Perilipin deficiency and autosomal dominant partial lipodystrophy

Abstract

Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.

Figure 1. Cosegregation of Heterozygous Frameshift PLIN1 Mutations with Insulin-Resistant Diabetes, Dyslipidemia, and Partial Lipodystrophy

In Panel A, photographs of Patient 1 (left) and her affected daughters (center and right) reveal the paucity of subcutaneous fat and the muscular appearance. Panel B shows the family pedigrees of Patients 1, 2, and 3 (the patients are designated by arrows), with the family members’ ages and genotypes. N denotes wild type, M1 mutation 1 (PLIN1 p.Leu404AlafsX158), and M2 mutation 2 (PLIN1 p.Val398GlyfsX166). There is complete concordance between features of severe insulin resistance and the presence of the PLIN1 mutations. Heterozygous family members were variably affected by additional features of the metabolic syndrome. Dyslipidemia was defined by triglyceride levels that were higher than 177 mg per deciliter (2 mmol per liter) and high-density-lipoprotein (HDL) cholesterol levels that were less than 39 mg per deciliter (1 mmol per liter). Panel C shows the effects of the PLIN1 p.Leu404AlafsX158 and p.Val398GlyfsX166 frameshift variants on the perilipin protein, indicating the predicted protein kinase A (PKA) sites in human perilipin. In Panel D, immunoblots show perilipin expression in samples of white adipose tissue from Patient 2, for the p.Val398GlyfsX166 mutation, and from Patient 1 and her two daughters, for the p.Leu404AlafsX158 mutation. Extracellular-signal-regulated kinase (ERK) 1/2 was used as a gel-loading control. Samples of abdominal subcutaneous adipose tissue from three healthy, lean, nondiabetic women were used as controls. Immunoblotting was done with N-terminal and C-terminal perilipin antibodies. The mutant isoform was recognized by the N-terminal antibody as a frameshifted band (arrow) just above the 62-kD molecular-weight (MW) marker. This extra band was not detected by the C-terminal antibody. Panel E (left) shows light micrographs of biopsy specimens of subcutaneous abdominal adipose tissue from a lean, healthy control subject and from Patients 1 and 2. Sirius red staining highlights the marked increase in fibrosis and CD68 immunostaining shows the increase in macrophage infiltration of the adipose tissue in the patients. The graphs at the right show the results of analyses that are in keeping with these findings. In the patients, as compared with the control, the fibrosis index (calculated as described in the Supplementary Appendix), transforming growth factor β1 (TGF-β1) messenger RNA (mRNA) levels, and hCD68 mRNA expression, a marker of proinflammatory macrophages, were significantly increased and the average adipocyte diameter (see the Supplementary Appendix) was significantly reduced. Results are given as means ±SD. hPPIA denotes peptidyl-prolyl isomerase A (cyclophilin A), used as an internal control for gene expression.

Figure 2. Functional Properties of the PLIN1 Variants

Preadipocytes (3T3L1) were retrovirally transfected with an empty vector (pBABE-puro or pLXSN [EV]) or with pBABE–wild-type PLIN1, pBABE–PLIN1 p.Leu404AlafsX158, or pBABE–PLIN1 p.Val398GlyfsX166, either alone or in combination with pLXSN–wild-type PLIN1. PLIN1 was N-terminally myc-tagged when expressed in pBABE and FLAG-tagged when expressed in pLXSN. Panel A shows intracellular localization of wild-type and mutant perilipin. Boron-dipyrromethene (BODIPY) was used to stain lipid droplets, and 4′,6′-diamidine-2-phenylindole dihydrochloride (DAPI) is a nuclear stain. Perilipin was stained with an N-terminal perilipin antibody that recognizes both wild-type and mutant perilipin. BODIPY staining suggested that the lipid droplets were smaller in cells that expressed mutant perilipin. In Panel B, automated assessment of lipid-droplet volume from five representative fields confirms the reduced size of lipid droplets in cells expressing the perilipin mutants, with an average of 355 lipid droplets per field. Panel C shows the biochemical measurement of triglyceride levels in preadipocytes expressing wild-type or mutant perilipin in response to treatment with oleic acid. Triglyceride levels were determined in duplicate in lysates from 12 to 18 dishes per cell line. An average of the duplicate measurements was used for each 100-mm dish. Data are mean values for the 12 to 18 dishes. Panel D shows the radiometric assessment of basal lipolysis in preadipocytes treated with ¹⁴C-labeled oleic acid. Data are mean values for nine independent measurements per cell line. In Panels B, C, and D, P<0.001 for the comparisons between empty vector and wild-type cells and between wild-type and mutant cells. Panel E shows triglyceride levels in preadipocytes coexpressing FLAG-tagged wild-type perilipin with myc-tagged wild-type or mutant perilipin. Triglyceride levels were determined in duplicate in lysates from 12 to 18 dishes (100 mm) per cell line. An average of the duplicate measurements was used for each 100-mm dish. Data are mean values for the 12 to 18 dishes. In Panel E, P<0.001 for the comparison between empty vector and FLAG-tagged wild-type cells, P<0.01 for the comparisons between FLAG-tagged and myc-tagged wild-type coexpressing cells and between FLAG-tagged wild-type and myc-tagged p.Leu404fs co-expressing cells, and P<0.001 for the comparisons between FLAG-tagged and myc-tagged wild-type coexpressing cells and between FLAG-tagged wild-type and myc-tagged p.Val398fs coexpressing cells. FLAG-tagged wild-type expressing cells were not statistically different from FLAG-tagged wild-type and myc-tagged mutant coexpressing cells. T bars in Panels B through E indicate standard deviations.