WO2020076175A2 - Diagnostic, prévention et/ou traitement d'affections liées à l'insulinorésistance - Google Patents

Diagnostic, prévention et/ou traitement d'affections liées à l'insulinorésistance Download PDF

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WO2020076175A2
WO2020076175A2 PCT/QA2019/050015 QA2019050015W WO2020076175A2 WO 2020076175 A2 WO2020076175 A2 WO 2020076175A2 QA 2019050015 W QA2019050015 W QA 2019050015W WO 2020076175 A2 WO2020076175 A2 WO 2020076175A2
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dnajb3
expression
ala
cells
stress
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Mohammed Dehbi
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Qatar Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Type 2 diabetes is a multifactorial metabolic disorder that represents a major health, economic and social challenge worldwide. It is characterized by chronic hyperglycemia secondary to either increased insulin resistance (IR) in peripheral organs, progressive failure of the pancreatic islet b-cells or both (Defronzo RA, Diabetes 2009; 58: 773-795).
  • IR insulin resistance
  • the etiology of the disease is complex and involves an intricate interplay between genetic susceptibility and environmental factors, including sedentary lifestyles and obesity (DeFronzo, et al., Nature reviews Disease primers 2015; 1 : 15019). This latter is recognized as a major independent risk factor for T2D through the development of IR (Upadhyay, et al., The Medical clinics of North America 2018; 102: 13-33).
  • Metabolic stress is a prominent hallmark underlying obesity, IR and T2D and it consists of a constellation of stress responses that are dysregulated in metabolically relevant sites. This includes chronic metaflammation (Hotamisligil G, Nature 2017; 542: 177-185), glucolipotoxicity (Poitout and Robertson 2008), increased oxidative stress (Houstis, et al., Nature 2006; 440: 944-948), mitochondrial dysfunction or biogenesis (Szendroedi, et al., Nature reviews Endocrinology 2011; 8: 92-103), and persistent ER stress (Engin and Hotamisligil, Diabetes, obesity & metabolism 20l0;l2 Suppl 2: 108-115) with the concomitant impairment of the anti-inflammatory response (Pirola and Ferraz, World J Biol Chem 2017;8: 120-128), anti-oxidant defense system (Picu, et al., Molecules (Basel, Switzerland) 2017; 22
  • JNK c-Jun NH2-terminal kinase
  • IKKb inhibitor of kappa B
  • both enzymes interfere with the insulin action by phosphorylating the inhibitory serine of the insulin receptor substrate (IRS) and thereby, converting it to a poor substrate for the activated insulin receptor (Aguirre, et ak, The Journal of biological chemistry 2000; 275: 9047-9054; Gao, et ak, The Journal of biological chemistry 2002; 277: 8115-48121).
  • IRS insulin receptor substrate
  • HSR heat shock proteins
  • HSP-25 and HSP-72 act as natural inhibitors of JNK and IKKb kinases, and accordingly, they exhibit anti-apoptotic, anti-inflammatory and anti-oxidative stress properties (Park, et ak, The EMBO journal 2001;20:446-456; Park, et ak, The Journal of biological chemistry 2003; 278: 35272-35278; Simar, et ak Cell stress & chaperones 2012; 17: 615-621). Interventions that activate the HSR system are being intensively explored as alternative strategies to mitigate damages resulting from various stressful conditions including metabolic diseases.
  • DNAJB3 is a member of the heat shock protein-40 (HSP-40) cochaperone that was identified to be downregulated in the adipose tissue biopsies and PBMCs isolated from obese non-diabetic (Abubaker et ak, PloS one 2013; 8: e692l7) and diabetic (Abu-Farha, et ak, Scientific reports 2015; 5: 14448) subjects, as well as in experimental animal model of high fat induced obesity (Aksu et ak, J. Appl Genet 2007; 48: 133-143; Mitugi et ak, Int J. Mol. Sci. 2015; 16: 14997-15008).
  • HSP-40 heat shock protein-40
  • DNAJB3 Low levels of DNAJB3 were associated with enhanced metabolic stress and poor clinical outcomes (Abu-Farha et ak, Scientific reports 2015; 5: 14448). Restoring the normal expression of DNAJB3 with a lifestyle intervention program (i.e., supervised physical exercise for at least 3 months) are associated improved outcomes (Abubaker et ak, PloS one 2013; 8: e692l7). Consistent with this, overexpression of DNAJB3 improved insulin signaling and glucose uptake in vitro in 3T3-L1 adipocytes (Abu-Farha et al., Scientific reports 2015; 5: 14448). More importantly, DNAJB3 interacts with both JNK1 and IKKb kinases in co-immunoprecipitation assays (Abubaker et al., PloS one 2013; 8: e692l7).
  • a-lipoic acid also called thioctic acid or l,2-dithiolane3-pentanoic acid
  • ALA thioctic acid
  • l,2-dithiolane3-pentanoic acid is a naturally occurring dithiol compound enzymatically synthesized from octanoic acid in the mitochondria with a powerful antioxidant property. It acts as a crucial cofactor of the mitochondrial a-ketoacid dehydrogenase complexes involved in carbohydrate metabolism (Packer, L., Witt, E. H. & Tritschler, H. J. alpha-Lipoic acid as a biological antioxidant. Free radical biology & medicine 19, 227-250 (1995)).
  • ALA elicits other biochemical activities such as scavenging free radicals, regenerating the cellular antioxidant agents such as GSH, vitamin C and E, and modulating several critical signal transduction pathways (Packer, L., Witt, E. H. & Tritschler, H. J. alpha-Lipoic acid as a biological antioxidant. Free radical biology & medicine 19, 227-250 (1995); Rochette, L., Ghibu, S., Muresan, A. & Vergely, C. Alpha-lipoic acid: molecular mechanisms and therapeutic potential in diabetes. Canadian journal of physiology and pharmacology 93, 1021- 1027, doi: l0.
  • Alpha-lipoic acid protects against cadmium-induced neuronal injury by inhibiting the endoplasmic reticulum stress eIF2alpha- ATF4 pathway in rat cortical neurons in vitro and in vivo. Toxicology 414, 1-13, doi: 10. l0l6/j .tox.2018.12.005 (2019)).
  • ALA is a commonly used and readily available dietary supplement. In humans and experimental animal models, administration of ALA proved its pharmacotherapeutic value with a great therapeutic window index against several chronic diseases associated with metabolic stress such as diabetes (Rochette, L., Ghibu, S., Muresan, A. & Vergely, C. Alpha-lipoic acid: molecular mechanisms and therapeutic potential in diabetes.
  • Diabetic Polyneuropathy in Type 2 Diabetes Mellitus Inflammation, Oxidative Stress, and Mitochondrial Function. Journal of diabetes research 2016, 3425617, doi: 10.1155/2017/3425617 (2016); Ziegler, D. et al. Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidant alpha-lipoic acid. A 3-week multicentre randomized controlled trial (ALADIN Study). Diabetologia 38, 1425-1433 (1995)), obesity (Namazi, N., Larijani, B. & Azadbakht, L. Alpha-lipoic acid supplement in obesity treatment: A systematic review and meta-analysis of clinical trials.
  • Alpha Lipoamide Ameliorates Motor Deficits and Mitochondrial Dynamics in the Parkinson's Disease Model Induced by 6-Hydroxy dopamine. Neurotoxicity research 33, 759- 767, doi: l0T007/sl2640-0l7-98l9-5 (2016)), and other vascular diseases Ozgun, E. etal. The effect of lipoic acid in the prevention of myocardial infarction in diabetic rats. Bratislavske lekarske listy 119, 664-669, doi: 10.4 l49/bll _ 2018 _ 119 (2016); Scaramuzza, A. et al. Alpha-
  • Lipoic Acid and Antioxidant Diet Help to Improve Endothelial Dysfunction in Adolescents with Type 1 Diabetes: A Pilot Trial. Journal of diabetes research 2015, 474561, doi: 10.1155/2015/474561 (2015); Tromba, L., Perla, F. M., Carbotta, G., Chiesa, C. & Pacifico, L. Effect of Alpha-Lipoic Acid Supplementation on Endothelial Function and Cardiovascular Risk Factors in Overweight/Obese Youths: A Double-Blind, Placebo- Controlled Randomized Trial. Nutrients 11, doi: 10.3390/nul 1020375 (2019); Zhao, L. & Hu, F. X.
  • Lipoic acid increases heat shock protein expression and inhibits stress kinase activation to improve insulin signaling in skeletal muscle from high-fat-fed rats. Journal of applied physiology (Bethesda, Md. : 1985) 106, 1425-1434, doi: 10. H52/japplphysiol.91210.2008 (2009); Henriksen, E. J. Exercise training and the antioxidant alpha-lipoic acid in the treatment of insulin resistance and type 2 diabetes. Free radical biology & medicine 40, 3-12, doi: l0. l0l6/j.freeradbiomed.2005.04.002 (2006); Henriksen, E. J. et al.
  • the antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GFUT4 translocation and GFUT4 activation: potential role of p38 mitogen-activated protein kinase in GFUT4 activation.
  • Diabetes 50, 1464-1471 2001; Kouzi, S. A., Yang, S., Nuzum, D. S. & Dirks-Naylor, A. J. Natural supplements for improving insulin sensitivity and glucose uptake in skeletal muscle. Frontiers in bioscience (Elite edition) 7, 94-106 (2015); Qin, Z. Y. et al.
  • alpha-Fipoic acid ameliorates impaired glucose uptake in FYRM1 overexpressing 3T3-F1 adipocytes through the IRS-l/Akt signaling pathway.
  • Antioxidants preserve redox balance and inhibit c-Jun-N-terminal kinase pathway while improving insulin signaling in fat-fed rats: evidence for the role of oxidative stress on IRS-l serine phosphorylation and insulin resistance.
  • Versatile cytoprotective activity of lipoic acid may reflect its ability to activate signalling intermediates that trigger the heat-shock and phase II responses .
  • Alpha-lipoic acid preserves the structural and functional integrity of red blood cells by adjusting the redox disturbance and decreasing O-GlcNAc modifications of antioxidant enzymes and heat shock proteins in diabetic rats.
  • Heat shock protein 60 response to exercise in diabetes effects of alpha-lipoic acid supplementation. Journal of diabetes and its complications 20, 257- 261, doi: l0. l0l6/j .jdiacomp.2005.07.008 (2006); Oksala, N. K. et al.
  • Alpha-lipoic Acid modulates heat shock factor- 1 expression in streptozotocin-induced diabetic rat kidney. Antioxidants & redox signaling 9, 497-506, doi: l0. l089/ars.2006. l450 (2007); Strokov, I. A. et al. The function of endogenous protective systems in patients with insulin-dependent diabetes mellitus and polyneuropathy: effect of antioxidant therapy.
  • DNAJB3/HSP-40 cochaperone is downregulated in obese humans and is restored by physical exercise. PloS one 8, e692l7, doi: 10. l37l/joumal.pone.0069217 (2013)). More recently, we provided evidence for a novel role of DNAJB3 in attenuating various forms of metabolic stress as well as in promoting insulin action and glucose uptake in 3T3-L1 adipocytes and C2C12 skeletal muscle cells (Abu-Farha, M. el al. DNAJB3/HSP-40 cochaperone improves insulin signaling and enhances glucose uptake in vitro through J K repression. Scientific reports 5, 14448, doi: l0.
  • DNAJB3 attenuates metabolic stress and promotes glucose uptake by eliciting Glut4 translocation.
  • DNAJB3 might represent a molecular intermediate through which AFA mediates its beneficial actions.
  • the present disclosure provides compositions and methods for diagnosing, prevention and/or treatment for conditions linked to insulin resistance (IR).
  • IR insulin resistance
  • the present disclosure provides a biomarker for diagnosis of a chronic condition linked to IR, the biomarker comprising a polypeptide encoded by DNAJB3, wherein the chronic condition is selected from the group consisting of diabetes, metabolic syndrome and their complications.
  • Overexpression of the DNAJB3 enhances basal and insulin- stimulated glucose uptake.
  • Overexpression of DNAJB3 elicits Glut4 translocation to the plasma membrane in C2C12 cells.
  • Overexpression of DNAJB3 alleviates basal ER stress and enhances the oxidative stress scavenging system.
  • DNAJB3 abrogated both J K1 and IKKb pathways.
  • DNAJB3 suppressed TNF-a-mediated IL-6 promoter activation and mRNA expression.
  • the present disclosure provides a method comprising using a biomarker comprising a polypeptide encoded by DNAJB3 to diagnose a chronic condition linked to IR, wherein the chronic condition is selected from the group consisting of diabetes, metabolic syndrome and their complications.
  • the biomarker is used as an early molecular signature to detect early cellular, molecular and biochemical aberrations underpinning IR and diabetes.
  • the present disclosure provides a pharmaceutical compound comprising AFA, wherein the pharmaceutical composition is configured to prevent and/or treat a chronic condition linked to IR, and the chronic condition is selected from the group consisting of diabetes, metabolic syndrome and their complications.
  • the present disclosure provides a method for preventing and/or treating a chronic condition linked to IR in a subject in need of same, the method comprising administering a compound comprising AFA to the subject, wherein the chronic condition is selected from the group consisting of diabetes, metabolic syndrome and their complications.
  • the AFA is used to induce the endogenous expression of DNAJB3 gene/protein or recapitulate the activity of DNAJB3.
  • C2C12 cells are pre-treated with the AFA to alleviate tunicamycin- induced ER stress.
  • the AFA is used to stimulate the expression of mitochondrial markers and the oxidative stress scavenging system in C2C12 cells.
  • Fig. 1A shows the effect of overexpression of DNAJB3 in HEK-293 cells on preventing the phosphorylation of JNK (P-JNK) in response to phorbol myristate acetate (PMA) as compared to pCMV and pCMV-HDAC4.
  • P-JNK phosphorylation of JNK
  • PMA phorbol myristate acetate
  • Fig. 1B shows the effect of overexpression of DNAJB3 in HEK-293 cells on abrogating PMA-mediated AP-l -dependent transactivation in luciferase assays.
  • Fig. 2A shows the effect of overexpression of DNAJB3 in C2C12 cells on preventing the activation of KB -dependent transactivation in response to PMA in luciferase assays.
  • Fig. 2B shows the effect of overexpression of DNAJB3 in C2C12 cells on abrogating TNF-a-mediated both KB- and IF-6 promoter-dependent luciferase activation.
  • Fig. 3A shows the effect of overexpression of DNAJB3 on reducing the endogenous expression of IF- 16 mRNA in response to TNF-a in C2C12.
  • Fig. 3B shows the effect of overexpression of DNAJB3 on reducing the endogenous expression of IF-16 mRNA in response to TNF-a in 3T3-F1 adipocytes.
  • Fig. 3C shows that silencing the expression of DNAJB3 in C2C12 myoblast with specific siRNA reduced significantly the expression of DNAJB3 mRNA in a dose dependent- manner. GAPDH gene was used as a reference for normalization.
  • Fig. 3D shows that knocking down the expression of DNAJB3 in C2C12 myoblasts with specific siRNA resulted in a significant increase in TNF-a-mediated IL-6 mRNA expression.
  • Fig. 3E shows the effect of overexpression of DNAJB3 in C2C12 myoblasts on reducing the translocation of p65 NF-kB to the nucleus in response to LPS treatment (1 pg/ml for 3 h). Full-length blots are displayed in Fig. 9.
  • Fig. 4A shows the effect of overexpression of DNAJB3 on preventing Tunicamycin - mediated ATF6 activation in C2C12 cells using a luciferase assay.
  • Fig. 4B shows the effect of overexpression of DNAJB3 on abrogating Tunicamycin- mediated mRNA expression of XBP-l in C2C12 cells.
  • Fig. 4C shows the effect of overexpression of DNAJB3 on abrogating Tunicamycin- mediated mRNA expression of GRP78 in C2C12 .
  • Fig. 4D shows the effect of overexpression of DNAJB3 in C2C12 cells on stimulating the endogenous mRNA expression of Catalase and Glutathione peroxidase 1 (GPX1) genes in response to 300 mM H202 treatment for 3 h.
  • GPX1 Glutathione peroxidase 1
  • Fig. 5 A shows dose response effect of insulin on glucose uptake in myoblasts (dashed box) and myotubes (black box).
  • Fig. 5B shows the expression pattern of DNAJB3 in myoblasts and myotubes as monitored by RT-PCR.
  • Fig. 5C shows the expression pattern of DNAJB3 in myoblasts and myotubes as monitored by Western blot.
  • Fig. 5D shows the effect of DNAJB3 overexpression on promoting both basal and insulin-stimulated glucose uptake in myoblasts as compared to pCMV.
  • Fig. 5E shows the effect of DNAJB3 overexpression on promoting glucose uptake in 3T3-L1 adipocytes.
  • Fig. 5F shows the effect DNAJB3 overexpression on promoting glucose uptake in HepG2 cells.
  • Fig. 5G shows that silencing the expression of DNAJB3 with 10 nM of specific siRNA blunted the expression of DNAJB3 mRNA in C2C12 myoblasts.
  • Fig. 5H shows that knocking down the expression of DNAJB3 expression with specific siRNA abrogated both basal and insulin-stimulated glucose uptake in C2C12 cells.
  • Fig. 6A shows the effect of DNAJB3 overexpression on the endogenous expression of Glutl and Glut4 mRNA in C2C12 cells.
  • Fig. 6B shows effect of DNAJB3 overexpression on Glut4 protein expression in C2C12 cells. Full-length blots are displayed in Fig. 10.
  • Fig. 6C is a schematic representation of HA-Glut4-GFP construct showing the exofacial HA epitope and the GFP tag at the C-terminal region.
  • Fig. 6D shows representative confocal microscopy images showing cell surface staining of tagged GLUT4 in response to stimulation with 100 nM of insulin in C2C12 cells.
  • Fig. 6E shows the cellular localization of HA-Glut4-GFP in cells transfected with pCMV and DNAJB3 at baseline (a,b and c versus g,h and i) and after stimulation with 100 nM of insulin (d,e and f versus j,k and 1).
  • Fig. 6F shows representative image illustrating the plasma membrane localization of HA-Glut4-GFP in C2C12 cotransfected DNAJB3 in the presence of insulin.
  • Fig. 6G shows the surface-to-total Glut4 ratio (HA/GFP) at baseline and after insulin simulation was determined by quantitative immunofluorescence and presented as percent change.
  • Figs. 7A and 7B are schematic representation for the role of DNAJB3 in mitigating metabolic stress and improving glucose uptake.
  • Fig. 8 shows the full-length western blots of Fig. 1A.
  • Fig. 9 shows the full-length western blots of Fig. 3E.
  • Fig. 10 shows the full-length western blots of Fig. 6B.
  • Fig. 11A shows RT-PCR data showing the effect of 0.3 mM ALA for 24h on the expression of representative components of the heat shock response in C2C12 cells.
  • Fig. 11B shows dose response effect of ALA on the expression of DNAJB3 mRNA in C2C12 cells. Full-length blots are displayed in Fig. 16.
  • Fig. 11C shows dose response effect of ALA on the expression of DNAJB3 protein in C2C12 cells. Full-length blots are displayed in Fig. 16
  • Fig. 11D shows ALA at 0.3 mM for 24h also increases the expression of DNAJB3 mRNA in HepG2 cells.
  • Fig. 11E shows heat shock treatment induces the expression of DNAJB3 mRNA in C2C12 cells.
  • Fig. 12A shows pre-treatment of C2C12 cells with 0.3 mM ALA abolishes significantly the mRNA expression of classical ER stress markers in response to tunicamycin stimulation.
  • Fig. 12B shows pre-treatment of C2C12 cells with 0.3 mM ALA abolishes significantly the mRNA expression of classical ER stress markers in response to tunicamy con/glucolipotoxicity stimulation .
  • Fig. 12C shows western blot confirming the effect of ALA on tunicamy cin-induced expression of GRP78 protein in C2C12 cells. Full-length blots are displayed in Fig. 17.
  • Fig. 12D shows ALA reduces ATF6-dependent luciferase activity in response to tunicamycin using a functional luciferase-based assay in C2C12 cells.
  • Fig. 12E shows ALA reduces ATF6-dependent luciferase activity in response to tunicamycin using a functional luciferase-based assay in HepG2 cells.
  • Fig. 13A shows ALA treatment triggers a significant increase in the expression involved in mitochondrial biogenesis and function.
  • Fig. 13B shows ALA stimulates the endogenous mRNA expression of Catalase, Superoxide dismutase 1 (SOD1) and Glutathione peroxidase 1 (GPX1) genes in response to 300 mM H202 treatment for 3 h.
  • SOD1 Superoxide dismutase 1
  • GPX1 Glutathione peroxidase 1
  • Fig. 14A shows knocking down the expression of DNAJB3 expression with 20 nM of specific siRNA blunted the endogenous expression of DNAJB3 in C2C12 cells. Actin gene was used as a reference control.
  • Fig. 14B shows ALA fails to protect cells from tunicamy cin-induced mRNA expression of ER stress markers in C2C12 cells transfected with siRNA specific for DNAJB3.
  • Fig. 14C shows ALA fails to protect cells from tunicamycin-induced ATF6-dependent luciferase activity in C2C12 cells transfected with siRNA specific for DNAJB3.
  • Fig. 15A shows effect of ALA on insulin-stimulated glucose uptake in C2C12 cells.
  • Fig. 15B shows silencing the expression of DNAJB3 abrogated the effect of ALA on insulin-stimulated glucose uptake as compared to scrambled siRNA control in C2C12 cells.
  • Fig. 16 shows the full-length western blots of Figs. 11B and 11C.
  • Fig. 17 shows the full-length western blots of Fig. 12C.
  • Fig. 18A shows the nucleotide sequence of the human DNAJB3 cDNA.
  • Fig. 18B shows the protein sequence of the human DNAJB3.
  • Fig. 18C is the Schematic representation of the NF-KB-dependent reporter plasmids used in Luciferase assays.
  • the term “patient” is understood to include an animal, especially a mammal, and more especially a human that is receiving or intended to receive treatment, as treatment is herein defined. While the terms “individual” and “patient” are often used herein to refer to a human, the present disclosure is not so limited. Accordingly, the terms “individual” and “patient” refer to any animal, mammal or human that can benefit from the treatment.
  • treatment and “treating” include any effect that results in the improvement of the condition or disorder, for example lessening, reducing, modulating, or eliminating the condition or disorder.
  • the term does not necessarily imply that a subject is treated until total recovery.
  • Non-limiting examples of "treating" or “treatment of' a condition or disorder include: (1) inhibiting the condition or disorder, i.e. arresting the development of the condition or disorder or its clinical symptoms and (2) relieving the condition or disorder, i.e. causing the temporary or permanent regression of the condition or disorder or its clinical symptoms.
  • a treatment can be patient- or doctor-related.
  • prevention or “preventing” mean causing the clinical symptoms of the referenced condition or disorder to not develop in an individual that may be exposed or predisposed to the condition or disorder but does not yet experience or display symptoms of the condition or disorder.
  • condition and “disorder” mean any disease, condition, symptom, or indication.
  • the present disclosure provides a novel drug target/biomarker to attenuate metabolic stress and prevent and/or treat IR in insulin-responsive sites and more particularly to prevent and/or treat chronic conditions linked to IR including diabetes, metabolic syndrome and their complications.
  • the present disclosure also provides methods of using such a novel drug target/biomarker to attenuate metabolic stress and prevent and/or treat IR in insulin-responsive sites and more particularly to prevent and/or treat chronic conditions linked to IR including diabetes, metabolic syndrome and their complications.
  • the inventors used a series of functional assays to investigate the in vitro role of DNAJB3 in modulating metabolic stress and improving glucose uptake in HEK-293, C2C12 and 3T3-L1 cells.
  • JNK1- and IKKb-dependent luciferase reporters the inventors observed a significant decrease in luciferase activity by DNAJB3 in response to phorbol myristate acetate (PMA) and tumor necrosis factor-a (TNF-a).
  • PMA phorbol myristate acetate
  • TNF-a tumor necrosis factor-a
  • TNF-a-mediated IF-6 promoter activation and the endogenous mRNA expression are significantly abrogated by DNAJB3.
  • DNAJB3 stimulates glucose uptake in C2C12 cells by eliciting Glut4 translocation to the plasma membrane.
  • the novel drug target/biomarker can include a polypeptide encoded by DNAJB3/HSP-40 with anti-inflammatory activity that acts as natural inhibitor of the inflammatory KB kinase b (IKKb).
  • IKKb is a crucial enzyme involved in the pathogenesis of IR, T2D mellitus and metabolic syndrome.
  • the human DNAJB3 gene encodes a DNAJ (Heat shock protein 40; Hsp40) homolog, subfamily B, member 3 chaperone protein (DNAJB3), which can be down-regulated in disease conditions, as observed in decreased expression of DNAJB3 mRNA in peripheral blood mononuclear cells (PBMC) of obese patients.
  • DNAJB3 Heat shock protein 40; Hsp40
  • PBMC peripheral blood mononuclear cells
  • the inventors surprisingly discovered the positive effects of DNAJB3 in enhancing glucose metabolism, eliciting the translocation of Glut4 transporter to the plasma membrane, and improving mitochondrial function/biogenesis.
  • the present disclosure further provides compositions comprising small molecules, such as ALA, for preventing and/or treating chronic conditions linked to IR including diabetes, metabolic syndrome and their complications.
  • small molecules such as ALA
  • the present disclosure also provides methods of using such small molecules as ALA for preventing and/or treating chronic conditions linked to IR including diabetes, metabolic syndrome and their complications.
  • small molecules, such as ALA can induce the endogenous expression of DNAJB3 gene/protein or recapitulate the activity of DNAJB3.
  • DNAJB3 as an early molecular signature or biomarker for diagnose the early cellular, molecular and biochemical aberrations underpinning IR and diabetes (e.g., T2D).
  • C2C12 myoblasts, 3T3-L1 preadipocytes, HEK-293 and HepG2 cells were all obtained from ATCC and maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% C02. Differentiation of C2C12 myoblasts to myotubes was done by replacing FBS with 2% horse serum with a daily change of the media for 7 days.
  • 3T3-L1 were differentiated from preadipocytes to adipocytes using isobutylmethylxanthine (IB MX), dexamethasone and insulin as we previously described. All the cells were used before the 25th passage.
  • IB MX isobutylmethylxanthine
  • pCMV-DNAJB3 and pCMV-HSPAlA plasmids were purchased from Origene (Origene Technologies, Inc., Rockvile, MD). They encode the human DNAJB3 and HSP-72, respectively.
  • pCMV6 empty vector was used as a negative control.
  • pHA-Glut4-GFP was a gift from Dr. MacGraw (Weill Cornell University, New York, NY) and consists of an exofacial HA epitope and a GFP tag located at the N-terminal and C-terminal of Glut4, respectively.
  • Reporter plasmids carrying firefly luciferase gene under the control of three copies of either wild type (3xwt-KB-Luc) or mutant (3xpKB-Luc) NF-kB binding site were described previously.
  • Reporter plasmid carrying the human IL-6 promoter (pIL6- Luc65 l) was obtained from Dr. Eickelberg (University of Colorado Denver, Aurora, CO).
  • Reporter plasmid carrying seven copies of AP-l binding site upstream of the firefly luciferase gene was obtained from Dr. Fahmi (Montreal University, Montreal, QC).
  • ATF6 -dependent reporter plasmid consisting of three copies of ATF6 response element upstream of the luciferase gene was purchased from Promega (Promega Corporation, Madison, WI). In all cases, Renilla Uuciferase vector under the control of CMV promoter (pRL-CMV; Promega Corporation, Madison, WI) was used as internal control. Three different siRNA molecules specific for DNAJB3 and the scrambled siRNA were used to knockdown the expression of DNAJB3 in C2C12 (#SR406762; Origene Technologies, Inc., Rockville, MD).
  • Luciferase assays HEK-293 and C2C12 were transfected with 5 pg of the reporter plasmid and 10 pg of either pCMV-DNAJB3 or pCMV and then, plated on 96-well plates at 1.104 cells/well followed by a 24-h incubation. Cells were then treated with 25 ng/ml of TNF- a (R&D Systems, Minneapolis, MN) or 5 pM PMA (Sigma Aldrich, St. Louis, MO) or 0.5 pg/ml Tunicamycin (Sigma Aldrich, St.
  • RT-PCR real-time PCR
  • the preparation of nuclear and cytoplasmic extracts from C2C12 myoblasts was carried out by using the Ready PrepTM Cytoplasmic/Nuclear Extraction Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Protein concentration was determined by Bradford assay (Biorad) at 595 nm using g- Globulin (Bio-Rad, Hercules, CA) as standard. Proteins were aliquoted and stored at -80 °C until assayed.
  • DNAJB3 and HSP-72 in whole cell extracts prepared from myoblasts and myotubes was also performed by western blot using anti-DNAJB3 (Proteintech Group, Inc., Chicago, IL) and anti-HSP-72 (ENZO Life Sciences, Inc., Farmingdale, NY) antibodies.
  • the endogenous expression of Glut4 in C2C12 overexpressing DNAJB3 (or control vector) was monitored by western bot using anti-Glut4 antibody (Abeam, Cambridge, UK).
  • Nuclear translocation of p65 NF-kB in C2C12 transfected with DNAJB3 or pCMV after LPS/TNF-a stimulation was carried out on cytoplasmic and nuclear fractions by western blot using anti-p65 antibody (Cell Signaling Technology, Inc., Danvers, MA).
  • Anti-GRP78 antibody (Cell Signaling Technology, Inc., Danvers, MA) was used to monitor the expression of GRP78 protein in response to Tunicamycin treatment using whole cell extracts from C2C12 transfected with DNAJB3 or pCMV.
  • GAPDH, b-Actin Cell Signaling Technology, Inc., Danvers, MA
  • g-Tubulin Acam, Cambridge, UK
  • Glucose uptake assay Cells were grown in 100 mm petri dishes until they reached 80% confluence and then, transfected with 7.5 pg of either pCMV-DNAJB3 or pCMV or 10 nM of DNAJB3-siRNA. The next day, they were plated on 96-well plates at 1.10 4 cells/well and then used to monitor glucose uptake using the fluorescent D-glucose analog (2-NBDG) (Cayman, Ann Arbor, MI) as we described previously, except that cells were glucose-starved for overnight while HepG2 cells were starved only for 3 h. After washes, the retained fluorescence was measured respectively at excitation and emission wavelengths of 485 nm and 535 nm with FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).
  • 2-NBDG fluorescent D-glucose analog
  • HA-Glut4-eGFP cells expressing the HA-Glut4-eGFP were randomly chosen in the GFP channel blinded to the expression of HA- Glut4-GFP on the plasma membrane (Alexa Fluor 594 channel). Images were collected in both GFP and Alexa Fluor 594 channels. To optimize the dynamic range of the assay, exposure times for the channels were independently set to maximize the signal while minimizing the number of cells with expression levels above saturation. Once set for each channel, all images in that channel were collected at the same exposure. The fluorescence intensities of GFP and Alexa 594 were quantified at the single-cell level. Mock- transfected cells were used in parallel to correct for fluorescence resulting from non-specific binding of the primary and/or secondary antibodies.
  • Fig. 1A shows that DNAJB3 acts as natural inhibitor of JNK1 stress kinase.
  • Transient overexpression of DNAJB3 in HEK-293 cells prevents the phosphorylation of JNK (P-JNK) in response to phorbol myristate acetate (PMA) as compared to pCMV and pCMV-HDAC4.
  • PMA phorbol myristate acetate
  • Total JNK and GAPDH were used as internal controls to monitor for protein loading differences.
  • the same membrane was stripped and probed with antibody against total JNK antibody. Full-length blots are displayed in Fig. 8.
  • Fig. 1B shows that DNAJB3 abrogates PMA-mediated AP-l -dependent transactivation in luciferase assays.
  • DMSO was used at 0.25% as a vehicle.
  • This data indicates that PMA treatment triggers 4-5 fold increase in luciferase activity as compared to the vehicle.
  • the luciferase activity was significantly reduced (P ⁇ 0.001), confirming thus the observed reduced levels in P-JNK triggered by DNAJB3.
  • DNAJB3 abrogates PMA and TNF-a-mediated IKKb activation. IKKb has also been shown to interact with DNAJB3, however, the functional consequence of such interaction was not explored.
  • the inventors interrogated whether DNAJB3 could interfere with NF-KB activation using our previously established Kb-dependent luciferase system. To this end, cells were co-transfected with p3xwtKB-Luc reporter and either pCMV-DNAJB3 or the pCMV and subsequently, they were stimulated with 5 mM of PMA or 25 nM TNF-a and then, the luciferase activity was monitored.
  • Fig. 2A shows transient overexpression of DNAJB3 in C2C12 cells prevents the activation of kB-dependent transactivation in response to phorbol myristate acetate (PMA) in luciferase assays.
  • PMA phorbol myristate acetate
  • Fig. 2B shows that DNAJB3 abrogates also TNF-a-mediated both KB- and IL-6 promoter-dependent luciferase activation.
  • DMSO at 0.25% and PBS were used as vehicles for PMA and TNF-a treatments, respectively.
  • a similar increase in luciferase activity following stimulation with TNF-a in cells transfected with p3xwtKB-Luc construct but not with p3xmutKB-Luc construct P ⁇ 0.01; Fig. 2B).
  • DNAJB3 overexpression abolished the KB- dependent luciferase activity triggered by TNF-a- (P ⁇ 0.001; Fig. 2B).
  • DNAJB3 assessed the effect of DNAJB3 in controlling NF-kB activity using a physiologically relevant context such as the IL-6 promoter whose activity is in part, regulated by NF-kB.
  • DNAJB3 reduced significantly the activity of IL-6 promoter following TNF-a stimulation (P ⁇ 0.001; Fig. 2B).
  • DNAJB3 acts upstream of the NF-kB signaling pathway and that IKKb is an interacting partner of DNAJB3. [00105] DNAJB3 modulate the expression of the IL-6 mRNA in response to TNF-a.
  • the inventors also investigated the effect of DNAJB3 on the endogenous expression of IL-6 mRNA by RT-PCR.
  • Figs. 3A-3E show that DNAJB3 is involved in TNF-a-mediated IL-6 mRNA expression.
  • Figs. 3A and 3B show overexpression of DNAJB3 reduces significantly the endogenous expression of IL-16 mRNA in response to TNF-a both in C2C12 (Fig. 3A) and 3T3-L1 adipocytes (Fig. 3B).
  • Fig. 3C shows silencing the expression of DNAJB3 in C2C12 myoblast with specific siRNA reduced significantly the expression of DNAJB3 mRNA in a dose dependent-manner. GAPDH gene was used as a reference for normalization.
  • Fig. 3A-3E show that DNAJB3 is involved in TNF-a-mediated IL-6 mRNA expression.
  • Figs. 3A and 3B show overexpression of DNAJB3 reduces significantly the endogenous expression of IL-16 mRNA in response to TNF-a both in C2C12 (Fig. 3A) and 3
  • FIG. 3D shows that knocking down the expression of DNAJB3 in C2C12 myoblasts resulted in a significant increase in TNF-a-mediated IL-6 mRNA expression.
  • Fig. 3E shows overexpression of DNAJB3 in C2C12 myoblasts reduces the translocation of p65 NF-kB to the nucleus in response to LPS treatment (1 pg/ml for 3 h). Full-length blots are displayed in Fig. 9. PBS was used as a vehicle. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; NS: Not significant.
  • Results displayed in Figs. 3A and 3B show a 2- to 2.5-fold increase in IL-6 mRNA expression upon stimulation with TNF-a as compared to the vehicle (P ⁇ 0.05) in C2C12 and 3T3-L1 cells, respectively.
  • Overexpression of DNAJB3 caused a significant reduction of IL-6 mRNA expression following TNF-a stimulation as compared to pCMV (P ⁇ 0.01) both in C2C12 myoblasts (Fig. 3A) and 3T3-L1 adipocytes (Fig. 3B).
  • HSP- 2 failed in preventing the response of IL-6 mRNA expression to TNF-a (Fig. 3A).
  • the inventors silenced the expression of DNAJB3 using specific siRNA.
  • the efficiency and specificity of these siRNA to abrogate the endogenous expression of DNAJB3 were first determined by RT-PCR in C2C12 myoblasts.
  • Transfection of cells with 10 nM DNAJB3 siRNA reduced the expression of DNAJB3 mRNA by 84% as compared to control siRNA (P ⁇ 0.0001; Fig. 3C).
  • the response of IL-6 mRNA expression to TNF-a under the conditions where DNAJB3 expression is silenced was investigated in C2C12 myoblasts and the finding is displayed in Fig. 3D. As shown, there was a slight increase in both basal and TNF-a induced IL-6 mRNA expression as compared to scrambled siRNA (P ⁇ 0.05).
  • DNAJB3 has a positive effect in alleviating ER stress and enhancing the oxidative stress scavenging system.
  • the contribution of persistent ER stress and enhanced oxidative stress to the pathogenesis of IR and T2D promoted the inventors to assess the effect of DNAJB3 on mitigating ER stress and oxidative stress.
  • the inventors used a luciferase reporter assay driven by multiple copies of ATF-6 transcription factor; the master transcription factor involved in the activation of ER stress.
  • C2C12 myoblasts were cotransfected with ATF-6 reporter and either pCMV-DNAJB3 or pCMV and then stimulated for overnight with 0.5pg/ml of Tunicamycin.
  • Figs. 4A-4D show overexpression of DNAJB3 alleviates basal ER stress and enhances the oxidative stress scavenging system.
  • Fig. 4A shows transient overexpression of DNAJB3 in C2C12 myoblasts cells reduces significantly the basal activity of ATF6 in Luciferase assays.
  • B-C DNAJB3 abrogates also Tunicamycin-mediated mRNA expression of both XBP-l (Fig. 4B) and GRP78 (Fig. 4C).
  • Fig. 4A-4D show overexpression of DNAJB3 alleviates basal ER stress and enhances the oxidative stress scavenging system.
  • Fig. 4A shows transient overexpression of DNAJB3 in C2C12 myoblasts cells reduces significantly the basal activity of ATF6 in Luciferase assays.
  • B-C DNAJB3 abrogates also Tunicamycin-mediated mRNA expression of both XBP-l (Fig. 4B
  • 4D shows overexpression of DNAJB3 in C2C12 cells stimulates the endogenous mRNA expression of Catalase and Glutathione peroxidase 1 (GPX1) genes in response to 300 mM H202 treatment for 3 h.
  • DMSO at 0.25% and PBS were used as vehicles for Tunicamycin and TNF-a treatments, respectively.
  • DNAJB3 reduces significantly (P ⁇ 0.01) the luciferase activity at both basal level and following Tunicamycin stimulation. This data is suggestive of a role of DNAJB3 in alleviating the ER stress. To complement this finding, we examined the effect of DNAJB3 on the endogenous expression of representative markers of ER stress; namely GRP78 and XPB1 in response to Tunicamycin. Data displayed in Figs. 4B-4C show a significant decrease in both XBP1 and GRP78 mRNA levels in DN A JB -transfected cells upon Tunicamycin treatment (P ⁇ 0.05).
  • DNAJB3 enhances basal and insulin-stimulated glucose uptake. Whether overexpression of DNAJB3 in C2C12 could enhance glucose uptake as investigated. The inventors initially compared the glucose uptake in differentiated (myotubes) and undifferentiated (myoblasts) C2C12 in response to insulin and the data shown in Fig. 5A revealed a subtle difference in insulin-stimulated glucose uptake between myoblasts and myotubes.
  • Figs. 5A-5G show that DNAJB3 promotes glucose uptake in C2C12 cells.
  • Fig. 5A shows that dose response effect of insulin on glucose uptake in myoblasts (dashed box) and myotubes (black box).
  • Fig. 5B shows RT-PCR data showing the expression of DNAJB3 in myoblasts and myotubes. GAPDH was used as a control.
  • Fig. 5C shows western blot showing the expression pattern of DNAJB3 myoblasts and myotubes. GAPDH was used as a reference for normalization.
  • Fig. 5D shows that DNAJB3 promotes both basal and insulin-stimulated glucose uptake in myoblasts as compared to pCMV.
  • Figs. 5E and 5F show overexpression of DNAJB3 promotes glucose uptake in 3T3-F1 adipocytes (Fig. 5E) and HepG2 cells (Fig. 5F).
  • Fig. 5G shows silencing the expression of DNAJB3 with 10 nM of specific siRNA blunted the expression of DNAJB3 mRNA in C2C12 myoblasts. Actin gene was used as a reference control.
  • Fig. 5H shows knocking down the expression of DNAJB3 expression with specific siRNA abrogated both basal and insulin- stimulated glucose uptake in C2C12 cells. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • DNAJB3 knocking down the expression of DNAJB3 reduced significantly both basal (P ⁇ 0.0001) and insulin stimulated (P ⁇ 0.01) glucose uptake as compared with scrambled siRNA.
  • DNAJB3 Overexpression of DNAJB3 elicits Glut4 translocation to the plasma membrane in C2C12 cells.
  • Glutl and Glut4 transporters have a central role in basal and insulin-mediated glucose uptake by the skeletal muscle, respectively.
  • Figs. 6A-6G show DNAJB3 elicits the translocation of Glut4 transporter to the plasma membrane in C2C12 cells without changing its expression.
  • Fig. 6A shows effect of DNAJB3 on the endogenous expression of Glutl and Glut4 mRNA.
  • Fig. 6B shows effect of DNAJB3 on Glut4 protein expression.
  • Full-length blots are displayed in Fig. 10.
  • Fig. 6C shows schematic representation of HA-Glut4-GFP construct showing the exofacial HA epitope and the GFP tag at the C-terminal region.
  • Fig. 6A-6G show DNAJB3 elicits the translocation of Glut4 transporter to the plasma membrane in C2C12 cells without changing its expression.
  • Fig. 6A shows effect of DNAJB3 on the endogenous expression of Glutl and Glut4 mRNA.
  • Fig. 6B shows effect of DNAJB3 on Glut
  • FIG. 6D shows representative confocal microscopy images showing cell surface staining of tagged GLUT4 in response to stimulation with 100 nM of insulin in C2C12 cells.
  • FIG. 6E shows cellular localization of HA-Glut4-GFP in cells transfected with pCMV and DNAJB3 at baseline (a,b and c versus g,h and i) and after stimulation with 100 nM of insulin (d,e and f versus j,k and 1). The images were captured using the tile scanning method; and each image represents 25 adjacent and overlapping fields acquired with a 40X objective. The quantification was done on individual cells and for each condition; we analyzed at least 100 cells.
  • FIG. 6F shows representative image illustrating the plasma membrane localization of HA-Glut4-GFP in C2C12 cotransfected DNAJB3 in the presence of insulin.
  • Fig. 6G shows the surface-to-total Glut4 ratio (HA/GFP) at baseline and after insulin simulation was determined by quantitative immunofluorescence and presented as percent change. **P ⁇ 0.01.
  • the surface Glut4 pool Upon expression of DNAJB3, the surface Glut4 pool is enriched to 48% (P ⁇ 0.01; Fig. 6G). In response to insulin, the Glut4 surface pool is increased to 52% in pCMV transfected cells and to 67% in cells overexpressing DNAJB3 (P ⁇ 0.01; Fig. 6G).
  • DNAJB3 has a role in modulating metabolic stress and its relationship to glucose metabolism. It has been demonstrated that DNAJB3: 1- Abrogated both JNK1 and IKKb pathways in functional assays, 2- Suppressed TNF-a- mediated IL-6 promoter activation and mRNA expression; 3- Reduced ER and oxidative stress and, 4- Enhanced glucose uptake and elicited Glut4 translocation. Altogether, the results provide for the first time a compelling evidence for a novel role of DNAJB3 in modulating metabolic stress; a prerequisite step that leads to IR and T2D.
  • DNAJB3 The pathophysiological role of DNAJB3 in glucose metabolism was investigated following the inventors’ observations that the levels of DNAJB3 are reduced in adipose tissue obtained from obese and diabetic subjects and they correlate with increased P-JNK1, enhanced inflammation and ER stress. More importantly, it has been shown that physical exercise training restored the normal expression of DNAJB3 while decreasing P-JNK1, inflammatory and ER stress responses. Interestingly, the decrease in DNAJB3 levels was more pronounced in obese-diabetic patients as compared to obese non-diabetic subjects (Abu-Farha, et al., Scientific reports 2015; 5: 14448).
  • DNAJB3 interacts with JNK1 and IKKb in co-immunoprecipitation assays (Abubaker, et al., PloS one 2013; 8: e692l7) and attenuates the activation of JNK in response to palmitate (Abu-Farha, et al., Scientific reports 2015; 5: 14448).A11 these observations demonstrate a protective role of DNAJB3 against obesity associated metabolic stress.
  • J K1 kinase pathway One of the pathways that are activated under metabolic stress conditions is the J K1 kinase pathway, which interferes with insulin signal transduction.
  • the findings of the inventors indicate that beside the role of DNAJB3 in attenuating the activation of J K1, it significantly reduces the phosphorylation of IRS-1S307 in response to palmitate while promoting the AKT survival pathway as monitored by increased phosphorylation of AKT protein in HEK-293 cells and 3T3-F1 adipocytes.
  • JNK1 plays a fundamental role in modulating gene expression by activating an array of transcription factors and other nuclear proteins involved in apoptosis, inflammation, DNA repair, mRNA stability and development.
  • DNAJB3 acts as a natural inhibitor of J K-l as it abrogates its ability to modulate gene transcription using functional assays, confirming thus the inventors’ demonstration that DNAJB3 binds to J K1 and reduces its activation in response to palmitate and PMA stressors (Figs. 1A-1B).
  • IKKb Besides J K1, pathological activation of the IKKb kinase has detrimental consequence on insulin signaling and glucose metabolism.
  • IKKb is also an inhibitor of IRS-l substrate as it phosphorylates its serine 307 residue.
  • IKKb is a master upstream kinase that activates the canonical pathway of NF-kB. Once activated, it turns on a complex transcription program driven by NF-kB that leads to inappropriate expression and release of an array of inflammatory mediators including cytokines, chemokines, metalloproteases and growth factors.
  • HSP-72 is one of the best-studied chaperones among all the HSPs in relationship to metabolic diseases. Its role in conferring protection against metabolic defects leading to IR and T2D in part by reducing the inflammation is extensively reported. However, HSP-72 does not seem to have a role in attenuating the expression of IL-6 mRNA in C2C12 cells under our experimental conditions (Fig. 3A).
  • DNAJB3 Another important aspect in this disclosure is the effect of DNAJB3 on glucose metabolism in skeletal muscle C2C12 as well as the molecular and biochemical determinants mediating such effect.
  • Glucose transport into muscle and fat cells is an important step in insulin action and is critical for the maintenance of glucose homeostasis.
  • the inventors have found that overexpression of DNAJB3 in 3T3-L1 adipocytes resulted in enhanced glucose uptake.
  • DNAJB3 has a positive impact on improving insulin signaling as it prevents IRS-1S307 phosphorylation while promoting its phosphorylation at tyrosine 612 (IRS-1Y612).
  • DNAJB3 enhances both basal and insulin-stimulated glucose uptake in C2C12 cells (Figs. 5A-5G).
  • the inventors observed a significant increase in glucose uptake in cells overexpressing DNAJB3 that was independent of insulin action (Fig. 5D).
  • An additive effect of insulin on glucose uptake was observed in C2C12 cells overexpressing DNAJB3 (Fig. 5D).
  • knocking down the expression of DNAJB3 by silencing RNA blunted the glucose uptake in response to insulin Fig. 5H.
  • DNAJB3 increases only the basal glucose uptake (Fig. 5F).
  • DNAJB3- mediated glucose uptake enhancement In the skeletal muscle, Glutl and Glut4 have a central role in basal and insulin-mediated glucose mobilization, respectively.
  • the observed effect of DNAJB3 on basal glucose uptake is consistent with the finding that DNAJB3 stimulates the expression of Glutl (Fig. 6A). Insulin elicits its metabolic action by activating multiple signaling cascades in metabolically relevant sites.
  • PI-3K phosphatidylinositol-3 -kinase
  • AS 160 Akt and its substrate 160
  • Akt and AS160 phosphorylation are impaired in skeletal muscle obtained from insulin-resistant patients (Karlsson, et ak, Diabetes 2005; 54: 1692-1697) as well as upon TNF-a stimulation.
  • the level P-AKT and P-AS 160 were significantly increased in 3T3-L1 adipocytes overexpressing DNAJB3.
  • the results indicate that DNAJB3 elicits both basal and insulin-stimulated Glut4 translocation in C2C12 (Figs. 6A- 6G).
  • DNAJB3 orchestrates its protective effects according to the model illustrated in Figs. 7A-7B.
  • excessive accumulation of free fatty acids, chronic hyperglycemia and inflammatory mediators lead to the persistent ER stress, oxidative stress and impaired expression of the HSR.
  • This toxic environment will lead to the activation of JNK-l and IKKb kinases that target the IRS-l and convert it to poor substrate of the insulin receptor and ultimately blocking the PI-3K/AKT pathway.
  • DNAJB3 has a protective role in mitigating metabolic stress by binding to JNK1 and IKKb enzymes and abrogating their activation in response to harmful stressors.
  • DNAJB3 has also a positive role in improving glucose uptake at least in part by enhancing Glut4 translocation to the plasma membrane in C2C 12.
  • the inventors have found DNAJB3 has a physiological role in glucose metabolism and insulin signaling. Identifying small molecules that induce the expression DNAJB3 or recapitulate its function could be leveraged as a possible novel strategy for the control and management of metabolic defects leading to IR andT2D.
  • the inventors further assessed the effect ALA treatment on the endogenous expression of DNAJB3 in metabolically relevant cells and determined the significance of such an effect on other forms of metabolic stress that trigger IR as well as on glucose uptake.
  • the inventors found that ALA triggers a significant increase in the expression of DNAJB3 in C2C12 and HepG2 cells.
  • the inventors investigated the significance of such activation on ER stress; one of the key hallmarks of obesity induced IR and T2D.
  • ALA pre-treatment significantly reduced the expression of ER stress markers namely, GRP78, XBP1, XBPls and ATF4 in response to both tunicamycin and glucolipotoxicity.
  • DNAJB3 is a key mediator of ALA-alleviated tunicamycin and gucolipotoxicity-induced ER stress.
  • the effect of ALA on insulin-stimulated glucose uptake is reduced significantly in C2C12 cells transfected with DNAJB3 siRNA.
  • Anti-DNAJB3 antibody was purchased from Proteintech (Proteintech Group, Inc., Chicago, IL).
  • Anti-HSP72 antibody was purchased from ENZO (ENZO Life Sciences, Inc., Farmingdale, NY).
  • Anti-GRP78 and anti-y-Tubulin were purchased from Abeam (Abeam, Cambridge, UK).
  • Anti-Actin and horseradish peroxidase-conjugated antibodies were purchased from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA).
  • ALA, tunicamycin, palmitic acid, H202 and insulin were purchased from Sigma (Sigma- Aldrich, St. Louis, MO).
  • C2C12 and HepG2 were purchased from ATCC (ATCC, Manassas, VA). Fluorescently labeled D-glucose analog (2-NBDG) was purchased from Cayman (Cayman, Ann Arbor, MI). Reporter plasmid carrying five copies of ATF6 binding site upstream of the Luc2P reporter gene was described previously. Scrambled and specific siRNA were purchased from Dharmacon (Dharmacon Inc., Lafayette, CO). Lipofectamine 3000 and lipofectamine RNAiMAX were purchased from Invitrogen (Invitrogen, Carlsbad, CA). Bright Glo Luciferase Assay kit was purchased from Promega (Promega Corporation, Madison, WI). PureLinkTM RNA Minikit and High-Capacity cDNA Reverse Transcription Kit were purchased from Invitrogen (Invitrogen, Carlsband, CA).
  • C2C12 and HepG2 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% C02. Differentiation of C2C12 from myoblasts to myotubes was done by replacing FBS with 2% horse serum with a daily change of the media for 5 days. For heat shock induction, ⁇ 85% confluent cells were incubated for lh at 43°C followed by a 4h recovery at 37°C and then harvested.
  • ALA For the effect of ALA on ER stress induced by glucolipotoxicity, cells were challenged with 25 mM of glucose and 400 uM of palmitate for overnight and treated with ALA or vehicle for 24h and afterwards, harvested for RNA extraction. Lipofectamine 3000 and RNAiMAX lipofectamine were used for transient DNA and siRNA transfection, respectively as recommended by the manufacturer. We used a smart pool Accell siRNA targeting DNAJB3 and Accell non-targeting control at 20 nM each. All the functional assays were analyzed at least in triplicate and a minimum of three independent experiments.
  • Luciferase assays C2C12 and HepG2 were grown in 100 mm petri dish and at -80% confluence, they were transfected with 7.5 pg of the reporter plasmid using lipofectamine 3000 and incubated overnight at 37°C. On next day, they were plated on 96-well plates at 1.104 cells/well in complete DMEM media containing 0.3 mM ALA or vehicle. After 8h of incubation, cells were stimulated 0.5 pg/ml tunicamycin or vehicle and incubated at 37°C for overnight and then harvested for luciferase assays using the Bright Glo Luciferase Assay kit.
  • DNAJB3 -siRNA or scrambled-siRNA or using lipofectmaine RNAiMAX protocol was carried out with DNAJB3 -siRNA or scrambled-siRNA or using lipofectmaine RNAiMAX protocol. The following day cells were transfected with 7.5 pg ATF6 reporter plasmid using Lipofectamine 3000 and incubated overnight at 37°C. Afterward, cells were then plated in 96-well plate at 1. 104 cells/well and pre-treated with ALA flowed by tunicamycin stimulation and luciferase activity.
  • DNAJB3 Mouse 5 '-AGGGGCTGTACCCTTCTCTA-3 ' 5 '-AGTTTCCTGG AG A ACCG A AG-3 ' SOD 1 Mouse 5 '-G A G A G GC ATGTTGG AG ACCT -3 ' 5 '-CC A CCTTTGCCC A AGTC ATC-3 ' Catalase Mouse 5 '-AGG AGGCAGA AACTTTCCCA-3 ' 5 '-GGCCCTG A AGC ATTTTGTC A-3’ GPX1 Mouse 5 '-ATC AGTTCGGAC ACCAGGAG-3 ' 5 '-GATGTA CTTGGGGTCGGTC A-3 '
  • HSP72 Mouse 5 '-GAC AAGAAGAAGGTGCTGGA-3 ' 5 '-TGGTACAGCCCACTGATGAT-3 '
  • Protein concentration was determined by the Bradford method using g-globulin as a standard, and 40- 80 pg of proteins were resolved on 10% SDS-PAGE gels. Proteins were then transferred onto
  • PVDF membranes blocked with 5% nonfat dried milk in Tris-buffered saline containing
  • Glucose uptake assay C2C12 cells at 80% confluence in 100 mm petri dishes were pre-treated with either 0.3mM ALA or vehicle for 8h and then, the media was replaced with glucose free media, containing either ALA or vehicle, for overnight starvation. The next day, the culture media was replaced with glucose free culture media containing Fluorescent tagged
  • D-glucose analog (2-NBDG) at a concentration of 150 pg/ml with or without insulin 100hM and incubated for lh at 37°C. Cells were then washed with Cell-Based Assay Buffer and transferred to 96-well assay plate at 1.104 cells/well. Finally, fluorescence was quantified on Glomax Multi+ Microplate Multimode Reader (Promega, Madison, WI). Monitoring glucose uptake in C2C12 where the expression of DNAJB3 has been silenced was done by transfecting cells with DNAJB3-siRNA or scrambled control and after 2 days, cells were pre-treated with ALA for 8h and the media was changed to glucose free media with ALA and starvation and glucose uptake was carried out as described above.
  • 2-NBDG D-glucose analog
  • ALA induces the endogenous expression of DNAJB3. It has been shown previously that ALA mediates its beneficial effects by activating the HSR. However, these investigations have focused on a limited set of HSPs; namely HSP25, HSP60, HSP72, HSF1 and GRP75. The in vivo and in vitro investigations confirmed a novel role of DNAJB3 in reducing metabolic stress, improving insulin signaling and promoting glucose uptake. These findings suggest that DNAJB3 may represent a relevant therapeutic target against IR and T2D. The investors then assess the effect of ALA on the endogenous expression of DNAJB3 together with other key representative genes of the HSR.
  • Figs. 11A-11E show ALA induces the endogenous expression of DNAJB3 in C2C12 and HepG2 cells.
  • Fig. 11A shows RT-PCR data showing the effect of 0.3 mM ALA for 24h on the expression of representative components of the heat shock response in C2C12 cells.
  • Figs. 11B-11C show dose response effect of ALA on the expression of DNAJB3 mRNA (Fig. 11B) and protein (Fig. 11C) in C2C12 cells. Full-length blots are shown in Fig. 16.
  • Fig. 16 shows that ALA induces the endogenous expression of DNAJB3 in C2Cl2cells. Cells were treated for 24h with increasing amounts of ALA. The levels of DNAJB3 protein were detected by western blot. g-Tubulin was used as internal control.
  • Fig. 11D shows ALA at 0.3 mM for 24h also increases the expression of DNAJB3 mRNA in HepG2 cells.
  • Fig. 11E shows heat shock treatment induces the expression of DNAJB3 mRNA.
  • HSP72 was used as a positive control.
  • Ethanol was used at 0.25% as a vehicle. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.00l .
  • Fig. 1 1A Data displayed in Fig. 1 1A confirmed indeed a significant increase (at least 4-fold increase) in the expression of DNAJB3 mRNA in C2C12 in response to treatment with 0.3 mM ALA (R ⁇ 0.001). Under the same experimental conditions, it was confirmed that the positive effect of ALA on modulating the expression of other heat shock related genes, although with different degrees, but the highest induction was observed for DNAJB3, flowed by HSP72 (Fig. 1A; P ⁇ 0.05). The inventors did a time course and a dose response with ALA and monitored the expression of DNAJB3 mRNA and protein. Data displayed in Figs. 11B and 11C indicate that ALA induces the expression of DNAJB3 mRNA (Fig. 1B) and protein (Fig.
  • Pre-treatment of C2C12 cells with ALA alleviates tunicamycin-induced ER stress.
  • the contribution of persistent ER stress to the pathogenesis of IR and T2D is well established.
  • Several studies reported the effectiveness of ALA in alleviating ER stress; however, none of these studies examined the effect ALA in skeletal muscle cells. The inventors therefore investigated whether pre-treatment of C2C12 cells with ALA could mitigate tunicamyin-induced ER stress by measuring the expression and activity of known ER stress markers.
  • Figs. 12A-12E show ALA alleviates tunicamycin and glucolipotixicity-induced ER stress.
  • Figs. 12A-12B show pre-treatment of C2C12 cells with 0.3 mM ALA abolishes significantly the mRNA expression of classical ER stress markers in response to both tunicamycin (Fig. 12A) and glucolipotoxicity (Fig. 12B) conditions.
  • Fig. 12C show western blot confirming the effect of ALA on tunicamycin-induced expression of GRP78 protein. Full- length blots are shown in Fig. 17.
  • Fig. 17 shows the ALA alleviates tunicamycin-induced expression of GRP78 protein in C2C12 cells as monitored by western blot.
  • b-Actin was used as internal control.
  • Figs. 12D-12E show ALA also reduces ATF6-dependent luciferase activity in response to tunicamycin using a functional luciferase-based assay both in C2C12 (Fig. 12D) and HepG2 (Fig. 12E) cells. Ethanol and DMSO were used at 0.25% as vehicles for ALA and tunicamycin, respectively. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.00l .
  • FIGs. 12A-12E Data displayed in Figs. 12A-12E indicate that overnight treatment of C2C12 cells with 0.5 pg/ml tunicamycin led to a marked increase in the endogenous expression of GRP78, XBP1 and it spliced form sXBPl and ATF4 as compared to the vehicle (Fig. 12A).
  • Fig. 12A In cells pre-treated with ALA, there was a significant reduction in tunicamycin-mediated effect on those markers (Fig. 12A).
  • the ability of ALA to alleviate ER stress has been confirmed in cells challenged to glucolipotoxicity (Fig. 12B).
  • the inventors confirmed the inhibitory effect of ALA on tunicamycin-induced activation of GRP78 protein (Fig. 12C).
  • Fig. 12D a functional luciferase-based assay to examine the effect of ALA on the activity of ATF6 in response to tunicamycin.
  • Fig. 12D a 5-fold increase in ATF6-dependent luciferase activity in response to 0.5 pg/ml tunicamycin was consistently observed.
  • Pre-treatment of cells with ALA reduced significantly the ATF6- dependent luciferase activity (Fig. 12D; P ⁇ 0.001).
  • Fig. 12E a similar pattern was also observed (Fig. 12E), although the magnitude of tunicamycin effect was less pronounced than in C2C 12.
  • ALA stimulates the expression of mitochondrial markers and the oxidative stress scavenging system in C2C12 cells.
  • Dysfunction of mitochondria and/or its biogenesis was linked to IR and T2D (Gonzalez-Franquesa, A. & Patti, M. E. Insulin Resistance and Mitochondrial Dysfunction. Advances in experimental medicine and biology 982, 465-520, doi: 10.1007/978-3-319-55330-6_25 (2017); Hesselink, M. K., Schrauwen-Hinderling, V. & Schrauwen, P. Skeletal muscle mitochondria as a target to prevent or treat type 2 diabetes mellitus. Nature reviews.
  • Figs. 13A shows that ALA improves mitochondrial function.
  • Fig. 13A shows that ALA treatment triggers a significant increase in the expression of TFAM (P ⁇ 0.05), PPARy and its coactivator PGCla (P ⁇ 0.00l), and cytochrome C (P ⁇ 0.05). No significant effect was observed for PPARy (Fig. 13A).
  • ALA effect of modulating the mRNA expression of genes encoding for representative antioxidant enzymes; namely catalase, glutathione peroxidase (GPX1) and superoxide dismutase 1 (SOD1).
  • results displayed in Fig. 13B indicate a significant increase in the mRNA expression levels of catalase (P ⁇ 0.00l) both at basal level and after H202 treatment.
  • a positive effect of ALA on the expression of SOD1 and GPX1 genes was observed but only under oxidative stress conditions (Fig. 11B; P ⁇ 0.05).
  • DNAJB3 abolished the beneficial effect of ALA on alleviating tunicamycin-induced ER stress.
  • the inventors found a negative regulation of DNAJB3 expression when ER stress is induced. Reciprocally, both basal and tunicamycin- induced ER stress are significantly reduced when DNAJB3 is overexpressed. This is suggestive of a mutual negative feedback regulation between DNAJB3 and ER stress.
  • the inventors knocked down the expression of DNAJB3 using siRNA and then, induced ER stress with tunicamycin in C2C12 cells pre-treated with ALA.
  • Figs. 14A-14C show silencing the expression of DNAB3 abolishes the protective effect of ALA on tunicamycin-induced ER stress.
  • Fig. 14A shows knocking down the expression of DNAJB3 expression with 20 nM of specific siRNA blunted the endogenous expression of DNAJB3 in C2C12 cells. Actin gene was used as a reference control.
  • Figs. 14B- 14C show ALA fails to protect cells from tunicamycin-induced mRNA expression of ER stress markers (Fig. 14B) and ATF6-dependent luciferase activity (Fig. 14C) in cells transfected with siRNA specific for DNAJB3.
  • DNAJB3 has a positive effect on enhancing both basal and insulin-stimulated glucose uptake.
  • the inventors assessed first the effect of ALA on glucose uptake in C2C 12 and then, investigated whether DNAJB3 is involved in such effect.
  • Figs. 15A-15B show silencing the expression of DNAB3 abrogated the effect of ALA on enhancing glucose uptake in C2C12 cells.
  • Fig. 15A shows effect of ALA on insulin- stimulated glucose uptake.
  • Fig. 15B shows silencing the expression of DNAJB3 abrogated the effect of ALA on insulin-stimulated glucose uptake as compared to scrambled siRNA control. *** P 0.00L
  • Fig. 15A Data displayed in Fig. 15A indicates that ALA enhanced insulin-stimulated glucose uptake by approximately 20% (PO.OOl). In cells where the expression of DNAJB3 has been silenced, ALA failed to promote glucose uptake elicited by insulin (Fig. 15B; P ⁇ 0.00l).
  • DNAJB3 1- induces the expression of DNAJB3 in C2C12 and HepG2 cell lines, 2- alleviates ER stress triggered by tunicamycin and glucolipotoxicity and improve glucose uptake, 3- knocking down the expression of DNAJB3 with siRNA abolished the beneficial effect of ALA in alleviating tunicamycin-induced ER stress and enhancing insulin-stimulated glucose uptake.
  • DNAJB3 as a molecular determinant that mediates the beneficial effect of ALA in attenuating metabolic stress.
  • the identification of DNAJB3 as a downstream target of ALA supports further the importance of the HSR in mitigating the key drivers of IR.
  • DNAJB3 previously known as MSJ-l was initially described in mice as a gene involved in male reproduction, but given the presence of a highly conserved and functionally critical J-domain, the gene was subsequently named DNAJB3; a member of the DNAJ/HSP40 cochaperone family that acts as an obligate partner and critical regulator of the activity and substrate specificity ofHSP70 chaperone. DNAJB members exert their role by stimulating the ATPase activity of HSP70 through their J-domain, thereby keeping the bound substrates in successive refolding cycles. The inventors found the novel in vitro role of DNAJB3 in insulin signaling and glucose metabolism.
  • DNAJB3 represents a potential therapeutic target for diseases associated with IR and proteotoxicity.
  • ALA treatment caused significant increase in DNAJB3 mRNA expression in C2C12 and HepG2 cells. Without being bound to the theory, the inventors believe that the most likely mechanism for HSPs induction would be activation of HSF1; the master transcription factor controlling the HSR. Under the experimental conditions, the inventors observed a slight, but significant increase in HSF1 mRNA levels in response to ALA (Fig. 11A) and this could play a role in DNAJB3 activation. Similar findings have been reported in streptozotocin-induced diabetic rat in response to ALA.
  • ALA supplementation showed no effect on HSF1 expression in the skeletal muscle of high fat fed rat model of IR, as well as under heat stress conditions in Caco-2 cells.
  • the dose, route of administration, biological context and duration of treatment could explain the discrepancy between those studies.
  • the DNA-binding activity of HSF1 has been shown to be potentiated with SIRT1 deacetylase.
  • SIRT1 expression is also positively regulated with ALA.
  • ALA could control the expression through the nuclear factor-erythroid 2 (Nrf2); another safeguard transcription factor that regulates the expression of anti -oxidant response genes, as well as HSR genes.
  • Nrf2 nuclear factor-erythroid 2
  • Nrf2 executes its task by binding to the antioxidant response element (ARE) in the promoter region of its downstream target genes and subsequently drives their expression.
  • ARE antioxidant response element
  • both mouse and human DNAJB3 promoters have a putative ARE.
  • Data show increased expression in DNAJB3 mRNA and protein in response to sulforaphane and resveratrol, two potent activators ofNrf2 (data not shown).
  • ER is a dispersed organelle throughout the cell that performs important homeostatic functions related to proteostasis, lipid metabolism and calcium storage. These processes rely on the protein folding activity of chaperone machinery densely populated in the ER. Disruption of ER homeostasis occurs when the folding capacity of the ER fails to accommodate the overwhelming load of misfolded and unfolded proteins, leading thus to ER stress. This elicits a potent adaptive response known as the unfolded protein response (UPR); an acute mechanism that assists in restoring ER activity and reestablishing cellular homeostasis.
  • UTR unfolded protein response
  • ER stress canonical transducers that act in concert to restore ER homeostasis: PKR-like ER kinase (PERK), inositol-requiring enzyme-la (IREla), and activating transcription factor-6 (ATF6).
  • PERK PKR-like ER kinase
  • IREla inositol-requiring enzyme-la
  • ATF6 activating transcription factor-6
  • PERK, IREla, and ATF6 interacts with GRP78 chaperone, however upon accumulation of unfolded proteins, GRP78 dissociates from these transducers, leading to their activation.
  • GRP78 dissociates from these transducers, leading to their activation.
  • Each of these transducers activates specific pathways and collectively leads to decreased overall protein synthesis, enhanced ER folding capacity and increased degradation of misfolded proteins, resulting in either recovery of ER homeostasis or cell death.
  • Tunicamycin is a well-known ER stress inducer both in vitro and in vivo.
  • DNAJB3 is one of the molecular targets through which ALA attenuates ER stress. Defects in the HSR and persistent ER stress are one of the critical aberrations underlying IR and T2D. Alleviation of ER stress and enhancement of the HSR have previously been considered as attractive potential therapeutic pathways against several chronic diseases. In L6 cells, the ability of ALA to prevent anisomycin-mediated INK activation is abolished upon inhibition of the HSR with KNK437 drug, supporting thus a role of the HSR as mediator of the metabolic actions elicited by ALA. The inventors found the negative regulation of DNAJB3 expression when ER stress is induced.
  • the inventors also found a reciprocal effect of DNAJB3 on both basal and tunicamycin- induced ER stress. This is suggestive of a mutual negative feedback regulation between DNAJB3 and ER stress that provides a functional crosstalk between both stress pathways.
  • DNAJB3 as a molecular determinant through which ALA alleviates ER stress
  • the inventors silenced the expression of DNAJB3 using siRNA and then, assessed the ability of ALA to reduce ER stress in response to tunicamycin.
  • the data show that DNAJB3 siRNA abolished the beneficial effect of ALA on tunicamycin-induced ER stress marker genes as compared to scrambled siRNA control (Fig. 14B; P ⁇ 0.05).
  • DNAJB3 exerts a direct effect on ER stress or indirectly via other pathways.
  • the close link between ER stress and mitochondrial homeostasis has been described.
  • ALA prevented ER stress-induced IR by enhancing mitochondrial function, but surpisingly; it failed to abrogate the expression of tunicamycin-induced ER stress markers.
  • the inventors observed a positive correlation between DNAJB3 levels and maximum oxygen consumption in human subjects.
  • overexpression of DNAJB3 stimulated the expression of conventional markers of mitochondrial biogenesis and function such as TFAM, PGCla, PPARy and OXPHOS subunits (unpublished data). Interestingly, this pattern was also observed in the current study.
  • DNAJB3 attenuates ER stress by promoting mitochondrial homeostasis. In this context, it will be important to assess the effect of DNAJB3 on ER stress under the conditions where the mitochondrial function is impaired (i.e., oligomycin). Alternatively, DNAJB3 could physically interact with one or several transducers of the UPR and trigger an inhibitory effect on their respective activities.
  • ALA may also attenuate ER stress via reduction of the oxidative stress through stimulating the anti-oxidant response genes as previously reported. ER stress and oxidative stress are important mechanisms of IR.
  • the inventors found the ability of DNAJB3 to mitigate oxidative stress induced with H202.
  • ALA treatment stimulated the expression of the oxidative stress scavenging genes Catalase, SOD1 and GPx-l in response to H202 (Fig. 13B), replicating previous findings in C2C12 cells.
  • C2C12 cells were treated with ALA and insulin-induced glucose uptake was performed. The results showed a significant increase in insulin-stimulated glucose uptake following ALA treatment, similar to a pattern following DANJB3 overexpression in C2C12 cells.
  • DNAJB3 To examine the specificity of DNAJB3 on the effect of ALA on insulin-stimulated glucose uptake, the ininventors silenced the expression of DNAJB3 using siRNA and then, assessed the effect of ALA on glucose uptake. The data show that inhibition of DNAJB3 abolished the beneficial effect of ALA on insulin-stimulated glucose uptake; suggesting that ALA improves glucose uptake through DNAJB3.

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Abstract

La présente invention concerne un biomarqueur pour le diagnostic d'une affection chronique liée à l'insulinorésistance (IR) et un procédé de diagnostic utilisant un tel biomarqueur. Le biomarqueur comprend un polypeptide codé par DNAJB3. L'affection liée à l'IR peut comprendre le diabète, le syndrome métabolique et leurs complications. L'invention concerne également un procédé d'utilisation du biomarqueur. Une composition pharmaceutique comprenant de l'acide lipoïque-α (ALA) peut être utilisée pour prévenir et/ou traiter une affection chronique liée à l'IR.
PCT/QA2019/050015 2018-10-11 2019-10-08 Diagnostic, prévention et/ou traitement d'affections liées à l'insulinorésistance Ceased WO2020076175A2 (fr)

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