Human Apo A-I Gene Transfer Influences Metabolic Parameters in Streptozotocin-Induced Diabetes Mellitus

Apo A-I transfer resulted in a 60 % increase in HDL cholesterol, which was paralleled with a significant decline in very-low-density lipoprotein (VLDL) cholesterol, intermediate-density lipoprotein (IDL) cholesterol, and triglycerides, whereas LDL cholesterol was unaffected (Van Linthout et al. 2008). Despite the well-described antidiabetic effects of HDL involving their ability to reduce pancreatic β-cell apoptosis (Abderrahmani et al. 2007; Fryirs et al. 2010; Rutti et al. 2009), apo A-I transfer did not reduce blood glucose levels (Van Linthout et al. 2008), potentially due to the severity of the streptozotocin model, which is associated with a remaining insulin production below 1 % (Hughes et al. 2001). The decline in triglycerides and in the triglyceride-rich lipoproteins VLDL and IDL (Sztalryd and Kraemer 1995) after apo A-I transfer suggests an HDL-mediated decrease in lipolysis in adipose tissue, leading to less free fatty acids in the circulation, less triglyceride synthesis in the liver, and subsequent less VLDL and IDL synthesis (Tunaru et al. 2003). This hypothesis is corroborated by the ability of HDL to reduce the expression of hormone-sensitive lipase (Van Linthout et al. 2010a), the rate-limiting enzyme of adipocyte lipolysis in abdominal fat (Sztalryd and Kraemer 1995), and to increase the phosphorylation of the PI3K downstream target Akt in abdominal fat (Van Linthout et al. 2010a). Akt is involved in the anti-lipolytic and lipogenic effects of insulin in adipose tissue (Whiteman et al. 2002) and in the regulation of the expression of the adipokine adiponectin (Cong et al. 2007; Pereira and Draznin 2005), which is known to improve insulin sensitivity under diabetes (Peterson et al. 2008). However, an involvement of HDL in the hepatic expression of genes involved in triglyceride metabolism may not be excluded. The decrease in cardiac glycogen content (Van Linthout et al. 2008) suggests that apo A-I transfer in streptozotocin rats partly restored glucose metabolism as an energy source in the heart instead of nearly exclusive reliance on fatty acid metabolism for production of ATP (Kota et al. 2011).

Human Apo A-I Gene Transfer Attenuates DiabetesAssociated Oxidative Stress, Cardiac Fibrosis,

and Endothelial Dysfunction

Hyperglycaemia induces oxidative stress via creating a disbalance between the generation of reactive oxygen species and their resolution by antioxidant enzymes, like superoxide dismutases, which convert O2anions into molecular oxygen and hydrogen peroxide (Nishikawa et al. 2000). Reactive oxygen species initiate diverse stress-signalling pathways including ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK, alter cellular proteins, and induce lipid peroxidation. In diabetic cardiomyocytes, reactive oxygen species are predominantly generated by mitochondria, due to mitochondrial oxidation of fatty acids (Kota et al. 2011), and by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Li et al. 2010b). The pathological importance of p38 MAPK in the diabetic heart follows from the observation that p38 MAPK inhibition reduces cardiac inflammation and improves left ventricular dysfunction in streptozotocin-induced diabetic mice (Westermann et al. 2006). Besides decreasing the activity of p38 MAPK, apo A-I transfer reduced cardiac oxidative stress via upregulating the expression of the diabetes-downregulated mitochondrial superoxide dismutase-2 and extracellularsuperoxide dismutase (Van Linthout et al. 2008). Both forms of superoxide dismutase are important for the heart as outlined in studies whereby cardiac overexpression of superoxide dismutase-2 protected the mitochondrial respiratory function and blocked apoptosis induction (Shen et al. 2006; Suzuki et al. 2002) and overexpression of extracellular-superoxide dismutase decreased macrophage infiltration and fibrosis and improved left ventricular dysfunction (Dewald et al. 2003). These studies postulate that the decrease in cardiac fibrosis after apo A-I transfer in streptozotocin-diabetic rats can be partly explained by the reduction in oxidative stress and inflammation, including downregulated expression of pro-fibrotic cytokines (inflammatory fibrosis) (Tschope et al. 2005), as well as by the decrease in cardiac apoptosis (cfr. supra) and subsequent replacement fibrosis, resulting in improved left ventricular function. The anti-inflammatory and antifibrotic effects of HDL are supported by findings in apo A-I knockout mice, which are associated with increased inflammatory cells and collagen deposition in the lung (Wang et al. 2010), and from observations with the mimetic apo A-I peptide 4F demonstrating an L-4F-mediated decreased endothelial cell matrix production (Ou et al. 2005). Since cardiac NADPH oxidases play a predominant role in the development of diabetic cardiomyopathy (Guo et al. 2007; Wold et al. 2006) and in diabetic cardiac remodelling (Li et al. 2010b), decreased cardiac NADPH oxidase activity following apo A-I transfer (Van Linthout et al. 2009) may be a critical mediator of reduced cardiac oxidative stress and subsequent fibrosis in the myocardium.

Systemically, apo A-I transfer decreased the oxidative stress in rats with streptozotocin-induced diabetes as indicated by the decline in serum levels of thiobarbituric acid reactive substances (TBARS) (Van Linthout et al. 2008), a marker of lipid peroxidation (Tschope et al. 2005). This finding is corroborated by Mackness et al. (1991), who reported that HDLs decrease the formation of TBARS on oxidised LDL. This is likely mediated by an increase in the activity of paraoxonase or platelet-activating factor-acetyl hydrolase, 2 enzymes with antioxidative features, which are associated with HDL and which are known to be induced after apo A-I gene transfer (De Geest et al. 2000).

In the vasculature of diabetic rats, apo A-I transfer decreased oxidative stress as indicated by the reduction in diabetes-enhanced NADPH oxidase activity and eNOS uncoupling (Van Linthout et al. 2009), a phenomenon occurring when eNOS produces O2rather than NO. Uncoupling of eNOS is, besides NADPH oxidases (Cai 2005), an important source of reactive oxygen species in diseased, including diabetic, blood vessels (Hink et al. 2001). Consistent with the demonstrated role of the angiotensin II receptor, type 1 (AT1R) in mediating increased NADPD oxidase activity, and eNOS uncoupling in diabetes (Oak and Cai 2007), the downregulation in diabetes-induced AT1R (Hodroj et al. 2007; Nyby et al. 2007) after apo A-I transfer (Van Linthout et al. 2009) was postulated to be the predominant mediator of reduced NADPH oxidase activity and eNOS uncoupling. This hypothesis is further supported by in vitro findings showing that the HDL-mediated reduction in AT1R expression in human aortic endothelial cells was associated with a decline in hyperglycaemia-induced oxidative stress and a reduced responsiveness to angiotensin II (Van Linthout et al. 2009). These observations underline the finding of Tolle et al. (2008), who showed that HDL reduce NADPH oxidase-dependent reactive oxygen species generation via inhibition of the activation of Rac1, which is a downstream AT1R-dependent mediator of angiotensin II (Ohtsu et al. 2006). The exact mechanism by which HDL affect AT1R regulation under diabetes mellitus requires further fundamental studies. However, since oxidised LDL (Li et al. 2000) and reactive oxygen species (Gragasin et al. 2003) play a role in the induction of the AT1R in human aortic endothelial cells, one may speculate that HDL via intrinsic anti-oxidative features (cfr. supra, via paraoxonase and platelet-activating factor-acetyl hydrolase) may contribute to the downregulation of the AT1R under diabetes mellitus, which results in less NADPH oxidase activity and reactive oxygen formation and in turn may decrease AT1R expression. Concomitant with the reduced vascular oxidative stress, including decreased eNOS uncoupling, apo A-I transfer in streptozotocin rats resulted in an enhanced NO bioavailability (Chalupsky and Cai 2005) and consequently in a decrease in endothelial dysfunction (Van Linthout et al. 2009), which is another hallmark of diabetic cardiomyopathy.

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