurogenesis. Interestingly, we found that higher untrained fitness level at week 12 showed positive correlations with more favorable brain profiles at week 28 in HFD-fed rats, both with and without caloric restriction or exercise. All of these findings suggested that higher untrained fitness exerts neuroprotection in HFD-induced obesity independently of caloric restriction or exercise. Therefore, targeting enhancement of untrained fitness may lead to more effective treatment of neurodegeneration in obese condition.

Arsenic induces the global hypophosphorylation of insulin receptor substrate proteins in differentiated human neuroblastoma SH-SY5Y cells

We recently reported that arsenic disrupted neuronal insulin signaling. Here, we further investigated the effect of arsenic on insulin receptor substrate (IRS) proteins, which are crucial downstream signaling molecules of insulin in differentiated human neuroblastoma SH-SY5Y cells. We also found that prolonged arsenic treatment accelerated the migration of IRS1 and IRS2 on SDS-PAGE. Treatment with phosphatases abolished the arsenic-induced increased mobility of IRS, suggesting that the electrophoretic mobility shift of IRS on SDS-PAGE by arsenic was phosphorylation-dependent. By using label-free mass spectrometry, the phosphorylation sites of IRS1 were found to be S24, S345, S636, T774, S1057, S1058, and S1070, while those of IRS2 were at S645, Y653, T657, S665, S667, S669, S672, S915, and S1203, which were at least 2-fold lower than found in the control. These findings indicated a global hypophosphorylation of IRS proteins after prolonged arsenic treatment. In addition, four novel phosphorylation sites were identified on IRS1 (T774, S1057, S1058, and S1070), with another two on IRS2 (S665 and S667). As basal IRS phosphorylation plays an important role in insulin signaling, the reduction of IRS phosphorylation on multiple residues may underlie arsenic-impaired insulin signaling in neurons.

Reduced insulin signaling in neurons induces sex-specific health benefits.

Reduced activity of insulin/insulin-like growth factor signaling (IIS) extends health and life span in mammals. Loss of the insulin receptor substrate 1 (Irs1) gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS-mediated longevity are currently unknown. Here, we measured survival and health span in mice lacking IRS1 specifically in liver, muscle, fat, and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of IRS1 in more than one tissue is required for life-span extension. Loss of IRS1 in liver, muscle, and fat did not improve health. In contrast, loss of neuronal IRS1 increased energy expenditure, locomotion, and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4, and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of aging in response to reduced IIS associated with improved health at old age.

Small molecule targeting long noncoding RNA GAS5 administered intranasally improves neuronal insulin signaling and decreases neuroinflammation in an aged mouse model

Shifts in normal aging set stage for neurodegeneration and dementia affecting 1 in 10 adults. The study demonstrates that lncRNA GAS5 is decreased in aged and Alzheimer’s disease brain. The role and targets of lncRNA GAS5 in the aging brain were elucidated using a GAS5-targeting small molecule NPC86, a frontier in lncRNA-targeting therapeutic. Robust techniques such as molecular dynamics simulation of NPC86 binding to GAS5, in vitro functional assays demonstrating that GAS5 regulates insulin signaling, neuronal survival, phosphorylation of tau, and neuroinflammation via toll-like receptors support the role of GAS5 in maintaining healthy neurons. The study demonstrates the safety and efficacy of intranasal NPC86 treatment in aged mice to improve cellular functions with transcriptomic analysis in response to NPC86. In summary, the study demonstrates that GAS5 contributes to pathways associated with neurodegeneration and NPC86 has tremendous therapeutic potential to prevent the advent of neurodegenerative diseases and dementias.

PP2Cα aggravates neuronal insulin resistance leading to AD-like phenotype in vitro

Neuronal insulin resistance is a major risk for development of Alzheimer's Disease (AD). Studies already reported few kinases participating in neuronal insulin signaling connected with progression of AD pathogenesis, yet complete information is missing. α isoform of Protein Phosphatase-2C (PP2C) is a Ser/Thr phosphatase, only known in 3T3-L1 adipocytes as a positive regulator of insulin signaling. However, many aspects of its function in neuronal insulin signaling and insulin resistance are unidentified. Recently, we reported that PP2Cα positively regulates neuronal glucose uptake possibly by a mechanism of dephosphorylation of IRS-1 at Ser522 and by inactivating AMPK, exacerbating hyperinsulinemia mediated neuronal insulin resistance. Since PP2Cα affected neuronal insulin signaling and AD is connected to neuronal insulin resistance, in the present study, we studied the role of PP2Cα in regulating activities of both isoforms of GSK3α and GSK3β (one of the leading kinases for AD progression). The results led us to test the role of PP2Cα on AD hallmarks. Silencing of PP2Cα caused hyperphosphorylation of a potential kinase Tau, leading to NFT formation and increased Aβ deposition. Our study thereby demonstrates escalation of hyperinsulinemia mediated neuronal insulin resistance leading to AD-like pathogenesis by PP2Cα in vitro and hints a novel molecule, PP2Cα, linking AD pathogenesis.

PROTECTIVE AND ANTIOXIDANT EFFECTS OF INSULIN ON RAT BRAIN CORTICAL NEURONS IN A MODEL OF OXYGEN AND GLUCOSE DEPRIVATION IN VITRO

Intranasal insulin is one of the most promising protectors in the treatment of neurodegenerative and other diseases associated with brain injuries. In these diseases, insulin levels in the brain (in contrast to its blood levels) are as a rule heavily reduced, which, along with the development of insulin resistance, leads to impaired insulin signaling in neurons. The aim of this work was to study the protective effect of insulin on cultured rat cortical neurons using an in vitro oxygen–glucose deprivation (OGD) model of ischemia–reperfusion brain injury followed by a resumption of oxygen and glucose supply to neurons. OGD exposure for 1 or 3 h with subsequent incubation of cultured rat cortical neurons in complete (oxygen- and glucose-containing) growth medium decreased neuronal viability and increased the production of reactive oxygen species, while the preincubation of neurons with insulin at micromolar concentrations had protective and antioxidant effects. One-hour OGD followed by incubation in complete growth medium led to downregulation of protein kinase B/Akt (decreased pAkt(Ser473)/Akt ratio) and upregulation of glycogen synthase kinase-3beta (GSK-3beta), one of the main Akt targets (decreased pGSK-3beta(Ser9)/GSK-3beta ratio). In contrast, preincubation with insulin activated Akt and inactivated GSK-3beta. Apparently, these effects of insulin significantly contribute to its neuroprotective action, because GSK-3beta activation leads to mitochondrial dysfunction and neuronal death. Insulin was shown to increase the neuronal activity of protein kinase regulated by extracellular signals (ERK1/2), which was diminished by OGD and subsequent exposure to growth medium containing glucose and oxygen.

PHLPP isoforms differentially regulate Akt isoforms and AS160 affecting neuronal insulin signaling and insulin resistance via Scribble

BackgroundThe aim of the present study was to determine the role of individual PHLPP isoforms in insulin signaling and insulin resistance in neuronal cells.MethodsPHLPP isoforms were either silenced or overexpressed individually, and the effects were observed on individual Akt isoforms, AS160 and on neuronal glucose uptake, under insulin sensitive and resistant conditions. To determine PHLPP regulation itself, we tested effect of scaffold protein, Scribble, on PHLPP isoforms and neuronal glucose uptake.ResultsWe observed elevated expression of both PHLPP1 and PHLPP2 in insulin resistant neuronal cells (Neuro-2A, mouse neuroblastoma; SHSY-5Y, human neuroblastoma) as well as in the whole brain lysates of high-fat-diet mediated diabetic mice. In insulin sensitive condition, PHLPP isoforms differentially affected activation of all Akt isoforms, wherein PHLPP1 regulated serine phosphorylation of Akt2 and Akt3, while PHLPP2 regulated Akt1 and Akt3. This PHLPP mediated Akt isoform specific regulation activated AS160 affecting glucose uptake. Under insulin resistant condition, a similar trend of results were observed in Akt isoforms, AS160 and glucose uptake. Over-expressed PHLPP isoforms combined with elevated endogenous expression under insulin resistant condition drastically affected downstream signaling, reducing neuronal glucose uptake. No compensation was observed amongst PHLPP isoforms under all conditions tested, indicating independent roles and pointing towards possible scaffolding interactions behind isoform specificity. Silencing of Scribble, a scaffolding protein known to interact with PHLPP, affected cellular localization of both PHLPP1 and PHLPP2, and caused increase in glucose uptake.ConclusionsPHLPP isoforms play independent roles via Scribble in regulating Akt isoforms differentially, affecting AS160 and neuronal glucose uptake.BQN7nd48EhvKr6o-7gBbL3Video abstract

Functional consequences of brain exposure to saturated fatty acids: From energy metabolism and insulin resistance to neuronal damage.

Saturated fatty acids (FAs) are the main component of high-fat diets (HFDs), and high consumption has been associated with the development of insulin resistance, endoplasmic reticulum stress and mitochondrial dysfunction in neuronal cells. In particular, the reduction in neuronal insulin signaling seems to underlie the development of cognitive impairments and has been considered a risk factor for Alzheimer's disease (AD). This review summarized and critically analyzed the research that has impacted the field of saturated FA metabolism in neurons. We reviewed the mechanisms for free FA transport from the systemic circulation to the brain and how they impact neuronal metabolism. Finally, we focused on the molecular and the physiopathological consequences of brain exposure to the most abundant FA in the HFD, palmitic acid (PA). Understanding the mechanisms that lead to metabolic alterations in neurons induced by saturated FAs could help to develop several strategies for the prevention and treatment of cognitive impairment associated with insulin resistance, metabolic syndrome, or type II diabetes.

Deletion of serine racemase reverses neuronal insulin signaling inhibition by amyloid-β oligomers.

Dysregulation of insulin signaling in the Alzheimer's disease (AD) brain has been extensively reported. Serine racemase (SR) modulates insulin secretion in pancreatic islets. This study aimed to examine whether SR regulates insulin synthesis and secretion in neurons, thereby modulating insulin signaling in the AD brain. Srr-knockout (Srr-/- ) mice generated with the CRISPR/Cas9 technique were used. Using immunofluorescence and fluorescence in situ hybridization, levels of insulin protein and insulin(ins2) mRNA were significantly increased in the hippocampal but not in hypothalamic sections of Srr-/- mice compared with WT mice. Real-time quantitative PCR revealed that ins2 mRNA from primary hippocampal neuronal cultures of Srr-/- mice was significantly increased compared with that from cultured neurons of WT mice. Notably, the secretion of proinsulin C-peptide was increased in Srr-/- neurons relative to WT neurons. By examining membrane fractional proteins with immunoblotting, Srr-/- neurons retained ATP-dependent potassium channels on plasmalemma and correspondingly contained higher levels of p-AMPK. After treatment with Aβ42, the phosphorylation levels of insulin receptor substrate at serine 616 636 (p-IRS1ser616,636 ) were significantly lower, whereas p-AKT308 and p-AKT473 were higher in Srr-/- neurons than in WT neurons, respectively. The phosphorylated form of c-Jun N-terminal kinase decreased in the cultured Srr-/- neurons relative to the WT neurons upon Aβ42 treatment. In contrast, phosphorylated protein kinase R remained at the same levels. Further, reactive oxygen species were reduced in cultured Srr-/- neurons under Aβ42 treatment relative to the WT neurons. Collectively, our study indicated that Srr deletion promoted insulin synthesis and secretion of proinsulin C-peptide, thereby reversing insulin resistance by Aβ42. This study suggests that targeting the neuronal SR may be utilized to enhance insulin signaling which is inhibited at the early stage of the AD brain.

Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism

Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

Ser/Thr phosphatases: One of the key regulators of insulin signaling.

Protein phosphorylation is an important post-translational modification that regulates several cellular processes including insulin signaling. The evidences so far have already portrayed the importance of balanced actions of kinases and phosphatases in regulating the insulin signaling cascade. Therefore, elucidating the role of both kinases and phosphatases are equally important. Unfortunately, the role of phosphatases is less studied as compared to kinases. Since brain responds to insulin and insulin signaling is reported to be crucial for many neuronal processes, it is important to understand the role of neuronal insulin signaling regulators. Ser/Thr phosphatases seem to play significant roles in regulating neuronal insulin signaling. Therefore, in this review, we discussed the involvement of Ser/Thr phosphatases in regulating insulin signaling and insulin resistance in neuronal system at the backdrop of the same phosphatases in peripheral insulin sensitive tissues.