Autophagic flux defect in diabetic kidney disease results in megamitochondria formation in podocytes.

Podocyte injury is an important factor in the pathogenesis of diabetic nephropathy. Podocytes are characterized by large numbers of mitochondria. However, mitochondrial dysfunction as it relates to kidney pathology remains poorly understood. The present study found that podocyte mitochondria in different animal models of diabetes mellitus became elongated with the development of albuminuria, suggesting a change in mitochondrial dynamics. We then treated cells with a combination of glucose, fatty acids, and angiotensin II (GFA) to mimic the diabetic milieu. Cultured podocytes exposed to GFA showed megamitochondria formation and decreased autophagosome degradation. We also found that GFA treatment decreased the binding of the autophagosome to the lysosome. Our results suggest that megamitochondria are common in podocytes during diabetic nephropathy and that insufficient autophagy flux may underlie this effect. These findings have expanded our understanding of the pathogenesis of diabetic nephropathy and identified a potential pharmacological target for treatment.

Inhibition of Ceramide Accumulation in Podocytes by Myriocin Prevents Diabetic Nephropathy.

BACKGROUND: Ceramides are associated with metabolic complications including diabetic nephropathy in patients with diabetes. Recent studies have reported that podocytes play a pivotal role in the progression of diabetic nephropathy. Also, mitochondrial dysfunction is known to be an early event in podocyte injury. Thus, we tested the hypothesis that ceramide accumulation in podocytes induces mitochondrial damage through reactive oxygen species (ROS) production in patients with diabetic nephropathy. METHODS: We used Otsuka Long Evans Tokushima Fatty (OLETF) rats and high-fat diet (HFD)-fed mice. We fed the animals either a control- or a myriocin-containing diet to evaluate the effects of the ceramide. Also, we assessed the effects of ceramide on intracellular ROS generation and on podocyte autophagy in cultured podocytes. RESULTS: OLETF rats and HFD-fed mice showed albuminuria, histologic features of diabetic nephropathy, and podocyte injury, whereas myriocin treatment effectively treated these abnormalities. Cultured podocytes exposed to agents predicted to be risk factors (high glucose, high free fatty acid, and angiotensin II in combination [GFA]) showed an increase in ceramide accumulation and ROS generation in podocyte mitochondria. Pretreatment with myriocin reversed GFA-induced mitochondrial ROS generation and prevented cell death. Myriocin-pretreated cells were protected from GFA-induced disruption of mitochondrial integrity. CONCLUSION: We showed that mitochondrial ceramide accumulation may result in podocyte damage through ROS production. Therefore, this signaling pathway could become a pharmacological target to abate the development of diabetic kidney disease.

Protective role of endogenous plasmalogens against hepatic steatosis and steatohepatitis in mice.

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat; the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of FC in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing sterol regulatory element-binding protein 2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat small interfering RNA treatment significantly decreased peroxisome proliferator-activated receptor α (Pparα) expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor, alkyl glycerol (AG), prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat+/- mice were more susceptible to hepatic lipid accumulation and less responsive to the preventive effect of fluvastatin on NASH development, suggesting that endogenous plasmalogens prevent hepatic steatosis and NASH. CONCLUSION: Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis through GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH. (Hepatology 2017;66:416-431).

Lipoic acid nanoparticles: effect of polymeric stabilizer on appetite suppression.

Alpha-lipoic acid (ALA), which is common in the human body, is efficacious in appetite suppression. However, its typical formulations of salt or micronized crystals cannot satisfy the desired bioavailability requirements for appetite suppression due to low absorption and a short plasma half-life. Herein, we describe a new ALA nanoparticulate formulation produced by nano-comminution using polymeric stabilizers, such as hydroxypropyl cellulose, Pluronic F127, and polyvinylpyrrolidone. Nanoparticles of similar sizes did not show any remarkable differences in the in vitro release profiles. However, the in vivo results from food intake studies in mice demonstrated that the hydroxypropyl cellulose case had the largest improved efficacy among the three polymeric stabilizer cases. Compared to the nanosuspension formulations, the powder formulations of nanoparticles had improved efficacy in reducing food intake for six hours, possibly because of the delayed release kinetics. Therefore, the ALA powder formulation of nanoparticles is a candidate to replace the current formulations to achieve proper appetite suppression.

Improved efficacy of appetite suppression by lipoic acid particles prepared by nanocomminution.

PURPOSE: This article was intended to improve the efficacy of alpha-lipoic acid (ALA) for appetite suppression by controlling the particle size and self-polymerization of ALA. METHODS: ALA was fabricated into micro- and nanoparticles, and the efficacy and in vitro release were investigated. Because of the self-polymerization of ALA into poly[3-(n-butane carboxylic acid)propyl]disulfide (PBCPD) by processing heat, low-speed rotation comminution was used to control PBCPD content. RESULTS: The ALA particle size initially decreased and then increased after 10 hours of nanocomminution, indicating aggregation related to PBCPD formation. The in vitro release of ALA was significantly reduced by the existence of PBCPD. Interestingly, the reduction was not followed by a decrease in efficacy. Alternatively, the food intake was significantly reduced by ALA particles containing more than 30 mol% PBCPD. CONCLUSIONS: When the particle size and self-polymerization of ALA were carefully controlled, the efficacy on appetite suppression could be superior to water-soluble ALA salt. The ALA particles might have a composite nanostructure of ALA and PBCPD.

Alpha-lipoic acid decreases hepatic lipogenesis through adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent pathways.

UNLABELLED: Fatty liver is common in obese subjects with insulin resistance. Hepatic expression of sterol regulatory element binding protein-1c (SREBP-1c), which plays a major role in hepatic steatosis, is regulated by multiple factors, including insulin, adenosine monophosphate-activated protein kinase (AMPK), liver X receptors (LXRs), and specificity protein 1. Alpha-lipoic acid (ALA), a naturally occurring antioxidant, has been shown to decrease lipid accumulation in skeletal muscle by activating AMPK. Here we show that ALA decreases hepatic steatosis and SREBP-1c expression in rats on a high fat diet or given an LXR agonist. ALA increased AMPK phosphorylation in the liver and in cultured liver cells, and dominant-negative AMPK partially prevented ALA-induced suppression of insulin-stimulated SREBP-1c expression. ALA also inhibited DNA-binding activity and transcriptional activity of both specificity protein 1 and LXR. CONCLUSION: These results show that ALA prevents fatty liver disease through multiple mechanisms, and suggest that ALA can be used to prevent the development and progression of nonalcoholic fatty liver disease in patients with insulin resistance.

AMPK activation increases fatty acid oxidation in skeletal muscle by activating PPARalpha and PGC-1.

AMP-activated protein kinase (AMPK) activation increases fatty acid oxidation in skeletal muscle by decreasing malonyl CoA concentrations. However, this may not explain the long-term effects of AMPK activation. Here we show that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) increases mRNA expression of PPARalpha target genes and PGC-1 in cultured muscle cells and mouse skeletal muscle, and that inhibition of PPARalpha and PGC-1 by siRNAs prevents AICAR-stimulated increase in fatty acid oxidation. These data suggest that a novel transcriptional regulatory mechanism involving PPARalpha and PGC-1 exists that is responsible for long-term stimulation of fatty acid oxidation in skeletal muscle by AICAR.

Alpha-lipoic acid prevents endothelial dysfunction in obese rats via activation of AMP-activated protein kinase.

OBJECTIVE: Lipid accumulation in vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis in obese subjects. We showed previously that alpha-lipoic acid (ALA) activates AMP-activated protein kinase (AMPK) and reduces lipid accumulation in skeletal muscle of obese rats. Here, we investigated whether ALA improves endothelial dysfunction in obese rats by activating AMPK in endothelial cells. METHODS AND RESULTS: Endothelium-dependent vascular relaxation was impaired, and the number of apoptotic endothelial cells was higher in the aorta of obese rats compared with control rats. In addition, triglyceride and lipid peroxide levels were higher, and NO synthesis was lower. Administration of ALA improved all of these abnormalities. AMPK activity was lower in aortic endothelium of obese rats, and ALA normalized it. Incubation of human aortic endothelial cells with ALA activated AMPK and protected cells from linoleic acid-induced apoptosis. Dominant-negative AMPK inhibited the antiapoptotic effects of ALA. CONCLUSIONS: Reduced AMPK activation may play an important role in the genesis of endothelial dysfunction in obese rats. ALA improves vascular dysfunction by normalizing lipid metabolism and activating AMPK in endothelial cells.

Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle.

Triglyceride accumulation in skeletal muscle contributes to insulin resistance in obesity. We recently showed that alpha-lipoic acid (ALA) reduces body weight and prevents the development of diabetes in diabetes-prone obese rats by reducing triglyceride accumulation in non-adipose tissues. AMP-activated protein kinase (AMPK) is a major regulator of cellular energy metabolism. We examined whether ALA lowers triglyceride accumulation in skeletal muscle by activating AMPK. Alpha2-AMPK activity was decreased in obese rats compared to control rats. Administration of ALA to obese rats increased insulin-stimulated glucose disposal in whole body and in skeletal muscle. ALA also increased fatty acid oxidation and activated AMPK in skeletal muscle. Adenovirus-mediated administration of dominant negative AMPK into skeletal muscle prevented the ALA-induced increases in fatty acid oxidation and insulin-stimulated glucose uptake. These results suggest that ALA-induced improvement of insulin sensitivity is mediated by activation of AMPK and reduced triglyceride accumulation in skeletal muscle.

Effects of recombinant adenovirus-mediated uncoupling protein 2 overexpression on endothelial function and apoptosis.

Increased oxidative stress in vascular cells plays a key role in the development of endothelial dysfunction and atherosclerosis. Uncoupling protein 2 (UCP2) is an important regulator of intracellular reactive oxygen species (ROS) production. This study was undertaken to test the hypothesis that, UCP2 functions as an inhibitor of the atherosclerotic process in endothelial cells. Adenovirus-mediated UCP2 (Ad-UCP2) overexpression led to a significant increase in endothelial nitric oxide synthase (eNOS) and decrease in endothelin-1 mRNA expression in human aortic endothelial cells (HAECs). Moreover, UCP2 inhibited the increase in ROS production and NF-kappaB activation, and apoptosis of HAECs induced by lysophophatidylcholine (LPC) and linoleic acid. LPC and linoleic acid caused mitochondrial calcium accumulation and transient mitochondrial membrane hyperpolarization, which was followed by depolarization. UCP2 overexpression prevented these processes. In isolated rat aorta, Ad-UCP2 infection markedly improved impaired vascular relaxation induced by LPC. The data collectively suggest that UCP2, functions as a physiologic regulator of ROS generation in endothelial cells. Thus, measures to increase UCP2 expression in vascular endothelial cells may aid in preventing the development and progression of atherosclerosis in patients with metabolic syndrome.