Glycogen Synthase Activity Research Articles

Melatonergic signalling instructs transcriptional inhibition of IFNGR2 to lessen interleukin-1β-dependent inflammation.

BackgroundImmunotransmitters (e.g., neurotransmitters and neuromodulators) could orchestrate diverse immune responses; however, the elaborated mechanism by which melatonergic activation governs inflammation remains less defined.MethodsPrimary macrophages, various cell lines, and Pasteurella multocida (PmCQ2)‐infected mice were respectively used to illustrate the influence of melatonergic signalling on inflammation in vitro and in vivo. A series of methods (e.g., RNA‐seq, metabolomics, and genetic manipulation) were conducted to reveal the mechanism whereby melatonergic signalling reduces macrophage inflammation.ResultsHere, we demonstrate that melatonergic activation substantially lessens interleukin (IL)‐1β‐dependent inflammation. Treatment of macrophages with melatonin rewires metabolic program, as well as remodels signalling pathways which depends on interferon regulatory factor (IRF) 7. Mechanistically, melatonin acts via membrane receptor (MT) 1 to increase heat shock factor (Hsf) 1 expression through lowering the inactive glycogen synthase kinase (GSK3) β, thereby transcriptionally inhibiting interferon (IFN)‐γ receptor (IFNGR) 2 and ultimately causing defective canonical signalling events [Janus kinase (JAK) 1/2‐signal transducer and activator of transcription (STAT) 1‐IRF7] and lower IL‐1β production in macrophages. Moreover, we find that melatonin amplifies host protective responses to PmCQ2 infection‐induced pneumonia.ConclusionsOur conceptual framework provides potential therapeutic targets to prevent and/or treat inflammatory diseases associating with excessive IL‐1β production.

INTERLEUKIN 21-INDUCED METABOLIC REPROGRAMMING DRIVES CD4 T CELL INFLAMMATORY RESPONSE DURING INTESTINAL INFLAMMATION

Abstract BACKGROUND Significant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL-21), which is known to support T helper (Th) cell function, is highly expressed within inflamed tissues of IBD patients compared to healthy controls. Also, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD, and the complete induction of IBD in mice has been shown to require IL-21. Given that healthy Tregs are critical for self-tolerance and prevention of IBD, we investigated the metabolic role of IL-21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolic pathways during IBD. METHODS Human Tregs as well as relevant control Th cells were generated from naïve CD4+ T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted transcriptional profiling. Immune phenotyping was assessed by fluorescence-activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry-based targeted metabolomics. Colitis was induced in Rag1-/- (T and B cell deficient) mice by adoptive transfer of pathogenic naïve CD4+ T cells. RESULTS Acute IL-21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed IL-21-induced alterations in the expression of genes associated with glycolysis and OXPHOS metabolism. Prolonged IL-21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of inflammatory Th cytokines such as interferon γ, tumor necrosis factor, IL-17A, and IL-17F. Exploring the mechanisms underlying IL-21-induced effects, we observed reduced expression of inactive glycogen synthase kinase 3 (GSK3) β, a protein known to prevent pyruvate entry into the mitochondria. This reduction in inactive GSK3β in response to IL-21 was accompanied by high abundance of intracellular pyruvate and lactate, as assessed by targeted metabolomics. Importantly, GSK3 inhibition or exogenous membrane-permeable methyl pyruvate abrogated IL-21-induced metabolic rewiring and Treg inflammatory response. Moreover, GSK3 inhibition diminished the metabolic and inflammatory function of Th cells. Lastly, GSK3 inhibition prevented pathogenic CD4+ T cell-induced colitis in mice, as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines. CONCLUSIONS IL-21 potently engages human Tregs in a metabolic state that subsequently augments an inflammatory phenotype. Therefore, desensitizing CD4+ T cells to detrimental cues, such as IL-21, may augment Treg function during human IBD.

Disruption of ER-mitochondria tethering and signalling in C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia.

Hexanucleotide repeat expansions in C9orf72 are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mechanisms by which the expansions cause disease are not properly understood but a favoured route involves its translation into dipeptide repeat (DPR) polypeptides, some of which are neurotoxic. However, the precise targets for mutant C9orf72 and DPR toxicity are not fully clear, and damage to several neuronal functions has been described. Many of these functions are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. ER‐mitochondria signalling requires close physical contacts between the two organelles that are mediated by the VAPB‐PTPIP51 ‘tethering’ proteins. Here, we show that ER‐mitochondria signalling and the VAPB‐PTPIP51 tethers are disrupted in neurons derived from induced pluripotent stem (iPS) cells from patients carrying ALS/FTD pathogenic C9orf72 expansions and in affected neurons in mutant C9orf72 transgenic mice. In these mice, disruption of the VAPB‐PTPIP51 tethers occurs prior to disease onset suggesting that it contributes to the pathogenic process. We also show that neurotoxic DPRs disrupt the VAPB‐PTPIP51 interaction and ER‐mitochondria contacts and that this may involve activation of glycogen synthase kinases‐3β (GSK3β), a known negative regulator of VAPB‐PTPIP51 binding. Finally, we show that these DPRs disrupt delivery of Ca2+ from ER stores to mitochondria, which is a primary function of the VAPB‐PTPIP51 tethers. This delivery regulates a number of key neuronal functions that are damaged in ALS/FTD including bioenergetics, autophagy and synaptic function. Our findings reveal a new molecular target for mutant C9orf72‐mediated toxicity.

Ferroptosis promotes microtubule-associated protein tau aggregation via GSK-3β activation and proteasome inhibition.

Ferroptosis is a form of regulated cell death resulting from iron accumulation and lipid peroxidation. Iron dyshomeostasis and peroxidation damage of neurons in some particular brain regions are closely related to a wide range of neurodegenerative diseases known as "tauopathies," in which intracellular aggregation of microtubule-associated protein tau is the common neuropathological feature. However, the relationship between ferroptosis and tau aggregation is not well understood. The current study demonstrates that erastin-induced ferroptosis can promote tau hyperphosphorylation and aggregation in mouse neuroblastoma cells (N2a cells). Moreover, ferroptosis inhibitor ferrostatin-1 can alleviate tau aggregation effectively. In-depth mechanism research indicates that activated glycogen synthase kinase-3β (GSK-3β) is responsible for the abnormal hyperphosphorylation of tau. More importantly, proteasome inhibition can exacerbate tau degradation obstacle and accelerate tau aggregation in the process of ferroptosis. Our results indicate that ferroptosis can lead to abnormal aggregation of tau protein and might be a promising therapeutic target of tauopathies.

Glucose metabolism in amyotrophic lateral sclerosis: it is bitter-sweet.

Glucose metabolism in amyotrophic lateral sclerosis: it is bitter-sweet.

Protein phosphatase 1 regulatory inhibitor subunit 14C promotes triple-negative breast cancer progression via sustaining inactive glycogen synthase kinase 3 beta.

Triple‐negative breast cancer (TNBC) is fast‐growing and highly metastatic with the poorest prognosis among the breast cancer subtypes. Inactivation of glycogen synthase kinase 3 beta (GSK3β) plays a vital role in the aggressiveness of TNBC; however, the underlying mechanism for sustained GSK3β inhibition remains largely unknown. Here, we find that protein phosphatase 1 regulatory inhibitor subunit 14C (PPP1R14C) is upregulated in TNBC and relevant to poor prognosis in patients. Overexpression of PPP1R14C facilitates cell proliferation and the aggressive phenotype of TNBC cells, whereas the depletion of PPP1R14C elicits opposite effects. Moreover, PPP1R14C is phosphorylated and activated by protein kinase C iota (PRKCI) at Thr73. p‐PPP1R14C then represses Ser/Thr protein phosphatase type 1 (PP1) to retain GSK3β phosphorylation at high levels. Furthermore, p‐PPP1R14C recruits E3 ligase, TRIM25, toward the ubiquitylation and degradation of non‐phosphorylated GSK3β. Importantly, the blockade of PPP1R14C phosphorylation inhibits xenograft tumorigenesis and lung metastasis of TNBC cells. These findings provide a novel mechanism for sustained GSK3β inactivation in TNBC and suggest that PPP1R14C might be a potential therapeutic target.

Ketamine Promotes Alzheimer’s-Like Neurodegeneration by Activating Glycogen Synthase Kinase 3 Beta and Inhibiting Protein Phosphatase 2A

To investigate the mechanism of ketamine promoting Alzheimer’s-like neurodegeneration in mice. 24 male 8 w old Kunming mice of specific pathogen-free grade were randomly divided into a blank control group (control group) and a ketamine intervention group (experimental group) according to the random number table, 12 mice in each group. The mice in the experimental group were given ketamine injection at the dose of 30 mg/kg. Mice in the control group were given an equal volume of normal saline injection. The intervention was conducted once a day for consecutive 6 mo. The behavioral changes of the mice in the two groups were compared. Western blot was used to detect the expression levels of tau (phospho Thr231), tau (phospho S396), tau (phospho Ser404), glycogen synthase kinase 3 beta, glycogen synthase kinase 3 beta (phospho ser9) and protein phosphatase 2A proteins in the brain tissue of hippocampus and the ratio of phosphorylated tau protein to tau protein was calculated. The expressions of beta-amyloid protein and tau protein in the hippocampus were observed by immunohistochemistry. The results of the Morris water maze showed that the escape latency of mice in both groups was gradually decreased and the escape latency of mice in the control group was lower than that in the experimental group. Besides, the times of crossing target platform and the proportion of activity time in target quadrant of mice in the experimental group were lower than those in the control group. At the same time, compared with the control group, the expression levels of tau (phospho Thr231) and tau (phospho Ser404) were decreased in the experimental group, and the expressions of tau (phospho S396), glycogen synthase kinase 3 beta, glycogen synthase kinase 3 beta (phospho ser9) and protein phosphatase 2A proteins were increased. Compared with the control group, the expression of beta-amyloid protein in the experimental group was significantly reduced and the opalescence density of beta-amyloid protein was significantly lower than that in the control group (p<0.05). Long-term use of ketamine can lead to up-regulated expression of beta-amyloid protein and tau protein in mice hippocampus, which may induce hyperphosphorylation of tau protein at Thr231, S396 and S404 by activating glycogen synthase kinase 3 beta and inhibiting protein phosphatase 2A, causing cognitive function decline in mice.

Depletion of Janus kinase-2 promotes neuronal differentiation of mouse embryonic stem cells

Janus kinase 2 (JAK2), a non-receptor tyrosine kinase, is a critical component of cytokine and growth factor signaling pathways regulating hematopoietic cell proliferation. JAK2 mutations are associated with multiple myeloproliferative neoplasms. Although physiological and pathological functions of JAK2 in hematopoietic tissues are well-known, such functions of JAK2 in the nervous system are not well studied yet. The present study demonstrated that JAK2 could negatively regulate neuronal differentiation of mouse embryonic stem cells (ESCs). Depletion of JAK2 stimulated neuronal differentiation of mouse ESCs and activated glycogen synthase kinase 3β, Fyn, and cyclin-dependent kinase 5. Knockdown of JAK2 resulted in accumulation of GTP-bound Rac1, a Rho GTPase implicated in the regulation of cytoskeletal dynamics. These findings suggest that JAK2 might negatively regulate neuronal differentiation by suppressing the GSK-3β/Fyn/CDK5 signaling pathway responsible for morphological maturation.

GSK-3β inhibition elicits a neuroprotection by restoring lysosomal dysfunction in neurons via facilitation of TFEB nuclear translocation after ischemic stroke

Lysosomal dysfunction is an essential pathogenesis of autophagic neuronal injury after ischemic stroke. As a result of cerebral ischemia, transcription factor EB (TFEB) is greatly phosphorylated by prominently activated glycogen synthase kinase-3β (GSK-3β). This increased TFEB phosphorylation decreases its nuclear translocation and subsequently leads to reduced lysosomal biosynthesis, which ultimately results in lysosomal dysfunction. The present study is to investigate whether the lysosomal dysfunction in neurons can be restored to alleviate post-stroke damage by GSK-3β inhibition.The GSK-3β activity was inhibited by pre-treatment with CHIR-99021 (CHIR) for 3 days before middle cerebral artery occlusion (MCAO) surgery in rats. Besides, the lysosomal capacity was altered by pre-administration with Bafilomycin A1 (Baf-A1) and EN6, respectively. Twenty-four hours after MCAO/reperfusion, the penumbral tissues were obtained to detect the GSK-3β, cytoplasmic and nuclear TFEB, and proteins in autophagic/lysosomal pathway by western blot and immunofluorescence, respectively. Meanwhile, the infarct volume, neurological deficits and neuron survival were assessed to evaluate the neurological outcomes elicited by GSK-3β inhibition. The results demonstrated that the neurological injury could be significantly mitigated by GSK-3β inhibition in MCAO + CHIR group, compared with that in MCAO group. Moreover, CHIR-facilitated TFEB nuclear translocation in neurons was coupled with reinforced lysosomal activities and attenuated autophagic substrates. However, GSK-3β inhibition-induced neuroprotection was greatly counteracted by Baf-A1-weakened lysosomal capacity. Conversely, EN6-reinforced lysosomal activities further ameliorated the autophagic/lysosomal signaling, and synergistically alleviated the neurological damage upon GSK-3β inhibition after MCAO/reperfusion. Our data suggests that GSK-3β inhibition-augmented neuroprotection against ischemic stroke is elicited by restoring the lysosomal dysfunction in neurons.

Systemic ablation of vitamin D receptor leads to skeletal muscle glycogen storage disorder in mice.

BackgroundVitamin D deficiency leads to pathologies of multiple organ systems including skeletal muscle. Patients with severe vitamin D deficiency exhibit muscle weakness and are susceptible to frequent falls. Mice lacking a functional vitamin D receptor (VDR) develop severe skeletal muscle atrophy immediately after weaning. But the root cause of myopathies when vitamin D signalling is impaired is unknown. Because vitamin D deficiency leads to metabolic changes as well, we hypothesized that the skeletal muscle atrophy in mice lacking VDR may have a metabolic origin.MethodsWe analysed wild‐type (WT) mice as well as vitamin D receptor null (vdr−/−) mice for skeletal muscle proteostasis, energy metabolism, systemic glucose homeostasis, and muscle glycogen levels. Dysregulation of signalling pathways as well as the glycogen synthesis and utilization machinery were also analysed using western blots. qRT–PCR assays were performed to understand changes in mRNA levels.ResultsSkeletal muscles of vdr−/− exhibited higher expression levels of muscle‐specific E3 ubiquitin ligases and showed increased protein ubiquitination, suggesting up‐regulation of protein degradation. Foxo1 transcription factor was activated in vdr−/− while Foxo3 factor was unaffected. Fasting protein synthesis as well as mTORC1 pathways were severely down‐regulated in vdr−/− mice. Skeletal muscle ATP levels were low in vdr−/− (0.58 ± 0.18 μmol/mL vs. 1.6 ± 0.0.14 μmol/mL, P = 0.006), leading to increased AMPK activity. Muscle energy deprivation was not caused by decreased mitochondrial activity as we found the respiratory complex II activity in vdr−/− muscles to be higher compared with WT (0.29 ± 0.007 mU/μL vs. 0.16 ± 0.005 mU/μL). vdr−/− mice had lower fasting blood glucose levels (95 ± 14.5 mg/dL vs. 148.6 ± 6.1 mg/dL, P = 0.0017) while they exhibited hyperlactataemia (7.42 ± 0.31 nmol/μL vs. 4.95 ± 0.44 nmol/μL, P = 0.0032), suggesting systemic energy deficiency in these mice. Insulin levels in these mice were significantly lower in response to intraperitoneal glucose injection (0.69 ± 0.08 pg/mL vs. 1.11 ± 0.09 pg/mL, P = 0.024). Skeletal muscles of these mice exhibit glycogen storage disorder characterized by increased glycogen accumulation. The glycogen storage disorder in vdr−/− muscles is driven by increased glycogen synthase activity and decreased glycogen phosphorylase activity. Increased glycogenin expression supports higher levels of glycogen synthesis in these muscles.ConclusionsThe results presented show that lack of vitamin D signalling leads to a glycogen storage defect in the skeletal muscles, which leads to muscle energy deprivation. The inability of vdr−/− skeletal muscles to use glycogen leads to systemic defects in glucose homeostasis, which in turn leads to proteostasis defects in skeletal muscles and atrophy.

Effect of cinnamon on blood sugar and anthropometric measurement in type two diabetes patients

There is some emerging evidence that suggests certain supplements help lower blood sugar levels. One of these is cinnamon, which exhibits characteristics that mimic insulin, such as the activity of biologically active substances to activate insulin receptor kinase, increasing glucose uptake, autophosphorylation of the insulin receptor, and glycogen synthase activity. To assess the effect of the cinnamon substance on lowering fasting blood sugar, modifying body weight, body mass index (B.M.I.), and waist circumference in type two diabetics. A randomized controlled intervention clinical trial. The study was conducted on 60 patients with type II diabetes mellitus; the study followed both gender patients for four weeks. The contributors were allocated randomly into a group that receives 2 g/d cinnamon substance (intervention group), and a group without cinnamon substance is given (control group). Fasting plasma glucose (F.P.G.) and anthropometrics measurement at the beginning (beforehand cinnamon supplementation and 4week at the end of the study duration). After 4 weeks of cinnamon taken, serum F.P.G. levels significantly improved (P≤0.0001). Anthropometric measurements (weight, height, B.M.I.) were reduced significantly (change mean ≤ P=0.001) for all. The Present study disclosed that supplementation of 2 gm cinnamon improves F.P.G. and has a good role in anthropometric indices (weight, waist circumference, B.M.I.).

Salbutamol Increases Leg Glucose Uptake and Metabolic Rate but not Muscle Glycogen Resynthesis in Recovery From Exercise.

Exercise blunts the effect of beta2-agonists on peripheral glucose uptake and energy expenditure. Whether such attenuation extends into recovery is unknown. To examine the effect of a beta2-agonist on leg glucose uptake and metabolic rate in recovery from exercise. Using leg arteriovenous balance technique and analyses of thigh muscle biopsies, we investigated the effect of a beta2-agonist (24 mg of oral salbutamol) vs placebo on leg glucose, lactate, and oxygen exchange before and during quadriceps exercise, and 0.5 to 5 hours in recovery from quadriceps exercise, as well as on muscle glycogen resynthesis and activity in recovery. Twelve healthy, lean, young men participated. Before exercise, leg glucose uptake was 0.42 ± 0.12 and 0.20 ± 0.02 mmol × min-1 (mean ± SD) for salbutamol and placebo (P = .06), respectively, while leg oxygen consumption was around 2-fold higher (P < .01) for salbutamol than for placebo (25 ± 3 vs 14 ± 1 mL × min-1). No treatment differences were observed in leg glucose uptake, lactate release, and oxygen consumption during exercise. But in recovery, cumulated leg glucose uptake, lactate release, and oxygen consumption was 21 mmol (95% CI 18-24, P = .018), 19 mmol (95% CI 16-23, P < .01), and 1.8 L (95% CI 1.6-2.0, P < .01) higher for salbutamol than for placebo, respectively. Muscle glycogen content was around 30% lower (P < .01) for salbutamol than for placebo in recovery, whereas no treatment differences were observed in muscle glycogen resynthesis or glycogen synthase activity. Exercise blunts the effect of beta2-agonist salbutamol on leg glucose uptake, but this attenuation diminishes in recovery. Salbutamol increases leg lactate release in recovery, which may relate to glycolytic trafficking due to excessive myocellular glucose uptake.

Role of hepatic PKCβ in nutritional regulation of hepatic glycogen synthesis.

The signaling mechanisms by which dietary fat and cholesterol signals regulate central pathways of glucose homeostasis are not completely understood. By using a hepatocyte-specific PKCβ-deficient (PKCβHep–/–) mouse model, we demonstrated the role of hepatic PKCβ in slowing disposal of glucose overload by suppressing glycogenesis and increasing hepatic glucose output. PKCβHep–/– mice exhibited lower plasma glucose under the fed condition, modestly improved systemic glucose tolerance and mildly suppressed gluconeogenesis, increased hepatic glycogen accumulation and synthesis due to elevated glucokinase expression and activated glycogen synthase (GS), and suppressed glucose-6-phosphatase expression compared with controls. These events were independent of hepatic AKT/GSK-3α/β signaling and were accompanied by increased HNF-4α transactivation, reduced FoxO1 protein abundance, and elevated expression of GS targeting protein phosphatase 1 regulatory subunit 3C in the PKCβHep–/– liver compared with controls. The above data strongly imply that hepatic PKCβ deficiency causes hypoglycemia postprandially by promoting glucose phosphorylation via upregulating glucokinase and subsequently redirecting more glucose-6-phosphate to glycogen via activating GS. In summary, hepatic PKCβ has a unique and essential ability to induce a coordinated response that negatively affects glycogenesis at multiple levels under physiological postprandial conditions, thereby integrating nutritional fat intake with dysregulation of glucose homeostasis.

Coffee Increases Post-Exercise Muscle Glycogen Recovery in Endurance Athletes: A Randomized Clinical Trial

Coffee is one of the most widely consumed beverages worldwide and caffeine is known to improve performance in physical exercise. Some substances in coffee have a positive effect on glucose metabolism and are promising for post-exercise muscle glycogen recovery. We investigated the effect of a coffee beverage after exhaustive exercise on muscle glycogen resynthesis, glycogen synthase activity and glycemic and insulinemic response in a double-blind, crossover, randomized clinical trial. Fourteen endurance-trained men performed an exhaustive cycle ergometer exercise to deplete muscle glycogen. The following morning, participants completed a second cycling protocol followed by a 4-h recovery, during which they received either test beverage (coffee + milk) or control (milk) and a breakfast meal, with a simple randomization. Blood samples and muscle biopsies were collected at the beginning and by the end of recovery. Eleven participants were included in data analysis (age: 39.0 ± 6.0 years; BMI: 24.0 ± 2.3 kg/m2; VO2max: 59.9 ± 8.3 mL·kg−1·min−1; PPO: 346 ± 39 W). The consumption of coffee + milk resulted in greater muscle glycogen recovery (102.56 ± 18.75 vs. 40.54 ± 18.74 mmol·kg dw−1; p = 0.01; d = 0.94) and greater glucose (p = 0.02; d = 0.83) and insulin (p = 0.03; d = 0.76) total area under the curve compared with control. The addition of coffee to a beverage with adequate amounts of carbohydrates increased muscle glycogen resynthesis and the glycemic and insulinemic response during the 4-h recovery after exhaustive cycling exercise.

Impaired non‐canonical transforming growth factor‐β signalling prevents profibrotic phenotypes in cultured peptidylarginine deiminase 4‐deficient murine cardiac fibroblasts

Transforming growth factor‐β (TGF‐β) becomes rapidly activated in the infarcted heart. Hence, TGF‐β‐mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis‐related genes was largely similar in cultured WT and PAD4‐/‐ CFs of passage 3, although collagen III was reduced in PAD4‐/‐ CFs. Exposure to TGF‐β inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4‐/‐ CFs. However, under baseline conditions, PAD4−/− CFs showed comparable functional characteristics as TGF‐β‐stimulated WT CFs. Although the SMAD‐dependent TGF‐β pathway was not disturbed in PAD4‐/‐ CFs, TGF‐β failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl‐amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase‐3β (GSK‐3β). Consequently, PAD4‐/‐ CFs did not upregulate collagen I and α‐smooth muscle actin (α‐SMA) expression after TGF‐β treatment. Thus, PAD4 is substantially involved in the regulation of non‐canonical TGF‐β signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.

Inhibition of dipeptidyl peptidase-4 averts free fatty acids deposition in the hearts of oral estrogen-progestin contraceptive-induced hyperinsulinemic female rats.

Free fatty acid (FFA) deposition in non-adipose tissues such as the heart is a characteristic of insulin resistant states which feature hyperinsulinemia and dipeptidyl peptidase-4 (DPP-4) activation. Estrogen-progestin oral contraceptives (OC) treatment reportedly increased DPP-4 activity in rat tissue, and DPP-4 inhibitors have anti-diabetic and anti-inflammatory properties. This study aims to investigate the effects of DPP-4 inhibition on cardiac FFA deposition in estrogen-progestin-treated female rats. From our data, estrogen-progestin OC exposure in female rats led to elevated plasma insulin, cardiac DPP-4 activity, FFA and triglyceride (TG) accumulation, TG/high-density lipoprotein cholesterol (TG/HDL-C) ratio, adenosine deaminase/xanthine oxidase/uric acid pathway (ADA/XO/UA), lipid peroxidation, glycogen synthase activity, and alanine phosphatase; whereas cardiac glucose-6-phosphate dehydrogenase, Na+/K+-ATPase and nitric oxide (NO) were decreased. However, DPP-4 inhibition resulted in decreased plasma insulin, cardiac DPP-4 activity, FFA, TG, TG/HDL-C ratio, and alkaline phosphatase. These were accompanied by reduced ADA/XO/UA pathway, lipid peroxidation, and augmented NO and Na+/K+-ATPase in estrogen-progestin OC-treated rats. DPP-4 inhibition attenuated cardiac lipid deposition accompanied by reduced activity in the ADA/XO/UA pathway in estrogen-progestin OC-treated female rats. DPP-4 is therefore a plausible therapeutic target in cardiometabolic disorders.

Move it or lose it: limb immobilisation results in impaired postprandial skeletal muscle glucose uptake.

Move it or lose it: limb immobilisation results in impaired postprandial skeletal muscle glucose uptake.

334-OR: Sex Differences in Protein Phosphorylation in Insulin Resistance in Humans at the Cellular Level

Insulin resistance in muscle precedes and predicts type 2 diabetes in offspring of diabetic parents and is also observed in ~25% of the general population. However, whether these changes represent cell autonomous defects or are secondary to changes in circulating factors is unclear. To define possible cell autonomous determinants of insulin resistance, we have utilized induced pluripotent stem cell (iPSC) differentiated into myoblasts from 20 nondiabetic individuals - 10 insulin sensitive (I-Sen) and 10 insulin resistant (I-Res) as assessed using the steady-state plasma glucose approach. While there was no difference in the ability of the iPSCs to differentiate into myoblasts (iMyos), cells from I-Res individuals show impaired insulin signaling, decreased insulin-stimulated glucose uptake, and glycogen synthase activity compared to iMyos from insulin sensitive individuals, indicating that these cells mirror in vitro the alterations seen in vivo. The global phosphoproteomic analysis using LC-MS/MS uncovered a large network of proteins whose phosphorylation was altered in association with insulin resistance, mostly outside the canonical insulin-signaling cascade. We also observed striking differences in the phosphoproteomic signature of iMyos derived from male versus female subjects. Analysis of the proteins involved reveals that male cells show enhanced phosphorylation of proteins associated with transcription, Rho GTPases, SUMOylation, mRNA splicing, and membrane trafficking, whereas females show a dominance of phosphorylation of proteins involved in the cell cycle, chromatin organization, gene expression, and protein ubiquitination. Taken together, these data demonstrate major alterations in cellular signaling underlying insulin resistance, even in the absence of diabetes, and provide evidence of a super-network of cell signaling differences in males and females that must be considered in understanding cellular regulation in health and disease. Disclosure N. Haider: None. J. Lebastchi: None. T. M. Batista: None. H. Pan: None. J. Dreyfuss: None. I. Carcamo-orive: None. J. Knowles: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences, Inc., Consultant; Self; Flagship Pioneering, Sana/Cobalt.

Increase of glycogen storage by sodium arsenite in rat cortical astrocytes through glycogen synthase activation and its association to toxicity

Increase of glycogen storage by sodium arsenite in rat cortical astrocytes through glycogen synthase activation and its association to toxicity

Dynamic insulin-stimulated mTOR/GSK3 signaling in peripheral immune cells: Preliminary evidence for an association with lithium response in bipolar disorder.

A key mechanism of lithium is the inhibition of glycogen synthase kinase-3β (GSK3β) and activation of mammalian target of rapamycin (mTOR), two contributors to insulin signaling. We explored the relationship between these markers and clinical response to lithium in bipolar disorder (BD). Thirty-four subjects with BD who had been taking lithium for ≥2years and had a maintenance lithium Alda score defined as either high (≥7; n=20) or low (≤2; n=14) were included in the study. Baseline protein expression of GSK3β and mTOR (total and phosphorylated (p)) was obtained from a buffy coat. Peripheral blood mononuclear cells (PBMCs) from a subset of each group (n=11) were stimulated with insulin (10µg) and change in protein expression was determined using Western blot. In buffy coat samples, significantly higher levels of pmTOR were present in subjects with an Alda score ≤2 (lithium non-responsive), relative to those with scores ≥7 (lithium-responsive). No differences were observed for pGSK3β. In contrast, functional PBMC responses to 5min of insulin stimulation demonstrated robust increases in pGSK3β (87.05±43.41%) and pmTOR (105.7±66.48%) in the lithium responsive group only. This contrasted observed decreases in pGSK3β (34.08±16.12%) and pmTOR (37.84±14.39%) 5mins post-insulin in non-responders. Dynamic increases in pmTOR and pGSK3β post-insulin stimulation may reflect an immunometabolic state that facilitates lithium response. Further prospective analyses are needed to replicate and extend these preliminary findings and further investigate the role of insulin signaling in lithium response in BD.