Additional file 1: of Gene expression of the zinc transporter ZIP14 (SLC39a14) is affected by weight loss and metabolic status and associates with PPARγ in human adipose tissue and 3T3-L1 pre-adipocytes

Primer sequences. Forward and reverse primer sequences are shown together with the annealing temperature. h, human primer; m, murine primer. In human adipose tissue, expression of the zinc transporter ZIP14 was investigated together with that of peroxisome proliferator-activated receptor γ isoform 1 (PPARγ1) and isoform 2 (PPARγ2). Low-density lipoprotein receptor-related protein 10 (LRP10) was used as a housekeeping gene. In 3T3-L1 cells, expression of the zinc transporter ZIP14 was investigated together with that of the adipocytic differentiation markers PPARγ and fatty-acid binding protein 4 (A-FABP). Cyclophilin A (Cyc-A), hypoxanthine guanine phosphoribosyl transferase (HPRT), and ubiquitin conjugase-7 (UBC-7) were used as housekeeping genes. (PDF 58 kb

Positron emission tomography.

<p>Myocardial glucose uptake (MGU) during normoglycemia and hypoglycemia (A and B). ¹⁸F-FDG clearance (K) during normoglycemia and hypoglycemia (C and D). Relation between placebo MGU and change in MGU during GLP-1 infusion in the normoglycemia study (E), placebo MGU and change in MGU during GLP-1 infusion in the hypoglycemia study (F). Relation between placebo K and change in K during GLP-1 infusion in the normoglycemia study (G), placebo K and change in K during GLP-1 infusion in the hypoglycemia study (H). HOMA 2IR and the change of MGU during GLP-1 infusion in the hypoglycemia study (I). HOMA 2IR and the change of K during GLP-1 infusion in the hypoglycemia study (J). Data are mean ± SD. Regression lines with 95% confidence intervals.</p

Influence of GLP-1 on Myocardial Glucose Metabolism in Healthy Men during Normo- or Hypoglycemia

<div><p>Background and Aims</p><p>Glucagon-like peptide-1 (GLP-1) may provide beneficial cardiovascular effects, possibly due to enhanced myocardial energetic efficiency by increasing myocardial glucose uptake (MGU). We assessed the effects of GLP-1 on MGU in healthy subjects during normo- and hypoglycemia.</p><p>Materials and Methods</p><p>We included eighteen healthy men in two randomized, double-blinded, placebo-controlled cross-over studies. MGU was assessed with GLP-1 or saline infusion during pituitary-pancreatic normo- (plasma glucose (PG): 4.5 mM, n = 10) and hypoglycemic clamps (PG: 3.0 mM, n = 8) by positron emission tomography with ¹⁸fluoro-deoxy-glucose (¹⁸F-FDG) as tracer.</p><p>Results</p><p>In the normoglycemia study mean (± SD) age was 25±3 years, and BMI was 22.6±0.6 kg/m² and in the hypoglycemia study the mean age was 23±2 years with a mean body mass index of 23±2 kg/m². GLP-1 did not change MGU during normoglycemia (mean (+/− SD) 0.15+/−0.04 and 0.16+/−0.03 µmol/g/min, P = 0.46) or during hypoglycemia (0.16+/−0.03 and 0.13+/−0.04 µmol/g/min, P = 0.14). However, the effect of GLP-1 on MGU was negatively correlated to baseline MGU both during normo- and hypoglycemia, (P = 0.006, r² = 0.64 and P = 0.018, r² = 0.64, respectively) and changes in MGU correlated positively with the level of insulin resistance (HOMA 2IR) during hypoglycemia, P = 0.04, r² = 0.54. GLP-1 mediated an increase in circulating glucagon levels at PG levels below 3.5 mM and increased glucose infusion rates during the hypoglycemia study. No differences in other circulating hormones or metabolites were found.</p><p>Conclusions</p><p>While GLP-1 does not affect overall MGU, GLP-1 induces changes in MGU dependent on baseline MGU such that GLP-1 increases MGU in subjects with low baseline MGU and decreases MGU in subjects with high baseline MGU. GLP-1 preserves MGU during hypoglycemia in insulin resistant subjects.</p><p>ClinicalTrials.gov registration numbers: <a href="http://clinicaltrials.gov/show/NCT00418288" target="_blank">NCT00418288:</a> (hypoglycemia) and <a href="http://clinicaltrials.gov/show/NCT00256256" target="_blank">NCT00256256:</a> (normoglycemia).</p></div

Hormones and metabolites - hypoglycemia study.

<p>Plasma glucose, glucose infusion rates (GIR), total GLP-1, insulin, cortisol, free fatty acid (FFA), glucagon and epinephrine concentrations during GLP-1 (black dots) and placebo infusion (white dots). Data are means ± SEM. * P≤0.05.</p

Study design.

<p>Design of the normoglycemic study (n = 10) (A) and the hypoglycemic study (n = 8) (B). The studies were conducted as randomized, double-blinded, placebo-controlled, crossover studies. X₁ and X₂: PET and clamp data.</p

Hormones and metabolites - normoglycemia study.

<p>Plasma glucose, glucose infusion rates (GIR), total GLP-1, insulin, cortisol, free fatty acid (FFA), glucagon and epinephrine concentrations during GLP-1 (black dots) and placebo infusion (white dots). Data are means ± SEM. * P≤0.05.</p

Relative gene expression of ZnT-3 and ZnT-8 after 24 hours of 100 µM DEDTC treatment.

<p>INS-1E cells were treated with DEDTC at 5 mM glucose. A) ZnT-3 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.05). N = 6. B) ZnT-8 gene expression normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6.</p

Fasting glucose levels after low-dose streptozotozin in ZnT-3−/− knock-out mice.

<p>ZnT-3−/− knock-out mice and control mice were treated with 50 mg/kg/day for five days. Data are mean and SEM (*p<0.01). N = 15.</p

ZnT-3 protein in INS-1E cells and mouse islets.

<p>A) Western blot of ZnT-3 knockout tissue, and normal background strain tissue using the anti-ZnT-3 polyclonal antibody (20 µg per lane). B) Western blot with ZnT-3 antibody. Lane one shows the protein marker in kDa. Subsequent lanes: Control rat brain (10 µg protein) (lanes 2–4), mock transfected INS1-E cells (50 µg protein) (lanes 5–7), 100 µM DEDTC-treated INS1-E cells (50 µg protein) (lanes 8–10), ZnT-3 siRNA transfected INS1-E cells (50 µg protein) (lanes 11–13). Insert shows the quantification, brain tissue values are original multiplied by 5. C) Light micrograph of INS-1E cells exposed to ZnT-3 antibody. Silver enhanced colloidal gold (10 nm) particles attached to secondary antibodies against the ZnT-3 primary antibody are seen within the cells. There was no background stain and controls were negative (insert). Bar = 20 µm. D) Demonstration of ZnT3 antibody positivity in intact mouse islets (lane 1), compared with INS-1E cells before (lane 2) and after (lane 3) treatment with 100 µM DEDTC and brain tissue (lane 4). Each upload with 20 µg protein.</p

Detection of apoptosis in INS-1E cells after 24 hours of hyperglycamia or zinc depletion.

<p>A–C) Glucose stimulation with 5 mM and 16 mM. A) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. B) Detection of intracellular DNA fragments in INS-1E cells (apoptosis) after 16 mM glucose stimulation. Data are mean and SEM. N = 4. C) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated 16 mM glucose. Data are mean and SEM (*p<0.05). N = 4. D–F) Zinc chelation with 100 µM DEDTC. D) Bax/Bcl-2 ratio of gene expression. Both genes were normalised to Cltc, HPRT and HSPcb. Data are mean and SEM (*p<0.01). N = 6. E) Detection of DNA fragments in INS-1E (apoptosis) after 100 µM DEDTC treatment. Data are mean and SEM (*p<0.01) N = 4. F) Detection of DNA fragments in medium from INS-1E cells (necrosis) treated with 100 µM DEDTC. Data are mean and SEM (*p<0.01). N = 4.</p