HEAT SHOCK PROTEINS: NOVEL THERAPEUTIC TARGETS AGAINST INSULIN RESISTANCE AND TYPE 2 DIABETES

Impaired insulin action, termed insulin resistance, is characteristic of type 2 diabetes, obesity and aging. Given the rising epidemic of diabetes, efforts to understand the mechanisms of insulin resistance and discover effective therapeutic interventions are urgent. Considerable evidence now implicates oxidative stress in the patho-physiology of insulin resistance, a condition prevalent in the elderly and obese. Oxidative stress is known to activate several signaling cascades. This includes pathways that activate the stress kinases c-Jun N-terminal kinase (JNK) and the inhibitor of kappa B kinase beta (IKK beta), which interact with and inhibit the insulin signaling cascade. The heat shock proteins HSP72 and HSP25 have been recently identified as natural inhibitors of JNK and IKK beta, respectively, and therefore represent novel therapeutic targets against insulin resistance. Overexpression of HSPs has been shown to protect against obesity-induced insulin resistance as well as age-related muscle damage. Skeletal muscle, the largest glucose disposing tissue, also contains large amounts of inducible HSPs. We hypothesized that heat shock protein overexpression in skeletal muscle could protect against insulin resistance in obesity and aging. We tested this hypothesis using aged male Fischer 344 rats (24-month-old) as the aging model of insulin resistance and male Wistar rats given a high fat diet (60% calories from fat) as the model of diet induced-insulin resistance. We examined the role of HSPs in insulin resistance by inducing HSP expression with both in vitro and in vivo heat treatments and anti-oxidant administration. Our results showed that reduced HSP expression in the aging muscles is associated with a higher degree of stress kinase activation and insulin resistance in fast-twitch muscles compared to slow-twitch muscles. Increasing HSP72 expression in the muscles of young and old animals via heat treatment inhibited JNK activation. Heat-mediated JNK inhibition was specific to HSP72 induction, as determined by HSP72-inhibition studies, and was mediated by a direct interaction between HSP72 and JNK. In contrast to the muscle, brain sections from aging rats showed a robust increase in HSP25 expression, suggesting a tissue-specific regulation of HSPs in aging. In the high fat diet model, alpha-lipoic acid (LA), a potent antioxidant, was administered to relieve oxidative stress associated with high fat feeding. LA treatment improved insulin signaling and glucose transport, reduced stress kinase activation and increased HSP expression. As another method of HSP-induction, heat treatment, given in parallel with a high fat diet, improved glucose tolerance, reduced hyperinsulinemia, and reduced epididymal fat storage. In skeletal muscles, heat treatment induced HSP72 expression, improved insulin sensitivity, and reduced stress kinase activities. Heat treatment also enhanced mitochondrial function in fast-twitch muscles, normalizing the compensatory changes in mitochondrial protein expression seen with high fat feeding. Studies in L6 myotubes showed that heat treatment improved oxygen consumption and fatty acid oxidation. Mechanistically, our results indicate that heat shock proteins can 1). improve insulin sensitivity, 2). directly inhibit stress kinase activities, and 3). protect and enhance mitochondrial function. Our studies provide strong evidence that HSP induction in skeletal muscle could be a potential therapeutic treatment for age-related and obesity-induced insulin resistance

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation

Plumbagin Elicits Cell-Specific Cytotoxic Effects and Metabolic Responses in Melanoma Cells

Melanoma is one of the most malignant skin cancers that require comprehensive therapies, including chemotherapy. A plant-derived drug, plumbagin (PLB), exhibits an anticancer property in several cancers. We compared the cytotoxic and metabolic roles of PLB in A375 and SK-MEL-28 cells, each with different aggressiveness. In our results, they were observed to have distinctive mitochondrial respiratory functions. The primary reactive oxygen species (ROS) source of A375 can be robustly attenuated by cell membrane permeabilization. A375 cell viability and proliferation, migration, and apoptosis induction are more sensitive to PLB treatment. PLB induced metabolic alternations in SK-MEL-28 cells, which included increasing mitochondrial oxidative phosphorylation (OXPHOS), mitochondrial ATP production, and mitochondrial mass. Decreasing mitochondrial OXPHOS and total ATP production with elevated mitochondrial membrane potential (MMP) were observed in PLB-induced A375 cells. PLB also induced ROS production and increased proton leak and non-mitochondria respiration in both cells. This study reveals the relationship between metabolism and cytotoxic effects of PLB in melanoma. PLB displays stronger cytotoxic effects on A375 cells, which exhibit lower respiratory function than SK-MEL-28 cells with higher respiratory function, and triggers cell-specific metabolic changes in accordance with its cytotoxic effects. These findings indicate that PLB might serve as a promising anticancer drug, targeting metabolism

The Role of Calcium Signaling in Melanoma

Calcium signaling plays important roles in physiological and pathological conditions, including cutaneous melanoma, the most lethal type of skin cancer. Intracellular calcium concentration ([Ca2+]i), cell membrane calcium channels, calcium related proteins (S100 family, E-cadherin, and calpain), and Wnt/Ca2+ pathways are related to melanogenesis and melanoma tumorigenesis and progression. Calcium signaling influences the melanoma microenvironment, including immune cells, extracellular matrix (ECM), the vascular network, and chemical and physical surroundings. Other ionic channels, such as sodium and potassium channels, are engaged in calcium-mediated pathways in melanoma. Calcium signaling serves as a promising pharmacological target in melanoma treatment, and its dysregulation might serve as a marker for melanoma prediction. We documented calcium-dependent endoplasmic reticulum (ER) stress and mitochondria dysfunction, by targeting calcium channels and influencing [Ca2+]i and calcium homeostasis, and attenuated drug resistance in melanoma management

AIBP Regulates Metabolism of Ketone and Lipids but Not Mitochondrial Respiration

Accumulating evidence indicates that the APOA1 binding protein (AIBP)—a secreted protein—plays a profound role in lipid metabolism. Interestingly, AIBP also functions as an NAD(P)H-hydrate epimerase to catalyze the interconversion of NAD(P)H hydrate [NAD(P)HX] epimers and is renamed as NAXE. Thus, we call it NAXE hereafter. We investigated its role in NAD(P)H-involved metabolism in murine cardiomyocytes, focusing on the metabolism of hexose, lipids, and amino acids as well as mitochondrial redox function. Unbiased metabolite profiling of cardiac tissue shows that NAXE knockout markedly upregulates the ketone body 3-hydroxybutyric acid (3-HB) and increases or trends increasing lipid-associated metabolites cholesterol, α-linolenic acid and deoxycholic acid. Paralleling greater ketone levels, ChemRICH analysis of the NAXE-regulated metabolites shows reduced abundance of hexose despite similar glucose levels in control and NAXE-deficient blood. NAXE knockout reduces cardiac lactic acid but has no effect on the content of other NAD(P)H-regulated metabolites, including those associated with glucose metabolism, the pentose phosphate pathway, or Krebs cycle flux. Although NAXE is present in mitochondria, it has no apparent effect on mitochondrial oxidative phosphorylation. Instead, we detected more metabolites that can potentially improve cardiac function (3-HB, adenosine, and α-linolenic acid) in the Naxe−/− heart; these mice also perform better in aerobic exercise. Our data reveal a new role of NAXE in cardiac ketone and lipid metabolism

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa