Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes

https://doi.org/10.1016/j.bbalip.2016.09.004Get rights and content

Highlights

  • Expression of chaperone-mediated autophagy signaling proteins is decreased in the obese and diabetic heart

  • Autophagic flux and lysosomal proteolysis is inhibited following obesity and diabetes

  • Obese and diabetic environment in vivo and nutrient overload ex vivo decrease myocyte TFEB

  • Palmitate but not oleate deplete TFEB protein and supress autophagic flux in myocytes

  • Loss of TFEB in the cardiomyocyte reduces lysosomal protein coentent and increases ER stress and cell death

Abstract

Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

Introduction

A subset of obese and diabetic patients suffers from cardiac muscle specific contractile dysfunction termed as cardiomyopathy [1], [2], [3], [4], [5]. Etiology in the progression of cardiomyopathy in rodent models of obesity and diabetes includes diastolic dysfunction [6], [7], myocyte hypertrophy [8], [9], [10], interstitial fibrosis [8], [11], [12], early-onset metabolic maladaptation [9], [10], [13], [14], and progressive lipid accumulation [9], [10], all of which precede heart failure. The underlying cause for metabolic inflexibility in cardiomyopathy is the lack of insulin or insulin action resulting in hyperglycemia and fatty acid overutilization, both of which lead to a condition known as “glucolipotoxicity” [1], [15], [16], [17]. Numerous studies have demonstrated that glucolipotoxic effects in the cardiomyocytes, originate or terminate primarily in the mitochondria and the endoplasmic reticulum (ER) [18], [19], [20], [21], [22].

A glucolipotoxic milieu in the cardiomyocyte impairs protein quality control, inducing ER stress and activating protein degradation pathway [23], [24]. To counter the damaging effects of glucolipotoxicity, proteasomal degradation is acutely activated to clear the cellular proteotoxic load [25]. Indeed, ubiquitin mRNA levels, caspase-3 and ATP-dependent proteasomal degradation are augmented acutely following streptozotocin (STZ)-induced type-1 diabetes [25]. However, sustained glucolipotoxicity exacerbates ER stress, saturating and impairing proteasomal protein degradation, causing toxic accumulation of misfolded proteins [26], [27]. In agreement with this theory, failing hearts from chronically obese humans with type-2 diabetes display significant accumulation of non-degraded proteins [28], suggesting that impaired proteasomal degradation in late stages of obesity and diabetes promotes a maladaptive buildup of cytotoxic proteins causing and/or exacerbating cardiomyopathy. Recent experimental evidence suggests that if the proteasome is impaired then damaged proteins must be degraded by the lysosomal machinery via autophagy to maintain cellular homeostasis [29], [30], [31]. Interestingly, islet dysfunction in ob/ob mice is exacerbated following treatment with lysosomal function inhibitors [32], suggesting that disruption in lysosomal function accelerates cell death. However, the underlying mechanisms by which glucolipotoxicity affects inter- and intra-lysosomal signaling, metabolism and function remain to be examined. Furthermore, whether clinically observed proteotoxicity and cardiomyopathy in obese and diabetic hearts involves negative targeting of lysosomes by glucolipotoxic substrates remains to be investigated.

Autophagy degrades short- and long-lived proteins in the lysosome [33], [34], [35] either via macroautophagy [36], [37], [38] or via chaperone-mediated autophagy (CMA) [39], [40]. Macroautophagy in the ER-cytosol interface requires lipidation of microtubule-associated protein 1 light chain B subtype 3 (LC3B-I) to form LC3B-II [37], [41] resulting in autophagosome formation, maturation and fusion with the lysosome to degrade proteins. The macroautophagy process utilizes polyubiquitin cargo-receptor, p62/SQSTM1 to engage in partial-selection of bulk load of intracellular protein and organelle content [23], [24] and therefore, changes in LC3B and p62/SQSTM1 signify changes in macroautophagy. Lysosomal CMA is a process by which cytosolic proteins targeted for degradation are delivered to lysosomal membrane protein-type 2A (LAMP-2A), which internalizes the protein cargo for lysosomal degradation [39], [40]. Notably, humans and mice with loss of function of LAMP-2 exhibit impaired autophagosome clearance, lysosomal dysfunction, and cardiomyopathy, suggesting that lysosomal autophagy is critical for cardiac function [42], [43], [44]. Expression of numerous lysosomal proteins responsible for autophagic processes are under the direct control of transcription factor EB (TFEB), a transcriptional regulator of lysosome autophagy and biogenesis [45], [46]. TFEB-action not only generates autophagosomes, but also accelerates their delivery and clearance by lysosomes via increases in lysosomal biogenesis [46]. It is plausible that changes in lysosome function and biogenesis could significantly impact cellular function in the setting of obesity, insulin resistance and diabetes, however, limited studies have examined the impact of glucolipotoxicity on TFEB and its downstream functions.

In this study, we examined whether glucolipotoxicity negatively targets TFEB, a transcriptional regulator of lysosome function, to impair autophagy and thereby render cardiomyocytes susceptible to proteotoxicity and cell death. Utilizing a mouse model of diet-induced obesity, type-1 diabetes and ex-vivo model of glucolipotoxicity (rat cardiomyofibroblasts and neonatal rat cardiomyocyte), we demonstrated that (1) baseline macroautophagy is reciprocally regulated in obesity and type-1 diabetes, (2) an obese and diabetic environment in-vivo suppressed lysosome signaling proteins, inhibited autophagic flux and reduced lysosomal proteolysis, (3) lysosomal protein suppression following ex-vivo myocyte nutrient overload is FA specific since palmitate or glucose/palmitate but not oleate or high glucose alone, reduces lysosomal protein content, (4) in the obese and diabetic heart, TFEB is decreased and this effect is recapitulated not only ex-vivo myocytes exposed to glucolipotoxic milieu but also in human heart tissue from patients with Class-1 obesity. Collectively, our data highlights a novel mechanism by which glucolipotoxicity targets TFEB to inhibit lysosomal integrity and render cardiomyocytes susceptible to proteotoxicity, injury and failure.

Section snippets

Animal models

All protocols involving rodents were approved by the Dalhousie University, Institutional Animal Care and Use Committee.

Heart from mice with diet induced obesity exhibit cellular stress with concomitant upregulation of cell growth pathways

To examine the impact of obesity and diabetes on cardiac autophagy, we fed C57BL/6J mice HFHS diet (45% kcal fat) for 16 weeks. This type of diet and length of feeding is reported to cause mild to moderate glucolipotoxicity in multiple tissues including the heart [9]. Consistent with previous diet-induced obesity studies [9], our mice fed HFHS diet exhibited increases in body weight (Fig. 1A) and fed glucose levels (Fig. 1B). Metabolic inflexibility in HFHS mice was evident from systemic glucose

Discussion

Protein degradation is severely impaired in the failing hearts of obese humans [28] and this impairment worsens with diabetes leading to buildup of cytotoxic proteins and causing cardiomyopathy. Experimental evidence suggests that cytotoxic proteins that are unable to be degraded by proteasome must be degraded by lysosomal autophagy to maintain cellular homeostasis [29], [30], [31]. Therefore, it is plausible that changes in lysosome function and biogenesis could significantly impact cellular

Conflict of interest

The authors declare that there is no conflict of interest.

Author contribution

T.P., P.C.T. and J.J.B. designed the research; P.C.T. and J.J.B. performed the experiments; P.C.T. and T.P. analyzed and interpreted the data and wrote the paper; L.J.P. generated mRNA data; P.C.K. provided intellectual inputs and technical assistance; K.R.B., J.L. & A.H assisted with clinical sample collection and provided intellectual inputs to clinical study.

Guarantor statement

Dr. Thomas Pulinilkunnil is the guarantor of this work, had full access to all the data, and takes full responsibility for the integrity of data and the accuracy of data analysis.

Transparency document

Transparency document

Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-03687), the Canadian Diabetes Association (NOD_OG-3-15-5037-TP) and the New Brunswick Health Research Foundation grants to T.P.; a Dalhousie Medicine New Brunswick graduate studentship to P.T. The clinical arm of this work was funded by a Chesley Grant (OPOS Study) to A.H. & P.C.K. in Saint John, NB, and by CIHR “REACH” grant to J.L. in Halifax, NS. We thank Dr. Gerard Karsenty for providing

References (94)

  • R. Guo et al.

    Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: role of autophagy

    Biochim. Biophys. Acta

    (2013)
  • B. Jaishy et al.

    Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity

    J. Lipid Res.

    (2015)
  • H.M. Shen et al.

    At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy

    Trends Biochem. Sci.

    (2014)
  • J.Y. Kong et al.

    Palmitate induces structural alterations in nuclei of cardiomyocytes

    Tissue Cell

    (1999)
  • R.T. Brookheart et al.

    As a matter of fat

    Cell Metab.

    (2009)
  • D.L. Medina et al.

    Transcriptional activation of lysosomal exocytosis promotes cellular clearance

    Dev. Cell

    (2011)
  • K.M. Mellor et al.

    Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice

    J. Mol. Cell. Cardiol.

    (2011)
  • J.L. Schneider et al.

    Deficient chaperone-mediated autophagy in liver leads to metabolic dysregulation

    Cell Metab.

    (2014)
  • G. Las et al.

    Fatty acids suppress autophagic turnover in beta-cells

    J. Biol. Chem.

    (2011)
  • L.R. Rega et al.

    Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells

    Kidney Int.

    (2016)
  • H. Martini-Stoica et al.

    The autophagy–lysosomal pathway in neurodegeneration: a TFEB perspective

    Trends Neurosci.

    (2016)
  • D. An et al.

    Role of changes in cardiac metabolism in development of diabetic cardiomyopathy

    Am. J. Physiol. Heart Circ. Physiol.

    (2006)
  • S. Boudina et al.

    Diabetic cardiomyopathy, causes and effects

    Rev. Endocr. Metab. Disord.

    (2010)
  • G. de Simone et al.

    Diabetes and incident heart failure in hypertensive and normotensive participants of the Strong Heart Study

    J. Hypertens.

    (2010)
  • D.L. Medina et al.

    Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

    Nat. Cell Biol.

    (2015)
  • R. Basu et al.

    Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function

    Am. J. Physiol. Heart Circ. Physiol.

    (2009)
  • T. Abe et al.

    Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats

    Am. J. Physiol. Heart Circ. Physiol.

    (2002)
  • I.G. Poornima et al.

    Diabetic cardiomyopathy: the search for a unifying hypothesis

    Circ. Res.

    (2006)
  • T. Pulinilkunnil et al.

    Cardiac-specific adipose triglyceride lipase overexpression protects from cardiac steatosis and dilated cardiomyopathy following diet-induced obesity

    Int. J. Obes.

    (2014)
  • T. Pulinilkunnil et al.

    Myocardial adipose triglyceride lipase overexpression protects diabetic mice from the development of lipotoxic cardiomyopathy

    Diabetes

    (2013)
  • H. Wakasaki et al.

    Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy

    Proc. Natl. Acad. Sci. U. S. A.

    (1997)
  • J. Buchanan et al.

    Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity

    Endocrinology

    (2005)
  • T. van de Weijer et al.

    Lipotoxicity in type 2 diabetic cardiomyopathy

    Cardiovasc. Res.

    (2011)
  • J.W. Kim et al.

    Glucolipotoxicity in pancreatic beta-cells

    Diabetes Metab. J.

    (2011)
  • S. Boudina et al.

    Mitochondrial uncoupling: a key contributor to reduced cardiac efficiency in diabetes

    Physiology (Bethesda)

    (2006)
  • L. Cai et al.

    Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway

    Diabetes

    (2002)
  • A. Gonzalez-Rodriguez et al.

    Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

    Cell Death Dis.

    (2014)
  • U. Karunakaran et al.

    Guards and culprits in the endoplasmic reticulum: glucolipotoxicity and beta-cell failure in type II diabetes

    Exp. Diabetes Res.

    (2012)
  • N. Mizushima et al.

    The role of Atg proteins in autophagosome formation

    Annu. Rev. Cell Dev. Biol.

    (2011)
  • M. Lippai et al.

    The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy

    Biomed. Res. Int.

    (2014)
  • J. Hu et al.

    Cardiac muscle protein catabolism in diabetes mellitus: activation of the ubiquitin-proteasome system by insulin deficiency

    Endocrinology

    (2008)
  • S. Casas et al.

    Impairment of the ubiquitin-proteasome pathway is a downstream endoplasmic reticulum stress response induced by extracellular human islet amyloid polypeptide and contributes to pancreatic beta-cell apoptosis

    Diabetes

    (2007)
  • S. Despa et al.

    Hyperamylinemia contributes to cardiac dysfunction in obesity and diabetes: a study in humans and rats

    Circ. Res.

    (2012)
  • J. Groenendyk et al.

    Biology of endoplasmic reticulum stress in the heart

    Circ. Res.

    (2010)
  • J. Groenendyk et al.

    Coping with endoplasmic reticulum stress in the cardiovascular system

    Annu. Rev. Physiol.

    (2013)
  • M. Hoyer-Hansen et al.

    Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium

    Cell Death Differ.

    (2007)
  • C. Broca et al.

    Proteasome dysfunction mediates high glucose-induced apoptosis in rodent beta cells and human islets

    PLoS One

    (2014)
  • Cited by (62)

    • Secreted frizzled-related protein 2 ameliorates diabetic cardiomyopathy by activating mitophagy

      2024, Biochimica et Biophysica Acta - Molecular Basis of Disease
    • Autophagy in the diabetic heart: A potential pharmacotherapeutic target in diabetic cardiomyopathy

      2021, Ageing Research Reviews
      Citation Excerpt :

      On the other hand, calcineurin (CaN) and protein phosphatase 2A (PP2A) can activate TFEB activity by triggering its dephosphorylation and nuclear translocation (Fig. 3) (Medina et al., 2015). Glucolipotoxicity has been revealed to suppress TFEB in cardiomyocytes and inhibits lysosomal autophagy during obesity and diabetes (Trivedi et al., 2016). ZKSCAN3 acts as a key transcriptional repressor of autophagy flux, particularly, in the nutrient abundant state (Chauhan et al., 2013).

    View all citing articles on Scopus
    View full text