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The occurrence of obesity has increased across the whole world. Many epidemiological studies have indicated that obesity strongly contributes to the development of cancer, cardiovascular diseases, type 2 diabetes, liver diseases and other disorders, accounting for a heavy burden on the public and on health-care systems every year. Excess energy uptake induces adipocyte hypertrophy, hyperplasia and formation of visceral fat in other non-adipose tissues to evoke cardiovascular disease, liver diseases. Adipose tissue can also secrete adipokines and inflammatory cytokines to affect the local microenvironment, induce insulin resistance, hyperglycemia, and activate associated inflammatory signaling pathways. This further exacerbates the development and progression of obesity-associated diseases. Although some progress in the treatment of obesity has been achieved in preclinical and clinical studies, the progression and pathogenesis of obesity-induced diseases are complex and unclear. We still need to understand their links to better guide the treatment of obesity and associated diseases. In this review, we review the links between obesity and other diseases, with a view to improve the future management and treatment of obesity and its co-morbidities., (© 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Background: Alzheimer disease (AD) is a neurodegenerative condition defined by the build-up of amyloid plaques in the brain and intraneuronal tangles of the protein tau. Autophagy is a cellular cleaning process involved in the degradation of proteins, including proteins directly responsible for amyloid plaques, but its activity is compromised in AD. The mechanistic target of rapamycin complex (mTORC) 1 inhibits autophagy when activated by amino acids., Objectives: We hypothesized that reducing amino acid intake by decreasing dietary protein could promote autophagy, which in turn could prevent amyloid plaque deposition in AD mice., Methods: Homozygote (2-mo-old) and heterozygote (4-mo-old) amyloid precursor protein NL-G-F mice, a model of brain amyloid deposition, were used in this study to test this hypothesis. Male and female mice were fed with isocaloric low-protein, control, or high-protein diets for 4 mo and killed for analysis. Locomotor performance was measured using the inverted screen test, and body composition was measured using EchoMRI. Samples were analyzed using western blotting, enzyme-linked immunosorbent assay, mass spectrometry, and immunohistochemical staining., Results: mTORC1 activity in the cerebral cortex was inversely covaried with protein consumption in both homozygote and heterozygote mice. Low-protein diet improved metabolic parameters and restored locomotor performance only in male homozygous mice. Dietary protein adjustment did not affect amyloid deposition in homozygous mice. However, in the heterozygous amyloid precursor protein NL-G-F mice, amyloid plaque was lower in male mice consuming the low protein compared with that in mice fed with the control diet., Conclusions: This study showed that reducing protein intake reduces mTORC1 activity and may prevent amyloid accumulation, at least in male mice. Moreover, dietary protein is a tool that can be used to change mTORC1 activity and amyloid deposition in the mouse brain, and the murine brain's response to dietary protein is sex specific., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Sustained fuel excess triggers low-grade inflammation that can drive mitochondrial dysfunction, a pivotal defect in the pathogenesis of insulin resistance in skeletal muscle., Objectives: This study aimed to investigate whether inflammation in skeletal muscle can be prevented by EPA, and if this is associated with an improvement in mitochondrial fusion, membrane potential, and insulin signaling., Methods: Human primary myotubes were treated for 24 h with palmitic acid (PA, 500 μM) under hyperglycemic conditions (13 mM glucose), which represents nutrient overload, and in the presence or absence of EPA (100 μM). After the treatments, the expression of peroxisome proliferator-activated receptor γ coactivator 1-α (PPARGC1A) and IL6 was assessed by q-PCR. Western blot was used to measure the abundance of the inhibitor of NF-κB (IKBA), mitofusin-2 (MFN2), mitochondrial electron transport chain complex proteins, and insulin-dependent AKT (Ser473) and AKT substrate 160 (AS 160; Thr642) phosphorylation. Mitochondrial dynamics and membrane potential were evaluated using immunocytochemistry and the JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) dye, respectively. Data were analyzed using 1-factor ANOVA followed by Tukey post hoc test., Results: Nutrient excess activated the proinflammatory NFκB signaling marked by a decrease in IKBA (40%; P < 0.05) and the upregulation of IL6 mRNA (12-fold; P < 0.001). It also promoted mitochondrial fragmentation (53%; P < 0.001). All these effects were counteracted by EPA. Furthermore, nutrient overload-induced drop in mitochondrial membrane potential (6%; P < 0.05) was prevented by EPA. Finally, EPA inhibited fuel surplus-induced impairment in insulin-mediated phosphorylation of AKT (235%; P < 0.01) and AS160 (49%; P < 0.05)., Conclusions: EPA inhibited NFκB signaling, which was associated with an attenuation of the deleterious effects of PA and hyperglycemia on both mitochondrial health and insulin signaling in human primary myotubes. Thus, EPA might preserve skeletal muscle metabolic health during sustained fuel excess but this requires confirmation in human clinical trials., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Heparan acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT) is a lysosomal multi-pass transmembrane protein whose deficiency may lead to an accumulation of heparan sulphate and the neurodegenerative lysosomal storage disorder mucopolysaccharidosis (MPS) IIIC. In this study, HGSNAT activity was detected in extracellular vesicles isolated from both human urine and culture medium conditioned with HEK 293T cells. We also demonstrate that HGSNAT co-immunoprecipitates with antibodies to ALIX, which is associated with the endosomal sorting complexes required for transport (ESCRT) proteins, and is implicated in the targeting of proteins to intraluminal vesicles of multivesicular bodies, the origin of exosomes. Furthermore, mutation of a putative LYPX n L-based binding site within HGSNAT for the V-domain of ALIX ablated association of HGSNAT with ALIX, post-translational maturation, and transport through the endo-lysosomal network. Unexpectedly, however, a mutation within the V-domain of ALIX demonstrated enhanced HGSNAT association, perhaps due to the actual involvement of other binding sites in this interaction. Indeed, HGSNAT still co-immunoprecipitates with truncations of ALIX lacking the V-domain. Interestingly, CRISPR/Cas9 mediated knock-down of ALIX did not inhibit HGSNAT trafficking through the endo-lysosomal network, suggesting that there is an alternative pathway for trafficking HGSNAT that does not require ALIX. Nonetheless, the targeting of HGSNAT to extracellular vesicles may provide a mechanism to subsequently transfer this enzyme extracellularly to provide a foundation for a therapy for MPS IIIC patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alzheimer's disease is the most important cause of dementia but there is no therapy that has been demonstrated to stop or slow disease progression. Amyloid precursor protein (APP) is the source of amyloid-β (Aβ), which aggregates in Alzheimer's disease to form toxic oligomeric species. The endo-lysosomal system can clear APP and Aβ from the cell if these molecular species are trafficked through to the lysosome. Currently, there are no easy methods available for the analysis of lysosomal APP trafficking. We therefore generated a fusion protein (tandem-fluorescent, or tf-APP) that allows detection of changes in APP trafficking using accessible techniques such as flow cytometry. This permits rapid analysis or screening of genes and compounds that alter APP processing in the cell. Using our novel molecular probe, we determined that starvation induces trafficking of APP and APP-carboxy-terminal fragments (APP-CTFs) to the degradative endo-lysosomal network. In line with this finding, suppression of mTOR signalling using AZD8055 also strongly induced trafficking of APP to the endo-lysosomal system. Remarkably, activation of mTOR signalling via RHEB over-expression inhibited the starvation-induced autophagy but did not affect trafficking of tf-APP. These results show tf-APP can be used to determine how APP is trafficked through the lysosomal system of the cell. This molecular probe is therefore useful for determining the molecular mechanism behind the commitment of APP to the degradative pathway or for screening compounds that can induce this effect. This is important as clearance of APP and APP-CTF provides an important potential therapeutic strategy for Alzheimer's disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Eukaryotic elongation factor 2 kinase (eEF2K) inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS) assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the small group of atypical 'α-kinases'. It phosphorylates and inhibits eukaryotic elongation factor 2, to slow down the elongation stage of protein synthesis, which normally consumes a great deal of energy and amino acids. The activity of eEF2K is normally dependent on calcium ions and calmodulin. eEF2K is also regulated by a plethora of other inputs, including inhibition by signalling downstream of anabolic signalling pathways such as the mammalian target of rapamycin complex 1. Recent data show that eEF2K helps to protect cancer cells against nutrient starvation and is also cytoprotective in other settings, including hypoxia. Growing evidence points to roles for eEF2K in neurological processes such as learning and memory and perhaps in depression., (Copyright © 2014. Published by Elsevier Ltd.)

Amino acids are the precursors for the synthesis of proteins. In humans, approximately half of the 20 different amino acids are essential, ie, must be obtained from the diet. Cells must therefore take account of amino acid availability to achieve sustainable rates of protein synthesis. One of the major mechanisms involved in this is signaling through a complex of proteins termed mammalian target of rapamycin complex (mTORC) 1, which is activated by amino acids. In turn, mTORC1 regulates the production of ribosomes, the molecular machines that make proteins, and the activity of other cellular components required for protein synthesis. mTORC1 signaling promotes the transcription of the genes for ribosomal RNAs and many other components involved in ribosome production. It also positively regulates the translation of the messenger RNAs (mRNAs) for ribosomal proteins. Indeed, recent studies have shown that mammalian target of rapamycin signaling drives the translation of mRNAs for many anabolic enzymes and other proteins involved in diverse cellular functions. The translational machinery is also regulated by the absence of amino acids through the protein kinase GCN2 (general control nonrepressed 2), which phosphorylates and in end-effect inhibits the translation initiation factor eIF2 (eukaryotic initiation factor 2). This process shuts down general protein synthesis to conserve amino acids.

The mammalian target of rapamycin (mTOR) is a large multidomain protein whose function is inhibited by the immunosuppressant drug rapamycin. mTOR (or its homologues in lower eukaryotes) plays roles in cell growth and the cell cycle, control of the cytoskeleton and nutrient transport, protein and RNA stability and transcription and translation. In mammalian cells, the best understood effectors of mTOR are proteins involved in controlling the translational machinery. Signalling through mTOR is stimulated by amino acids and by hormones and mitogens. On the other hand, mTOR signaling is impaired in response to a range of stressful stimuli. These include DNA damage, nutrient withdrawal and depletion of cellular energy, as well as hypoxia. In response, e.g. to DNA damage, impairment of mTOR signaling appears to precede the commitment of cells to apoptosis. The mechanisms by which these stressful conditions still remain largely unclear. However, these responses make physiological sense, as impairment of mTOR signalling under these conditions will tend to inhibit anabolic processes and cell growth and division.

The mammalian-target-of-rapamycin (mTOR) is a multidomain protein that is important in regulating several components of the translational machinery. mTOR signalling is stimulated by hormones (e.g., insulin) and by amino acids. Our recent data suggest that TOR signalling responds to intracellular amino acids rather than to external amino acid levels. The translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is regulated through mTOR and undergoes phosphorylation at multiple sites, which affects its function. It contains two regulatory motifs: the C-terminal TOS motif interacts with the mTOR-binding partner, raptor, and mediates phosphorylation of specific sites in 4E-BP1. However, the N-terminal RAIP motif affects a larger range of mTOR-regulated sites. Since this motif does not bind raptor, mTOR must signal to 4E-BP1 via additional mechanisms that are independent of raptor. The kinase that phosphorylates and inhibits elongation factor 2 (eEF2 kinase) is inactivated by insulin via mTOR. Insulin decreases the ability of eEF2 kinase to bind calmodulin, its essential activator, and this effect requires mTOR signalling and a novel phosphorylation site in eEF2 kinase, Ser78. Ser78 is not phosphorylated by known components of the mTOR pathway implying the existence of novel mTOR-regulated kinases that control eEF2 kinase.

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide-exchange factor which mediates the exchange of GDP (bound to initiation factor eIF2) for GTP, thus regenerating the active [eIF2.GTP] complex that is required for peptide-chain initiation. The activity of eIF2B is a key control point for eukaryotic protein synthesis and is altered in response to viral infection, hormones, nutrients, growth factors and certain stresses. It may be regulated directly by its own phosphorylation, phosphorylation of its substrate, eIF2 and by allosteric effectors. Understanding its regulation will lead to a better knowledge of insulin action, regulation of the immune system and of insulin production in the pancreas.

p70 S6 kinase (p70 S6k) is important in regulating a variety of cellular functions including mRNA translation and cell cycle progression and is activated by mitogens and hormones. Unexpectedly, we have found that, in adult rat cardiomyocytes, arsenite, which generally induces stress responses, markedly and rapidly activates p70 S6k. This activation of p70 S6k is completely blocked by rapamycin but only partially prevented by inhibitors of phosphatidylinositol 3-kinase. In trying to delineate the mechanism underlying this effect, we found that arsenite did not activate protein kinase B, JNK or MAP kinase, but did activate p38 MAP kinase in cardiac myocytes. A specific inhibitor of p38 MAP kinase (SB203580) partially attenuated the stimulation of p70 S6k by arsenite. These data indicate that the activation of p70 S6k by arsenite involves p38 MAP kinase and phosphatidylinositol 3-kinase but not PKB.

In this study we describe the characterization and use of new peptide substrates for assaying glycogen synthase kinase-3 (GSK-3) which are based on the sequence around the single GSK-3 phosphorylation site in the translation factor eIF2B. The new peptides offer important advantages over previous substrates, which were based on the sequence around the multiple GSK-3 phosphorylation sites in glycogen synthase (GS), for the assay of GSK-3 in cell extracts. In particular, decreases in GSK-3 activity following, e.g., insulin treatment, are partially or completely masked when the GS-based peptides are used but are readily measured using the new, eIF2B-based, peptides. The new peptides, unlike those based on GS, are therefore suitable for the assay of changes in GSK-3 activity in cell extracts without the need for prior immunoprecipitation or ion-exchange chromatography.

Eukaryotic initiation factor (eIF)-2B, the guanine nucleotide exchange factor for eIF-2, consists of five distinct subunits in both mammals and the yeast Saccharomyces cerevisiae. The exchange reaction mediated by eIF-2B can be regulated by phosphorylation of eIF-2 on its alpha-subunit. This represents a key control point in the initiation of translation. The functions of the individual subunits of the eIF-2B complex remain unclear. Mutational analysis in Saccharomyces cerevisiae suggested that the smallest subunit (the alpha) is dispensable for exchange, but required for the inhibition of eIF-2B by eIF-2(alphaP). Here we present evidence that, in mammalian cells, eIF-2Balpha is essential for the activity of the complex, since preparations of eIF-2B lacking this subunit are not active in nucleotide exchange in vitro, although the complex still contains the beta, gamma, delta and epsilon subunits.

Initiation factor eIF-2 (a trimer of subunits alpha, beta and gamma) attaches the initiator Met-tRNA to the ribosome during the initiation of translation in eukaryotic cells. Both the alpha and beta subunits can be phosphorylated although the sites in the beta-subunit have not previously been fully identified. Here we identify the sites at which eIF-2 beta is phosphorylated in vitro by three well-characterised protein kinases, casein kinase-2 (which phosphorylates serine residues-2 and -67), protein kinase C (serine-13) and cAMP-dependent protein kinase (serine-218). This constitutes an essential prerequisite for studying the phosphorylation of eIF-2 beta in vivo. Indeed, we present evidence that at least one of these sites (serine-67) is phosphorylated in reticulocytes. The major kinase activity against eIF-2 beta in reticulocyte lysates appears in CK-2 and protein phosphatase-2A is the principal enzyme responsible for dephosphorylation of eIF-2 beta phosphorylated by this kinase.

A peptide P(45-56) corresponding to residues 45-56 (sequence: ILLSELSRRRIR) of eIF-2 alpha was synthesised. It was phosphorylated by both of the well characterised eIF-2 alpha kinases viz.; the heme-controlled repressor (HCR) and the double stranded RNA-dependent inhibitor (dsI). Of four other protein kinases tested only protein kinase C (PKC) phosphorylated P(45-56), with complete dependence on phosphatidylserine. Only the residue corresponding to serine-51 in eIF-2 alpha was phosphorylated by HCR, dsI or PKC. The phosphorylation of the peptide by dsI and the phosphorylation of eIF-2 alpha by dsI or HCR showed sigmoidal kinetics with respect to substrate concentration.

We have examined the phosphorylation of the alpha-subunit of initiation factor-2 (eIF-2 alpha) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2 alpha, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2 alpha in isoelectric focusing/immunoblotting analyses of lysates treated as described above.

Hepatic phosphofructokinase, isolated in a medium containing 100 mM (NH4)2SO4, can be activated by ATP. This metabolite-induced activation was investigated in view of the suggestion that it is related to phosphorylation of phosphofructokinase. The results obtained do not support this interpretation. Inhibitors of protein phosphatases (NaF) and kinases (the Mg++-chelator, ethylene diamine tetraacetic acid) did not affect the recovery of phosphofructokinase. In contrast, media of high ionic strength reduced the phosphofructokinase activity and rendered the enzyme sensitive to ATP-induced activation. Activation was also induced by other known effectors of phosphofructokinase (nucleoside triphosphates, fructose bisphosphates) and was not dependent on Mg++-ions. It is suggested that activation represents ligand-induced reversal of the inactivation of phosphofructokinase which occurs at high ionic strength. The differential sensitivity of phosphofructokinase from fed or starved animals to inactivation and reactivation is discussed.