Burn control, an adipocyte-specific function for galectin-12

Families of molecules often have similar functions—think of kinases, transcription factors, chemokines, or Toll-like receptors. The galectins, an evolutionarily ancient family of carbohydrate binding proteins found in organisms from multicellular fungi to humans, are different (1, 2). Galectins are a family of lectins; the family designation is based on highly conserved structural features, particularly the conserved carbohydrate recognition domains (CRDs) that define the proteins as lectins. In contrast, the pleiotropic functions of galectins do not seem to be conserved. Based on the cell type investigated, galectins have been reported to regulate cell proliferation, migration, adhesion, differentiation, survival, and death, and they also positively and negatively regulate host defense against microbial pathogens. Extracellular functions of galectins typically depend on the carbohydrate binding properties of the lectins, but many different glycoprotein receptors can be recognized by various galectins if the glycoprotein receptors bear the appropriate glycan ligands, resulting in the vast array of cellular functions triggered by galectin binding. In addition, galectins are also found intracellularly, where for example, protein–protein interactions at the cytoplasmic face of the plasma membrane, nucleus, and mitochondria promote association of galectin-3 with K-Ras, β-catenin, and Bcl-2 family members to regulate oncogenesis and cell death (3–5). In PNAS, the work by Yang et al. (6) reports an unexpected function for a galectin, galectin-12, at an unexpected subcellular location.

Galectins in Adipocytes

The work by Yang et al. (7) identified galectin-12 as a galectin family member in 2001, and the work by Hotta et al. (8) identified expression of galectin-12 in adipocytes shortly thereafter (7, 8). However, except for one paper in 2004 suggesting that galectin-12 regulated preadipocyte differentiation (9), the function of galectin-12 remained unknown. Now, the work by Yang et al. (6) shows that galectin-12 localizes to lipid droplets in adipocytes. Adipocytes lacking galectin-12 had an increased rate of lipolysis, and loss of galectin-12 promoted protein kinase A-dependent recruitment of lipases to lipid droplets. Moreover, galectin-12 null mice had a significant reduction in adiposity, with ∼40% reduction in whole-body lipid content, and reduced adipocyte size, a characteristic determined by the number and volume of lipid droplets in the cell. Galectin-12 deficiency in mice also ameliorated insulin resistance associated with weight gain.

How does galectin-12 participate in lipolysis? Despite the striking physiologic findings in galectin-12 null mice, the precise mechanism of lipolysis control is not clear. However, an important first step in answering this question was to determine where galectin-12 is acting. Although many galectins are both intracellular and

The work by Yang et al. suggests that galectin-12 deficiency increases the efficiency of cAMP signaling at the lipid droplet.

secreted, the work by Yang et al. (6) finds abundant intracellular galectin-12 but insignificant secretion of galectin-12 in adipocytes; thus, galectin-12 must exert its antilipolytic effect from a cytosolic location. Although galectins can be both intracellular and secreted, secreted galectins do not enter the classical secretory pathway but are exported directly from the cytosol to the extracellular milieu (10). Thus, intracellular galectins would never encounter the glycoprotein receptors that secreted galectins could bind on the cell surface, and cytosolic galectins must interact with partners through a mechanism other than glycan binding; as mentioned above, galectins found at intracellular locations, including the cytosolic face of the plasma membrane, nuclei, and mitochondria, interact with partners through protein–protein interactions. In adipocytes, localization of galectin-12 to lipid droplets was not dependent on recognition of a glycan ligand, a not unexpected finding for a cytosolic galectin; however, it is not yet clear with what galectin-12 associates to retain it in a subset of lipid droplets. Intriguingly, the work by Yang et al. (6) shows that the galectin-12 polypeptide, unlike other galectins but similar to perilipin protein, which also localizes to lipid droplets, has several hydrophobic regions, suggesting that galectin-12 could localize to lipid droplets either through protein–protein interactions with other hydrophobic proteins in the droplets or possibly through direct association with lipids. The critical next step in understanding how galectin-12 participates in lipolysis will be to determine how galectin-12 localizes to lipid droplets and identify the molecules at the droplets that directly interact with galectin-12.

The two key physiologic signaling pathways that control lipolysis are the adrenergic receptor–cAMP pathway (which promotes it) and the insulin signaling pathway (which inhibits it). Overall, the work by Yang et al. (6) suggests that galectin-12 deficiency increases the efficiency of cAMP signaling at the lipid droplet, perhaps by altering the recruitment or function of a phosphodiesterase. However, it is premature to conclude that galectin-12 is itself a regulatory protein in the strictest sense. It is not yet clear whether galectin-12 is a regulated component of either the cAMP or insulin pathways. The work by Yang et al. (6) does not show that galectin-12 expression or localization is modulated by lipolytic or antilipolytic stimuli. Therefore, it is possible that galectin-12 functions as a scaffolding protein that is required for proper localization of other proteins that transduce cAMP- and insulin-dependent signals at the lipid droplet.

Galectins As Scaffolding Proteins Outside and Inside the Cell

If galectins localize to many sites and bind many different partners, is there a common theme to their functions? Secreted galectins bind to a variety of glycan ligands on cell surface glycoproteins. Galectins are typically multivalent, and therefore, the relatively low-affinity interaction of a single galectin with a single glycan is multiplied and can result in high-avidity binding to highly glycosylated, abundant cell surface proteins; this type of interaction has been proposed to result in formation of galectin–glycoprotein clusters or lattices that can regulate a range of activities from organization of lipid rafts to control of residence time of glycoprotein receptors on the cell surface to association of accessory molecules with signaling complexes (11, 12). Thus, on the cell surface, galectins act as scaffolding proteins. In the cytosol, K-Ras binds galectin-3 to recruit galectin-3 to the cytosolic face of the plasma membrane; there, multivalent galectin-3 can also act as a scaffolding protein to retain multiple K-Ras molecules in clusters and promote K-Ras activation (13). Therefore, acting as scaffolding proteins may be a common theme for both intracellular and extracellular galectins. Whether galectin-12 at lipid droplets serves as a scaffold or multivalent organizing protein to promote interaction of proteins that control lipolysis remains to be determined. However, it is tempting to speculate that galectin-12, a tandem repeat galectin with N- and C-terminal CRDs connected by a linker peptide, may multimerize into higher-order oligomers, which has been shown for tandem repeat galectins-8 and -9 that oligomerize through protein–protein interactions of the CRDs (14, 15). Such oligomerization would promote a scaffolding or cross-linking function of galectin-12.

Perhaps the nonclassical secretion pathway of galectins evolved to maintain the flexibility of galectins to either stay in the cytosol or be released into the extracellular space, and to traffic to different intracellular sites. However, the factors that control subcellular localization of galectins are very poorly understood. There is one example of a posttranslational modification, phosphorylation of a single serine residue in galectin-3, that appears to regulate mitochondrial vs. nuclear localization of galectin-3 (16), but little else is known that determines where a galectin localizes and whether it is secreted. The work by Yang et al. (6) proposes that the hydrophobic domains of galectin-12 contribute to localization to lipid droplets in adipocytes. In the 2001 paper describing isolation and characterization of galectin-12, galectin-12 transcripts were found in a variety of tissues, including heart, spleen, thymus, and peripheral blood leukocytes (7). The function of galectin-12 in these tissues is not known, but it is clearly not regulation of lipolysis; thus, if galectin-12 is localized intracellularly in these cells and tissues, it must have different binding partners than those binding partners found in lipid droplets.

A Target to Promote Lipolysis?

The observation that galectin-12 deficiency had a profound effect on adiposity in mice fed a standard diet as well as mice challenged with a high-fat diet suggests that, unlike some other galectins, galectin-12 plays a nonredundant role primarily in one type of tissue, fat. Other galectin family members, such as galectin-1, -3, and -9, are expressed in a variety of cell types in many different tissues. Additionally, although KOs of galectins-1, -3, and -9 have been informative regarding the roles of these galectins in immune development and inflammatory disease, these studies typically required some type of cell- or tissue-specific challenge to uncover the effects of galectin deficiency. In contrast, although galectin-12 transcripts have been found in other tissues, galectin-12 seems to have a primary and significant role in controlling lipolysis in adipocytes. Thus, it may be productive to focus future KO experiments on galectins that have expression restricted to a particular cell type, such as galectin-10, which is expressed primarily in eosinophils and may also act as an intracellular scaffolding protein to regulate activity of an associated enzyme (17).

The work by Yang et al. (6) raises several provocative questions. How does galectin-12 localize to lipid droplets in adipocytes, and what are the binding partners at the droplets? Does this interaction involve a scaffolding function of galectin-12, and what does that scaffold look like? What are the structural features of galectin-12 that determine subcellular localization and interactions with binding partners? How does this galectin-12 interaction retard lipolysis, and what is the mechanism of lipolysis activation in the absence of galectin-12? Does galectin-12 have other functions and other scaffolding partners in other cell types? The answers to these questions may inform development of adipocyte-specific antigalectin-12 therapeutics that could increase lipolysis and reduce insulin resistance in the increasing numbers of patients with obesity and type II diabetes. Given the epidemic of obesity and diabetes in developed countries, new strategies to increase the burn would be welcome.