Autophagy in Adipocyte Differentiation.

Adipose tissue regulates metabolic homeostasis by acting as an endocrine organ and energy reservoir. Adipose tissue development and functional maintenance are dependent on adipocyte differentiation, in which autophagy plays an important role. It has been shown that autophagy deficiency dampens adipocyte differentiation, compromises adipose tissue development, dysregulates adipocytokine secretion, and even causes sudden death in young animals. Therefore, accurate assessment of autophagy in adipocyte is critical for the study of adipose biology or pathology of metabolic diseases. In this chapter, we described the procedure of autophagy analysis during adipocyte differentiation, and discussed the power of steady-state autophagy protein (e.g., beclin 1, LC3, and p62) levels versus autophagy flux to reflect autophagy activity.

Estradiol signaling mediates gender difference in visceral adiposity via autophagy.

Excessive adiposity (particularly visceral fat mass) increases the risks of developing metabolic syndrome. Women have lower deposit of visceral fat than men, and this pattern becomes diminished postmenopausally, but the underlying mechanism remains largely unknown. Here, we show that the gender difference in visceral fat distribution is controlled by an estradiol-autophagy axis. In C57BL/6J and wild-type control mice, a higher visceral fat mass was detected in the males than in the females, which was associated with lower expression of estrogen receptor α (ERα) and more active autophagy in males vs. females. However, deletion of ERα normalized autophagy activity and abolished the gender difference in visceral adiposity. In line with the adiposity-reducing effect of the ERα-autophagy axis, we found that downregulation of ERα and increased autophagy activity were required for adipogenesis, while induction of estradiol signaling dampened autophagy and drastically prevented adipogenesis. Mechanistically, the estradiol-ERα signaling activated mTOR, which phosphorylated and inhibited ULK1, thereby suppressing autophagy and adipogenesis. Together, our study suggests that the lower visceral adiposity in the females (vs. the males) arises from a more active estradiol-ERα signaling, which tunes down autophagy and adipogenesis.

Isolation of Mouse Stromal Vascular Cells for Monolayer Culture.

Positive energy balance contributes to adipose tissue expansion and dysfunction, which accounts largely for obesity and related metabolic disorders. Thermogenic fat can dissipate energy, activation or induction of which may promote energy balance and address the pressing health issues. Recent studies have shown that stromal vascular fraction (SVF) from white adipose tissue (WAT) can develop both white and brown-like adipocyte phenotypes, thus serving as a unique model to study adipogenesis and thermogenesis. Here, we describe a protocol for effective isolation of mouse SVF from WAT, induction of differentiation, and detection of adipogenesis. Success tips for isolation and culture of SVF are also discussed.

FoxO1 interacts with transcription factor EB and differentially regulates mitochondrial uncoupling proteins via autophagy in adipocytes.

Mitochondrial uncoupling proteins (UCPs) are inducible and play an important role in metabolic and redox homeostasis. Recent studies have suggested that FoxO1 controls mitochondrial biogenesis and morphology, but it remains largely unknown how FoxO1 may regulate mitochondrial UCPs. Here we show that FoxO1 interacted with transcription factor EB (Tfeb), a key regulator of autophagosome and lysosome, and mediated the expression of UCP1, UCP2 and UCP3 differentially via autophagy in adipocytes. UCP1 was down-regulated but UCP2 and UCP3 were upregulated during adipocyte differentiation, which was associated with increased Tfeb and autophagy activity. However, inhibition of FoxO1 suppressed Tfeb and autophagy, attenuating UCP2 and UCP3 but increasing UCP1 expression. Pharmacological blockade of autophagy recapitulated the effects of FoxO1 inhibition on UCPs. Chromatin immunoprecipitation assay demonstrated that FoxO1 interacted with Tfeb by directly binding to its promoter, and silencing FoxO1 led to drastic decrease in Tfeb transcript and protein levels. These data provide the first line of evidence that FoxO1 interacts with Tfeb to regulate autophagy and UCP expression in adipocytes. Dysregulation of FoxO1→autophagy→UCP pathway may account for metabolic changes in obesity.

Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment.

Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1), was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam) in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker) and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy). The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas). Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.

Mitochondrial Epigenetic Changes Link to Increased Diabetes Risk and Early-Stage Prediabetes Indicator.

Type 2 diabetes (T2D) is characterized by mitochondrial derangement and oxidative stress. With no known cure for T2D, it is critical to identify mitochondrial biomarkers for early diagnosis of prediabetes and disease prevention. Here we examined 87 participants on the diagnosis power of fasting glucose (FG) and hemoglobin A1c levels and investigated their interactions with mitochondrial DNA methylation. FG and A1c led to discordant diagnostic results irrespective of increased body mass index (BMI), underscoring the need of new biomarkers for prediabetes diagnosis. Mitochondrial DNA methylation levels were not correlated with late-stage (impaired FG or A1c) but significantly with early-stage (impaired insulin sensitivity) events. Quartiles of BMI suggested that mitochondrial DNA methylation increased drastically from Q1 (20 < BMI < 24.9, lean) to Q2 (30 < BMI < 34.9, obese), but marginally from Q2 to Q3 (35 < BMI < 39.9, severely obese) and from Q3 to Q4 (BMI > 40, morbidly obese). A significant change was also observed from Q1 to Q2 in HOMA insulin sensitivity but not in A1c or FG. Thus, mitochondrial epigenetic changes link to increased diabetes risk and the indicator of early-stage prediabetes. Further larger-scale studies to examine the potential of mitochondrial epigenetic marker in prediabetes diagnosis will be of critical importance for T2D prevention.

FoxO1 antagonist suppresses autophagy and lipid droplet growth in adipocytes.

Obesity and related metabolic disorders constitute one of the most pressing heath concerns worldwide. Increased adiposity is linked to autophagy upregulation in adipose tissues. However, it is unknown how autophagy is upregulated and contributes to aberrant adiposity. Here we show a FoxO1-autophagy-FSP27 axis that regulates adipogenesis and lipid droplet (LD) growth in adipocytes. Adipocyte differentiation was associated with upregulation of autophagy and fat specific protein 27 (FSP27), a key regulator of adipocyte maturation and expansion by promoting LD formation and growth. However, FoxO1 specific inhibitor AS1842856 potently suppressed autophagy, FSP27 expression, and adipocyte differentiation. In terminally differentiated adipocytes, AS1842856 significantly reduced FSP27 level and LD size, which was recapitulated by autophagy inhibitors (bafilomycin-A1 and leupeptin, BL). Similarly, AS1842856 and BL dampened autophagy activity and FSP27 expression in explant cultures of white adipose tissue. To our knowledge, this is the first study addressing FoxO1 in the regulation of adipose autophagy, shedding light on the mechanism of increased autophagy and adiposity in obese individuals. Given that adipogenesis and adipocyte expansion contribute to aberrant adiposity, targeting the FoxO1-autophagy-FSP27 axis may lead to new anti-obesity options.

Insulin resistance is associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans.

BACKGROUND: Mitochondrial alterations have been observed in subjects with metabolic disorders such as obesity and diabetes. Studies on animal models and cell cultures suggest aberrant glucose and lipid levels, and impaired insulin signaling might lead to mitochondrial changes. However, the molecular mechanism underlying mitochondrial aberrance remains largely unexplored in human subjects. RESULTS: Here we show that the mitochondrial DNA copy number (mtDNAn) was significantly reduced (6.9-fold lower, p < 0.001) in the leukocytes from obese humans (BMI >30). The reduction of mtDNAn was strongly associated with insulin resistance (HOMA-IR: -0.703, p < 0.05; fasting insulin level: -0.015, p < 0.05); by contrast, the correlation between fasting glucose or lipid levels and mtDNAn was not significant. Epigenetic study of the displacement loop (D-loop) region of mitochondrial genome, which controls the replication and transcription of the mitochondrial DNA as well as organization of the mitochondrial nucleoid, revealed a dramatic increase of DNA methylation in obese (5.2-fold higher vs. lean subjects, p < 0.05) and insulin-resistant (4.6-fold higher vs. insulin-sensitive subjects, p < 0.05) individuals. CONCLUSIONS: The reduction of mtDNAn in obese human subjects is associated with insulin resistance and may arise from increased D-loop methylation, suggesting an insulin signaling-epigenetic-genetic axis in mitochondrial regulation.