Adipocyte HSL is required for maintaining circulating vitamin A and RBP4 levels during fasting.

Vitamin A (retinol) is distributed via the blood bound to its specific carrier protein, retinol-binding protein 4 (RBP4). Retinol-loaded RBP4 is secreted into the circulation exclusively from hepatocytes, thereby mobilizing hepatic retinoid stores that represent the major vitamin A reserves in the body. The relevance of extrahepatic retinoid stores for circulating retinol and RBP4 levels that are usually kept within narrow physiological limits is unknown. Here, we show that fasting affects retinoid mobilization in a tissue-specific manner, and that hormone-sensitive lipase (HSL) in adipose tissue is required to maintain serum concentrations of retinol and RBP4 during fasting in mice. We found that extracellular retinol-free apo-RBP4 induces retinol release by adipocytes in an HSL-dependent manner. Consistently, global or adipocyte-specific HSL deficiency leads to an accumulation of retinoids in adipose tissue and a drop of serum retinol and RBP4 during fasting, which affects retinoid-responsive gene expression in eye and kidney and lowers renal retinoid content. These findings establish a novel crosstalk between liver and adipose tissue retinoid stores for the maintenance of systemic vitamin A homeostasis during fasting.

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored. METHODS: The expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured. RESULTS: Results showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1. CONCLUSIONS: NCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

Characterization of lysosomal acid lipase in Ly6G+ and CD11c+ myeloid-derived suppressor cells.

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) induces the differentiation of myeloid lineage cells into myeloid-derived suppressor cells (MDSCs), which promotes tumor growth and metastasis. This protocol provides detailed procedures for assessment of various LAL biochemical and physiological activities in Ly6G+ and CD11c+ MDSCs, including isolation of Ly6G+ and CD11c+ cells from the bone marrow and blood of mice, assays of LAL-D-induced cellular metabolic and mitochondrial activities, assessment of LAL-D-induced pathogenic immunosuppressive activity and tumor stimulatory activity. Pharmacological inhibition of the LAL activity was also described in both murine myeloid cells and human white blood cells.

Metformin improves obesity-related oligoasthenospermia via regulating the expression of HSL in testis in mice.

Researches have proposed that obesity might contribute to development of oligoasthenospermia. This study was performed to confirm whether obesity contributes to oligoasthenospermia as well as the underlying mechanisms in mice fed with a high fat diet (HFD). Meanwhile, the actions of metformin, a drug of well-known weight-lowering effect, on sperm quality in obese mice were investigated. Our results showed that HFD feeding reduced sperm quality and steroid hormone levels in mice, associated with disruptions in testicular histomorphology and spermatogenesis. Moreover, obesity increased sperm apoptosis. These effects could be prevented by metformin treatment in HFD-fed mice. Mechanistically, an increasement in lipid contents associated with decreased hormone-sensitive lipase (HSL) protein expression in testes in HFD-fed mice was observed, which could be improved by metformin treatment. Then, the model of TM4 mouse Sertoli cells stimulated with palmitic acid (PA) was used to investigate the potential effect of lipid retention on testicular apoptosis and sperm quality reduction. In consistent, PA exposure elevated lipid contents as well as apoptosis in TM4 cells, which could also be improved by metformin treatment. Of note, the protein expression of HSL was reduced stimulated by PA in TM4 cells, also rescued by metformin. Then, anti-apoptosis effect of metformin would be lost with the deficiency of HSL. In summary, our study propose that obesity contributes to oligoasthenospermia by increasing sperm apoptosis induced by impaired lipid hydrolysis due to HSL down-regulation, which could be prevented with metformin treatment via regulating the expression of HSL in testis in mice.

Regional Differences in the Small Intestinal Proteome of Control Mice and of Mice Lacking Lysosomal Acid Lipase.

The metabolic contribution of the small intestine (SI) is still unclear despite recent studies investigating the involvement of single cells in regional differences. Using untargeted proteomics, we identified regional characteristics of the three intestinal tracts of C57BL/6J mice and found that proteins abundant in the mouse ileum correlated with the high ileal expression of the corresponding genes in humans. In the SI of C57BL/6J mice, we also detected an increasing abundance of lysosomal acid lipase (LAL), which is responsible for degrading triacylglycerols and cholesteryl esters within the lysosome. LAL deficiency in patients and mice leads to lipid accumulation, gastrointestinal disturbances, and malabsorption. We previously demonstrated that macrophages massively infiltrated the SI of Lal-deficient (KO) mice, especially in the duodenum. Using untargeted proteomics (ProteomeXchange repository, data identifier PXD048378), we revealed a general inflammatory response and a common lipid-associated macrophage phenotype in all three intestinal segments of Lal KO mice, accompanied by a higher expression of GPNMB and concentrations of circulating sTREM2. However, only duodenal macrophages activated a metabolic switch from lipids to other pathways, which were downregulated in the jejunum and ileum of Lal KO mice. Our results provide new insights into the process of absorption in control mice and possible novel markers of LAL-D and/or systemic inflammation in LAL-D.

Genetic deletion of hormone-sensitive lipase in mice reduces cerebral blood flow but does not aggravate the impact of diet-induced obesity on memory.

Hormone-sensitive lipase (HSL) is active throughout the brain and its genetic ablation impacts brain function. Its activity in the brain was proposed to regulate bioactive lipid availability, namely eicosanoids that are inflammatory mediators and regulate cerebral blood flow (CBF). We aimed at testing whether HSL deletion increases susceptibility to neuroinflammation and impaired brain perfusion upon diet-induced obesity. HSL-/-, HSL+/-, and HSL+/+ mice of either sex were fed high-fat diet (HFD) or control diet for 8 weeks, and then assessed in behavior tests (object recognition, open field, and elevated plus maze), metabolic tests (insulin and glucose tolerance tests and indirect calorimetry in metabolic cages), and CBF determination by arterial spin labeling (ASL) magnetic resonance imaging (MRI). Immunofluorescence microscopy was used to determine coverage of blood vessels, and morphology of astrocytes and microglia in brain slices. HSL deletion reduced CBF, most prominently in cortex and hippocampus, while HFD feeding only lowered CBF in the hippocampus of wild-type mice. CBF was positively correlated with lectin-stained vessel density. HSL deletion did not exacerbate HFD-induced microgliosis in the hippocampus and hypothalamus. HSL-/- mice showed preserved memory performance when compared to wild-type mice, and HSL deletion did not significantly aggravate HFD-induced memory impairment in object recognition tests. In contrast, HSL deletion conferred protection against HFD-induced obesity, glucose intolerance, and insulin resistance. Altogether, this study points to distinct roles of HSL in periphery and brain during diet-induced obesity. While HSL-/- mice were protected against metabolic syndrome development, HSL deletion reduced brain perfusion without leading to aggravated HFD-induced neuroinflammation and memory dysfunction.

p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL.

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.

Exploring the inhibitory potential of KPHs-AL-derived GLLF peptide on pancreatic lipase and cholesterol esterase activities.

The effective modulation of pancreatic lipase and cholesterol esterase activities proves critical in maintaining circulatory triglycerides and cholesterol levels within physiological boundaries. In this study, peptides derived from KPHs-AL, produced through the enzymatic hydrolysis of skipjack tuna dark muscle using alkaline protease, have a specific inhibitory effect on pancreatic lipase and cholesterol esterase. It is hypothesized that these peptides target and modulate the activities of enzymes by inducing conformational changes within their binding pockets, potentially impacting the catalytic functions of both pancreatic lipase and cholesterol esterase. Results revealed these peptides including AINDPFIDL, FLGM, GLLF and WGPL, were found to nestle into the binding site groove of pancreatic lipase and cholesterol esterase. Among these, GLLF stood out, demonstrating potent inhibition with IC50 values of 0.1891 mg/mL and 0.2534 mg/mL for pancreatic lipase and cholesterol esterase, respectively. The kinetics studies suggested that GLLF competed effectively with substrates for the enzyme active sites. Spectroscopic analyses, including ultraviolet-visible, fluorescence quenching, and circular dichroism, indicated that GLLF binding induced conformational changes within the enzymes, likely through hydrogen bond formation and hydrophobic interactions, thereby increasing structural flexibility. Molecular docking and molecular dynamics simulations supported these findings, showing GLLF's stable interaction with vital active site residues. These findings position GLLF as a potent inhibitor of key digestive enzymes, offering insights into its role in regulating lipid metabolism and highlighting its potential as functional ingredient.

Benzyl isothiocyanate inhibits TNFα-driven lipolysis via suppression of the ERK/PKA/HSL signaling pathway in 3T3-L1 adipocytes.

Tumor necrosis factor α (TNFα), an inflammatory cytokine, induces lipolysis and increases circulating concentrations of free fatty acids. In addition, TNFα is the first adipokine produced by adipose tissue in obesity, contributing to obesity-associated metabolic disease. Given that benzyl isothiocyanate (BITC) is a well-known anti-inflammatory agent, we hypothesized that BITC can ameliorate TNFα-induced lipolysis and investigated the working mechanisms involved. We first challenged 3T3-L1 adipocytes with TNFα to induce lipolysis, which was confirmed by increased glycerol release, decreased protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and perilipin 1 (PLIN1), and increased phosphorylation of ERK, protein kinase A (PKA), and hormone-sensitive lipase (HSL). However, inhibition of ERK or PKA significantly attenuated the lipolytic activity of TNFα. Meanwhile, pretreatment with BITC significantly ameliorated the lipolytic activity of TNFα; the TNFα-induced phosphorylation of ERK, PKA, and HSL; the TNFα-induced ubiquitination of PPARγ; the TNFα-induced decrease in PPARγ nuclear protein binding to PPAR response element; and the TNFα-induced decrease in PLIN1 protein expression. Our results indicate that BITC ameliorates TNFα-induced lipolysis by inhibiting the ERK/PKA/HSL signaling pathway, preventing PPARγ proteasomal degradation, and maintaining PLIN1 protein expression.

Follistatin-like 1 is a myokine regulating lipid mobilization during endurance exercise and recovery.

OBJECTIVE: The aim of this study was to investigate the role of the follistatin-like 1 (Fstl1) and disco-interacting protein 2 homolog A (DIP2a) axis in relation to lipid metabolism during and after endurance exercise and to elucidate the mechanisms underlying the metabolic effects of Fstl1 on adipocytes, considering its regulation by exercise and muscle mass and its link to obesity. METHODS: Twenty-nine sedentary males participated in endurance exercise, and blood samples were collected during and after the exercise. Body composition, Fstl1, glycerol, epinephrine, growth hormone, and atrial natriuretic peptide were measured. 3T3-L1 adipocytes, with or without DIP2a knockdown, were treated with Fstl1 to assess glycerol release, cyclic AMP/cyclic GMP production, and hormone sensitive lipase phosphorylation. The association between DIP2a gene expression levels in human adipose tissues and exercise-induced lipolysis was examined. RESULTS: Fstl1 levels significantly increased during endurance exercise and following recovery, correlating with lean body mass and lipolysis. In 3T3-L1 adipocytes, Fstl1 increased glycerol release, cyclic GMP production, and hormone sensitive lipase activation, but these effects were attenuated by DIP2a knockdown. DIP2a gene expression in human adipose tissues correlated with serum glycerol concentrations during endurance exercise. CONCLUSIONS: Fstl1 is a myokine facilitating lipid mobilization during and after endurance exercise through DIP2a-mediated lipolytic effects in adipocytes.

Extracellular macrophage migration inhibitory factor (MIF) downregulates adipose hormone-sensitive lipase (HSL) and contributes to obesity.

Attenuation of adipose hormone sensitive lipase (HSL) may impair lipolysis and exacerbate obesity. We investigate the role of cytokine, macrophage migration inhibitory factor (MIF) in regulating adipose HSL and adipocyte hypertrophy. Extracellular MIF downregulates HSL in an autocrine fashion, by activating the AMPK/JNK signaling pathway upon binding to its membrane receptor, CD74. WT mice fed high fat diet (HFD), as well as mice overexpressing MIF, both had high circulating MIF levels and showed suppression of HSL during the development of obesity. Blocking the extracellular action of MIF by a neutralizing MIF antibody significantly reduced obesity in HFD mice. Interestingly, intracellular MIF binds with COP9 signalosome subunit 5 (Csn5) and JNK, which leads to an opposing effect to inhibit JNK phosphorylation. With global MIF deletion, adipocyte JNK phosphorylation increased, resulting in decreased HSL expression, suggesting that the loss of MIF's intracellular inhibitory action on JNK was dominant in Mif-/- mice. Adipose tissue from Mif-/- mice also exhibited higher Akt and lower PKA phosphorylation following HFD feeding compared with WT, which may contribute to the downregulation of HSL activation during more severe obesity. Both intracellular and extracellular MIF have opposing effects to regulate HSL, but extracellular actions predominate to downregulate HSL and exacerbate the development of obesity during HFD.

SCD1-related epigenetic modifications affect hormone-sensitive lipase (Lipe) gene expression in cardiomyocytes.

Stearoyl-CoA desaturase 1 (SCD1) is an enzyme that is involved in the regulation of lipolysis in the heart. SCD1 also affects epigenetic mechanisms, such as DNA and histone modifications, in various tissues. Both epigenetic modifications and changes in lipid metabolism are involved in the heart's response to hypoxia. The present study tested the hypothesis that SCD1 and epigenetic modifications interact to control lipolysis in cardiomyocytes under normoxic and hypoxic conditions. We found that the inhibition of SCD1 activity and loss of SCD1 expression reduced global DNA methylation levels, DNA methyltransferase (DNMT) activity, and DNMT1 expression in HL-1 cardiomyocytes and the mouse heart. We also found that the inhibition of adipose triglyceride lipase is involved in the control of global DNA methylation levels in cardiomyocytes in an SCD1-independent manner. Additionally, SCD1 inhibition reduced expression of the hormone-sensitive lipase (Lipe) gene through an increase in methylation of the Lipe gene promoter. Under hypoxic conditions, SCD1 inhibition abolished hypoxia-inducible transcription factor 1α, likely through decreases in histone deacetylase, protein kinase A, and abhydrolase domain containing 5 protein levels, leading to the attenuation of DNA hypomethylation by DNMT1. Hypoxia led to demethylation of the Lipe promoter in cardiomyocytes with SCD1 inhibition, which increased Lipe expression. These results indicate that SCD1 is involved in the control of epigenetic mechanisms in the heart and may affect Lipe expression through changes in methylation in its promoter region. Therefore, SCD1 may be considered a key player in the epigenetic response to normoxia and hypoxia in cardiomyocytes.

Loss of lysosomal acid lipase results in mitochondrial dysfunction and fiber switch in skeletal muscles of mice.

OBJECTIVE: Lysosomal acid lipase (LAL) is the only enzyme known to hydrolyze cholesteryl esters (CE) and triacylglycerols in lysosomes at an acidic pH. Despite the importance of lysosomal hydrolysis in skeletal muscle (SM), research in this area is limited. We hypothesized that LAL may play an important role in SM development, function, and metabolism as a result of lipid and/or carbohydrate metabolism disruptions. RESULTS: Mice with systemic LAL deficiency (Lal-/-) had markedly lower SM mass, cross-sectional area, and Feret diameter despite unchanged proteolysis or protein synthesis markers in all SM examined. In addition, Lal-/- SM showed increased total cholesterol and CE concentrations, especially during fasting and maturation. Regardless of increased glucose uptake, expression of the slow oxidative fiber marker MYH7 was markedly increased in Lal-/-SM, indicating a fiber switch from glycolytic, fast-twitch fibers to oxidative, slow-twitch fibers. Proteomic analysis of the oxidative and glycolytic parts of the SM confirmed the transition between fast- and slow-twitch fibers, consistent with the decreased Lal-/- muscle size due to the "fiber paradox". Decreased oxidative capacity and ATP concentration were associated with reduced mitochondrial function of Lal-/- SM, particularly affecting oxidative phosphorylation, despite unchanged structure and number of mitochondria. Impairment in muscle function was reflected by increased exhaustion in the treadmill peak effort test in vivo. CONCLUSION: We conclude that whole-body loss of LAL is associated with a profound remodeling of the muscular phenotype, manifested by fiber type switch and a decline in muscle mass, most likely due to dysfunctional mitochondria and impaired energy metabolism, at least in mice.

Vitisin A-13-O-ß-D-glucoside and Vitisin A from Iris lactea inhibit lipogenesis and promote lipolysis via the PKA/HSL pathway during adipogenic transdifferentiation of C2C12 cells.

Oligostilbenes are a group of natural products derived from the polymerization of stilbene monomers. Despite the demonstration of their activities in regulating lipid metabolism, the function of oligostilbenes in the adipogenic transdifferentiation of multipotent myoblast cells remains unknown. Hence, the five oligostilbenes from Iris lactea were tested for their regulatory effects on adipogenic transdifferentiation of C2C12 myoblast cells. As a result, it was shown that Vitisin A-13-O-ß-D-glucoside (VitAOG), Vitisin A (VitA) and Hopeaphenol (Hop) can greatly inhibit the adipogenic transdifferentiation of C2C12 cells by reducing lipid accumulation and downregulating the expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein beta (C/EBPß) and fatty acid binding proteins 4 (FABP4). In contrast, Vitisin D (VitD) and Isohopeaphenol (Isohop) promote adipogenic transdifferentiation of C2C12 cells by increasing lipid accumulation and upregulating the expression of adipogenesis and lipogenesis markers. Further research found that the lipolytic protein levels of adipocyte triglyceride lipase (ATGL) and phosphorylation of hormone-sensitive lipase (HSL) were elevated by VitAOG and VitA. Additionally, VitAOG and VitA maintain lipid homeostasis by improving mitochondrial function. Taken together, our study reveals an effect of oligostilbenes on lipid metabolism in C2C12 cells, and VitAOG and VitA can be regarded as potential candidates for the treatment of obesity and other disorders of lipid metabolism.

Metabolic changes and propensity for inflammation, fibrosis, and cancer in livers of mice lacking lysosomal acid lipase.

Lysosomal acid lipase (LAL) is the sole lysosomal enzyme responsible for the degradation of cholesteryl esters and triacylglycerols at acidic pH. Impaired LAL activity leads to LAL deficiency (LAL-D), a severe and fatal disease characterized by ectopic lysosomal lipid accumulation. Reduced LAL activity also contributes to the development and progression of non-alcoholic fatty liver disease (NAFLD). To advance our understanding of LAL-related liver pathologies, we performed comprehensive proteomic profiling of livers from mice with systemic genetic loss of LAL (Lal-/-) and from mice with hepatocyte-specific LAL-D (hepLal-/-). Lal-/- mice exhibited drastic proteome alterations, including dysregulation of multiple proteins related to metabolism, inflammation, liver fibrosis, and cancer. Global loss of LAL activity impaired both acidic and neutral lipase activities and resulted in hepatic lipid accumulation, indicating a complete metabolic shift in Lal-/- livers. Hepatic inflammation and immune cell infiltration were evident, with numerous upregulated inflammation-related gene ontology biological process terms. In contrast, both young and mature hepLal-/- mice displayed only minor changes in the liver proteome, suggesting that loss of LAL solely in hepatocytes does not phenocopy metabolic alterations observed in mice globally lacking LAL. These findings provide valuable insights into the mechanisms underlying liver dysfunction in LAL-D and may help in understanding why decreased LAL activity contributes to NAFLD. Our study highlights the importance of LAL in maintaining liver homeostasis and demonstrates the drastic consequences of its global deficiency on the liver proteome and liver function.

A Form of Metabolic-Associated Fatty Liver Disease Associated with a Novel LIPA Variant.

BACKGROUND: The LIPA gene on chromosome 10q23.31 contains 10 exons and encodes lipase A, the lysosomal acid lipase (LAL) containing 399 amino acids. Pathogenic variants in the LIPA result in autosomal recessive Wolman disease and cholesteryl ester storage disease (CESD). Here, we report a novel missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of LIPA in an Iranian family with fatty liver disease identified by whole-exome sequencing and confirmed by Sanger sequencing. METHODS: A 28-year-old woman referred with lean NASH cirrhosis and extremely high cholesterol levels. Fatty liver disease was found in six of her family members using vibration-controlled transient elastography (VCTE). Baseline routine laboratory tests were performed and whole-exome sequencing and confirmation by Sanger sequencing were done. RESULTS: The index case had severe dyslipidemia and cirrhosis despite a body mass index of 21.09 kg/m2 . Six other family members had dyslipidemia and fatty liver or cirrhosis. A homozygous missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of LIPA which caused LAL-D was found to be associated with fatty liver disease and/or cirrhosis. CONCLUSION: A homozygous missense variant (NM_001127605.3:c.928T>A, p.Trp310Arg) of the LIPA gene which caused LAL-D was found to be associated with dyslipidemia, fatty liver disease and/or cirrhosis in six members of an Iranian family. These results should be confirmed by functional studies and extending the study to at least three families.

Enzymatic Characterization of a Novel HSL Family IV Esterase EstD04 from Pseudomonas sp. D01 in Mealworm Gut Microbiota.

Pseudomonas sp. D01, capable of growing in tributyrin medium, was isolated from the gut microbiota of yellow mealworm. By using in silico analyses, we discovered a hypothesized esterase encoding gene in the D01 bacterium, and its encoded protein, EstD04, was classified as a bacterial hormone-sensitive lipase (bHSL) of the type IV lipase family. The study revealed that the recombinant EstD04-His(6x) protein exhibited esterase activity and broad substrate specificity, as it was capable of hydrolyzing p-nitrophenyl derivatives with different acyl chain lengths. By using the most favorable substrate p-nitrophenyl butyrate (C4), we defined the optimal temperature and pH value for EstD04 esterase activity as 40 °C and pH 8, respectively, with a catalytic efficiency (kcat/Km) of 6.17 × 103 mM-1 s-1 at 40 °C. EstD04 demonstrated high stability between pH 8 and 10, and thus, it might be capably used as an alkaline esterase in industrial applications. The addition of Mg2+ and NH4+, as well as DMSO, could stimulate EstD04 enzyme activity. Based on bioinformatic motif analyses and tertiary structural simulation, we determined EstD04 to be a typical bHSL protein with highly conserved motifs, including a triad catalytic center (Ser160, Glu253, and His283), two cap regions, hinge sites, and an oxyanion hole, which are important for the type IV enzyme activity. Moreover, the sequence analysis suggested that the two unique discrete cap regions of EstD04 may contribute to its alkali mesophilic nature, allowing EstD04 to exhibit extremely distinct physiological properties from its evolutionarily closest esterase.

Exercise Equals the Mobilization of Visceral versus Subcutaneous Adipose Fatty Acid Molecules in Fasted Rats Associated with the Modulation of the AMPK/ATGL/HSL Axis.

Combining exercise with fasting is known to boost fat mass-loss, but detailed analysis on the consequential mobilization of visceral and subcutaneous WAT-derived fatty acids has not been performed. In this study, a subset of fasted male rats (66 h) was submitted to daily bouts of mild exercise. Subsequently, by using gas chromatography-flame ionization detection, the content of 22 fatty acids (FA) in visceral (v) versus subcutaneous (sc) white adipose tissue (WAT) depots was compared to those found in response to the separate events. Findings were related to those obtained in serum and liver samples, the latter taking up FA to increase gluconeogenesis and ketogenesis. Each separate intervention reduced scWAT FA content, associated with increased levels of adipose triglyceride lipase (ATGL) protein despite unaltered AMP-activated protein kinase (AMPK) Thr172 phosphorylation, known to induce ATGL expression. The mobility of FAs from vWAT during fasting was absent with the exception of the MUFA 16:1 n-7 and only induced by combining fasting with exercise which was accompanied with reduced hormone sensitive lipase (HSL) Ser563 and increased Ser565 phosphorylation, whereas ATGL protein levels were elevated during fasting in association with the persistently increased phosphorylation of AMPK at Thr172 both during fasting and in response to the combined intervention. As expected, liver FA content increased during fasting, and was not further affected by exercise, despite additional FA release from vWAT in this condition, underlining increased hepatic FA metabolism. Both fasting and its combination with exercise showed preferential hepatic metabolism of the prominent saturated FAs C:16 and C:18 compared to the unsaturated FAs 18:1 n-9 and 18:2 n-6:1. In conclusion, depot-specific differences in WAT fatty acid molecule release during fasting, irrelevant to their degree of saturation or chain length, are mitigated when combined with exercise, to provide fuel to surrounding organs such as the liver which is correlated with increased ATGL/ HSL ratios, involving AMPK only in vWAT.

Paeonia lactiflora root decreases lipid accumulation through the induction of lipolysis and thermogenesis via AMPK activation in 3T3­L1 cells.

Obesity is associated with high risk of mortality globally because obesity is associated with development of diseases such as diabetes, dyslipidemia, fatty liver disease, hypertension, and cancer. The present study aimed to identify the mechanism of action related to the anti­obesity activity of Paeonia lactiflora root (PLR) based on its effects on lipid droplet accumulation. The inhibitory activity on lipid accumulation was analyzed through Oil­Red O staining, and the changes in levels of lipid accumulation­related proteins were analyzed using Western blot analysis. And the contents of triacylglycerol and free glycerol were analyzed using an ELISA Kit. PLR significantly inhibited the accumulation of lipid droplets and triacylglycerol in differentiating 3T3­L1 cells. PLR increased phosphorylated­hormone sensitive lipase (HSL), HSL and adipose triglyceride lipase (ATGL) and decreases perilipin­1 in differentiating and fully differentiated 3T3­L1 cells. Furthermore, treatment of fully differentiated 3T3­L1 cells with PLR resulted in increased free glycerol levels. PLR treatment increased levels of peroxisome proliferator­activated receptor­gamma coactivator­1 alpha (PGC­1α), PR domain containing 16 (PRDM16) and uncoupling protein 1 (UCP­1) in both differentiating and fully differentiated 3T3­L1 cells. However, the PLR­mediated increase in lipolytic, such as ATGL and HSL, and thermogenic factors, such as PGC­1a and UCP­1, were decreased by inhibition of AMP­activated protein kinase (AMPK) with Compound C. Taken together, these results suggest that PLR exerted anti­obesity effects by regulating lipolytic and thermogenic factors via AMPK activation. Therefore, the present study provided evidence that PLR is a potential natural agent for the development of drugs to control obesity.

Habitual nappers and non-nappers differ in circadian rhythms of LIPE expression in abdominal adipose tissue explants.

Background and purpose: Napping is a widespread practice worldwide and has in recent years been linked to increased abdominal adiposity. Lipase E or LIPE encodes the protein hormone-sensitive lipase (HSL), an enzyme that plays an important role in lipid mobilization and exhibits a circadian expression rhythm in human adipose tissue. We hypothesized that habitual napping may impact the circadian expression pattern of LIPE, which in turn may attenuate lipid mobilization and induce abdominal fat accumulation. Methods: Abdominal adipose tissue explants from participants with obesity (n = 17) were cultured for a 24-h duration and analyzed every 4 h. Habitual nappers (n = 8) were selected to match non-nappers (n = 9) in age, sex, BMI, adiposity, and metabolic syndrome traits. Circadian LIPE expression rhythmicity was analyzed using the cosinor method. Results: Adipose tissue explants exhibited robust circadian rhythms in LIPE expression in non-nappers. In contrast, nappers had a flattened rhythm. LIPE amplitude was decreased in nappers as compared with non-nappers (71% lower). The decrease in amplitude among nappers was related to the frequency of napping (times per week) where a lower rhythm amplitude was associated with a higher napping frequency (r = -0.80; P = 0.018). Confirmatory analyses in the activity of LIPE's protein (i.e., HSL) also showed a significant rhythm in non-nappers, whereas significance in the activity of HSL was lost among nappers. Conclusion: Our results suggest that nappers display dysregulated circadian LIPE expression as well as dysregulated circadian HSL activity, which may alter lipid mobilization and contribute to increased abdominal obesity in habitual nappers.