Protocol for using artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins.

Here, we present a protocol to construct artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins. We provide procedures to construct adiposomes and prepare recombinant lipid droplet-associated proteins. Then we describe approaches to measure the number density of perilipin 2 on natural lipid droplets, construct artificial lipid droplets, and determine the binding affinity of perilipin 2 on artificial lipid droplets. This protocol can be adapted to determine the binding properties of various lipid droplet-associated proteins. For complete details on the use and execution of this protocol, please refer to Ma et al. (2021).

Hypothyroidism induces uterine hyperplasia and inflammation related to sex hormone receptors expression in virgin rabbits.

AIMS: In women, uterine alterations have been associated with sex steroid hormones. Sex hormones regulate the expression of thyroid hormone receptors (TRs) in the uterus, but an inverse link is unknown. We analyzed the impact of hypothyroidism on histological characteristics, vascular endothelial growth factor (VEGF-A), progesterone receptors (PR), estrogen receptors (ER), thyroid hormone receptors (TRs), perilipin (PLIN-A), and lipid content in the uterus of virgin rabbits. MAIN METHODS: Twelve Chinchilla-breed adult female rabbits were grouped into control (n = 6) and hypothyroid (n = 6; 0.02% of methimazole for 30 days). The thickness of endometrium and myometrium, number of uterine glands, and infiltration of immune cells were analyzed. The expression of VEGF-A, PR, ERα, and PLIN-A was determined by RT-PCR and western blot. The uterine content of triglycerides (TAG), total cholesterol (TC), and malondialdehyde (MDA) was quantified. KEY FINDINGS: Hypothyroidism promoted uterine hyperplasia and a high infiltration of immune cells into the endometrium, including macrophages CD163+. It also increased the expression of VEGF-A, TRA, and ERα-66 but reduced that of PR and ERα-46. The uterine content of PLIN-A, TAG, and TC was reduced, but that of MDA was augmented in hypothyroid rabbits. SIGNIFICANCE: Our results suggest that uterine hyperplasia and inflammation promoted by hypothyroidism should be related to changes in the VEGF-A, PR, ER, and TRs expression, as well as to modifications in the PLIN-A expression, lipid content, and oxidative status. These results suggest that hypothyroidism should affect the fertility of females.

Influence of expression of UCP3, PLIN1 and PPARG2 on the oxidation of substrates after hypocaloric dietary intervention.

BACKGROUND & AIMS: In addition to environmental and psychosocial factors, it is known that genetic factors can also influence the regulation of energy metabolism, body composition and determination of excess weight. The objective of this study was to evaluate the influence of UCP3, PLIN1 and PPARG2 genes on the substrates oxidation in women with grade III obesity after hypocaloric dietary intervention. SUBJECTS/METHODS: This is a longitudinal study with 21 women, divided into two groups: Intervention Group (G1): 11 obese women (Body Mass Index (BMI) ≥40 kg/m2), and Control Group (G2): 10 eutrophic women (BMI between 18.5 kg/m2 and 24.9 kg/m2). Weight (kg), height (m), BMI (kg/m2), substrate oxidation (by Indirect Calorimetry) and abdominal subcutaneous adipose tissue were collected before and after the intervention. For the dietary intervention, the patients were hospitalized for 6 weeks receiving 1200 kcal/day. RESULTS: There was a significant weight loss (8.4 ± 4.3 kg - 5.2 ± 1.8%) and reduction of UCP3 expression after hypocaloric dietary intervention. There was a positive correlation between carbohydrate oxidation and UCP3 (r = 0.609; p = 0.04), PLIN1 (r = 0.882; p = 0.00) and PPARG2 (r = 0.791; p = 0.00) expression before dietary intervention and with UCP3 (r = 0.682; p = 0.02) and PLIN1 (r = 0.745; p = 0.00) genes after 6 weeks of intervention. There was a negative correlation between lipid oxidation and PLIN1 (r = -0.755; p = 0.00) and PPARG2 (r = 0.664; p = 0.02) expression before dietary intervention and negative correlation with PLIN1 (r = 0.730; p = 0.02) expression after 6 weeks of hypocaloric diet. CONCLUSION: Hypocaloric diet reduces UCP3 expression in individuals with obesity and the UCP3, PLIN1 and PPARG2 expression correlate positively with carbohydrate oxidation and negatively with lipid oxidation.

Prognostic significance of PLIN1 expression in human breast cancer.

Breast cancer is a heterogeneous disease associated with diverse clinical, biological and molecular features, presenting huge challenges for prognosis and treatment. Here we found that perilipin-1 (PLIN1) mRNA expression is significantly downregulated in human breast cancer. Kaplan-Meier analysis indicated that patients presenting with reduced PLIN1 expression exhibited poorer overall metastatic relapse-free survival (p = 0.03). Further Cox proportional hazard models analysis revealed that the reduced expression of PLIN1 is an independent predictor of overall survival in estrogen receptor positive (p < 0.0001, HR = 0.87, 95% CI = 0.81-0.92, N = 3,600) and luminal A-subtype (p = 0.02, HR = 0.88, 95% CI = 0.78-0.98, N = 1,469) breast cancer patients. We also demonstrated that the exogenous expression of PLIN1 in human breast cancer MCF-7 and MDA-MB-231 cells significantly inhibits cell proliferation, migration, invasion and in vivo tumorigenesis in mice. Together, these data provide novel insights into a prognostic significance of PLIN1 in human breast cancer and reveal a potentially new gene therapy target for breast cancer.