Neuronal insulin signaling and resistance: a balancing act of kinases and phosphatases.

Insulin signaling cascade in peripheral insulin-sensitive tissues regulates whole-body glucose metabolism. Any deregulation in this pathway leads to insulin resistance, ultimately leading to metabolic diseases like type 1 diabetes, type 2 diabetes, and obesity. Insulin signaling in the brain has also been studied for many decades and associated with many primary functions like maintenance of synaptic plasticity, regulation of cognition, and circadian rhythm. Importantly, neuronal insulin signaling has also been associated with the regulation of neuronal glucose uptake. Any impairment in neuronal insulin signaling affecting neuronal glucose uptake has been associated with neurodegenerative disorders like Alzheimer's disease, the process now being termed as type 3 diabetes. Since the criticality lies in proper signaling cascade, determining important points of deregulation is important. In this review, we have discussed some critical points of such deregulation, dividing them into two classes of enzymes: kinases and phosphatases. We have highlighted their individual roles in neuronal insulin signaling, along with their possible implications in neuronal insulin resistance. Future strategies targeting these nodes in neuronal insulin signaling might be helpful in exploring potential therapeutic opportunities to overcome neuronal insulin resistance and related neurodegenerative diseases.

PKCα Isoform Inhibits Insulin Signaling and Aggravates Neuronal Insulin Resistance.

Overexpression of PKCα has been linked to inhibit insulin signaling disrupting IRS-1 and Akt phosphorylations in skeletal muscle. PKCα inhibits IRS-1 and Akt phosphorylations, but not required for insulin-stimulated glucose transport in skeletal muscles. Inhibition of PKCα increased whereas in some studies decreased GLUT-4 levels at the plasma membrane in skeletal muscles and adipocytes. Controversial studies have reported opposite expression pattern of PKCα expression in insulin-resistant skeletal muscles. These findings indicate that the role of PKCα on insulin signaling is controversial and could be tissue specific. Evidently, studies are required to decipher the role of PKCα in regulating insulin signaling and preferably in other cellular systems. Utilizing neuronal cells, like Neuro-2a, SHSY-5Y and insulin-resistant diabetic mice brain tissues; we have demonstrated that PKCα inhibits insulin signaling, through IRS-Akt pathway in PP2A-dependent mechanism by an AS160-independent route involving 14-3-3ζ. Inhibition and silencing of PKCα improves insulin sensitivity by increasing GLUT-4 translocation to the plasma membrane and glucose uptake. PKCα regulates GSK3 isoforms in an opposite manner in insulin-sensitive and in insulin-resistant condition. Higher activity of PKCα aggravates insulin-resistant neuronal diabetic condition through GSK3ß but not GSK3α. Our results mechanistically explored the contribution of PKCα in regulating neuronal insulin resistance and diabetes, which opens up new avenues in dealing with metabolic disorders and neurodegenerative disorders.

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

BACKGROUND: PP1γ 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. RESULTS: Results 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 GSK3ß via AKT2 while phosphorylation of GSK3α via MLK3. Imbalance in this regulation results into AD-like phenotype. CONCLUSION: PP1γ acts as a linker, regulating two pathophysiological conditions, neuronal insulin resistance and AD. Video Abstract.

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 GSK3ß (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 Aß 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.

Cellular and Molecular Regulation of Exercise-A Neuronal Perspective.

The beneficial effects of exercise on the proper functioning of the body have been firmly established. Multi-systemic metabolic regulation of exercise is the consequence of multitudinous changes that occur at the cellular level. The exercise responsome comprises all molecular entities including exerkines, miRNA species, growth factors, signaling proteins that are elevated and activated by physical exercise. Exerkines are secretory molecules released by organs such as skeletal muscle, adipose tissue, liver, and gut as a function of acute/chronic exercise. Exerkines such as FNDC5/irisin, Cathepsin B, Adiponectin, and IL-6 circulate through the bloodstream, cross the blood-brain barrier, and modulate the expression of important signaling molecules such as AMPK, SIRT1, PGC1α, BDNF, IGF-1, and VEGF which further contribute to improved energy metabolism, glucose homeostasis, insulin sensitivity, neurogenesis, synaptic plasticity, and overall well-being of the body and brain. These molecules are also responsible for neuroprotective adaptations that exercise confers on the brain and potentially ameliorate neurodegeneration. This review aims to detail important cellular and molecular species that directly or indirectly mediate exercise-induced benefits in the body, with an emphasis on the central nervous system.

Emerging roles of PHLPP phosphatases in the nervous system.

It has been more than a decade since the discovery of a novel class of phosphatase, the Pleckstrin Homology (PH) domain Leucine-rich repeat Protein Phosphatases (PHLPP). Over time, they have been recognized as crucial regulators of various cellular processes, such as memory formation, cellular survival and proliferation, maintenance of circadian rhythm, and others, with any deregulation in their expression or cellular localization causing havoc in any cellular system. With the ever-growing number of downstream substrates across multiple tissue systems, a web is emerging wherein the central point is PHLPP. A slight nick in the normal signaling cascade of the two isoforms of PHLPP, namely PHLPP1 and PHLPP2, has been recently found to invoke a variety of neurological disorders including Alzheimer's disease, epileptic seizures, Parkinson's disease, and others, in the neuronal system. Improper regulation of the two isoforms has also been associated with various disease pathologies such as diabetes, cardiovascular disorders, cancer, musculoskeletal disorders, etc. In this review, we have summarized all the current knowledge about PHLPP1 (PHLPP1α and PHLPP1ß) and PHLPP2 and their emerging roles in regulating various neuronal signaling pathways to pave the way for a better understanding of the complexities. This would in turn aid in providing context for the development of possible future therapeutic strategies.

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

BACKGROUND: The aim of the present study was to determine the role of individual PHLPP isoforms in insulin signaling and insulin resistance in neuronal cells. METHODS: PHLPP 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. RESULTS: We 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. CONCLUSIONS: PHLPP isoforms play independent roles via Scribble in regulating Akt isoforms differentially, affecting AS160 and neuronal glucose uptake. Video abstract.

PP2Cα positively regulates neuronal insulin signalling and aggravates neuronal insulin resistance.

PP2Cα is one of the newly identified isoforms of metal-dependent protein phosphatases (PPM). The role of this phosphatase in neuronal insulin signalling is completely unknown. In the present study, we show insulin-mediated rapid upregulation of a protein of the insulin signalling cascade, PP2Cα, in mouse N2a cells and human SH-SY5Y cells. By contrast, such PP2Cα upregulation is not observed in insulin-resistant conditions despite insulin stimulation. Here, we report that, under insulin-sensitive and insulin-resistant conditions, the translation of PP2Cα was regulated by insulin through c-Jun N-terminal kinase. PP2Cα in turn dephosphorylated a novel inhibitory site of insulin receptor substrate-1 at Ser522 and AMP-activated protein kinase, hence positively regulating neuronal insulin signalling and insulin resistance.

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.

CDK5 inhibition improves glucose uptake in insulin-resistant neuronal cells via ERK1/2 pathway.

Role of CDK5 and its inhibition in various neuronal processes and functions are well established. However, role of CDK5 and its inhibition in neuronal insulin-signaling and-resistance is not yet explored. In the present study, we investigated the effect of CDK5 inhibition in neuronal insulin signaling, specifically insulin-stimulated glucose uptake. CDK5 expression in neuro-2a cells was increased under insulin-resistant state, developed by chronic treatment of insulin, confirming the crucial role of CDK5 in insulin resistance in neuronal cells. However, whether increased expression of CDK5 in hyperinsulinemia-mediated insulin-resistant conditions is a cause or a consequence, is still an unanswered question. We showed that CDK5 inhibition did not affect basal insulin signaling; however, insulin-stimulated glucose uptake enhanced in insulin-resistant cells. Moreover, CDK5 inhibition could improve glucose uptake, the ultimate outcome of insulin signaling, in insulin-resistant neuro-2a cells. We first time showed that CDK5 inhibition by roscovitine could ameliorate insulin resistance and increase glucose uptake in neuronal cells via ERK1/2 pathway. Our study provides intriguing insights about the effect of CDK5 inhibition on neuronal insulin resistance and opens up a new paradigm to develop new therapeutic strategies for neuronal insulin resistance and associated pathophysiological conditions.

Role of Akt isoforms in neuronal insulin signaling and resistance.

The aim of the present study was to determine the role of Akt isoforms in insulin signaling and resistance in neuronal cells. By silencing Akt isoforms individually and in pairs, in Neuro-2a and HT22 cells we observed that, in insulin-sensitive condition, Akt isoforms differentially reduced activation of AS160 and glucose uptake with Akt2 playing the major role. Under insulin-resistant condition, phosphorylation of all isoforms and glucose uptake were severely affected. Over-expression of individual isoforms in insulin-sensitive and resistant cells differentially reversed AS160 phosphorylation with concomitant reversal in glucose uptake indicating a compensatory role of Akt isoforms in controlling neuronal insulin signaling. Post-insulin stimulation Akt2 translocated to the membrane the most followed by Akt3 and Akt1, decreasing glucose uptake in the similar order in insulin-sensitive cells. None of the Akt isoforms translocated in insulin-resistant cells or high-fat-diet mediated diabetic mice brain cells. Based on our data, insulin-dependent differential translocation of Akt isoforms to the plasma membrane turns out to be the key factor in determining Akt isoform specificity. Thus, isoforms play parallel with predominant role by Akt2, and compensatory yet novel role by Akt1 and Akt3 to regulate neuronal insulin signaling, glucose uptake, and insulin-resistance.

PKCα: Prospects in Regulating Insulin Resistance and AD.

Protein kinase C alpha (PKCα) is known to participate in various signaling pathways due to its ubiquitous and dynamic characteristics. Previous studies report that PKCα abrogates peripheral insulin resistance, and recent publications show that it takes part in regulating Alzheimer's disease (AD). Based on evidence in the literature, we have highlighted how many of the substrates of PKCα in its signal transduction cascades are common in AD and diabetes and may have the capability to regulate both diseases simultaneously. Signaling pathways crosslinking these two diseases by PKCα have not been explored. Understanding the complexities of PKCα interactions with common molecules will deepen our understanding of its regulation of relevant pathophysiologies and, in the future, may broaden the possibility of using PKCα as a therapeutic target.

AKT ISOFORMS-AS160-GLUT4: The defining axis of insulin resistance.

The Akt isoforms-AS160-GLUT4 axis is the primary axis that governs glucose homeostasis in the body. The first step on the path to insulin resistance is deregulated Akt isoforms. This could be Akt isoform expression, its phosphorylation, or improper isoform-specific redistribution to the plasma membrane in a specific tissue system. The second step is deregulated AS160 expression, its phosphorylation, improper dissociation from glucose transporter storage vesicles (GSVs), or its inability to bind to 14-3-3 proteins, thus not allowing it to execute its function. The final step is improper GLUT4 translocation and aberrant glucose uptake. These processes lead to insulin resistance in a tissue-specific way affecting the whole-body glucose homeostasis, eventually progressing to an overt diabetic phenotype. Thus, the relationship between these three key proteins and their proper regulation comes out as the defining axis of insulin signaling and -resistance. This review summarizes the role of this central axis in insulin resistance and disease in a new light.

Type-2 diabetes, a co-morbidity in Covid-19: does insulin signaling matter?

Type-2 Diabetes is associated with one of the co-morbidities due to SARS-Coronavirus 2 (SARS-Cov2) infection. Clinical studies show out of control glucose levels in SARS-Cov2 infected patients with type-2 diabetes. There is no experimental evidence suggesting aberrant molecular pathway(s) that explains why SARS-Cov2 infected patients with type-2 diabetes have uncontrolled glucose homeostasis and are co-morbid. In this article, we have highlighted major proteins involved in SARS-Cov2 infection, like, ACE 2, proteases like, TMPRSS2, Furin and their connectivity to insulin signaling molecules like, PI3K, Akt, AMPK, MAPK, mTOR, those regulate glucose homeostasis and the possible outcome of that cross-talk. We also raised concerns about the effect of anti-SARS-Cov2 drugs on patients with type-2 diabetes with reference to insulin signaling and the outcome of their possible cross-talk. There are no studies to decipher the possibilities of these obvious cross-talks. The major objective of this article is to urge the scientific community to explore the possibility of determining whether derangement of insulin signaling could be one of the possible causes of the patients with type-2 diabetes being co-morbid due to SARS-Cov2 infection.

Tankyrase inhibition augments neuronal insulin sensitivity and glucose uptake via AMPK-AS160 mediated pathway.

Tankyrase, a member of poly (ADP-ribose) polymerase (PARP) family, regulates various cellular pathways including wnt signaling, telomere maintenance and mitosis, has become a prime target for the development of cancer therapeutics. Inhibition of tankyrase, which leads to its increased cellular accumulation, reveal the role of tankyrase in the regulation of Glucose transporter type 4 (GLUT4) translocation and glucose homeostasis in peripheral insulin responsive tissues. While in adipocytes inhibition of tankyrase improves insulin sensitivity and glucose uptake, its inhibition in skeletal muscle leads to development of insulin resistance. Evidently further studies are required to determine the broader perspective of tankyrase in other cellular systems in regulating insulin signaling and insulin resistance. Role of tankyrase in neuronal tissues/cells has not been tested. In the present study, we investigated the effect of tankyrase inhibition in insulin-sensitive and insulin-resistant Neuro-2a cells. Here, we report that XAV939 treatment, a tankyrase inhibitor, improves insulin-stimulated glucose uptake in insulin-sensitive as well as in insulin-resistant neuronal cells via AMP-activated protein kinase (AMPK) - AKT Substrate of 160 kDa (AS160) mediated pathway without affecting the phosphorylation/activation of AKT. AMPK inhibition by Compound C repressed XAV939 treatment mediated increase in glucose uptake, confirming the role of tankyrase in glucose uptake via AMPK. We show for the first time that inhibition of tankyrase significantly improves glucose uptake and insulin sensitivity of insulin-resistant neuronal cells via AMPK-AS160 mediated pathway. Our study demonstrates new mechanistic insights of tankyrase mediated regulation of insulin sensitivity as well as glucose uptake in neuronal cells.

Protein Kinase C Attenuates Insulin Signalling Cascade in Insulin-Sensitive and Insulin-Resistant Neuro-2a Cells.

Protein kinase C (PKC) family of enzymes is known to be a feedback regulator of insulin signalling pathway in peripheral insulin-responsive tissues. Insulin signalling is reported to be required for maintaining cognitive abilities in brain. PKCs are involved in innumerable neuronal processes including differentiation, apoptosis, survival, maintaining synaptic plasticity, long-term potentiation and memory formation. In the present study, we made an attempt to elucidate the role of PKC, if any, in regulating insulin signalling and insulin resistance in Neuro-2a (N2a) cells in vitro. We show that phorbol 12-myristate 13-acetate (PMA) -activated PKC inhibited Akt activation in neuronal cell, N2a. In the process of inhibiting Akt, PMA-activated PKC decreased downstream insulin signalling proteins like Akt substrate 160 kDa (AS160) and glycogen synthase kinase (GSK3ß), followed by a decrease of glucose uptake in N2a cells. PKC activation caused insulin resistance in N2a cells and worsened the resistant state of already insulin-resistant cells. Hence, our study demonstrated that the activation of PKC attenuates insulin signalling cascade and make N2a cells insulin-resistant.

Effect of inhibition of axonemal dynein ATPases on the regulation of flagellar and ciliary waveforms in Leishmania parasites.

Trypanosomes of the genus Leishmania swim by undulating motions of a single flagellum driven by axonemal dynein ATPases, essential for parasite survival and infectivity. The flagellum possesses two waveforms; flagellar (tip-to-base) responsible for forward movements and ciliary (base-to-tip) possibly responsible for reorientation in response to changes in surroundings. However, the role of dyneins in regulating the two waveforms remains unknown. Moreover, the unpredictable nature of the parasite ciliary waveform makes it difficult to study. We have previously reported a detergent-extracted, ATP-reactivated model ideal for investigating flagellar motility regulation in Leishmania that allows one to generate reactivated Leishmania flagella with constitutively beating ciliary waves in presence of cyclic-AMP. Here, using three dynein inhibitors [erythro-9-(2-hydroxy-3-nonyl) adenine, ciliobrevin A and vanadate] we investigated the role of dyneins in regulating the two waveforms of Leishmania. Using high speed videomicroscopy we observed differential inhibition of beat frequencies and waveforms of flagellar and ciliary beats in live (in vivo) and ATP-reactivated (in vitro) parasites. Beat frequency of flagellar waveform was more strongly reduced than ciliary waveform. Surprisingly, inhibition of the ciliary waveform led to an altered phenotype with the distal half of the flagellum paralysed. ATPase assays confirmed that dynein activity of flagellar cells was more strongly inhibited compared to ciliary cells irrespective of the mechanism of inhibition. Possibly the two different waveforms are an outcome of changes in the mechanical properties of axonemal dyneins present at the tip of the flagellum that contains a sliding resistive structure. Our study suggests that dyneins responsible for the two waveforms in Leishmania bear different structural and functional conformations. Moreover, during ciliary beating, there is heterogeneity along the flagellum.

The p38 MAP kinase inhibitor, PD 169316, inhibits flagellar motility in Leishmania donovani.

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni. However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani.

Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Characterization of ciliobrevin A mediated dynein ATPase inhibition on flagellar motility of Leishmania donovani.

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250µM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.