Alpha-lipoic acid alleviates NAFLD and triglyceride accumulation in liver via modulating hepatic NLRP3 inflammasome activation pathway in type 2 diabetic rats

Chih-Yuan Ko, Yangming Martin Lo, Jian-Hua Xu, Wen-Chang Chang, Da-Wei Huang, James Swi-Bea Wu, Cho-Hua Yang, Wen-Chung Huang, Szu-Chuan Shen
March 2021

Abstract

The occurrence of nonalcoholic fatty liver disease (NAFLD) is associated with type 2 diabetes mellitus (T2DM). The activation of nucleotide-binding domain and leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in the liver may lead to hepatic fat accumulation. Alpha-lipoic acid (ALA) has been reported to improve IR in a T2DM rodent model. We investigated the effects of ALA on NLRP3 inflammasome activation and fat accumulation in the liver of a high-fat diet (HFD) plus streptozotocin (STZ)-induced T2DM rats. The HFD/STZ-induced T2DM rats were orally administered ALA (50, 100, or 200 mg/kg BW) once a day for 13 weeks. The results showed that the liver triglyceride contents of T2DM rats were 11.35 ± 1.84%, whereas the administration of 50, 100, and 200 mg/kg BW ALA significantly reduced the liver triglyceride contents of T2DM rats to 4.14 ± 0.59%, 4.02 ± 0.41%, and 3.01 ± 1.07%, respectively. Moreover, 200 mg/kg BW ALA significantly decreased the hepatic levels of NLRP3 inflammasome activation-related proteins NLRP3, caspase-1, and interleukin-1β expression by 40.0%, 60.1%, and 24.5%, respectively, in T2DM rats. Furthermore, the expression levels of hepatic fat synthesis-related proteins decreased, namely a 45.4% decrease in sterol regulatory element-binding protein-1c, whereas the expression of hepatic lipid oxidation-related proteins increased, including a 27.5% increase in carnitine palmitoyltransferase, in T2DM rats after 200 mg/kg BW ALA treatment. We concluded that ALA treatment may suppress hepatic NLRP3 inflammasome activation, consequently alleviating NAFLD and excess hepatic lipid accumulation in HFD/STZ-induced T2DM rats.

INTRODUCTION

Diabetes mellitus (DM) is a worldwide, chronic, noncommunicable disease with a complex etiology. To date, the popularity of Westernized diet, reduced physical activity, and aging populations appears to lead to the increasing prevalence of DM. Up to 2017, the number of people with DM has increased to 422 million and is expected to grow to 642 million over the next 25 years (Ogurtsova et al., 2017). Insulin resistance (IR) is the primary trait of type 2 DM (T2DM), which is known to result in the inability of peripheral cells to effectively uptake glucose, hence leading to hyperglycemia (Samuel & Shulman, 2012).

A chronic liver disease characterized as universal liver fat deposition without alcohol intake (Chalasani et al., 2012), nonalcoholic fatty liver disease (NAFLD) is one of the most common comorbidities of T2DM. Epidemiological research has reported that the prevalence of NAFLD in Western countries ranges from 20% to 30% versus 5%–18% in Asia (Benedict & Zhang, 2017). Clinically, 70%–90% of NAFLD patients are also diagnosed with T2DM or IR (Kotronen & Yki-Järvinen, 2008). The progression of NAFLD has been associated with hepatic inflammation due to hyperglycemia. Lipid accumulation in the liver leads to hepatic fatty lesions, promotes the expression of inflammatory factors and IR, and eventually results in irreversible damage to the liver tissues (Shoelson et al., 2006).

Hepatic Kupffer cells are specialized macrophages that eliminate viruses and bacteria and rapidly trigger liver inflammation (Olteanu et al., 2014). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) is the most fully characterized inflammasome among others such as the adaptor protein apoptosis-associated speck-like protein (ASC) and the proinflammatory caspase, caspase-1. The activation of the NLRP3 inflammasome stimulates Kupffer cells to secrete inflammatory cytokines, including interleukin (IL)-1β, in the liver (Odegaard & Chawla, 2008). In addition, the activation of NLRP3 has also been associated with chronic diseases such as T2DM and NAFLD (Wang et al., 2018; Zhu et al., 2018).

Alpha-lipoic acid (ALA), a vitamin-like compound, is a thioctic acid and a sulfur-containing organic compound that is enriched in the livers, kidneys, and hearts of animals. It can also be found in whole grains, spinach, cauliflower, and yeast (Singh & Jialal, 2008). ALA has been demonstrated to improve hyperglycemia, metabolic disorders, and liver inflammation (Abdelhalim et al., 2018; Castro et al., 2019). However, investigations of the effects of ALA on hepatic NLRP3 inflammasome activation and NAFLD have been limited. The present study aimed to elucidate the effects of ALA on hepatic NLRP3 inflammasome activation, fat accumulation, and NAFLD using a high-fat diet (HFD) plus streptozotocin (STZ)-induced T2DM model rats.

DISCUSSION

Excessive calories or fat intake can cause lipid cells apoptosis, promote the activation of the inflammatory c-Jun N-terminal kinase 1 pathway, increase the secretion of cytokines, such as tumor necrosis factor (TNF)-α or IL-1β, and block insulin signaling in liver tissue, consequently resulting in compensatory insulin production by pancreatic β-cells and the subsequent development of hyperinsulinemia (He et al., 2011). NAFLD is defined as the presence of macrovesicular steatosis that TG level exceeding 5% in liver tissue of nondrinking individuals (Huang et al., 2020; Loomba & Sanya, 2013). The results of the present study indicate that NAFLD was induced in the HFD/STZ-treated rats. Patients with metabolic syndrome or T2DM are commonly accompanied by the high prevalence of NAFLD (Kotronen & Yki-Järvinen, 2008), while the occurrence of hepatic IR or lipid accumulation has been associated with the progression of T2DM and NAFLD (Chalasani et al., 2012). The results of this study also suggested that ALA treatments were able to improve hyperinsulinemia, reduce hepatic TG contents, and alleviate NAFLD in an HFD plus STZ-induced diabetic rat model.

The NLRP3 inflammasome in hepatic Kupffer cells, a specific macrophage found in liver tissue, has been reported to be associated with the progression of NAFLD (Camellet al., 2015). The activation of the NLRP3 inflammasome stimulates the production of the proinflammatory cytokine IL-1β and causes the occurrence of IR and inflammation (Maedler et al., 2009; Schroder et al., 2010). NLRP3 inflammasome activation requires two stimulation steps: (a) Damage-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) molecules trigger NLRP3 protein expression, and the NLRP3 protein combines with ASC and pro-caspase-1 to form the NLRP3 inflammasome; and (b) the NLRP3 inflammasome is further activated by free radicals, large crystals, or DAMPs/PAMPs, to promote the formation of caspase-1, which then converts pro-IL-1β into IL-1β, subsequently resulting in the inflammatory response (Shao et al., 2015).

To date, the literature regarding the inhibitory effects of ALA on NLRP3 has been limited. In this study, treatments with ALA significantly were found to decrease the protein expression levels of NLRP3, caspase-1, and IL-1β in the livers of T2DM model rats. In addition, increased NF-κB protein expression may promote the activation of the NLRP3 inflammasome in the liver. The expression level of the upstream transcription factor NF-κB was also significantly decreased by ALA treatment in the livers of T2DM model rats. ALA treatments did not affect hepatic ASC protein expression in T2DM model rats. The combination of NLRP3 protein and ASC, a downstream binding protein of NLRP3, may cause oligomerization, attracting downstream pro-caspase-1 to form the NLRP3 inflammasome, further promoting inflammatory reactions. However, when NLRP3 protein expression decreased, NLRP3 protein levels were insufficient for oligomerization with free ASC, resulting in decreased downstream caspase-1 expression and IL-1β production. The results of the present study indicated that ALA may inhibit the expression of NF-κB, suppressing the activation of the NLRP3 inflammasome, and subsequently reducing cytokine production and inflammatory responses in the livers of T2DM model rats.

Hepatic IR promotes TG accumulation in the liver (Brown & Goldstein, 2008). The present study suggested that ALA alleviated hepatic IR and liver-free fatty acid contents by inhibiting the secondary activation signal of the NLRP3 inflammasome and reducing cytokine production in the livers of T2DM model rats. The PI3K/Akt pathway is a critical pathway for insulin signal transduction. Excessive HFD consumption may result in lipid accumulation in liver cells and decreased hepatic PI3K/Akt protein expression, contributing to NAFLD formation in rats (Han et al., 2010). The cytokine IL-1β not only destroys pancreatic β-cells, but it also inhibits PI3K/Akt expression in the insulin signaling pathway (Stienstra et al., 2010). The administration of ALA increased the expression levels of hepatic PI3K/Akt, thus reducing IR in the livers of T2DM model rats.

Accumulation of lipids in the liver may accelerate the occurrence of NAFLD. Hepatic TG contents have been associated with lipid synthesis and oxidation in the liver. SREBP-1c is an important transcription factor associated with fat synthesis that promotes lipid synthesis by converting acetyl-CoA to malonyl-CoA in the nucleus (Chao et al., 2019). The overexpression of SREBP-1c in the liver accelerates the occurrence of NAFLD in T2DM. In this study, ALA treatments blocked the PI3K/Akt pathway, inhibited SREBP-1c expression, and, subsequently, reduced adipogenesis in the livers of T2DM model rats. CPT-1 is the primary regulatory enzyme for fatty acid β-oxidation. ALA treatments also promoted hepatic CPT-1 protein expression levels, indicating accelerated lipid metabolism in the livers of T2DM model rats. In addition, the generation of free radicals is important for secondary signal activation during the progression of NAFLD. Free radicals are primarily produced by mitochondria, such that mitochondrial dysfunction promotes increased free radical formation. AST has been reported to be released when mitochondria are damaged in the liver (Botros & Sikaris, 2013). Clinically, the AST/ALT ratio has been used to assess liver inflammation. An AST/ALT ratio above 1 indicates chronic inflammation in the liver (Botros & Sikaris, 2013). In the current study, T2DM model rats exhibited the highest AST/ALT ratio, whereas ALA treatment reduced the AST/ALT ratio, indicating that ALA may reduce the secondary signaling associated with hepatic NLRP3 inflammasome activation in the livers of T2DM model rats. The above observations suggested that ALA may reduce inflammatory cytokine secretion by inhibiting hepatic NLRP3 inflammasome activation, suppressing IR and lipid accumulation in the liver, and, subsequently, alleviating the occurrence of NAFLD in T2DM.

CONCLUSIONS

This study demonstrated that ALA treatment for 13 weeks was able to inhibit the activation of the hepatic NLRP3 inflammasome in T2DM rats and could be attributed to (a) decreased expression of transcription factor NF-κB, which reduced the expression levels of NLRP3 and caspase-1; and (b) decreased ALT/AST ratio, which reduced the production of the inflammatory cytokine IL-1β in the liver. Moreover, ALA treatment increased PI3K/Akt protein expression levels, suppressed expression of lipid synthesis transcription factor SREBP-1c, and increased the protein expression of lipid oxidative enzyme CPT-1, subsequently alleviating TG accumulation in the livers of T2DM rats (Figure 6). The results of this study also suggested that ALA might be used as a health supplement for alleviation of NAFLD progression associated with T2DM.