J Cell Physiol. 2017 Dec;232(12):3209-3217. doi: 10.1002/jcp.25867.

Palmitate induces mitochondrial superoxide generation and activates AMPK in podocytes

Eugene Lee, Jin Choi and Hyun Soon Lee

Renal Pathology Lab, Hankook Kidney and Diabetes Institute, Seoul, Korea

 

Abstract 

Studies have shown that high levels of serum free fatty acids (FFAs) are associated with lipotoxicity and type 2 diabetes. Palmitic acid (PA) is the predominant circulating saturated FFA, yet its role in the pathogenesis of diabetic nephropathy (DN) is not clear. Recently, one study suggested that mitochondrial superoxide production is related to AMP-activated protein kinase (AMPK) activity in diabetic mice kidneys. To elucidate the link between PA and oxidative stress and AMPK activity in DN, we compared the cultured murine podocytes exposed to PA and oleic acid (OA). Incubation of cells with 250 µM PA or OA induced a translocation of CD36, a fatty acid transport protein, with intracellular lipid accumulation. PA, but not OA, induced mitochondrial superoxide and hydrogen peroxide (H2O2) generation in podocytes, as shown by enhanced fluorescence of MitoSOX Red and dichlorofluorescein (DCF), respectively. Costimulation of PA-treated cells with the H2O2 scavenger catalase abolished the PA-induced DCF fluorescence. Only PA induced mitochondrial damage as shown by electron microscopy. The AMPK activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK (p-AMPK) to total AMPK. Only PA significantly increased the p-AMPK levels compared with controls. Addition of catalase to PA-treated cells did not affect the PA-stimulated p-AMPK levels. Collectively, our results indicate that PA induces mitochondrial superoxide and H2O2 generation in cultured podocytes, which may not be directly linked to AMPK activation. Given that, PA seems to play an important role in the pathogenesis of DN through lipotoxicity initiated by mitochondrial superoxide overproduction.

 

Supplement 

Excess carbohydrate, which cannot be converted to glycogen, is converted to triglyceride (TG) and stored in adipose tissue. Serum free fatty acids (FFAs) are liberated by lipolysis of TG. In obesity and type 2 diabetes, chronically high levels of serum TG and FFAs contribute to intracellular fatty acid accumulation (Lee, 2011). Excess lipid deposition in non-adipose tissues leads to lipotoxicity, which may contribute to the pathogenesis of type 2 diabetes. Palmitic acid (PA) (C16:0) is the predominant circulating saturated FFA, while oleic acid (OA) (C18:1) is a nontoxic mono-unsaturated fatty acid.

A master regulator of fatty acid ß-oxidation is AMP-activated protein kinase (AMPK). In chronically diabetic mice kidneys, mitochondrial superoxide production is reportedly related to AMPK activity (Dugan et al., 2013).

Podocyte damage seems to play a pivotal role in the pathogenesis of diabetic nephropathy (DN) (Lee, 2013). To elucidate the link between PA and oxidative stress and AMPK activity in DN, we compared the cultured murine podocytes exposed to PA and OA.

PA induces mitochondrial superoxide generation in podocytes

Using MitoSOX, no fluorescence was detected in control podocytes. When podocytes were incubated with 250 µM PA for 5 h, there was an intense MitoSOX fluorescence. The fluorescence colocalized with the mitochondria as detected using mitoTracker. In contrast, no MitoSOX fluorescence was shown in cells incubated with OA (Fig. 1).

PA induces intracellular H2O2 production in podocytes

Intracellular DCF fluorescence was markedly increased in podocytes exposed to 250 µM PA, whereas cells incubated with OA showed only minimal fluorescence. Coincubation of PA-treated cells with catalase or OA almost totally suppressed the PA-induced DCF signal. In cells exposed to high glucose, DCF fluorescence was increased (Fig. 2).

PA causes AMPKa phosphorylation in podocytes

Sixteen hours following PA exposure, the levels of p-AMPKa (pThr172) were significantly increased as compared to controls in podocytes, whereas OA did not induce the changes in pThr172 levels. When catalase was added to PA, no significant difference was shown in pThr172 levels as compared with those induced by PA alone (Fig. 3).

In summary, our study shows that PA, but not OA, induces mitochondrial superoxide and subsequently H2O2 generation in podocytes. In addition, only PA induces AMPK activation in podocytes, which is not directly linked to mitochondrial reactive oxygen species (ROS) generation.

Superoxide is membrane impermeable, whereas H2O2 easily penetrates the mitochondrial membranes increasing cytoplasmic H2O2 levels. DCFH is an H2O2-sensitive fluorescence probe. Thus, an enhanced cellular DCF signal by PA in the present study is consistent with increased H2O2 dismutated from the mitochondrial superoxide.

The cause of the AMPK activation by PA is not clear in this study. We conjecture that PA, as a metabolic poison, can lead to AMPK activation in podocytes.

Our demonstration of PA-induced mitochondrial superoxide generation in podocytes is important to corroborate the “unifying hypothesis”, suggesting that excess generation of mitochondrial superoxide plays a central role in the initiation and progression of DN. Furthermore, our study suggests that intracellular accumulation of PA in podocytes of diabetic patients could induce mitochondrial ROS overproduction to activate latent TGF-ß. Activated TGF-ß by ROS in podocytes may slowly lead to thickening of the glomerular basement membrane and mesangial matrix accumulation, culminating in the florid phase of DN (Lee, 2012, 2013).

 

 

Figure 1. Palmitate (PA)-induced mitochondrial O2 generation in podocytes. In control cells, no MitoSOX fluorescence is seen; using the probe mitoTracker, mitochondria are observed mainly in perinuclear locations. In cells incubated with 250 µM PA for 5 h, intense MitoSOX fluorescence is present; the fluorescence colocalized with the mitochondria as detected using the probe mitoTracker; merged image showing multiple yellow aggregates (arrows). Incubation of cells with oleate induced no MitoSOX fluorescence (Data adapted from Lee et al. (2017). J Cell Physiol. 232(12):3209-3217).

 

 

Figure 2. A) In podocytes incubated with 250 µM OA for 5 h and exposed to carboxy-DCFH DA-AM for 2.5 h, no DCF fluorescence is shown. By contrast, cells incubated with 250 µM PA show markedly increased DCF fluorescence. Coincubation of PA-treated cells with catalase abolished the PA-induced DCF signal. B) Dual bright field and fluorescence microscopic view of podocytes exposed to the same experimental condition. C) In cells coincubated with OA and PA for 5 h, intracellular DCF fluorescence is markedly decreased as compared to PA alone. Incubation of cells with high glucose (HG) shows increased DCF fluorescence (Data adapted from Lee et al. (2017). J Cell Physiol. 232(12):3209-3217).

 

 

Figure 3. Immunoblots showing phosphorylation (pThr172) and expression of AMPKa in podocytes in response to 250 µM PA, OA, or PA + catalase. Sixteen hours following PA exposure, levels of pThr172 are significantly increased (A), whereas exposure to OA shows no significant changes as compared to controls (B). When catalase was added to PA, no significant difference is shown as compared with those induced by PA alone (C). (D) Quantitative analysis of pThr172 (A-C) is shown. Results are mean ± SD. (*P < 0.05 vs. control, #P < 0.05 vs. OA) (Data adapted from Lee et al. (2017). J Cell Physiol. 232(12):3209-3217).

 

References

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Lee HS. 2011. Mechanisms and consequences of hypertriglyceridemia and cellular lipid accumulation in chronic kidney disease and metabolic syndrome. Histol Histopathol 26:1599-1610.

Lee HS. 2012. Mechanisms and consequences of TGF-ß overexpression by podocytes in progressive podocyte disease. Cell Tissue Res 347:129-140.

Lee HS. 2013. Pathogenic role of TGF-ß in diabetic nephropathy. J Diabetes Metab doi:10.4172/2155-6156.S9-008.

Lee E, Choi J, Lee HS. 2017. Palmitate induces mitochondrial superoxide generation and activates AMPK in podocytes. J Cell Physiol 232:3209-3217.