DDT mRNA expression in the adipose tissues.

<p><b>A</b> Correlation between DDT mRNA levels in adipocytes fractionated from visceral and subcutaneous adipose tissues (AT) and each obesity-related clinical parameter in 19 subjects. The normalized DDT mRNA levels are blotted relative to the highest level of DDT mRNA in visceral AT from a patient. <b>B</b> DDT mRNA levels in adipocytes (Ad) and SVF cells (SVF) fractionated from visceral and subcutaneous AT of 19 patients. The normalized DDT mRNA levels are shown relative to those in visceral adipocytes. Data are the mean ± SEM (n = 19). *<i>P</i><0.05.</p

Effects of DDT knockdown on the expression of genes related to lipid metabolism.

<p><b>A, B</b> DDT mRNA (<b>A</b>) and protein (<b>B</b>) expression in adipocytes differentiated from SGBS cells infected with adenovirus expressing shDDT or shNC. mRNA and protein levels were detected by RT-qPCR and Western blotting using anti-DDT antibody, respectively. The normalized DDT mRNA levels are shown relative to those in cells expressing shNC. The graph is representative of 5 independent experiments. Data are the mean ± SEM (n = 3). *<i>P</i><0.05. <b>C</b> At 9 days after adipogenic induction, total RNA was extracted from cells subjected to infection with the adenovirus expressing shNC (white bars) or shDDT (black bars). DDT knockdown adipocytes were treated with 2 nM rDDT for 24 h and total RNA was extracted (gray bars). The mRNA levels of lipid metabolism-related genes in the cells were measured by RT-qPCR. The expression of each gene was normalized to that of GAPDH mRNA. The normalized DDT mRNA levels are shown relative to those in cells expressing shNC. The graph is representative of 5 independent experiments. Data are the mean ± SEM (n = 3). *<i>P</i><0.05. <b>D</b> FABP4 protein expression in adipocytes. Western blot analysis of lysate prepared from cells infected with the adenovirus expressing shNC or shDDT was performed with an FABP4 antibody. The graph represents band density ratio of FABP4 to β-actin. The levels are shown relative to those in cells expressing shNC. The graph is representative of 3 independent experiments. Data are the mean ± SEM (n = 3). *<i>P</i><0.05.</p

DDT expression in adipocytes and preadipocytes.

<p><b>A</b> Development of lipid droplets in differentiated adipocytes from SGBS cells. No lipid droplets were observed in confluent SGBS cells (day 0). After differentiation was induced, lipid droplets started appearing at 6 days, and were enlarged by 9 days and 12 days. <b>B</b> DDT and adiponectin mRNA levels during differentiation of SGBS cells into adipocytes. The normalized mRNA levels are shown relative to those at day 0. The graph is representative of 3 independent experiments. Data are the mean ± SEM (n = 3). <b>C</b> Detection of endogenous DDT in adipocytes using an anti-DDT antibody. Lysates of adipocytes derived from SGBS cells infected with the adenovirus expressing β-gal (β-GAL) or FLAG tagged DDT (DDT-FLAG) were subjected to SDS-PAGE and Western blotting using the anti-DDT antibody. <b>D</b> Immuncytochemistry using the anti-DDT antibody or normal rabbit IgG as a negative control in SGBS adipocytes. DAPI (blue); DDT (Alexa 488, green); Sudan III (red). Endogenous DDT was detected only in cells with lipid droplets. <b>E</b> Secretion of DDT from adipocytes. Western blot analysis of the concentrated CM (fifty times) from SGBS preadipocytes (PA) and adipocytes differentiated from SGBS cells (AD) was performed.</p

Effect of rDDT injection in db/db mice on glucose intolerance.

<p><b>A</b> mouse DDT (Ddt) mRNA levels in the epididymal white adipose tissue (WAT) and liver from wild-type mice (white bars) and db/db mice (black bars). mRNA levels of Ddt were measured by RT-qPCR. The normalized Ddt mRNA levels are shown relative to those in WAT from wild-type mice. <b>B</b> A GTT in db/db mice injected with PBS (white circles) or rDDT (black circles) for 2 weeks. These mice fasted for 16 h were injected intraperitoneally with 2 g/kg of glucose and blood glucose was monitored at the indicated times. <b>C</b> An ITT in db/db mice treated with PBS (white circles) or rDDT (black circles). These mice fasted for 4 h was injected intraperitoneally with 0.75 IU/kg of insulin and blood glucose was monitored at the indicated times. <b>D, E</b> Fasting serum insulin (<b>D</b>) and FFA (<b>E</b>) levels in db/db mice treated with PBS (white bar) or rDDT (black bar). <b>F</b> FABP4 levels in the epididymal adipose tissue from db/db mice injected with PBS or rDDT. The WAT was removed from each mouse 1 h after the last PBS/rDDT injection and a Western blot analysis of FABP4 and ~αβ-tubulin was performed. The image is representative of 3 independent experiments. The graph represents mean of band density ratio of FABP4 to ˜αβ tubulin in WAT from each mice. The levels are shown relative to those in WAT from wild-type mice. <b>A–F</b> Each graph is representative of 3 independent experiments. Data are the mean ± SEM (n = 4). *<i>P</i><0.05.</p

Inhibition of lipolysis by DDT in adipocytes.

<p>PKA inactivation by DDT brings about increased AMPK activity and decreased HSL activity. The AMPK activation induces further inhibition of HSL activity, followed by inhibiting hydrolysis of triacylglycerol to FFA that are then released from the adipocytes.</p

Effects of DDT knockdown on phosphorylation of AMPKα at Thr-172, HSL at Ser-565, and ACC at Ser-79.

<p><b>A</b> At 9 days after adipogenic induction, adipocytes differentiated from SGBS cells infected with an adenovirus expressing shNC or shDDT were used. These cells were cultivated for 30 min in the presence or absence of AICAR. Protein lysate was prepared and probed with the indicated antibodies. An image shown is representative of 3 independent experiments. The graphs represents band density ratio of each phosphorylated protein to total protein in a left image. <b>B</b> Cells treated in the same manner as in (<b>a</b>) were used. The cells treated with 2 nM rDDT in serum-free medium were cultivated for the period indicated. Protein lysate was prepared and probed with the indicated antibodies. An image shown is representative of 3 independent experiments. The graphs represents band density ratio of each phosphorylated protein to total protein in a left image.</p

Phosphorylation of HSL, AMPK, and PKA's substrates in the adipose tissues from rDDT-treated db/db mice.

<p><b>A</b> Phosphorylation of HSL and AMPK in the epididymal adipose tissues removed from each mouse 1 h after the injection of PBS or rDDT. Western blot analysis using indicated antibodies was performed. An image shown is representative of 3 independent experiments. The graph represents band density ratio of each phosphorylated protein to total protein obtained from a left image. White and black bars denote mice injected PBS and rDDT, respectively. The graph represents mean of band density ratio of each phosphorylated protein to total protein in the adipose tissue from db/db mice injected with PBS (white bars) or rDDT (black bars). <b>B</b> Phosphorylation of PKA's substrates in the epididymal adipose tissues removed from each mouse 1 h after the injection of PBS or rDDT. For detection of the phosphorylation of PKA's substrates, antibody recognizing phosphorylated Ser or Thr in the RRXS/T motif was used.</p

Additional file 1: Table S1. of Characteristics, treatments, and outcomes of severe sepsis of 3195 ICU-treated adult patients throughout Japan during 2011–2013

Epidemiological information from previous reports after 2005. (DOC 48 kb