Individuals diagnosed with T2DM are susceptible to hepatocyte injury and fibrosis, even when presenting with isolated steatosis in the initial stages. This progression underscores the significance of managing T2DM-induced hepatic steatosis to forestall the development of fibrosis and the onset of MAFLD. The presence of liver fibrosis, often a precursor to cirrhosis, is notably more prevalent among individuals with diabetes, highlighting the urgent need for effective therapeutic interventions (48, 49). However, the current clinical landscape is marked by a dearth of FDA-approved drugs specifically tailored for treating MAFLD (32). Addressing this gap, the present study delves into the protective potential of EA against T2DM-induced hepatic steatosis and ensuing fibrosis. Utilizing a T2DM rat model, this investigation scrutinizes the comparative efficacy of EA, metformin, and a combination thereof in mitigating the progression of hepatic complications. Metformin, a cornerstone medication in T2DM management, operates through the activation of the AMPK pathway, thereby enhancing liver function and insulin sensitivity (50, 51). The study endeavored to elucidate whether the combined administration of EA and metformin yields synergistic effects, potentially offering a novel therapeutic approach for ameliorating hepatic dysfunction in T2DM. Our studies indicated that hyperglycemia, hyperlipidemia and IR induced by high-fat diet (HFD) and injection of STZ promoted lipid accumulation, pathological and functional damage, and resulting in hepatic steatosis and fibrosis. Meanwhile, we found that EA, Met, and EA + Met enhanced the activation of AMPK, which in turn promoted the expression of PPARα/CPT1A pathway, inhibited the expression of SREBP1c and PGC-1α/PCK2 pathway, futher improved levels of autophagy mediated by AMPK/mTOR pathway and thus suppressed TGFβ1/Smad2/3 signaling pathway, ultimately exerting its effect on ameliorating hepatic steatosis and fibrosis in T2DM rats. In a nutshell, our study revealed that the three treatment prominently alleviated T2DM-induced MAFLD, the mechanism was likely associated with the regulation of glycolipid metabolism and autophagy mediated by AMPK signaling pathway in liver.
The interplay between T2DM and MAFLD entails intricate bidirectional regulation mechanisms, influencing each other's progression and severity (52). T2DM and IR instigate hepatic triglyceride accumulation, exacerbating MAFLD, while lipid toxicity and hyperglycemia further fuel the development of MAFLD (52). Conversely, the presence of MAFLD aggravates insulin resistance, establishing a detrimental cycle of mutual reinforcement. The AMPK pathway emerges as a pivotal player in bridging the pathophysiological connection between T2DM and MAFLD, orchestrating glycolipid metabolism and insulin resistance (33, 36). In our study using T2DM rats induced by HFD + STZ treatment, we observed that intervention with EA treatment in T2DM rodent models exhibited hepatoprotective effects by bolstering AMPK activity. Numerous studies emphasized AMPK's pivotal role in T2DM and MAFLD development and progression (53). AMPK, comprising one catalytic subunit (α1 or α2) and two regulatory subunits, serves as a vital regulator of energy balance in liver, muscle, and adipose tissues (54–56). AMPK conserves ATP and modulates energy metabolism by activating catabolic pathways and inhibiting anabolic pathways, including fatty acid oxidation, hepatic lipogenesis, glucose uptake and output, and insulin sensitivity regulation (36). AMPK phosphorylation inhibits transcription factors that induce gluconeogenesis and lipogenic programs, notably PCK2 and SREBP1, reducing liver glucose and fat accumulation (33, 57). Additionally, AMPK promotes fatty acid entry into mitochondria and oxidation through the CPT1 system via PPARα (37), as shown in Fig. 6. Regular exercise enhanced whole-body insulin sensitivity by activating AMPK, as evidenced by epidemiological data indicating lower prevalence of metabolic syndrome diseases among physically active individuals (58, 59). Strategies aimed at activating AMPK hold significant promise for both the prevention and treatment of T2DM and MAFLD. To delve deeper into the mechanism underlying EA, we conducted experiments utilizing T2DM rat models subjected to EA treatment, assessing the expression of molecular markers associated with AMPK. Throughout our rigorous investigation, stark disparities were observed between the control group and the T2DM rats. Specifically, levels of FBG, serum lipid components (including TG, TC, LDL), and HOMA-IR significantly escalated in the T2DM group. Conversely, serum HDL and HOMA-ISI levels notably declined. These findings underscored the manifestation of evident IR and dysregulation in glycolipid metabolism within the T2DM rat models. Following a 6-week intervention period, all three treatment modalities not only induced a substantial decrease in FBG levels from the fourth to sixth week but also precipitated a remarkable reduction in TC, TG, LDL, and HOMA-IR levels, coupled with a significant elevation in HDL and HOMA-ISI levels. These salutary effects were consonant with the activation of AMPK, culminating in the upregulation of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), and glucose transporter type 4 (GLUT4), alongside the inhibition of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), phosphoenolpyruvate carboxykinase 2 (PCK2), and sterol regulatory element-binding protein 1c (SREBP1c). Consequently, these cascades led to diminished adipogenesis and gluconeogenesis, augmented fatty acid oxidation, and heightened insulin sensitivity, thereby substantiating the therapeutic potential of AMPK activation in ameliorating the metabolic derangements associated with T2DM and MAFLD. The regulatory impact of EA, when combined with Met intervention, on the expression of hepatic AMPK surpassed that of both EA and Met administration. This superiority underscored the potential advantage of integrating EA with Met therapy in clinical practice.
To assess the impact of enhanced glycolipid metabolism on the liver, we examined liver function and pathological changes. Our observations revealed elevated levels of ALT and AST, alongside reduced ALB levels in T2DM rats compared to the control group. Notably, the livers of T2DM rats exhibited inflammation, lipid vacuoles, ballooning, and significant fat accumulation, indicating liver metabolic dysfunction and hepatocyte steatosis. Furthermore, our results indicated that the three treatments effectively alleviated liver injury induced by IR. This was evidenced by decreased AST and ALT levels, increased ALB levels in serum, and improvements in hepatic morphology, along with reduced areas of positive Oil Red O staining. Moreover, electron microscope analysis revealed a notable presence of autophagosomes in liver tissue across the three treatment groups.
Autophagy, an essential system for maintaining cellular homeostasis, mitigates liver injury, oxidative stress, and inflammation by breaking down and recycling misfolded proteins and damaged organelles resulting from lipid peroxidation. This process thereby prevents the onset of hepatic steatosis (24) (28). Lipophagy, a subtype of autophagy, targets intracellular lipid droplets (LDs) for degradation, aiming to regulate liver fat stores (25, 26, 60). Research indicated that compromised autophagic flux contributed to lipid droplet accumulation in hepatocytes, exacerbating hepatic steatosis and damage in T2DM rats. LC3-II, a protein crucial for autophagosome formation and maturation, serves as a reliable marker for assessing autophagic activity (61, 62). In the present study, both the protein and mRNA expression levels of LC3-II were significantly reduced in the T2DM group, suggesting suppressed autophagic activation. This finding correlated with the observed increase in hepatic lipid deposition and morphological alterations. Conversely, LC3B-II levels were markedly elevated, accompanied by a reduction in LDs following the three treatments compared to the T2DM group. This suggested that enhanced autophagy facilitated lipid clearance in the liver. Consequently, EA, Met, and EA + Met treatments may alleviate hepatic lipid accumulation by inducing autophagy in T2DM. Central to the regulation of autophagy in MAFLD is the AMPK /mTOR pathway, which has been extensively documented (63). AMPK, a highly conserved serine/threonine-protein kinase across evolutionary scales, serves as a pivotal energy sensor, thereby regulating metabolic homeostasis and amplifying autophagy (38, 39). Conversely, mammalian target of rapamycin (mTOR), a central regulator of cell growth, inhibits autophagy by integrating nutrient signals and growth factors. mTORC1, a complex of mTOR (64), phosphorylates ULK1 at Ser757, impeding its interaction with AMPK, thus halting autophagy. AMPK, as the upstream regulator of mTOR, negatively modulates mTOR activation either directly by phosphorylating mTORC1 or by phosphorylating various components of mTORC1. Moreover, AMPK fosters autophagy by directly activating ULK1 through phosphorylation at Ser555 and Ser777, facilitating the release of ULK1 from mTORC1. Consequently, the activated ULK1 kinase complex recruits other autophagy proteins, instigating autophagy induction (63, 65–68). However, in conditions such as steatosis and hypernutrition, autophagy initiation is hindered due to alterations in AMPK and mTOR signaling. The current study revealed a significant decrease in the p-AMPK/AMPK ratio and AMPK mRNA alongside a remarkable increase in the p-mTOR/mTOR ratio and mTOR mRNA. This was coupled with the inhibition of autophagy, as evidenced by the notable decrease in LC3-II levels in the T2DM group. However, autophagy was restored in the groups treated with EA, Met, and EA + Met, achieved through elevated AMPK expression and reduced mTOR expression levels, consequently leading to increased LC3-II levels. Moreover, our results showed that EA-induced autophagy and improvement of hepatic lipid droplet accumulation and glycolipid metabolism was partly disrupted by Compound C, an inhibitor of AMPK, in the EA + Compd C group. Taken together, these results suggest that the beneficial effects of EA may stem from enhanced autophagy via the AMPK/mTOR pathway.
Chronic liver injury caused by T2DM and metabolic disorders gradually progresses to fibrosis, which is characterized by excess deposition of collagen between hepatocytes and hepatic sinusoids to repair the liver damage (69). Collagen, a pivotal structural protein within the extracellular matrix (ECM), serves as a fundamental component in maintaining tissue integrity and function. In the liver, hepatic stellate cells (HSCs) emerge as the primary source of collagen synthesis, playing a critical role in the dynamic equilibrium of ECM turnover and regulation. In response to liver injury, HSCs undergo a phenotypic transformation into activated myofibroblast-like cells (MFCs), which are proficient in ECM production, notably collagen types I and III, and formate α-smooth muscle actin (α-SMA) stress fibers (70, 71). This transition marks a pivotal event in the pathogenesis of liver fibrosis, characterized by excessive ECM deposition and aberrant tissue remodeling. Central to the fibrotic cascade is the multifaceted cytokine transforming growth factor-beta (TGF-β), which orchestrates various cellular processes, including HSCs activation and proliferation. Within the liver microenvironment, TGF-β1 emerges as a key isoform, synthesized by both liver parenchymal cells and activated HSCs, exerting its effects through a Smad3-dependent signaling pathway (72, 73). Upon binding to the TGF-β II receptor on the cell membrane of HSCs, TGF-β1 initiates a signaling cascade culminating in the activation of intracellular mediators, notably Smad proteins (74). Subsequently, the Smad complex, comprising Smad2, Smad3, and Smad4, translocates into the nucleus, where it modulates gene transcription, including the upregulation of collagen expression. By directly binding to the collagen promoter region, the Smad complex exerts transcriptional control, thereby fostering ECM production and deposition, thereby perpetuating the fibrotic response in the liver (75, 76). Understanding the intricate interplay between HSCs, TGF-β signaling, and collagen synthesis offers crucial insights into the molecular mechanisms underpinning liver fibrosis. Targeting these pathways holds promise for the development of novel therapeutic strategies aimed at mitigating fibrotic progression and restoring hepatic homeostasis. Our results showed that the expressions of α-SMA, a fibroblast marker, were abundantly increased in the T2DM group. The pathological characteristics of T2DM in massson staining and electron-microscope exhibited an increase in hepatic fibrosis, indicating activation of HSCs. We also found that HFD + STZ treatment distinctly increased the expressions of TGFβ1, smad2, and smad3. These data prompted that T2DM-induced aberrant glycolipid metabolism possess a critical role in liver injury and fibrosis in T2DM rats. It is worth mentioning that the three treatments could attenuated this effect and significantly ameliorate hepatic fibrosis and decrease the expression of TGFβ1, smad2, and smad3 in T2DM rats, suggesting that the antifibrotic activity of three treatments may be associated, at least partially, with TGFβ1/smad2/3 signaling pathway activity attenuation.
However, there arised a question: was the upregulation of autophagy and glycolipid metabolism by EA linked to the downregulation of TGFβ1/SMAD3 signaling, or was it an independent molecular event requiring further investigation? As previously mentioned, AMPK-mediated glycolipid metabolism and autophagy contributed to restoring liver structure and function, thereby preventing liver fibrosis by mitigating factors causing liver damage. Rangnath Mishra investigated the interplay between AMPK and the TGFβ1/SMAD3 pathway. Their study revealed that pharmacologically activating AMPK inhibited TGFβ-induced secretion of collagen types I and IV and fibronectin, driven by Smad3-binding cis-elements. In our present study, as anticipated, the introduction of Compound C, an AMPK inhibitor, suppressed AMPK activation and partially reversed the beneficial effects of EA on the TGFβ1/SMAD3 signaling pathway and liver fibrosis. This finding confirmed a direct relationship between AMPK activation and the anti-fibrotic effects of EA treatment. Considering the aforementioned studies, the AMPK signaling pathway mediatedEA's beneficial effects in delaying the progression of MAFLD in T2DM rats.
In conclusion, to the best of our knowledge, the results of the present study showed that EA played a critical role in ameliorating in MAFLD in T2DM rats. Specifically, EA intervention improved FBG, serum lipids, insulin sensitivity and hepatic function, and inhabited liver steatosis and fibrosis of T2DM rats. Furthermore, we demonstrated insights that EA treatment was likely through the mechanisms of the up-regulating of hepatic AMPK signaling pathway, which mediated glycolipid metabolism and autophagy, inhibited TGFβ1/SMAD3 pathway-induced fibrosis. Moreover, the above effects of EA were consistent with metformin. The combination of EA and metformin had significant advantages in increasing hepatic AMPK expression, improving liver morphology, lipid droplet infiltration, fibrosis, and reducing serum ALT levels. Taken together, our findings indicated that EA might be taken as an effective therapeutic strategy and improving the efficacy of metformin in MAFLD. Therefore, the combined approach of EA and Met in clinical practice offers a personalized and holistic treatment strategy that considers the individual's constitutional differences and the dynamic nature of metabolic disorders. Through synergistic interactions, acupuncture may enhance the efficacy of pharmacotherapy while minimizing adverse effects and promoting overall well-being. Future research endeavors should focus on elucidating the underlying mechanisms responsible for the superior regulatory effect of EA combined with Met on liver AMPK expression. Additionally, multiple animal studies and larger clinical trials are warranted to validate the therapeutic efficacy and safety of this integrated approach in diverse patient populations with metabolic disorders. Ultimately, integrating EA with Met holds promise for advancing precision medicine and improving patient outcomes in the management of metabolic diseases.