Frequency of rs1051338 and rs116928232 Variants in Individuals from Northwest Mexico.

BACKGROUND: LIPA, situated on chromosome 10q23.2-q23.3, encodes the enzyme lysosomal acid lipase (LAL) (EC 3.1.1.13). Genetic alterations in LIPA lead to lysosomal acid lipase deficiency (LALD), an inborn error causing lipid metabolism anomalies and impairing cholesterol and triacylglyceride degradation. Over 40 LIPA variants have been documented, yet this study focuses on just two. The rs1051338 variant (NM_000235:c.46A>C) affects the signal peptide in Exon 2, whereas rs116928232, located in Exon 8, alters the splice site (NM_000235:c.894G>A), impacting lysosomal acid lipase activity. Considering the diverse clinical manifestations of LALD and the rising hepatic steatosis prevalence in Mexican population, mainly due to diet, these variants were investigated within this demographic to uncover potential contributing factors. This study aimed to reveal the frequency of rs1051338 and rs116928232 among healthy mestizo individuals in Northwest Mexico, marking a significant genetic exploration in this demographic. METHODS: Three hundred ten healthy mestizo individuals underwent PCR-RFLP analysis for both variants, and Sanger sequencing was performed for variant rs116928232. Bioinformatic analysis was also performed to predict protein changes. RESULTS: Allele frequencies for rs1051338 (FA = 0.39, p value = 0.15) and rs116928232 (FA = 0.0016, p value = 0.49) aligned with reported data, while bioinformatic analysis allowed us to identify the protein alteration observed in both variants; finally, the variants showed no linkage between them (normalized D' = 1.03, p value = 0.56). CONCLUSIONS: Allelic frequencies closely matched reported data, and protein structure analysis confirmed variant impacts on LAL enzyme function. Notably, this study marks the first analysis of rs1051338 and rs116928232 in a healthy Mexican mestizo population.

Lipid droplet-associated hydrolase mobilizes stores of liver X receptor sterol ligands and protects against atherosclerosis.

Foam cells in atheroma are engorged with lipid droplets (LDs) that contain esters of regulatory lipids whose metabolism remains poorly understood. LD-associated hydrolase (LDAH) has a lipase structure and high affinity for LDs of foam cells. Using knockout and transgenic mice of both sexes, here we show that LDAH inhibits atherosclerosis development and promotes stable lesion architectures. Broad and targeted lipidomic analyzes of primary macrophages and comparative lipid profiling of atheroma identified a broad impact of LDAH on esterified sterols, including natural liver X receptor (LXR) sterol ligands. Transcriptomic analyzes coupled with rescue experiments show that LDAH modulates the expression of prototypical LXR targets and leads macrophages to a less inflammatory phenotype with a profibrotic gene signature. These studies underscore the role of LDs as reservoirs and metabolic hubs of bioactive lipids, and suggest that LDAH favorably modulates macrophage activation and protects against atherosclerosis via lipolytic mobilization of regulatory sterols.

ANGPTL4 Suppresses Clear Cell Renal Cell Carcinoma via Inhibition of Lysosomal Acid Lipase.

Renal cell carcinoma (RCC), the most common form of kidney cancer, is a heterogeneous disease with clear cell RCC (ccRCC) being the most prevalent and aggressive subtype. While most ccRCC tumors have elevated expression of angiopoietin-like4 (ANGPTL4), in our study we identified a significant subset of patients whose cancers show no increase in ANGPTL4 expression. These patients have a worse prognosis compared to the patients with high expression of ANGPTL4. These ANGPTL4-low cancers are characterized by the increased frequency of wild-type Von Hippel-Lindau(WT VHL), a gene that is commonly mutated in ccRCC, and an enrichment for genes associated with lipid metabolism. Using RCC tumor models with WT VHL, we demonstrate that ANGPTL4 behaves as a tumor suppressor. The loss of ANGPTL4 in ccRCC cell lines results in increased tumor growth and colony formation in a lysosomal acid lipase (LAL)-dependent manner, a phenotype rescued by the expression of N-terminus ANGPTL4. At the mechanistic level, the loss of ANGPTL4 increases LAL activity in ccRCC cells. These data suggest that ANGPTL4 enacts its tumor-suppressive effects in ccRCC by regulating LAL activity. Importantly, the identified patient cohort with low ANGPTL4 expression may exhibit increased reliance on lipid metabolism, which can be a point of target for future therapy. SIGNIFICANCE: Our data indicate angiopoietin-like 4 (ANGPTL4) acts as a tumor suppressor in clear cell renal cell carcinoma via regulating lipid metabolism and identifies a cohort of patients with lower expression of ANGPTL4 that are correlated with shorter survival.

Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis.

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 202 dipeptides and 203 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity.

Evaluation of 73 Enlisted Patients for Liver Transplant with Unknown Etiology Reveals a Late-Diagnosed Case of Lysosomal Acid Lipase Deficiency.

Lysosomal acid lipase deficiency (LALD) varies from a severe infantile-onset form (Wolman disease) to a late-onset form known as cholesteryl ester storage disease (CESD), both of which are autosomal recessive disorders caused by biallelic LIPA pathogenic variants. We evaluated seventy-three patients enlisted for liver transplant (LT) at Instituto da Criança (HCFMUSP-Brazil) who were subjected to LAL activity measurement and LIPA Sanger sequencing analysis, resulting in a positive LALD diagnosis for only one of these individuals. This LALD patient presented recurrent diarrhea, failure to thrive, hepatomegaly, and dyslipidemia at the age of 4 months and liver failure by the age of 13 years. The LALD diagnosis confirmation was conducted at 24 years old due to low levels of LAL enzyme activity. The causal homozygous variant LIPA(NM_000235.4):c.266T>C(p.Leu89Pro) was identified, but the patient had already undergone his first LT at 18 years with several rejection episodes. Despite beginning treatment with sebelipase alfa at 26 years old (total of five infusions), this patient died at 28 years from complications after his second liver transplant. LALD is an important differential diagnosis in cases presenting with hepatomegaly, elevated liver enzymes, and dyslipidemia. Detecting low/absent LAL activity and identifying the LIPA causal variant are essential for diagnosis and specific treatment, as well as for appropriate genetic counseling. Early diagnosis, along with sebelipase alfa therapy, may improve the prognosis of affected patients.

Glycerol Handling in Paired Visceral and Subcutaneous Adipose Tissues in Women with Normal Weight and Upper-Body Obesity.

In adipose tissue, reduced expression of the glycerol channel aquaporin 7 (AQP7) has been associated with increased accumulation of triglyceride. The present study determines the relative protein abundances of lipolytic enzymes, AQP7, and cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in paired mesenteric and omental visceral adipose tissue (VAT) and abdominal and femoral subcutaneous adipose tissue (SAT) in women with either normal weight or upper-body obesity. No differences in the expression of hormone-sensitive lipase (HSL) or AQP7 were found between the two groups in the four depots. The expression of adipocyte triglyceride lipase (ATGL) and HSL were higher in omental VAT and femoral SAT than in mesenteric VAT in both groups of women. Similarly, AQP7 expression was higher in omental VAT than in mesenteric VAT. The expression of PEPCK-C was lower in omental VAT than in femoral SAT. No correlation between the expression of AQP7 and the mean adipocyte size was observed; however, the expression of PEPCK-C positively correlated with the mean adipocyte size. In conclusion, a depot-specific protein expression pattern was found for ATGL, HSL, AQP7, and PEPCK-C. The expression pattern supports that the regulation of AQP7 protein expression is at least in part linked to the lipolytic rate. Furthermore, the results support that the synthesis of glycerol-3-phosphate via glyceroneogenesis contributes to regulating triglyceride accumulation in white adipose tissue in women.

Reducing the lipase LIPE in mutant α-synuclein mice improves Parkinson-like deficits and reveals sex differences in fatty acid metabolism.

Impaired lipid metabolism is a risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) ratio toward aggregation prone monomers. A resultant increase in phospho-serine 129+ αS monomers associating with excess mono- and polyunsaturated fatty acids contributes to the αS aggregation. We previously reported that decreasing the release of monounsaturated fatty acids (MUFAs) by reducing or inhibiting the hormone sensitive lipase (LIPE) reversed pathologic αS phosphorylation and improved soluble αS homeostasis in cultured αS triplication PD neurons and reduced DAergic neurodegeneration in a C.elegans αS model. However, assessing LIPE as a potential therapeutic target for progressive PD motor phenotypes has not been investigated. 3K αS mice, representing a biochemical and neuropathological amplification of the E46K fPD-causing mutation, have decreased αS T:M ratios, lipidic aggregates, and a L-DOPA responsive PD-like motor syndrome. Here, we reduced LIPE by crossings of 3K mice with LIPE null mice, which attenuated motor deficits in male LIPE+/- knockdown (LKD)-3K mice. Heterozygous LIPE reduction was associated with an improved αS T:M ratio, and dopaminergic neurotransmitter levels and fiber densities. In female 3K-LKD mice, an increase in pS129+ and larger lipid droplets (LDs) likely decreased the benefits seen in males. Reducing LIPE decreased MUFA release from neutral lipid storage, thereby reducing MUFA in phospholipid membranes with which αS interacts. Our study highlights fatty acid turnover as a therapeutic target for Lewy body diseases and support LIPE as a promising target in males. LIPE regulation represents a novel approach to mitigate PD and DLB risk and treat disease.

Impaired Fat Absorption from Intestinal Tract in High-Fat Diet Fed Male Mice Deficient in Proglucagon-Derived Peptides.

(1) Background: Proglucagon-derived peptides (PDGPs) including glucagon (Gcg), GLP-1, and GLP-2 regulate lipid metabolism in the liver, adipocytes, and intestine. However, the mechanism by which PGDPs participate in alterations in lipid metabolism induced by high-fat diet (HFD) feeding has not been elucidated. (2) Methods: Mice deficient in PGDP (GCGKO) and control mice were fed HFD for 7 days and analyzed, and differences in lipid metabolism in the liver, adipose tissue, and duodenum were investigated. (3) Results: GCGKO mice under HFD showed lower expression levels of the genes involved in free fatty acid (FFA) oxidation such as Hsl, Atgl, Cpt1a, Acox1 (p < 0.05), and Pparα (p = 0.05) mRNA in the liver than in control mice, and both FFA and triglycerides content in liver and adipose tissue weight were lower in the GCGKO mice. On the other hand, phosphorylation of hormone-sensitive lipase (HSL) in white adipose tissue did not differ between the two groups. GCGKO mice under HFD exhibited lower expression levels of Pparα and Cd36 mRNA in the duodenum as well as increased fecal cholesterol contents compared to HFD-controls. (4) Conclusions: GCGKO mice fed HFD exhibit a lesser increase in hepatic FFA and triglyceride contents and adipose tissue weight, despite reduced ß-oxidation in the liver, than in control mice. Thus, the absence of PGDP prevents dietary-induced fatty liver development due to decreased lipid uptake in the intestinal tract.

Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

Twice weekly dosing with Sebelipase alfa (Kanuma®) rescues severely ill infants with Wolman disease.

BACKGROUND: Sebelipase alfa (Kanuma®) is approved for patients with Wolman disease (WD) at a dosage of 3-5 mg/kg once weekly. Survival rates in the second of two clinical trials was greater, despite recruiting more severely ill patients, probably related to higher initial and maximal doses. We aimed to evaluate the effective pharmacokinetics and pharmacodynamics of Sebelipase alfa when administered to patients with severe WD at 5 mg/kg twice weekly, an intensive regimen which was not assessed in the trials. METHODS: We recruited 3 patients receiving Sebelipase alfa 5 mg/kg twice weekly. We measured LAL activity in leukocytes and plasma oxysterol concentration in two patients and LAL activity in fibroblasts in one patient. Clinical follow up was also assessed. RESULTS: Analyses of LAL activity and oxysterols demonstrate that there is short-lived enzyme activity post-dosing which is associated with the release of stored lipids. Clinical data demonstrate that 5 mg/kg twice weekly dosing is well tolerated and effective. CONCLUSION: 5 mg/kg twice weekly dosing with Sebelipase alfa rescues severely ill infants with WD by increasing substrate clearance. There is biologically relevant lipid accumulation in the 'trough' periods before the next dosing, even with this intensive regimen.

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.

Pedigree Analysis of Nonclassical Cholesteryl Ester Storage Disease with Dominant Inheritance in a LIPA I378T Heterozygous Carrier.

BACKGROUND: Cholesterol ester storage disorder (CESD; OMIM: 278,000) was formerly assumed to be an autosomal recessive allelic genetic condition connected to diminished lysosomal acid lipase (LAL) activity due to LIPA gene abnormalities. CESD is characterized by abnormal liver function and lipid metabolism, and in severe cases, liver failure can occur leading to death. In this study, one Chinese nonclassical CESD pedigree with dominant inheritance was phenotyped and analyzed for the corresponding gene alterations. METHODS: Seven males and eight females from nonclassical CESD pedigree were recruited. Clinical features and LAL activities were documented. Whole genome Next-generation sequencing (NGS) was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations, and qPCR detected LIPA mRNA expression. RESULTS: Eight individuals of the pedigree were speculatively thought to have CESD. LAL activity was discovered to be lowered in four living members of the pedigree, but undetectable in the other four deceased members who died of probable hepatic failure. Three of the four living relatives had abnormal lipid metabolism and all four had liver dysfunctions. By liver biopsy, the proband exhibited diffuse vesicular fatty changes in noticeably enlarged hepatocytes and Kupffer cell hyperplasia. Surprisingly, only a newly discovered heterozygous mutation, c.1133T>C (p. Ile378Thr) on LIPA, was found by gene sequencing in the proband. All living family members who carried the p.I378T variant displayed reduced LAL activity. CONCLUSIONS: Phenotypic analyses indicate that this may be an autosomal dominant nonclassical CESD pedigree with a LIPA gene mutation.

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.

Kinetic and thermodynamic-based studies on the interaction mechanism of novel R. roxburghii seed peptides against pancreatic lipase and cholesterol esterase.

Pancreatic lipase (PL) and cholesterol esterase (CE) are vital digestive enzymes that regulate lipid digestion. Three bioactive peptides (LFCMH, RIPAGSPF, YFRPR), possessing enzyme inhibitory activities, were identified in the seed proteins of R. roxburghii. It is hypothesized that these peptides could inhibit the activities of these enzymes by binding to their active sites or altering their conformation. The results showed that LFCMH exhibited superior inhibitory activity against these enzymes compared to the other peptides. The inhibition mechanisms of the three peptides were identified as either competitive or mixed, according to inhibition models. Further studies have shown that peptides could bind to the active sites of enzymes, thus affecting their spatial conformation and restricting substrate entry into the active site. Molecular simulation further proved that hydrogen bonds and hydrophobic interactions played a vital role in the binding of peptides to enzymes. This study enriches our understanding of interaction mechanisms of peptides on PL and CE.

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.