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Apelin, a newly identified adipokine up regulated by insulin and obesity

The results presented herein demonstrate that apelin is expressed and secreted by both human and mouse adipocytes. Apelin mRNA levels in isolated adipocytes are close to other cell types present in white adipose tissue or other organs known to express apelin such as kidney, heart, and to a lesser extent brown adipose tissue. Apelin expression is increased during adipocyte differentiation stage. A comparison of four different models of obesity in mice showed a large increase in both apelin expression in fat cells and apelin plasma levels in all the hyperinsulinemia-associated obesities and clearly demonstrated that obesity or high fat feeding are not the main determinants of the rise of apelin expression. The lack of insulin in streptozotocin-treated mice is associated with a decreased expression of apelin in adipocytes. Furthermore, apelin expression in fat cells is strongly inhibited by fasting and recovered after refeeding, in a similar way to insulin. A direct regulation of apelin expression by insulin is observed in both human and mouse adipocytes and clearly associated with the stimulation of PI3K, PKC and MAPK. These data provide evidence that insulin exerts a direct control on apelin gene expression in adipocytes. In obese patients, both plasma apelin and insulin levels were significantly higher suggesting that the regulation of apelin by insulin could influence blood concentrations of apelin. The present work identifies apelin as a novel adipocyte endocrine secretion and focuses on its potential link with obesity-associated variations of

Boucher J., et al. Endocrinology. First published January 27, 2005 as doi:10.1210/en.2004-1427

Apelin was quantified with either the non selective apelin-12 EIA or the specific human RIA kit (Phoenix Pharmaceuticals, Belmont, CA) following manufacturer’s instructions.

A- Apelin mRNA in Human isolated adipocytes (adip, black bars) and stroma vascular fraction (SVF, white bars), results are mean values ± SEM from 7 subjects; BImmuno- histochemical staining without (upper panel) or with (lower panel) an immune serum directed against the 13-COOH terminal aminoacids region of apelin (bar represents 10µm). Cexpression of apelin and adipocyte specific genes in SVF (white bars) and adipocytes (black bars) from either female or male subcutaneous (SC) or intra-abdominal (INT) fat pads. Results are mean values ± SEM from 5 mice; D-apelin mRNA in various mouse tissues (WAT: white adipose tissue, Kidn: Kidney, Mus: Muscle, BAT: brown adipose tissue).

A- Effect of fasting/refeeding on transcriptional regulation of apelin in C57Bl6/J mice adipocytes. 12-week-old female mice were divided into three groups. One group was allowed free access to food, the other groups were fasted for a 24h period. At the end of the cycle one group served as a fasting group whereas the other was refed for 24 hours. Plasma insulin levels (white bars) and adipocyte apelin mRNA levels (black bars) were quantified in the 3 groups. Results are mean values ± SEM from 6 mice per group. * P < 0.05 when compared with fed group; § P < 0.05 when compared with fasted group. B- Apelin mRNA levels were quantified in isolated adipocytes from streptozotocin-treated FVB/n mice (STZ) and compared with buffer citrate treated mice (CONT). Results are mean values ± SEM from 4 mice per group. * P < 0.05 when compared with controls. C- Plasma insulin levels and apelin mRNA from isolated adipocytes in leptin receptor deficient mice C57BlKS homozygous db/db (circles), heterozygous db/+ (squares) and wild type +/+ (triangles) littermates, n = 8, 4 and 6 respectively.

Plasma apelin levels in humans of nine controls and eight age matched drug free obese. Blood samples were drawn in the morning under fasting conditions and collected on EDTA. * P < 0.05 when compared with control subjects.

Apelin-12-ir in rat hypothalamus Tissue Sample Rat lateral hypothalamus Fixative 10% Formalin Embedding Paraffin Negative control No primary antibody Pretreatment Target Retrieval 25 min (Steam) Blocking 2% Normal Goat Serum Primary Antibody Rabbit Anti-Apelin-12 (Human, Bovine) Antiserum (Catalog No.:H-057-23) Optimal Dilution 1:3000 (1hour at RT) Secondary Antibody Goat anti-Rabbit IgG, Biotinylated (1:400) Amplification  Streptavidin-HRP (Vector), 1:400, 30 min Detection system HRP Substrate DAB (Sigma) Counterstained Hematoxylin

Apelin-12-ir in rat  medial forebrain boundle Tissue Sample Rat medial forebrain boundle Fixative 10% Formalin Embedding Paraffin Negative control No primary antibody Pretreatment Target Retrieval 25 min (Steam) Blocking 2% Normal Goat Serum Primary Antibody Rabbit Anti-Apelin-12 (Human, Bovine) Antiserum (Catalog No.:H-057-23) Optimal Dilution 1:3000 (1hour at RT) Secondary Antibody Goat anti-Rabbit IgG, Biotinylated (1:400) Amplification  Streptavidin-HRP (Vector), 1:400, 30 min Detection system HRP Substrate DAB (Sigma) Counterstained Hematoxylin

Apelin Related Products

	Binding Characteristics of [125I]-[Pyr1]-Apelin-13 in Human Heart Tissue Kd (nM) Bmax (fmol/mg Protein) Left Ventricle 0.35 ± 0.08  4.3 ± 0.9  Right Atria 0.33 ± 0.09 3.1 ± 0.6 Distribution of APJ Orphan Receptor in Human Cardiovascular Tissue Tissue Binding Density of [125I]-[Pyr1]-Apelin-13 (autography) Heart (LV & RA) ++++ Coronary artery +++ Aorta +++ Saphenous Vein Grafts +++ Distribution of APJ Orphan Receptor in Rat Tissues Tissue Binding Density of [125I]-[Pyr1]-Apelin-13 (autography) Cerebellum +++ Lung +++ Heart +++ Kidney Cortex + Katugampola, S.D. et al. Br. J. Pharmacol. 132(6), 1255-1260 (2001)

New Apelin-12 EIA Kit is available for cardiovascular research!!!

Specificity   Peptide % Crossreactivity Apelin-12 (Human) 100 Apelin-13 (Human) 100 Apelin-36 (Human) 100

Specificity   Peptide % Crossreactivity Apelin-36 (Human) 100 Apelin-16 (Human) 0.10 Apelin-13 (Human) 0.27

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