Project description:Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.
Project description:Global obesity rates have reached pandemic proportions, increasing the risk of metabolic complications for hundreds of millions of individuals worldwide. Gaining insight on adipose tissue biology and understanding how fat pads behave during obesity is critical to investigate metabolic syndromes. Elucidation of cellular signaling pathways engaged by adipose tissue both in health and disease requires standardized protocols for protein extraction that yield consistently pure samples. A recurrent problem of currently available protocols is lipid or detergent contamination in extracted protein samples, which renders protein quantification inaccurate and, as a consequence, consistency and reproducibility of protein loading become unreliable. To overcome this problem, we improved the process of adipose tissue protein extraction by improving tissue lysis and decreasing lipid contamination. Here we describe the Removal of Excess Lipids (RELi) protocol to obtain increased yields of total proteins extracted from adipose tissue. The RELi protocol allows accurate and reproducible adipose tissue sample preparation for Western blot analysis and other investigative techniques requiring adipose tissue-derived proteins.
Project description:Several studies suggest a role of extracellular adenine nucleotides in regulating adipose tissue functions via the purinergic signaling network. Metabolic studies in mice with global deletion of the purinergic receptor P2X7 on the C57BL/6 background indicate that this receptor has only a minor role in adipose tissue for diet-induced inflammation or cold-triggered thermogenesis. However, recent data show that a polymorphism (P451L) present in C57BL/6 mice attenuates P2X7 receptor function, whereas BALB/c mice express the fully functional P451 allele. To determine the potential role of P2rx7 under metabolic and thermogenic stress conditions, we performed comparative studies using male P2rx7 knockout (KO) and respective wild-type controls on both BALB/c and C57BL/6 backgrounds. Our data show that adipose P2rx7 mRNA levels are increased in obese mice. Moreover, P2rx7 deficiency results in reduced levels of circulating CCL2 and IL6 with a moderate effect on gene expression of pro-inflammatory markers in white adipose tissue and liver of BALB/c and C57BL/6 mice. However, P2X7 expression does not alter body weight, insulin resistance, and hyperglycemia associated with high-fat diet feeding on both genetic backgrounds. Furthermore, deficiency of P2rx7 is dispensable for energy expenditure at thermoneutral and acute cold exposure conditions. In summary, these data show that-apart from a moderate effect on inflammatory cytokines-P2X7 plays only a minor role in inflammatory and thermogenic effects of white and brown adipose tissue even on the BALB/c background.
Project description:We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms.
Project description:Type 2 diabetes (T2D) is characterized by a general dysregulation of postprandial energy substrate partitioning. Although classically described in regard to glucose metabolism, it is now evident that metabolic inflexibility of plasma lipid fluxes is also present in T2D. The organ that is most importantly involved in the latter metabolic defect is the white adipose tissue (WAT). Both catecholamine-induced nonesterified fatty acid mobilization and insulin-stimulated storage of meal fatty acids are impaired in many WAT depots of insulin-resistant individuals. Novel molecular imaging techniques now demonstrate that these defects are linked to increased dietary fatty acid fluxes toward lean organs and myocardial dysfunction in humans. Recent findings also demonstrate functional abnormalities of brown adipose tissues in T2D, thus suggesting that a generalized adipose tissue dysregulation of energy storage and dissipation may be at play in the development of lean tissue energy overload and lipotoxicity.
Project description:Cyclin-dependent kinases (CDKs) are emerging regulators of adipose tissue metabolism. Here we aimed to explore the role of CDK7 in thermogenic fat. We found that CDK7 brown adipose tissue (BAT)-specific knockout mice (Cdk7bKO) have decreased BAT mass and impaired β3-adrenergic signaling and develop hypothermia upon cold exposure. We found that loss of CDK7 in BAT disrupts the induction of thermogenic genes in response to cold. However, Cdk7bKO mice do not show systemic metabolic dysfunction. Increased expression of genes of the creatine metabolism compensates for the heat generation in the BAT of Cdk7bKO mice in response to cold. Finally, we show that CDK7 is required for beta 3-adrenergic agonist-induced browning of white adipose tissue (WAT). Indeed, Cdk7 ablation in all adipose tissues (Cdk7aKO) has impaired browning in WAT. Together, our results demonstrate that CDK7 is an important mediator of beta-adrenergic signaling in thermogenic brown and beige fat.
Project description:Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP-based cell lineage tracing in mice, we have identified a population of aP2-expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2-lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFR?. When isolated and grown in culture, the aP2-expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild-type mice, the aP2-lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.
Project description:Classical brown adipocytes such as those found in interscapular brown adipose tissue (iBAT) represent energy-burning cells, which have been postulated to play a pivotal role in energy metabolism. Brown adipocytes can also be found in white adipose tissue (WAT) depots [e.g., inguinal WAT (iWAT)] following adrenergic stimulation, and they have been referred to as "beige" adipocytes. Whether the presence of these adipocytes, which gives iWAT a beige appearance, can confer a white depot with some thermogenic activity remains to be seen. In consequence, we designed the present study to investigate the metabolic activity of iBAT, iWAT, and epididymal white depots in mice. Mice were either 1) kept at thermoneutrality (30°C), 2) kept at 30°C and treated daily for 14 days with an adrenergic agonist [CL-316,243 (CL)], or 3) housed at 10°C for 14 days. Metabolic activity was assessed using positron emission tomography imaging with fluoro-[(18)F]deoxyglucose (glucose uptake), fluoro-[(18)F]thiaheptadecanoic acid (fatty acid uptake), and [(11)C]acetate (oxidative activity). In each group, substrate uptakes and oxidative activity were measured in anesthetized mice in response to acute CL. Our results revealed iBAT as a major site of metabolic activity, which exhibited enhanced glucose and nonesterified fatty acid uptakes and oxidative activity in response to chronic cold and CL. On the other hand, beige adipose tissue failed to exhibit appreciable increase in oxidative activity in response to chronic cold and CL. Altogether, our results suggest that the contribution of beige fat to acute-CL-induced metabolic activity is low compared with that of iBAT, even after sustained adrenergic stimulation.
Project description:Gene expression profiling of supraclavicular brown, interscapular brown, inguinal white, and epididymal white adipose tissues from C57BL/6 male mice was performed by RNA-sequencing.
Project description:Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.