Autolysosomal Dysfunction in Obesity-induced Metabolic Inflammation and Related Disorders

Obesity induces numerous cellular stresses and inflammatory signaling pathways by ectopic accumulation of fat in various tissues, leading to insulin resistance. Among the metabolic organs, adipose tissue (AT) and the liver are especially implicated in energy imbalance and obesity-related pathology [22, 23, 24]. Adipocytes store excessive energy in the form of triglyceride, and AT functions as an endocrine organ that secretes adiponectin, leptin, and pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNF), and monocyte chemoattractant protein-1 (MCP1). In adipocytes from obese individuals, lysosomal function and autophagy function is suppressed [22, 25, 26, 27]. Autophagy inhibition further leads to increased endoplasmic reticulum stress, which leads to an upregulation of inflammation-related genes, including MCP-1, IL-6, and interleukin-1β (IL-1β) [25]. Pharmacological inhibition of autophagy using 3-methyladenine further exacerbated inflammatory gene expression, underscoring autophagy’s critical role in regulating adipocyte inflammation [22, 25, 26]. Obese adipose-derived stem cells (O-ADSCs) isolated from liposuction specimens of obese donors exhibited weaker Lysotracker Red staining, indicating elevated lysosomal pH and reduced autophagic flux [27]. Moreover, O-ADSCs demonstrated significant mitochondrial dysfunction, including increased fragmentation and reduced organelle size, reduced mitochondrial membrane potential, increased oxidative stress, and impaired energy metabolism [27]. Additionally, these cells showed an increased expression of CD36 (fatty acid translocase) and CD106, markers associated with inflammation [27]. Lysosomal dysfunction has also been observed in AT from obese individuals, as indicated by elevated lysosomal gene expression in mesenteric AT compared to lean subjects [28].

Under conditions of overnutrition, AT undergoes dynamic remodeling, which not only contributes to local inflammation but also promotes systemic inflammation through the recruitment of specialized immune cells that facilitate the clearance of dysfunctional adipocytes and their replacement with new fat cells [24, 29]. Among these immune cells, lipid-associated macrophages (LAMs) play a crucial role in obesity and lipid stress-related inflammation. LAMs selectively express transmembrane 4L six family member 19 (TM4SF19), a lysosomal protein that interacts with the lysosomal V-ATPase, leading to reduced lysosomal acidification. Inactivation of TM4SF19 has been shown to enhance lysosomal acidification, reduce LAM accumulation, accelerate the clearance of apoptotic adipocytes in vitro and in HFD mice, and mitigate inflammation and immune cell infiltration, thereby improving systemic insulin sensitivity [29]. Increased CD36 expression has been observed in human visceral AT, preadipocytes from HFD mice, and 3 T3L1 preadipocytes treated with free fatty acids (FFA) [30, 31]. CD36 overexpression can activate inositol (1,4,5)-trisphosphate receptor 1, leading to lysosomal calcium overload, lysosomal membrane permeabilization (LMP), and decreased V-ATPase activity, resulting in increased lysosomal pH [30, 31]. Additionally, CD36 functions as an inflammatory receptor in adipose tissue [31, 32], with its upregulation by FFAs in preadipocytes triggering increased expression of MCP-1, IL-6, and IL-1β, further propagating systemic inflammation [31].

Exposure to an HFD or FFAs induces LMP in AT, leading to the release of lysosomal proteases such as cathepsin B (CTSB) [30, 33, 34, 35, 36]. Increased cytosolic CTSB contributes to mitochondrial dysfunction [30, 33, 34, 35], which further exacerbates lysosomal acidification impairment [37, 38]. Notably, coincubation of adipocytes with palmitic acid and a selective CTSB inhibitor almost completely prevented the loss of lysosomal acidification induced by palmitic acid [33]. CTSB activation not only promotes adipocyte cell death but also recruits immune cells to AT, triggering the release of inflammatory cytokines that propagate systemic inflammation [22, 30, 33, 34]. In HFD mice, increased LMP has also been associated with the release of cathepsin D, leading to mitochondrial dysfunction and adipocyte cell death [35].

Despite evidence linking lysosomal dysfunction to obesity, few studies have specifically explored strategies to restore lysosomal acidification in adipocytes. Most research has focused on enhancing autophagic function as a whole. For instance, an 8-week treadmill exercise regimen in HFD mice increased autophagic turnover in AT, accompanied by upregulation of LAMP2, a key regulator of autophagosome-lysosome fusion [39]. Similarly, exercise-induced improvements in autophagic machinery were associated with reduced cytosolic CTSB release in HFD mice [40]. However, the impact of exercise on autophagy varies among different adipose depots. While endurance exercise increased autophagy in inguinal and subcutaneous visceral AT, the effect was less pronounced in epididymal AT [41]. Dietary interventions, particularly those rich in unsaturated fatty acids, may also serve as potential therapeutic strategies to restore autophagic function. Long-term consumption of a monounsaturated fatty acid-rich diet upregulated autophagy-related genes BECN1 and Atg7 in subcutaneous AT of obese individuals [42]. Similarly, omega-3 supplementation improved autophagic flux in AT of obese rats [43].

Lipotoxicity is a pathological state wherein excessive lipid accumulation in non-adipose tissues perturbs cellular homeostasis and increases cell death [22]. Inadequate lipid droplet biogenesis or storage capacity leads to sustained elevation of circulating free fatty acids, which are ectopically deposited in insulin-sensitive tissues such as skeletal muscle, liver, and pancreas [22]. This aberrant lipid deposition disrupts mitochondrial function, promotes endoplasmic reticulum stress, and activates inflammatory and apoptotic signaling cascades, culminating in insulin resistance and the progression of metabolic disorders [22]. Sarcopenic obesity is characterized by a simultaneous decline in muscle mass and function, accompanied by an increase in adipose tissue mass [44]. This condition is a growing concern among older adults due to its significant health risks, including the development of comorbidities and geriatric syndromes [44]. Maintaining proper protein metabolism is essential for preserving skeletal muscle mass and function. Under obesity, primary human skeletal muscle myotubes exhibit reduced autophagic flux, leading to impaired carbohydrate and lipid metabolism and contributing to muscle atrophy, and insulin resistance [44]. In overweight elderly individuals, elevated expression of autophagy-related proteins such as Atg5, Atg12, LC3-I, LC3-II, and Beclin-1 suggests increased autophagy initiation and autophagosome formation. However, the accumulation of p62 within skeletal muscles indicates impaired autophagic flux, preventing proper degradation and recycling of cellular components [45]. Recent study comparing 49 BMI-discordant monozygotic twin pairs revealed that the more obese co-twins exhibited dysregulated lysosomal metabolic networks, indicative of lysosomal dysfunction, alongside elevated inflammatory pathways [46].

In HFD mice, suppression of autophagy has been observed in gastrocnemius [47] and soleus muscles [48], leading to reduced myofiber cross-sectional area and structural and functional impairments [47, 48]. Treatment of C2 C12 differentiated myotubes with increasing doses of PA resulted in elevated expression of p62 and LC3, signifying autophagic dysfunction. Additionally, GFP-mRFP-LC3-transfected C2C12 myotubes showed an increase in yellow puncta without a corresponding rise in red puncta, indicating impaired autophagosome-lysosome fusion and defective autophagic degradation [47]. Despite growing evidence linking lysosomal dysfunction to skeletal muscle impairment, studies specifically examining how modulating lysosomal acidification affects muscle homeostasis remain limited, underscoring the need for further research in this area.

Exercise interventions have been widely employed to enhance autolysosomal function in skeletal muscle under obesity. Moderate-intensity swim training has been shown to restore autophagic function in the gastrocnemius muscles of HFD mice [49]. Hypoxia training, administered for durations ranging from one to four weeks, increased Beclin-1 and LC3 expression in the gastrocnemius muscle of mice, suggesting enhanced autophagic activity [50]. Additionally, a single bout of exercise stimulation was sufficient to reverse autophagic inhibition in the soleus muscle of mice fed a high-fat, high-sucrose diet [48]. Liraglutide, a GLP-1 receptor agonist, has been shown to enhance autophagy in rat skeletal muscle cells by upregulating sestrin2 via the GLUT4–pAKT–mTOR signaling pathway [51]. Similarly, geniposide activates the GLP-1 receptor and promotes autophagy through the AMPK/mTOR pathway [52]. Moreover, GLP-1 overexpression in skeletal muscle improves glucose metabolism and mitochondrial biogenesis by increasing autophagy [53].

Obesity is associated with impaired intestinal barrier function and dysbiosis of the gut microbiota [54]. Autophagy plays an essential role in maintaining gut homeostasis, and dysfunctional autophagy has been linked to gut microbiota imbalance and intestinal dysbiosis [55, 56]. In patients with obesity and T2D, the gut microbiome is enriched in harmful taxa such as Clostridium leptum, Clostridium coccoides, Enterobacteriaceae, and Turicibacter sp., while beneficial bacteria like Butyricicoccus sp., Akkermansia muciniphila, and Faecalibacterium prausnitzii are significantly reduced [55, 57, 58]. Similarly, in HFD mice, there is an increased abundance of Butyricimonas, a reduction in Akkermansia, and decreased expression of the tight junction protein occludin, indicating compromised intestinal barrier function [55, 59, 60]. Supporting the role of autophagy, mice with colonic epithelial cell-specific deletion of the autophagy gene Atg7 display altered fecal microbiota composition, with increased total bacterial load and enrichment of Clostridium leptum, Eubacterium cylindroides, and Bacteroides fragilis, compared to wild-type controls [61]. Overall, gut dysbiosis, particularly the loss of Akkermansia muciniphila, is correlated with increased intestinal permeability and endotoxemia-driven systemic inflammation in obesity [62, 63].

Targeting autophagy has emerged as a promising strategy to restore gut barrier integrity and metabolic health [56, 64]. Spermidine, an autophagy inducer, improves insulin resistance in both humans and mice [54]. Mechanistically, spermidine treatment protects gut barrier function by activating autophagy, attenuating apoptosis in colonic and intestinal cells, and increasing the number of mucus-secreting goblet cells and mucin secretion in HFD mice [54]. Similarly, treatment with nuciferine promotes autophagosome formation and autolysosomal fusion in HFD mice, reducing hyperglycemia and intestinal permeability [59]. Furthermore, resveratrol-mediated inhibition of mTORC1, which results in activated autophagy, reduced the abundance of obesity-associated gut microbiota, including Lactococcus, Clostridium XI, Oscillibacter, and Hydrogenoanaerobacterium, and alleviated intestinal inflammation in diet-induced obese mice [65]. Probiotics have also shown beneficial effects, helping to rebalance the microbial population by enhancing autophagic function [66, 67].

The gut plays a central role in inter-organ communication, influencing the liver, brain, and other systems [17, 68, 69]. Autolysosomal dysfunction is increasingly seen across these organs, contributing to systemic pathology [17]. In HFD-fed mice, there is an increased Firmicutes/Bacteroidetes ratio and reduced Akkermansia levels, which were associated with hepatic steatosis. Treatment with apple polyphenol extract reversed these microbiota changes through upregulating autophagy-related genes, leading to reduced hepatic steatosis [70]. In obesity, dysbiosis-driven increases in lipid absorption and circulating metabolites promote mitochondrial and endolysosomal dysfunction in neurons, contributing to AD pathogenesis [71]. The breakdown of the gut barrier facilitates endotoxemia, triggering a pro-inflammatory cascade that disrupts metabolic pathways in the brain [72]. Notably, HFD-induced microbiome alterations can be ameliorated by dimethyl itaconate supplementation, which enriches propionate- and butyrate-producing bacteria. Furthermore, fecal microbiota transplantation from dimethyl itaconate-treated mice improves cognitive function and hippocampal synaptic structure, indicating restoration of brain metabolic health [73].

During obesity, excessive FFAs surpass the liver’s capacity to store them as lipid droplets, leading to lipotoxicity, increased liver inflammation, and impaired hepatic function [74]. Liver biopsy specimens from patients with MASLD show significantly reduced expression of lysosomal cathepsins (e.g., CTSB, CTSD, and CTSL) and increased p62 accumulation, indicative of autolysosomal dysfunction [75]. Elevated serum alanine aminotransferase (ALT) levels further suggest hepatic inflammation [75]. Additionally, liver biopsies from morbidly obese patients undergoing bariatric surgery exhibit impaired autophagy, along with increased hepatic apoptosis and pyroptosis [76]. In three different murine models of MASLD, reduced numbers of acidic organelles and lower levels of the lysosomal enzyme CTSD point to defective lysosomal acidification [77].

In hepatocytes treated with PA, elevated lysosomal pH disrupts autophagic degradation and mitochondrial function, leading to lipid droplet accumulation and increased insulin resistance [14]. PA-treated primary hepatocytes also exhibit downregulation of lysosomal V-ATPase subunit ATP6V1A and LAMP1, indicating impaired lysosomal acidification [78]. Furthermore, TFEB expression is reduced in the livers of HFD mice, signifying compromised lysosomal function [78, 79]. Obesity-induced lysosomal dysfunction is exacerbated by increased nitric oxide production, which promotes nitrosative stress and S-nitrosylation, further impairing lysosomal function and exacerbating hepatic inflammation in HFD mice [80]. Treatment of cultured hepatocytes with both oleate and PA results in increased lipid droplet accumulation and impaired autophagic clearance [81].

Exercise has been shown to enhance lysosomal function, as indicated by increased LAMP1 and LAMP2 levels in HFD mice [82]. Voluntary wheel running [83] and swim training [84] exercises both improved autophagic activity and reduced hepatic lipid accumulation in MASLD mouse models [85]. Dietary supplementation with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) mitigated lipid accumulation and cell death in FFA-exposed L02 liver cells [86]. The combined effects of DHA supplementation and aerobic exercise synergistically enhanced autophagic function in HFD mice [87]. In HFD mice, oleic acid supplementation upregulated lysosomal TFEB expression and enhanced lysosomal acidification [88]. Treatment with phillygenin, a lignan derived from Forsythia suspensa, promoted TFEB dephosphorylation, enhancing lysosomal biogenesis and attenuating NLRP3 inflammasome activation, thereby reducing liver inflammation [78]. Similarly, baicalein supplementation in HFD mice increased the expression of lysosomal V-ATPase V1 subunits, improving lysosomal acidification and function while reducing LMP and hepatic inflammation [89]. Tetrahydrocurcumin similarly upregulated lysosomal biogenesis through TFEB nuclear translocation, mediated via mTORC1 inhibition [90]. The GLP-1 receptor agonist liraglutide restores autophagic activity in HFD mice by promoting lysosomal biogenesis and function [91], and enhances the expression of cytosolic lipolysis-related proteins, thereby reducing intracellular lipid accumulation [92]. Semaglutide, an incretin mimetic, increased autophagic function and reduced lipid accumulation in steatotic hepatocytes [93]. Finally, recent work has shown that lysosome-targeting acidic nanoparticles (NPs) composed of poly(tetrafluorosuccinate-co-succinate) ester (PEFSU NPs) successfully increased lysosomal acidification and autophagic degradation, reducing lipid droplet accumulation and hepatic inflammation in HFD mice [14].

When the pancreas is exposed to high amounts of lipid in obesity, impairment of autophagic flux and lysosomal function are found in the pancreatic β-cells, as well as reduced insulin secretion and cellular viability [18, 37, 38]. In islets isolated from T2D human subjects, there is downregulation of lysosomal V-ATPase subunit genes ATP6V1B2, ATP6AP2, ATP6V1A, ATP6V1H, ATP6V1G1, ATP6V0E1 and ATP6V0B [18]. In HFD mouse model of T2D, autolysosomal dysfunction was present in the mice pancreas [18]. Studies in other diabetic mouse models have shown a reduction in lysosomal subunit ATP6V1A in mouse islets [94, 95]. Similarly, in INS-1 832/13 rat insulinoma cells treated with palmitate, autolysosomal function is impaired, consequently leading to reduced glucose-stimulated insulin secretion [18, 96, 97]. Expression levels of inflammatory markers TNF, IL-1β and IL-6 were also increased in INS-1 cells under palmitate [98]. While underexplored, a study has shown that pancreatic alpha cells from HFD mice show changes in protein degradative pathways, including lysosomal pathways [99].

Lysosome-targeted nanoparticles have emerged as a promising strategy for restoring lysosomal acidification defects in T2D cellular and animal models. These include photo-activated nanoparticles that release acids upon UV-light exposure to lower lysosomal pH [96], and poly(tetrafluorosuccinate)-based nanoparticles (TFSA NPs), which release acidic components within the lysosomal lumen to restore acidification in pancreatic β-cells [18]. Quercetin, a natural flavonoid, has been shown to reverse lysosomal dysfunction and enhance autophagosome-lysosome fusion in pancreatic β-cells under palmitate treatment [100]. Additionally, supplementation with unsaturated fatty acids such as docosahexaenoic acid has been reported to enhance autophagy in palmitic acid-treated mouse insulinoma 6 cells via modulation of the mTOR/GPR120 axis [101]. Omega-3 fatty acid enrichment has also been demonstrated to improve autophagic flux in pancreatic β-cells of STZ-treated fat-1 mice, thereby reducing diabetes-related β-cell damage [102].

Lifestyle interventions such as physical exercise have been explored as potential strategies to restore pancreatic function. High-intensity training has been found to enhance pancreatic β-cell function in both T2D patients [103] and healthy individuals following a high-fat meal [104]. Additionally, physical exercise has been shown to improve islet morphology, increase β-cell number, and reduce β-cell apoptosis in obese rats [105], though the precise mechanisms regulating β-cell activity remain unclear. Another lifestyle intervention, caloric restriction, has been demonstrated to enhance autophagic flux in obese mice and rats [106, 107]. Intermittent fasting (IF) has also been shown to reduce the accumulation of autophagic substrates and increase TFEB expression, indicating improved lysosomal function [108]. Furthermore, IF promotes β-cell proliferation, potentially through activation of the autophagy–lysosome pathway and upregulation of Neurogenin-3 (Ngn3) expression [109, 110]. Notably, the protective effects of IF, including inhibition of β-cell mortality and promotion of Ngn3 expression, were absent in obese mice with lysosomal deficiencies or autophagosome dysfunctions, underscoring the critical role of the autophagy–lysosome pathway in these processes [108].

The systemic inflammation driven by increased adipokine secretion from adipose tissue exacerbates lipid accumulation in multiple organs, including the kidneys, contributing to glomerular hypertrophy and segmental sclerosis, which impairs renal function [111, 112]. Studies in both human and experimental models have demonstrated that impaired lysosomal function and disrupted autophagic flux play a crucial role in obesity-related kidney disease by promoting lipotoxicity and inflammation [112, 113]. In patients with chronic kidney disease (CKD), a higher body mass index has been linked to increased vacuolation and decreased nuclear TFEB levels in proximal tubules, indicating autophagy dysregulation [112]. Additionally, kidney biopsy samples from obese CKD patients revealed LAMP1-positive vacuoles and p62 accumulation in renal proximal tubular epithelial cells (PTECs), further suggesting impaired autolysosomal function [19, 112]. In vitro studies using palmitate-treated PTECs have also shown reduced lysosomal acidification, reinforcing the role of autolysosomal dysfunction in obesity-associated renal pathology [19]. This reduction in autolysosomal activity has been linked to mitochondrial dysfunction, increased macrophage infiltration, and inflammasome activation, all of which contribute to renal fibrosis and disease progression [19].

In patients with diabetic nephropathy, renal biopsy tissues exhibited reduced TFEB expression, accompanied by lipid accumulation and increased apoptosis, indicating lysosomal and autophagic dysfunction [114]. Similarly, HFD rats displayed lower nuclear TFEB levels compared to those on a low-fat diet, further suggesting impaired lysosomal function [114]. Additionally, HFD rats showed autophagy suppression alongside elevated serum cystatin C levels and increased urinary N-acetyl-β-d-glucosaminidase activity, both markers of kidney injury [115]. Proximal tubular epithelial cell–specific mice fed on HFD have increased phospholipid accumulation in enlarged lysosomes indicative of lysosomal dysfunction, and reduced autophagic function [112]. In HK-2 cells, palmitate treatment induced TFEB phosphorylation at Ser211, which impaired its nuclear translocation, thereby disrupting TFEB-mediated lysosomal biogenesis and function [115]. Another study using the same cellular model demonstrated that lysosomal V-ATPase subunit ATP6V1D protein expression was significantly reduced, indicating a decline in lysosomal acidification and function [114]. Moreover, palmitate exposure was found to promote TFEB dephosphorylation and nuclear translocation by inhibiting the mTOR pathway in a Rag GTPase–dependent manner, further linking autophagic dysfunction to metabolic stress in renal cells [112].

Few studies have specifically targeted lysosomal acidification and function within the kidney, with most focusing on enhancing autophagic activity more broadly. Sodium-glucose co-transporter inhibitors have been shown to mitigate autophagy suppression in the kidney by reducing mTOR expression in HFHS mice, thereby restoring autophagic function [116]. Epigallocatechin-3-gallate (EGCG) has demonstrated the ability to enhance autophagy in palmitate-treated HK-2 cells, while also increasing AMP-activated protein kinase (AMPK) phosphorylation in the kidneys of HFD rats and palmitate-treated HK-2 cells, contributing to improved autophagic activity [115]. Additionally, supplementation with eicosapentaenoic acid, a polyunsaturated fatty acid, has been found to rescue autophagy impairment in renal PTECs [117]. Exercise-based interventions have also shown promise, with endurance exercise in obese mice activating ULK-1, increasing Beclin-1 and LAMP1 levels, and promoting AMPK activation, leading to enhanced autophagic and lysosomal function in kidney tissues [118]. Similarly, treadmill exercise in obese rats activated AMPK while suppressing mTOR signaling in kidney tissues, further supporting the role of exercise in improving autophagic function [119].

Obese and overweight individuals with AT hypertrophy exhibit significant alterations in cardiac structure and function, increasing the risk of cardiomyopathy, hypertrophy, atrial fibrillation, and arrhythmia. AT hypertrophy is associated with the secretion of atherogenic cytokines that impair autolysosomal function in cardiac cells [20]. In the right atrial appendage cardiac tissues of obese individuals, TFEB expression is significantly reduced compared to healthy individuals, indicating compromised lysosomal acidification and function [120]. Similarly, in mice fed a HFD or a HFHS diet, cardiomyocytes show decreased TFEB content [120, 121, 122], along with a significant increase in diacylglycerol and triacylglycerol levels, indicating lipid metabolism dysregulation and cardiomyocyte cell death [123]. Additionally, cardiomyocytes in obese models exhibit reduced lysosomal V-ATPase activity and elevated lysosomal pH, indicative of impaired lysosomal acidification [124]. In both HFD mice and palmitate-treated H9C2 cardiomyocytes, lysosomal acidification and autophagosome clearance are significantly suppressed [125]. This dysfunction in lysosomal acidification within cardiomyocytes contributes to a marked reduction in sarcomere shortening, leading to contractile dysfunction and the progression of cardiomyopathy [124, 126].

Palmitate treatment of cardiomyocytes increased s-nitrosylation of a highly conserved cysteine residue (Cys-277) of the ATP6V1A1 subunit, which could impair V-ATPase function and reduce lysosomal acidification [125]. Similar phenomena were also observed in cardiac cells including induced pluripotent stem cell cardiomyocytes, HL-1 and aRCM, where palmitate treatment reduced lysosomal V-ATPase function through dissociation of the V-ATPase [124, 126, 127]. In addition, palmitate induced activation of NADPH oxidase (Nox-2), which leads to superoxide production that impairs lysosome acidification and enzyme activity [125]. Exposure to palmitate acid has also been shown to decrease TFEB expression in various cell types including H9C2 [120, 121, 122, 123], HL-1 [123], adult mouse cardiomyocytes [123] and neonatal rat cardiomyocytes [121], as well as reduce lysosomal enzyme activity [121, 123].

Therapeutic strategies targeting lysosomal acidification have shown promise in restoring autophagic function in cardiac cells. Poly(lactic-co-glycolic) acid (PLGA) NPs have been utilized to reverse lysosomal de-acidification in palmitate treated H9C2 cardiomyocytes, aiding in cellular homeostasis [128]. Additionally, eicosapentaenoic acid (EPA) supplementation has been found to activate AMPK, leading to mTOR inhibition and ULK1 activation, thereby promoting autophagy initiation in palmitate-treated H9C2 cardiomyocytes [129, 130] Canagliflozin, a sodium-glucose cotransporter 2 inhibitor, has demonstrated efficacy in inhibiting mTOR and enhancing autophagic activity in both HFD/STZ-induced mouse hearts and palmitate-treated HL-1 cardiomyocytes [131]. Regular aerobic exercise has also been shown to activate AMPK in cardiac muscle, supporting autophagic processes [132, 133]. A recent study highlighted that rhythmic handgrip exercises improved key autophagy markers, including increased Beclin1, LC3B, Atg3, and LAMP2 levels, along with reduced p62 levels in endothelial cells from the human radial artery, suggesting enhanced autophagic function [134]. Furthermore, calorie restriction has been associated with increased autophagosome formation in heart muscle, indicating improved autophagic flux [135]. Intermittent fasting has also been reported to stimulate lysosomal function in cardiac tissues, further supporting its potential as a metabolic intervention [136].

Obesity-induced metabolic disorders can result in an increase in systemic inflammation, which can potentially propagate and induce neuroinflammation [17, 137]. There are increased levels of palmitic acid in the plasma of obese individuals. Hypothalamus, a brain region with a key role in the regulation of energy balance, food intake, insulin sensitivity and glucose homeostasis, has the greatest susceptibility to changes in the amount of palmitic acid derived from diets [138]. In rodent models of obesity, the hypothalamus has the highest amount of palmitic acid accumulation under HFD [138, 139, 140]. Mice with exposure to 16 weeks of HFD have decreased protein levels of Beclin-1 and LC3-II along with increased levels of p62, indicating reduced autophagic flux in the hypothalamus [141]. In addition, there is increased inflammation and ER stress as indicated by increased IKKβ/NF-kB activation and EIF2α expression [141]. This trend is also observed in mice hippocampus [142, 143, 144] where HFD feeding induced accumulation of LC3, Atg3, Beclin1 and p62 in the hippocampus indicative of reduced autophagic flux, leading to hippocampal amyloidosis [142]. HFD feeding also increased the activation of microglia and astrocytes, and increased NLRP3 inflammasome levels, leading to neuroinflammation [144].

Exposure of embryonic mouse hypothalamus cell line, N43-5, to palmitate resulted in the activation of free fatty acid receptor 1 leading to decreased autophagic flux, leading to insulin resistance [139]. In another study, palmitate treatment to N43-5 resulted in LC3 accumulation and lowered its co-localization with LAMP1 indicating impairment of autophagosome-lysosome fusion, potentially due to reduced lysosomal acidification, leading to increased neuroinflammation [138]. Palmitate treatment to other neuronal cell lines such as CLU-189 [141] and BV2 [142] have also shown reduction in autophagic flux. In cholesterol-enriched SH-SY5Y cells and primary neurons, high intracellular cholesterol levels stimulated mitochondrial PINK1 accumulation and mitophagosomes formation, indicative of reduced autophagic flux [145]. This trend is also seen in an AD mouse model [145].

Dietary supplementation with unsaturated fatty acids and exercise have demonstrated restorative effects in brain cells under obesity or lipotoxicity. Using Fat-1 transgenic mice which produces endogenous levels of n-3 polyunsaturated fatty acids, it was shown that there is an increased expression of ATG7 and LC3II, as well as reduced p62, indicative of increased autophagic flux in the hypothalamus [146]. Oleic acid ameliorates the impairment in fusion between autophagosomes and lysosomes within mHypoE-43/5 neuronal cells under palmitate treatment [147]. Autophagic function was also restored in the brain of obese rats after weight maintenance following short-term caloric restriction, long-term caloric restriction, and long-term exercise [148]. In addition, GLP-1 mimetic exendin-4 enhances autophagy and protects against T2D-induced apoptosis. It increases cortical GLP-1 and IGF-1 levels in mice and activates PKA and PI3K/Akt signaling pathways, leading to upregulation of autophagy and lysosomal markers such as Atg7 and LAMP-1, thereby reducing apoptotic cell death [149]. Other therapeutic agents that restore autophagic and cellular function in CNS cells under obesity have been reviewed elsewhere [150, 151].