Triglycerides are stored in specialized organelles called lipid droplets. Numerous proteins have been shown to be physically associated with lipid droplets and govern their function. Previously, the protein hypoxia-inducible lipid droplet-associated (HILPDA) was localized to lipid droplets and was suggested to inhibit triglyceride lipolysis in hepatocytes. We confirm the partial localization of HILPDA to lipid droplets and show that HILPDA is highly abundant in adipose tissue, where its expression is controlled by the peroxisome proliferator-activated receptor γ and by β-adrenergic stimulation. Levels of HILPDA markedly increased during 3T3-L1 adipocyte differentiation. Nevertheless, silencing of Hilpda using small interfering RNA or overexpression of Hilpda using adenovirus did not show a clear impact on 3T3-L1 adipogenesis. Following β-adrenergic stimulation, the silencing of Hilpda in adipocytes did not significantly alter the release of nonesterified fatty acids (NEFA) and glycerol. By contrast, adenoviral-mediated overexpression of Hilpda modestly attenuated the release of NEFA from adipocytes following β-adrenergic stimulation. In mice, adipocyte-specific inactivation of Hilpda had no effect on plasma levels of NEFA and glycerol after fasting, cold exposure, or pharmacological β-adrenergic stimulation. In addition, other relevant metabolic parameters were unchanged by adipocyte-specific inactivation of Hilpda. Taken together, we find that HILPDA is highly abundant in adipose tissue, where its levels are induced by peroxisome proliferator-activated receptor γ and β-adrenergic stimulation. In contrast to the reported inhibition of lipolysis by HILPDA in hepatocytes, our data do not support an important direct role of HILPDA in the regulation of lipolysis in adipocytes in vivo and in vitro.

Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor B (NF-B) and c-Jun N-terminal kinase (JNK).The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425-439), which also recruits TRAF2 and activates NF-B. In addition to that, human TNFR2 contains a conserved region (amino acids 338 -379) which is responsible for TRAF2 degradation and therefore of terminating NF-B signaling. TRAF2 degradation and the lack of NF-B activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation. Tumor necrosis factor receptor 2 (TNFR2)5 is one of the two receptors known to bind TNF, this cytokine can be found as a transmembrane (mTNF) or a soluble (sTNF) form. Whereas both mTNF and sTNF activate TNFR1, TNFR2 is mainly activated by mTNF (1, 2), which upon binding to the receptor, causes its trimerization and activation. The fact that mTNF is the optimal activator of TNFR2 has implied serious limitations in the study of this receptor. Because the activation of TNFR2 depends on its aggregation, receptor activation can be forced by its overexpression or by the use of specific antibodies against the receptor (3, 4).TNFR2 lacks any intrinsic catalytic activity within its cytoplasmic tail, thus any signal emerging from the receptor depends on the recruitment of adaptor proteins. TNFR2 can bind directly TRAF2 and through this interaction signals for NF-B and JNK activation, as the expression of a dominant negative form of the adaptor protein (TRAF2dn) can suppress both the activation of NF-B and JNK (5).Seven different TRAF proteins have been identified so far (6). All of them share a highly conserved TRAF domain at the protein C terminus and, with the exception of TRAF1, a N-terminal-RING finger domain followed by five to seven zinc-finger motifs (7,8). TRAF proteins were initially considered as adaptor proteins between TNFRs and the kinases implicated in the activation of JNK or IB kinase IKK (9). It was then described that TRAF proteins, because of their RING finger domain, might act as E3 ubiquitin ligases, which catalyze K63-linked ubiquitination (10). In the case of TRAF2 this requires the interaction with cellular inhibitor of apoptosis 1 (cIAP1) and 2 (cIAP2) (11). More recently, it has been suggested that TRAF2 is by itself unable to ac...

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Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia-inducible lipid droplet-associated protein (HILPDA)/hypoxia-inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL. HILPDA inhibits ATGL activity in a dose-dependent manner with an IC50 value of ∼2 μM. ATGL inhibition depends on the direct physical interaction of both proteins and involves the N-terminal hydrophobic region of HILPDA and the N-terminal patatin domain-containing segment of ATGL. Finally, confocal microscopy combined with Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA and ATGL colocalize and physically interact intracellularly. These findings provide a rational biochemical explanation for the tissue-specific increased TAG accumulation in HILPDA-overexpressing transgenic mouse models.

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