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  • Quantification of lipid droplets and associated proteins in cellular models of obesity via high-content/high-throughput microscopy and automated image analysis. 19895345

    Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-(c) agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc's CyteSeer((R)) image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z' values of 0.54-0.71). Regarding colocalization with lipid droplets, Pearson's correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and -0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z's greater than 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.
    Document Type:
    Reference
    Product Catalog Number:
    AP180C
    Product Catalog Name:
    Donkey Anti-Goat IgG Antibody, Cy3 conjugate, Species Adsorbed

  • Ablation of TRIP-Br2, a regulator of fat lipolysis, thermogenesis and oxidative metabolism, prevents diet-induced obesity and insulin resistance. 23291629

    Obesity develops as a result of altered energy homeostasis favoring fat storage. Here we describe a new transcription co-regulator for adiposity and energy metabolism, SERTA domain containing 2 (TRIP-Br2, also called SERTAD2). TRIP-Br2-null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of these knockout mice showed greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and β3-adrenergic (Adrb3) receptors. The knockout mice also have higher energy expenditure because of increased adipocyte thermogenesis and oxidative metabolism caused by upregulating key enzymes in their respective processes. Our data show that a cell-cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data, together with the observation that TRIP-Br2 expression is selectively elevated in visceral fat in obese humans, suggests that this transcriptional co-regulator is a new therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.
    Document Type:
    Reference
    Product Catalog Number:
    17-409
    Product Catalog Name:
    EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

  • Coordination of lipid droplet-associated proteins during the transition period of Holstein dairy cows. 21426973

    Dairy cows often experience negative energy balance with the onset of lactation, and severe or prolonged negative energy balance can contribute to declines in overall fitness. Energy stores, in the form of adipose tissue triacylglycerides, are mobilized during times of energy deficit, and recent research has implicated several proteins associated with the lipid droplet as lipolytic regulators. The objective of this study was to determine if these novel proteins associated with lipolytic regulation are altered with the changing metabolic demands of lactation. Weekly blood samples were collected from 26 Holstein cows from 21 d before expected parturition through 28 d postpartum, and again at 150 d postpartum. Serum nonesterified fatty acids, glycerol, and β-hydroxybutyrate were measured. Energy balance was calculated from daily feed intake and milk yield, weekly body weight, and monthly milk component measurements. Adipose tissue biopsies were taken 21 d before expected parturition (-21 d) and at 5, 21, and 150 d postpartum. Semiquantitative Western blotting was used to measure abundance of hormone-sensitive lipase (HSL), phosphorylated HSL, perilipin, phosphorylated perilipin (PPLIN), adipose triglyceride lipase (ATGL), and comparative gene identity-58 (CGI-58). Abundance of ATGL was less at 5 and 21 d in milk (DIM) compared with -21 and 150 DIM, even though cows were in negative energy balance and experiencing increased rates of lipolysis in early lactation. In contrast, phosphorylated HSL and PPLIN increased with increasing lipolysis immediately after parturition. Additionally, PPLIN was negatively correlated with milk yield at 5, 21, and 150 d postpartum, and negatively correlated with feed intake and energy balance at 21 d postpartum. This result is consistent with the hypothesis that phosphorylation of perilipin is responsive to signals for increased triaclyglyceride mobilization. Finally, a consistent negative correlation between abundance of perilipin and CGI-58 proteins was observed throughout the transition period. These results confirm that novel lipolytic proteins in adipose tissue are regulated at the level of protein abundance and phosphorylation during the periparturient period and into mid lactation.Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    AB10200

  • Caveolin-1 deficiency leads to increased susceptibility to cell death and fibrosis in white adipose tissue: characterization of a lipodystrophic model. 23049990

    Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1(-/-) mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1(-/-) adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1(+/+) mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1(-/-) mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1501
    Product Catalog Name:
    Anti-Actin Antibody, clone C4

  • mTORC1 inhibition via rapamycin promotes triacylglycerol lipolysis and release of free fatty acids in 3T3-L1 adipocytes. 21042876

    Signaling by mTOR complex 1 (mTORC1) promotes anabolic cellular processes in response to growth factors, nutrients, and hormonal cues. Numerous clinical trials employing the mTORC1 inhibitor rapamycin (aka sirolimus) to immuno-suppress patients following organ transplantation have documented the development of hypertriglyceridemia and elevated serum free fatty acids (FFA). We therefore investigated the cellular role of mTORC1 in control of triacylglycerol (TAG) metabolism using cultured murine 3T3-L1 adipocytes. We found that treatment of adipocytes with rapamycin reduced insulin-stimulated TAG storage ~50%. To determine whether rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of rapamycin and isoproterenol, a ?2-adrenergic agonist that activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented isoproterenol-induced lipolysis without altering cAMP levels. Rapamycin enhanced the isoproterenol-stimulated phosphorylation of hormone sensitive lipase (HSL) on Ser-563 (a PKA site), but had no effect on the phosphorylation of HSL S565 (an AMPK site). Additionally, rapamycin did not affect the isoproterenol-mediated phosphorylation of perilipin, a protein that coats the lipid droplet to initiate lipolysis upon phosphorylation by PKA. These data demonstrate that inhibition of mTORC1 signaling synergizes with the ?-adrenergic-cAMP/PKA pathway to augment phosphorylation of HSL to promote hormone-induced lipolysis. Moreover, they reveal a novel metabolic function for mTORC1; mTORC1 signaling suppresses lipolysis, thus augmenting TAG storage.
    Document Type:
    Reference
    Product Catalog Number:
    AB10200

  • Myotubes from severely obese type 2 diabetic subjects accumulate less lipids and show higher lipolytic rate than myotubes from severely obese non-diabetic subjects. 25790476

    About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several severely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to fundamental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexibility were examined in human myotubes derived from severely obese subjects without (BMI 44±7 kg/m2) and with type 2 diabetes (BMI 43±6 kg/m2). Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to severely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also similar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher lipolysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1628
    Product Catalog Name:
    Anti-Myosin Antibody, slow muscle, clone NOQ7.5.4D

  • Lipopolysaccharide significantly influences the hepatic triglyceride metabolism in growing pigs. 26121977

    In the practical commercial pig farms, inflammation is a perennial problem, yet most of studies on inflammation are focused on immune response. Actually, inflammation can induce body metabolism disorder which will finally influence animals' growth. In this study, we investigated the effect of acute inflammation on the triglyceride (TG) metabolism in the liver of growing pigs and the possible underlying mechanisms.Twelve male growing pigs were randomly divided into two groups, a control group (received saline) and a LPS group (intramuscular injected with 15 μg/kg LPS). Six hours after LPS injection, the pigs were euthanized and sampled. Biochemical indexes, inflammation factors, lipid metabolism related parameters and mitochondrial function were evaluated. The relationship between glucocorticoid receptor (GR) and the key enzymes of de novo lipogenesis were also investigated by chromatin immunoprecipitation assay (ChIP).LPS induced a serious inflammation in the liver of growing pigs proved by liver morphologic changes, the up-regulated plasma cortisol, tumor necrosis factor-α (TNF-α) content and gene expression of inflammation related genes in liver. For de novo lipogenesis, LPS significantly decreased the gene expression of fatty acid synthase (FAS), Acetyl-CoA carboxylase-1 (ACC-1) and Stearoyl-CoA desaturase-1 (SCD-1), and the protein expression of ACC-1 and SCD-1. For lipolysis, only the gene expression of adipose triglyceride lipase (ATGL) was decreased. LPS did nothing to the gene expression of hormone-sensitive lipase (HSL) and the lipolytic enzymes activities. For β-oxidation, LPS significantly increased the protein expression of CPT-1α, but the gene expression of mitochondrial DNA-encoded genes and the activities of mitochondrial complex IV and V demonstrated no obviously changes. Furthermore, ChIP results showed that LPS significantly decreased the level of GR binding to ACC-1 promoter.LPS infection has a profound impact on hepatic TG metabolism. This impact is mainly demonstrated by the significantly deceased de novo lipogenesis, and GR may involve in its regulation.
    Document Type:
    Reference
    Product Catalog Number:
    12-370
    Product Catalog Name:
    Normal Rabbit IgG

  • Transcriptional control of adipose lipid handling by IRF4. 21356515

    Adipocytes store triglyceride during periods of nutritional affluence and release free fatty acids during fasting through coordinated cycles of lipogenesis and lipolysis. While much is known about the acute regulation of these processes during fasting and feeding, less is understood about the transcriptional basis by which adipocytes control lipid handling. Here, we show that interferon regulatory factor 4 (IRF4) is a critical determinant of the transcriptional response to nutrient availability in adipocytes. Fasting induces IRF4 in an insulin- and FoxO1-dependent manner. IRF4 is required for lipolysis, at least in part due to direct effects on the expression of adipocyte triglyceride lipase and hormone-sensitive lipase. Conversely, reduction of IRF4 enhances lipid synthesis. Mice lacking adipocyte IRF4 exhibit increased adiposity and deficient lipolysis. These studies establish a link between IRF4 and the disposition of calories in adipose tissue, with consequences for systemic metabolic homeostasis.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3408
    Product Catalog Name:
    Anti-Tubulin Antibody, beta, clone KMX-1

  • Sphingosine-1-phosphate rapidly increases cortisol biosynthesis and the expression of genes involved in cholesterol uptake and transport in H295R adrenocortical cells. 21864647

    In the acute phase of adrenocortical steroidogenesis, adrenocorticotrophin (ACTH) activates a cAMP/PKA-signaling pathway that promotes the transport of free cholesterol to the inner mitochondrial membrane. We have previously shown that ACTH rapidly stimulates the metabolism of sphingolipids and the secretion of sphingosine-1-phosphate (S1P) in H295R cells. In this study, we examined the effect of S1P on genes involved in the acute phase of steroidogenesis. We show that S1P increases the expression of steroidogenic acute regulatory protein (StAR), 18-kDa translocator protein (TSPO), low-density lipoprotein receptor (LDLR), and scavenger receptor class B type I (SR-BI). S1P-induced StAR mRNA expression requires Gα(i) signaling, phospholipase C (PLC), Ca(2+)/calmodulin-dependent kinase II (CamKII), and ERK1/2 activation. S1P also increases intracellular Ca(2+), the phosphorylation of hormone sensitive lipase (HSL) at Ser(563), and cortisol secretion. Collectively, these findings identify multiple roles for S1P in the regulation of glucocorticoid biosynthesis.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple