Original article
Sulforaphane prevents the development of cardiomyopathy in type 2 diabetic mice probably by reversing oxidative stress-induced inhibition of LKB1/AMPK pathway

https://doi.org/10.1016/j.yjmcc.2014.09.022Get rights and content

Highlights

  • Compared with mice fed a high-fat diet, diabetes aggravated heart damage.

  • We have shown that sulforaphane (SFN) prevents T2DM cardiomyopathy.

  • SFN prevents cardiac dilation and dysfunction in T2DM mice.

  • SFN prevents diabetes-induced cardiac lipotoxicity.

  • SFN prevents diabetic oxidative stress-induced inhibition of LKB1/AMPK activity.

Abstract

Type 2 diabetes mellitus (T2DM)-induced cardiomyopathy is associated with cardiac oxidative stress, inflammation, and remodeling. Sulforaphane (SFN), an isothiocyanate naturally presenting in widely consumed vegetables, particularly broccoli, plays an important role in cardiac protection from diabetes. We investigated the effect of SFN on T2DM-induced cardiac lipid accumulation and subsequent cardiomyopathy. Male C57BL/6J mice were fed a high-fat diet for 3 months to induce insulin resistance, followed by a treatment with 100 mg/kg body-weight streptozotocin to induce hyperglycemia; we referred to it as the T2DM mouse model. Other age-matched mice were fed a normal diet as control. T2DM and control mice were treated with or without 4-month SFN at 0.5 mg/kg daily five days a week. At the study's end, cardiac function was assessed. SFN treatment significantly attenuated cardiac remodeling and dysfunction induced by T2DM. SFN treatment also significantly inhibited cardiac lipid accumulation, measured by Oil Red O staining, and improved cardiac inflammation oxidative stress and fibrosis, shown by down-regulating diabetes-induced PAI-1, TNF-α, CTGF, TGF-β, 3-NT, and 4-HNE expression. Elevated 4-HNE resulted in the increase of 4-HNE-LKB1 adducts that should inhibit LKB1 and subsequent AMPK activity. SFN upregulated the expression of Nrf2 and its downstream genes, NQO1 and HO-1, decreased 4-HNE-LKB1 adducts and then reversed diabetes-induced inhibition of LKB1/AMPK and its downstream targets, including sirtuin 1, PGC-1α, phosphorylated acetyl-CoA carboxylase, carnitine palmitoyl transferase-1, ULK1, and light chain-3 II. These results suggest that SFN treatment to T2DM mice may attenuate the cardiac oxidative stress-induced inhibition of LKB1/AMPK signaling pathway, thereby preventing T2DM-induced lipotoxicity and cardiomyopathy.

Introduction

Type 2 diabetes mellitus (T2DM) is the most widespread metabolic disease in the world. It is estimated that 347 million people worldwide have diabetes [1], of which 90–95% is T2DM [2]. Cardiovascular disease is the major cause of mortality in diabetic patients [3]. Although coronary atherosclerosis likely contributes to the onset of heart failure in diabetic patients [4], diabetes-related cardiomyopathy appears to be a major initiating factor [4]. As first reported by Rubler et al. in 1972 [5], diabetic cardiomyopathy is characterized by cardiac dysfunction without underlying coronary artery disease and/or hypertension. A number of mechanisms for the development of diabetic cardiomyopathy have been proposed, including oxidative stress, inflammation and extracellular fibrosis [6], all of which may be related to cardiac lipotoxicity [7], [8]. However, the involvement of intramyocardial lipid accumulation in the pathogenesis of diabetic cardiomyopathy remains incompletely understood.

AMP-activated protein kinase (AMPK), which is an α,β,γ-heterotrimer complex, is an important sensor that regulates lipid metabolism [9]. Liver kinase B1 (LKB1), the major upstream kinase of AMPK, phosphorylates Thr-172 and activates AMPK [10]. Activation of AMPK in the heart stimulates lipid oxidation to produce energy through its downstream targets. First, activated AMPK upregulates autophagy [11]. Reportedly lipid droplets (LDs) can be selectively degraded by the lysosomal pathway of macroautophagy, which is termed lipophagy [12]. In T2DM, elevated myocardial lipids stored as triglycerides (TGs) in LDs were observed [13], [14]. Lipophagy contributes to myocardial TGs catabolizing into fatty acids via the induction of lysosomal acid lipase [15]. Second, AMPK phosphorylates and inactivates acetyl-CoA carboxylase (ACC), resulting in the down-regulation of malonyl-CoA levels. The product of ACC, malonyl-CoA, is an inhibitor of carnitine palmitoyl transferase-1 (CPT-1), which is a key protein involved in mitochondrial uptake of fatty acids. Therefore, AMPK activation will increase mitochondrial fatty acid uptake [16]. Third, AMPK enhances NAD+-dependent type III deacetylase sirtuin 1 (Sirt1) activity [17]. Activation of AMPK and Sirt1 increases the expression of peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) [17], [18]. PGC-1α is a key regulator of fatty acid oxidation, ATP synthesis and lipid homeostasis in mitochondria.

Sulforaphane (SFN), a naturally occurring isothiocyanate compound, is isolated from cruciferous vegetables such as broccoli and cabbage. Several studies indicate that SFN prevents diabetes-induced cardiac [19] and aortic damage [20] and testicular apoptotic cell death [21]. It was found that SFN activates nuclear factor erythroid 2 related factor 2 (Nrf2) to upregulate cellular antioxidants against oxidative stress and damage [19]. Recently, it was reported that SFN can attenuate high fat diet (HFD)-induced visceral adiposity, adipocyte hypertrophy and lipid accumulation in the liver [22]. SFN inhibition of adipogenesis through suppressing lipogenesis is mediated by activating AMPK to inhibit ACC in the adipose tissue of HFD-fed mice. However, the mechanism of AMPK activity by SFN is still unclear.

There are several key pathogenic abnormalities, including hyperglycemia, hyperlipidemia, insulin resistance, and abnormal insulin secretion caused by impaired β-cell function in patients with T2DM [23]. However, there is no genetic animal model of T2DM that includes all above features [24], [25], [26]. Several approaches to make T2DM animal models have been explored [21], [27], [28], one of which is the non-genetic model of T2DM [HFD feeding with a single dose of streptozotocin (STZ) treatment], which mimics the metabolic abnormalities seen in human T2DM [21], [29]. We have also used this model of T2DM to investigate diabetic complications including diabetic cardiomyopathy [27] and male germ cell death [21].

In the present study, therefore, we used our previously reported animal model of T2DM [21] to determine (a) whether HFD feeding induces cardiac lipotoxicity; (b) whether T2DM further worsens cardiac lipotoxicity compared to HFD-induced obesity alone; and (c) whether and how SFN prevents T2DM-induced cardiac lipotoxicity and the development of cardiomyopathy by activation of the AMPK pathway.

Section snippets

Animals

C57BL/6J male mice, 8–10 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, Maine). The mice were housed at the University of Louisville Research Resources Center at 22 °C with a 12 h light/dark cycle with free access to food and tap water.

Generation of diabetic mouse model and treatment with SFN

Experimental procedures, animal treatment and tissue collection information have been described in a recent study [21]. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of

T2DM general features and the effect of SFN on heart weight of DM mice

Mice fed with HFD exhibited all of the features of insulin resistance compared with control mice [21], characterized by an increased area under the curve of the glucose tolerance test, hyperinsulinemia, mild hyperglycemia, hypertriglyceridemia, and hypercholesterolemia (Table 1). DM mice further increased insulin resistance, along with elevations in the levels of fasting blood glucose, triglyceride, and cholesterol. SFN treatment for 4 months did not change these diabetes-induced increases

Discussion

In our present study, we observed that HFD feeding induced cardiac hypertrophy but not cardiac dysfunction. We have successfully established a non-genetic rodent model of T2DM, the HFD + STZ induced diabetic mouse [20], [21]. In this animal model, diabetic mice exhibited significant insulin resistance along with increased levels of fasting blood glucose, insulin, triglyceride, and cholesterol. We demonstrated here that DM mice showed a decrease of cardiac wall thickness, EF and FS along with an

Disclosures

None.

Acknowledgements

This study was supported in part by grants from the American Diabetes Association (1-14-IN-38, to N.M. & L.C.; 7-14-BS-018, to L.C.) and the National Natural Science Foundation of China (No. 81370318, to Y.Z.).

Author contribution

Z.Z., S.W., S.Z., X.Y., Y.W., J.C., J.G., N.M., and Y.T. researched data. Z.Z., J.C., M.K. and Y.T. analyzed the data and reviewed the article. N.M., Y.Z., and L.C. contributed initial discussion of the project and reviewed the article. N.M. and L.C. wrote and edited

References (59)

  • K.M. Choi et al.

    Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice

    J Nutr Biochem

    (2014)
  • S.I. Taylor

    Deconstructing type 2 diabetes

    Cell

    (1999)
  • H. Chen et al.

    Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice

    Cell

    (1996)
  • R.S. Danda et al.

    Kidney involvement in a nongenetic rat model of type 2 diabetes

    Kidney Int

    (2005)
  • G. Zhou et al.

    Metallothionein suppresses angiotensin II-induced nicotinamide adenine dinucleotide phosphate oxidase activation, nitrosative stress, apoptosis, and pathological remodeling in the diabetic heart

    J Am Coll Cardiol

    (2008)
  • T. Inoue et al.

    Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice

    Biochem Biophys Res Commun

    (2013)
  • R.S. Guleria et al.

    Activation of retinoid receptor-mediated signaling ameliorates diabetes-induced cardiac dysfunction in Zucker diabetic rats

    J Mol Cell Cardiol

    (2013)
  • L.J. Rijzewijk et al.

    Altered myocardial substrate metabolism and decreased diastolic function in nonischemic human diabetic cardiomyopathy: studies with cardiac positron emission tomography and magnetic resonance imaging

    J Am Coll Cardiol

    (2009)
  • J. Nagendran et al.

    AMPK signalling and the control of substrate use in the heart

    Mol Cell Endocrinol

    (2013)
  • M. Shibata et al.

    The MAP1–LC3 conjugation system is involved in lipid droplet formation

    Biochem Biophys Res Commun

    (2009)
  • N. Kudo et al.

    Characterization of 5′AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion following ischemia

    Biochim Biophys Acta

    (1996)
  • K. Uchida

    4-Hydroxy-2-nonenal: a product and mediator of oxidative stress

    Prog Lipid Res

    (2003)
  • T.M. Wagner et al.

    Reactive lipid species from cyclooxygenase-2 inactivate tumor suppressor LKB1/STK11: cyclopentenone prostaglandins and 4-hydroxy-2-nonenal covalently modify and inhibit the AMP-kinase kinase that modulates cellular energy homeostasis and protein translation

    J Biol Chem

    (2006)
  • T. Ishii et al.

    Roles of Nrf2 in activation of antioxidant enzyme genes via antioxidant responsive elements

    Methods Enzymol

    (2002)
  • A.D. Association

    Diagnosis and classification of diabetes mellitus

    Diabetes Care

    (2014)
  • A.A. Lteif et al.

    Diabetes and heart disease an evidence-driven guide to risk factors management in diabetes

    Cardiol Rev

    (2003)
  • J.R. Ussher

    The role of cardiac lipotoxicity in the pathogenesis of diabetic cardiomyopathy

    Expert Rev Cardiovasc Ther

    (2014)
  • Z.Y. Fang et al.

    Diabetic cardiomyopathy: evidence, mechanisms, and therapeutic implications

    Endocr Rev

    (2004)
  • E.A. Dunlop et al.

    The kinase triad, AMPK, mTORC1 and ULK1, maintains energy and nutrient homoeostasis

    Biochem Soc Trans

    (2013)
  • Cited by (162)

    • The improvement of sulforaphane in type 2 diabetes mellitus (T2DM) and related complications: A review

      2022, Trends in Food Science and Technology
      Citation Excerpt :

      SFN can also reduce the expressions of fibrogenic mediators (CTGF and TGF-β1) and prevent the fibrosis of the aortic induced by DM(Bai & Cui et al., 2013; Miao & Bai et al., 2012). Inflammation and oxidative damage are two of the main pathogenic factors of myocardial fibrosis, while SFN could significantly reduce the accumulation of 4-HNE and 3-NT in aortic (Miao & Bai et al., 2012; Zhang & Wang et al., 2014), and prevent inflammation (TNF- α and PAI-1) caused by DM(Bai & Cui et al., 2013). The prevalence of hypertension in T2DM patients increased significantly with age.

    View all citing articles on Scopus
    View full text