Cooperative lipolytic control of neuronal triacylglycerol by spastic paraplegia-associated enzyme DDHD2 and ATGL.

Intracellular lipolysis-the enzymatic breakdown of lipid droplet-associated triacylglycerol (TAG)-depends on the cooperative action of several hydrolytic enzymes and regulatory proteins, together designated as lipolysome. Adipose triglyceride lipase (ATGL) acts as a major cellular TAG hydrolase and core effector of the lipolysome in many peripheral tissues. Neurons initiate lipolysis independently of ATGL via DDHD domain-containing 2 (DDHD2), a multifunctional lipid hydrolase whose dysfunction causes neuronal TAG deposition and hereditary spastic paraplegia. Whether and how DDHD2 cooperates with other lipolytic enzymes is currently unknown. In this study, we further investigated the enzymatic properties and functions of DDHD2 in neuroblastoma cells and primary neurons. We found that DDHD2 hydrolyzes multiple acylglycerols in vitro and substantially contributes to neutral lipid hydrolase activities of neuroblastoma cells and brain tissue. Substrate promiscuity of DDHD2 allowed its engagement at different steps of the lipolytic cascade: In neuroblastoma cells, DDHD2 functioned exclusively downstream of ATGL in the hydrolysis of sn-1,3-diacylglycerol (DAG) isomers but was dispensable for TAG hydrolysis and lipid droplet homeostasis. In primary cortical neurons, DDHD2 exhibited lipolytic control over both, DAG and TAG, and complemented ATGL-dependent TAG hydrolysis. We conclude that neuronal cells use noncanonical configurations of the lipolysome and engage DDHD2 as dual TAG/DAG hydrolase in cooperation with ATGL.

Impact of (intestinal) LAL deficiency on lipid metabolism and macrophage infiltration.

OBJECTIVE: To date, the only enzyme known to be responsible for the hydrolysis of cholesteryl esters and triacylglycerols in the lysosome at acidic pH is lysosomal acid lipase (LAL). Lipid malabsorption in the small intestine (SI), accompanied by macrophage infiltration, is one of the most common pathological features of LAL deficiency. However, the exact role of LAL in intestinal lipid metabolism is still unknown. METHODS: We collected three parts of the SI (duodenum, jejunum, ileum) from mice with a global (LAL KO) or intestine-specific deletion of LAL (iLAL KO) and corresponding controls. RESULTS: We observed infiltration of lipid-associated macrophages into the lamina propria, where neutral lipids accumulate massively in the SI of LAL KO mice. In addition, LAL KO mice absorb less dietary lipids but have accelerated basolateral lipid uptake, secrete fewer chylomicrons, and have increased fecal lipid loss. Inflammatory markers and genes involved in lipid metabolism were overexpressed in the duodenum of old but not in younger LAL KO mice. Despite the significant reduction of LAL activity in enterocytes of enterocyte-specific (iLAL) KO mice, villous morphology, intestinal lipid concentrations, expression of lipid transporters and inflammatory genes, as well as lipoprotein secretion were comparable to control mice. CONCLUSIONS: We conclude that loss of LAL only in enterocytes is insufficient to cause lipid deposition in the SI, suggesting that infiltrating macrophages are the key players in this process.

Small-Molecule Inhibitors Targeting Lipolysis in Human Adipocytes.

Chronically elevated circulating fatty acid levels promote lipid accumulation in nonadipose tissues and cause lipotoxicity. Adipose triglyceride lipase (ATGL) critically determines the release of fatty acids from white adipose tissue, and accumulating evidence suggests that inactivation of ATGL has beneficial effects on lipotoxicity-driven disorders including insulin resistance, steatohepatitis, and heart disease, classifying ATGL as a promising drug target. Here, we report on the development and biological characterization of the first small-molecule inhibitor of human ATGL. This inhibitor, designated NG-497, selectively inactivates human and nonhuman primate ATGL but not structurally and functionally related lipid hydrolases. We demonstrate that NG-497 abolishes lipolysis in human adipocytes in a dose-dependent and reversible manner. The combined analysis of mouse- and human-selective inhibitors, chimeric ATGL proteins, and homology models revealed detailed insights into enzyme-inhibitor interactions. NG-497 binds ATGL within a hydrophobic cavity near the active site. Therein, three amino acid residues determine inhibitor efficacy and species selectivity and thus provide the molecular scaffold for selective inhibition.

Metabolic regulation of the lysosomal cofactor bis(monoacylglycero)phosphate in mice.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid, is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endolysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and parenchymal and nonparenchymal cells. Tissue samples were obtained from fed, fasted, 2 h refed, and insulin-treated mice, as well as from mice housed at 5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles that were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels, indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue- and cell-type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP plays a role in the functional adaption to nutrient starvation and ambient temperature.