Perilipin 1 Antibodies in Patients With Acquired Generalized Lipodystrophy

Perilipin 1 is an intracellular lipid droplet surface protein almost exclusively present in white adipocytes. Genetic variants in the PLIN1 gene have previously been associated with partial lipodystrophy and fat distribution. Now two groups report on the presence of perilipin 1 antibodies in patients with acquired generalized lipodystrophy (AGL). The data suggest that the antibodies are a useful diagnostic biomarker for a subset of AGL cases and hint at the possibility that they might be involved in mediating the lipodystrophy, though further work will be required to robustly establish the latter.

Perilipin 1 is one of five perilipin proteins found in humans (1). Among these it is the one most exclusively expressed in adipocytes with the greatest affinity for association with the surface of lipid droplets. Here it is thought to be involved in stabilizing the phospholipid surface monolayer of the droplet as well as playing a key role in regulating lipolysis, the sequential series of enzymatic reactions that release fatty acids from triacylglycerol stored within the droplet. More specifically, perilipin 1 binds and sequesters ABHD5 (abhydrolase domain containing 5) a key activator of adipose triglyceride lipase (ATGL), thereby inhibiting basal lipolysis and priming the adipocyte for a response to lipolytic stimuli (2). Lipolytic simulation typically involves β-adrenergic GPCR (G-protein–coupled receptor) signaling, which activates protein kinase A (PKA). PKA phosphorylates perilipin 1 on several sites, leading to 1) the release of ABHD5, which duly activates ATGL, and 2) activation of hormone-sensitive lipase, which catalyzes the second step in triacylglycerol hydrolysis (1) (Fig. 1).

Robust links between perilipin 1 and human metabolic disease first emerged from the discovery that heterozygous frameshift mutations affecting the COOH terminus of the protein cosegregated with a partial lipodystrophy phenotype (3,4). However, this relationship is not as straightforward as it initially appeared, as subsequent work by Laver et al. (5) suggested that other heterozygous frameshift mutations predicted to result in nonsense-mediated decay, and thus effectively in loss of perilipin expression from one of the two PLIN1 alleles, were not associated with a lipodystrophic phenotype. Very recently results of gene burden analyses in the UK Biobank study have confirmed the latter finding and have in fact revealed that heterozygous loss-of-function mutations, most of which are expected to result in nonsense-mediated decay, are in fact associated with a favorable impact on fat distribution (or waist-to-hip ratio) and reduced risk of metabolic disease (6,7).

Two articles in this issue of Diabetes (8,9) now suggest that antibodies targeted against perilipin 1 are present in some patients with AGL. The existence of perilipin 1 antibodies was first reported by Corvillo et al. (10) in 2018. In that earlier publication, the authors detected perilipin 1 antibodies in three of five patients with AGL. Collectively these data suggest that perilipin 1 antibodies constitute a useful biomarker for AGL and may even be involved in the pathogenesis of many cases of AGL. So how convincing are the data?

First, are the antibodies really targeted against perilipin 1, widely considered to be an exclusively intracellular protein? The antibodies were originally shown to bind to a protein present in human adipocyte lysates with the molecular weight of perilipin 1 and immunostained the periphery of adipocytes in human adipose samples in a manner consistent with what has been widely reported previously with perilipin antibodies as well as the periphery of lipid droplets in human mesenchymal stem cell–derived adipocytes (10). Corvillo et al. also showed that the antibodies bound to recombinant perilipin 1 and went on to try and identify the antigenic epitopes involved (9). Mandel-Brehm et al. (8) independently identified antibodies that bound to phage-displayed peptides derived from the Plin1 gene in sera from Aire^−/− mice. They validated these antibodies by showing that they immunoprecipitated recombinantly expressed tagged mouse perilipin 1 and that these antibodies colocalized with commercially available Plin1 antibodies around lipid droplets in murine omental fat samples (8). They then detected perilipin 1 antibodies in a patient with AIRE mutations (i.e., autoimmune polyglandular syndrome type 1 [APS1]) and AGL using a radioactive ligand binding assay (8). Collectively these findings provide compelling evidence for the presence of perilipin 1 antibodies, though the work on the target epitopes is somewhat less convincing in my view as perilipin 1 is largely unstructured in solution and so the precision of studies of in vitro binding to peptide fragments will require further validation.

Allied to this point is the issue of how the generation of perilipin 1 antibodies might be triggered? In this regard, the fact that many of the patients, in whom the antibodies were detected, manifested evidence of other autoimmune disorders suggests that they had an underlying inherited predisposition to generating autoantibodies. Interestingly, the only instances where perilipin 1 antibodies were present in patients not reported to have AGL were a few patients with APS1. APS1 is caused by biallelic mutations in the AIRE gene, whose protein product (known as autoimmune regulator) is highly expressed in the thymus where it plays a key role in the development of immune tolerance. Intriguingly, Mandel-Brehm et al. (8) showed that perilipin 1 is expressed in the thymus, suggesting that AIRE deficiency may be involved in priming patients with APS1 to develop perilipin 1 antibodies. Careful follow-up of APS1 cases where lipodystrophy has not been reported would be helpful, as if patients do progress to manifest lipodystrophy, this would at least be consistent with the idea that the antibodies might be involved in causing lipodystrophy rather than simply reflecting its presence. These authors also reported the presence of perilipin 1 antibodies in some Aire null mice that are not known to manifest lipodystrophy, arguing against a primary pathogenic role of the perilipin 1 antibodies present in that species at least. However, further work may be required to establish whether some Aire null mice do manifest lipodystrophy—a phenotype that can be difficult to detect in mice.

Second, are the antibodies useful as a biomarker for AGL? In the original study these antibodies were detected in three of five patients with AGL and it was shown that they were not present in samples from 20 healthy control subjects and 8 patients with acquired partial lipodystrophy, a distinct highly stereotyped disorder characterized by a cephalocaudal pattern of fat loss in patients who may also manifest mesangiocapillary glomerulonephritis type II and whose serum often contains C3 nephritic factor antibodies (11). In their more recent article, Corvillo et al. (9) reported that they detected perilipin 1 antibodies in 20 of 40 cases of AGL, whereas Mandel-Brehm et al. (8) detected them in 17 of 46 cases of AGL (giving a rough prevalence of ∼44% of patients with AGL). Both groups went on to test further healthy control subjects and patients with acquired partial lipodystrophy, all of whom were negative for perilipin 1 antibodies. Similarly, the antibodies were not detected in cohorts of patients with type 1 diabetes, type 2 diabetes, or obesity. Three subtypes of AGL have been described hitherto, namely, those associated with panniculitis, those associated with other autoimmune phenotypes, and an idiopathic cluster. The presence of perilipin 1 antibodies did not precisely overlap with any of these categories and may well preempt refinement of the existing classification in due course. An interesting possibility is that the precise mode of adipocyte cell death in patients with AGL might lead to perilipin “exposure” to the immune system in a manner that triggers antibody formation. If this were the case, careful review and follow-up of affected cases may help with better defining this subtype of AGL.

Third, are the antibodies involved in causing AGL? Corvillo et al. (9) argue that they are involved in the pathogenesis and even suggest that they do so by interfering with the ability of perilipin 1 to tether ABHD5 and thereby inhibit basal lipolysis. Data supporting this assertion include those from epitope mapping studies suggesting that the perilipin 1 antibodies most frequently target a region that incorporates the amino acids previously reported to mediate the interaction between perilipin 1 and ABHD5, in vitro studies suggesting that serum from affected case subjects inhibits the interaction between perilipin 1 and ABHD5, and finally a series of studies in which murine 3T3L1 preadipocytes were incubated with serum from patients with perilipin 1 antibodies. The latter included data suggesting that this serum increased basal lipolysis in some, but not all cases, in addition to confocal images suggesting that the perilipin 1 antibodies might even localize on the surface of lipid droplets in these cells. In my view, these data are not yet wholly convincing for the following reasons. First, I remain concerned about the validity of the in vitro ELISA-based binding studies given the fact that full-length perilipin 1 is insoluble and almost certainly misfolded in these analyses. Second, the authors have yet to explain how the perilipin 1 antibodies could access endogenous perilipin 1 on the surface of a lipid droplet. Third, the clinical pattern of lipodystrophy present in patients with AGL is very different from that reported in patients with partial lipodystrophy due to PLIN1 mutations, and the lipodystrophy in AGL is largely reported to be irreversible. If increased lipolysis were the sole mechanism for the observed phenotype, one might expect the lipodystrophy to reverse to a large extent when antibody levels fall as they typically do in many autoimmune disorders. It is also difficult to reconcile panniculitis, which typically manifests in an initially localized manner, with the presence of circulating antibodies proposed to be triggering increased lipolysis.

In conclusion, these reports strongly suggest that perilipin 1 antibodies are a useful biomarker and diagnostic marker for a subset of AGL cases and may well lead to more precise subclassification of AGL cases in the future. However, further work will be required to establish whether they are causally involved. If they are causally involved, one wonders whether their early detection might prompt therapeutic efforts to remove them or suppress their production prior to the development of generalized lipodystrophy.