Sequences of primers (5′–3′).

<p>XBP1s: X-box-binding protein 1 spliced, XBP1u: X-box-binding protein 1 unspliced, CHOP: C/EBP homologous, RPL19: Ribosomal protein L19, CPHN: Cyclophilin, HPRT1: Hypoxanthine phosphoribosyltransferase 1, IL-6: interleukin-6, IL-1β: interleukin-1β TNFα: tumor necrosis factor-α.</p

OGTT and circulating NEFA after a high-fat diet in wild-type and TLR4^−/− mice.

<p>(A) Plasma glucose concentration following an oral glucose load. Wild-type mice upon chow (○), wild-type mice upon a high-fatd diet (•), TLR4 knockout mice upon chow (▿), TLR4 knockout mice upon a high-fat diet (▾). The <i>inset</i> represents the area under the curve (AUC) expressed in arbitrary units (A.U.). (B) Fasting NEFA concentrations expressed in mmol.L⁻¹.</p

TLR4 deficiency protects from obesity without reducing energy intake.

<p>(A) Body weight gain calculated as the body weight difference between the end (18 weeks) and the beginning of the diet. (B) Mean energy intake expressed in kcal.week⁻¹ (C) Subcutaneous adipose tissue weight expressed in percentage of body weight. (D) Visceral adipose tissue weight expressed in percentage of body weight. Values are expressed as means ± SEM (n = 8–10), * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Changes in MAPK phosphorylation after a high-fat diet in wild-type and TLR4^−/− mice.

<p>(A) Phosphorylation state of p38, JNK and ERK1/2 in skeletal muscle, liver and subcutaneous adipose tissue of wild-type (WT) and TLR4 knockout (TLR4^−/−) mice fed with a standard (chow) or a high-fat diet (HFD). Results are presented as means ± SEM (n = 8–10), * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. (B) Illustration of the data presented in panel A.</p

ER stress protein markers are not induced in TLR4^−/− mice after a high-fat diet.

<p>(A) Protein expression of BiP and phosphorylated eIF2α in skeletal muscle, liver and subcutaneous adipose tissue of wild-type (WT) and TLR4 knockout (TLR4^−/−) mice fed with a standard (chow) or a high-fat diet (HFD). Results are presented as means ± SEM (n = 8–10), * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. (B) Illustration of the data presented in panel A.</p

ER stress mRNA markers are not up-regulated in TLR4^−/− mice after a high-fat diet.

<p>mRNA level of XBP1u (X-box-binding protein 1 unspliced), XBP1s (X-box-binding protein 1 spliced) and CHOP (C/EBP homologous) in skeletal muscle, liver and subcutaneous adipose tissue of wild-type (WT) and TLR4 knockout (TLR4^−/−) mice fed with a standard (chow) or a high-fat diet (HFD). Results are expressed as means ± SEM (n = 8–10), * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Validation of HTRF insulin assay conditions.

<p><b>(A)</b> pH significantly affected the ratiometric HTRF signal across a range of human insulin concentrations [F(2, 40) = 21.35, p<0.001]. Antibody incubation at pH 7 yielded the greatest HTRF signal compared to other pHs (p<0.001). <b>(B)</b> 2 h antibody incubation was sufficient to produce a robust HTRF signal, with longer antibody incubation times (12 and 48 h) further increasing HTRF signal. <b>(C)</b> There was a temperature-dependent difference in HTRF values when antibodies were incubated for 2 h at room temperature (RT, 25°C) versus 37°C [F(1,29) = 16.57, p<0.001] with higher signal observed at RT. <b>(D)</b> There was no significant difference in HTRF signal between human, rodent, porcine and bovine insulin across a range of concentrations (0.01–10 nM; p>0.05). <b>Panels A-C:</b> Data are represented as the mean emitted HTRF ratio ± SEM. <b>Panel D:</b> Data are represented as %ΔF of the HTRF signal for the respective species. For all panels, the data are from experiments performed in triplicate in 384-well plates.</p

Advantages of the HTRF-based insulin assay compared to ELISA and RIA approaches.

<p>Here we show the main advantages of an HTRF insulin assay over comparable RIA and ELISA-based methods. Though all three methods are similarly sensitive and specific for insulin detection, the homogenous nature of the HTRF assay eliminates numerous reagents and mixing, washing and blocking steps, making the assay shorter, less expensive and more amenable to medium and high-throughput screens.</p

Comparison between HTRF and ELISA insulin detection assays.

<p>Supernatants collected from glucose-stimulated INS-1E cells (20 mM glucose, 90 min, 37°C) were measured concurrently with HTRF or ELISA insulin assays. The respective HTRF and ELISA assay-derived insulin concentration values were plotted. A linear regression curve of the data showed close correlation of the insulin values from the two methods (slope = 1.15 ± 0.16, R² = 0.84). Results are represented as mean insulin concentrations ± SEM performed in triplicate in 3 independent experiments.</p

HTRF measurement of dopamine and bromocriptine effects on insulin secretion in cells and islets.

<p><b>(A)</b> Increasing concentrations of dopamine (DA) caused dose-dependent inhibition of GSIS in INS-1E cells, which was best fit to a sigmoidal curve (IC₅₀ = 1.28 ± 0.06 μM, R² = 0.93). <b>(B)</b> Similarly, treatment of wildtype mouse islets with 10 μM DA significantly and comparably inhibited GSIS (p<0.001) by 70.7 ± 6.8%. Consistent with a role for dopaminergic signaling as a negative mediator of GSIS, treatment of islets with10 μM bromocriptine inhibited GSIS by 67.4 ± 8.1%. For INS-1E cell-based and mouse islet experiments (<b>Panels A and B</b>, respectively), data are represented as % maximal insulin secretion based on mean HTRF values ± SEM from n≥3 independent experiments. For all panels, HTRF measurements were performed in 96-well plates with INS-1E cell secretion experiments performed in triplicate and mouse islet experiments performed in hextuplicate.</p