Journal: Nutrition research (New York, N.Y.)

Article Title: A single dose of c9,t11 or t10,c12 conjugated linoleic acid isomers perturbs vitamin A metabolism in mice

doi: 10.1016/j.nutres.2011.09.013

Figure Lengend Snippet: Serum retinol levels (μg/dL) upon a single dietary administration of CLA isomers

Article Snippet: CLA c9,t11 and t10,c12 (purity greater than 98%) were purchased from Matreya (Pleasant Gap, PA).

Techniques:

Journal: Nutrition research (New York, N.Y.)

Article Title: A single dose of c9,t11 or t10,c12 conjugated linoleic acid isomers perturbs vitamin A metabolism in mice

doi: 10.1016/j.nutres.2011.09.013

Figure Lengend Snippet: (A) Representative Western blot analysis of serum RBP levels in wild-type mice given an oral gavage of [3H]-retinol + olive oil (olive oil) or either CLA isomer (olive oil + t10,c12 or olive oil + c9,t11). A rabbit polyclonal anti-rat RBP antiserum [28] was used for RBP immunodetection. Albumin (Alb) detected by a rabbit polyclonal anti-Albumin antibody (Abcam) was used as a loading control for tissue samples. The molecular weight of each detected protein is indicated on the left side of the panels. Sera from a RBP knockout mouse (RBP-/-) and from a wild-type mouse (WT), were used as controls. (B) Quantification of Western blots performed by densitometry of the bands detected upon Western analysis. Results presented as means ± SD are shown as a fold change. n= 3-4 mice per group.

Article Snippet: CLA c9,t11 and t10,c12 (purity greater than 98%) were purchased from Matreya (Pleasant Gap, PA).

Techniques: Western Blot, Immunodetection, Control, Molecular Weight, Knock-Out

Journal: Nutrition research (New York, N.Y.)

Article Title: A single dose of c9,t11 or t10,c12 conjugated linoleic acid isomers perturbs vitamin A metabolism in mice

doi: 10.1016/j.nutres.2011.09.013

Figure Lengend Snippet: Triglyceride serum levels of mice gavaged with olive oil and either CLA isomer (mg/dL)

Article Snippet: CLA c9,t11 and t10,c12 (purity greater than 98%) were purchased from Matreya (Pleasant Gap, PA).

Techniques:

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: The PLC inhibitor U73122 attenuates 18:2t10,c12-mediated insulin resistance and suppression of lipogenic protein or gene expression in human adipocytes. (A) Human primary adipocytes were incubated in serum-free, low-glucose DMEM media for 24 h and pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 48 h. Insulin-stimulated glucose uptake was measured by scintillation radioactivity counting on the day of assay (n = 4/treatment). (B) Cultures were pretreated as in A, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control treatment for 24 h. The protein abundance of PPARγ was measured by immunoblotting (n = 3/treatment). (C) Cultures were pretreated as in A, followed by treatment of 50 μmol/L 18:2t10,c12 or BSA vehicle control for 18 h. The expression of PPARγ, GLUT4, ACC-1, and SCD-1 was measured by qPCR (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A–C are representative of at least 3 independent experiments. ACC, acetyl-CoA carboxylase; GLUT4, insulin-dependent glucose transporter 4; PLC, phospholipase C; SCD, stearoyl-CoA desaturase; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Expressing, Incubation, Radioactivity, Western Blot

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: U73122 inhibits 18:2t10,c12-mediated inflammatory signaling in human adipocytes. (A) Human primary adipocytes were pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 18 h. Gene expression of CaMK2-β, COX-2, MCP-1, IL-6, and IL-8 was measured by qPCR (n = 3–4/treatment). (B) Cultures were pretreated as in A, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control treatment for 24 h. Media was collect to measure secreted proinflammatory markers (n = 3–4/treatment). (C) Cultures were used from B. Activation of inflammatory mediators, including ERK, JNK, c-Jun, and GAPDH, were measured by immunoblotting (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A–C are representative of at least 3 independent experiments. CaMK, Ca2+/calmodulin-dependent protein kinase; COX, cyclooxygenase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; MCP, monocyte chemoattractant protein; P, phosphorylated; T, total; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Expressing, Activation Assay, Western Blot

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: U73122 attenuates 18:2t10,c12-mediated induction of HSPs in human adipocytes. (A) Human primary adipocytes were treated with 50 μmol/L 18:2c9,t11, 18:2t10,c12, or BSA vehicle control (B) for 3, 6, 12, or 24 h. Gene expression of HSPA1A, HSPA6, and HSPH1 was measured by qPCR (n = 3–4/treatment). (B) Cultures were pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by treatment with 50 μmol/L 18:2t10,c12 or BSA vehicle control (B) for 12 h. Gene expression of HSPA1A, HSPA6, and HSPH1 were measure by qPCR (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A,B are representative of 2–3 independent experiments. 18:2c9,t11, cis-9, trans-11; HSP, heat shock protein; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Expressing

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: U73122 attenuates 18:2t10,c12-mediated intracellular calcium accumulation in human adipocytes. Human primary adipocytes were incubated in 5 μmol/L Fluo-3 for 30 min, then treated with 5 or 10 μmol/L U73122 for 10 min and followed by injection of 150 μmol/L 18:2t10,c12, 5 μmol/L thapsigargin (Tg), a positive control for stimulating calcium release from the ER, or vehicle (NT). A kinetic intracellular calcium curve was generated by measuring the change in intensity of fluorescence over time. Data (n = 4–6/treatment) are representative of 3 independent experiments. ER, endoplasmic reticulum; F/Fo, changes in the ratio of calcium-dependent fluorescence to pre-stimulus background fluorescence. 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Incubation, Injection, Positive Control, Generated, Fluorescence

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: 18:2t10,c12 increases mRNA levels of PLCδ4 and the translocation of PLCγ1 protein in human adipocytes. (A) Human primary adipocytes were treated with 50 μmol/L 18:2c9,t11, 18:2t10,c12, or BSA vehicle control (B) for 3, 6, 12, or 24 h. Gene expression of PLCδ4 and PLCγ1 was measured by qPCR (n = 3/treatment). (B) Another set of cells was pretreated with 5, 10, or 15 μmol/L U73122 for 30 min, followed by 50 μmol/L 18:2t10,c12 or BSA vehicle control (B). Gene expression of PLCδ4 and PLCγ1 were measured by qPCR (n = 3/treatment) (C). For measuring protein abundance, cultures were treated with vehicle control (B), 50 μmol/L 18:2t10,c12 (C), or 5 μmol/L thapsigargin (T) for 1, 3, or 6 min. Plasma membranes were then isolated and candidate PLC isomers were measured by immunoblotting (n = 3–4/treatment). Means without a common letter differ, P < 0.05. Data in A,B are representative of at least 2 independent experiments. Cav-1, caveolin-1; 18:2c9,t11, cis-9, trans-11; PLC, phospholipase C; 18:2t10,c12, trans-10, cis-12; U73122, 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Translocation Assay, Expressing, Isolation, Western Blot

Journal: The Journal of Nutrition

Article Title: The Phospholipase C Inhibitor U73122 Attenuates trans -10, cis -12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes ^{1 2}

doi: 10.3945/jn.112.173161

Figure Lengend Snippet: Working model in human adipocytes. 18:2t10,c12 activates within seconds the specific cell surface receptors, including GPCRs, GPRs, or protein tyrosine kinases. Activation of these receptors stimulates the translocation of specific isoforms of PLC (i.e., PLCγ1) to the plasma membrane within 3 min, thereby generating DAG and IP3 from PIP2. DAG is rapidly converted to PA, and along with IP3, triggers calcium release from ER within 3 min. The 18:2t10,c12-mediated increase in intracellular calcium, in turn: 1) increases the transcription of HSPs within 6–12 h; 2) upregulates the transcription of calcium-specific isoforms of PLC (e.g., PLCδ4) within 12 h; and 3) activates inflammatory signaling within 24 h. Inflammatory signaling subsequently antagonizes PPARγ abundance and activity within 24 h, thereby suppressing insulin-stimulated glucose uptake and lipogenesis within 48 h. Together, these 18:2t10,c12-mediated events cause adipocyte delipidation. DAG, diacylglycerol; ER, endoplasmic reticulum; GPCR, G protein coupled receptor; GPR, G protein receptor; IP3, inositol-3-phosphate; PA, phosphatidic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

Article Snippet: 18:2c9,t11 and 18:2t10,c12 (+98% pure) were purchased from Matreya.

Techniques: Activation Assay, Translocation Assay, Activity Assay