Consistency of the hyperbolic relationship (DI) between insulin sensitivity and insulin secretion.

<p>Solid black hyperbolic curve =  average DI value for week 0; black solid square  =  average DI for week 0; open square  =  average DI for week 2; black solid triangle  =  average DI for week 4; open triangle  =  average DI for week 6. The hyperbola represents the curve on which the animals would have to remain to maintain a constant DI (DI = AIRg.SI = constant) and it is calculated as extrapolation of the average DI for week 0 over a range of insulin sensitivities.</p

Weekly changes in body weight and body composition.

<p>(A) Body weight increased by week 2 and remained elevated throughout the six-week hypercaloric high-fat feeding period. (B) Visceral (white bar) and subcutaneous (black bar) adipose tissue calculated as area percent of total tissue. *P<.05 vs. week0; **P<.01 vs. week 0; ***P<.001 vs. week 0.</p

Glucose and FFA plasma levels at three time periods during 24-hr before and after fat feeding.

<p>(A) Plasma glucose and (B) plasma FFAs during 2–4 AM (hash bars), 8–8:30 AM (black bars), and 6–8 PM (white bars). *P<.05 vs. week0; **P<.01 vs. week 0; ***P<.001 vs. week 0.</p

Spearman’s rank correlation between fasting insulin and fasting FFA.

<p>Spearman’s rank correlation between fasting insulin and fasting FFA.</p

Weekly estimates of insulin sensitivity, insulin secretion and whole-body insulin clearance.

<p>(A) SI, (B) AIRg, and (C) MCR of insulin. *P<.05 vs. week 0; **P<.01 vs. week 0; ***P<.001 vs week 0.</p

Observed fasting plasma insulin, glucose and FFAs.

<p>Fasting levels of (A) insulin, (B) glucose, and (C) FFA before and after fat feeding. *P<.05 vs. week0; **P<.01 vs. week 0; ***P<.001 vs. week 0.</p

Profile changes in dogs (n = 9) maintained on a hypercaloric high-fat diet for 22 weeks.

<p>Values are means±S.E.M. 2-AG, 2-arachidonoyl-glycerol; C-peptide slope, changes in plasma C-peptide relative to plasma glucose during hyperglycemic clamp; Insulin slope, changes in plasma insulin relative to plasma glucose during hyperglycemic clamp; LEA, linoleoyl-ethanolamide; NEFA, non-esterified fatty acids; OEA, oleoyl-ethanolamide; PEA, palmitoyl-ethanolamide; SI, whole-body insulin sensitivity assessed by the euglycemic hyperinsulinemic clamp.</p><p>* Fasting plasma values.</p><p>Profile changes in dogs (n = 9) maintained on a hypercaloric high-fat diet for 22 weeks.</p

Anandamide significantly enhances insulin and glucagon secretion.

<p>Insulin, glucagon, and somatostatin concentrations were measured in batches of 100 islets from control-diet animals (n = 7) during static incubation with anandamide 10 μmol/L for 1 h. 3G, 3 mmol/L; 15G, 15 mmol/L glucose. Data are mean±S.E.M.</p

Supraphysiologic concentrations of anandamide enhance <i>in vitro</i> insulin secretion.

<p>(<b>A</b>) Anandamide stimulates basal insulin secretion in control-diet (n = 7) and high-fat diet (HFD) animals (n = 6) at 3 mmol/L glucose (3G). (<b>B</b>) Anandamide also potentiates glucose-stimulated insulin secretion in both groups at 15 mmol/L glucose (15G). Islets were incubated with anandamide or CB1R antagonist rimonabant (R) at 10 μmol/L for 1 h. Experiments on every animal were done in quadruplicate. Data are mean±S.E.M. (<b>C</b>) At the doses tested to stimulate insulin secretion, anandamide did not impair islet viability. Green and red colors represent viable and non-viable cells, respectively. Staining of islet batches from a same animal is representative of 3 independent experiments. Note the complete loss of islet viability (red stain) at 1000 μmol/L, consistent with a massive release of insulin as measured by ELISA (data not shown). Total magnification: 100X. (<b>D</b>) Relative mRNA expression of CB1R, CB2R, and TRPV1 to 18S in intact islets from control (n = 4) and HFD animals (n = 6–7).</p

Physiological concentrations of anandamide enhance <i>in vitro</i> insulin secretion.

<p>(<b>A</b>) In islets from control-diet dogs (n = 7), anandamide significantly increased basal insulin secretion at 3 mmol/L glucose (3G) and GSIS at 15 mmol/L glucose (15G). Islets were incubated for 1 h with either anandamide (10 nmol/L) or cannabinoid receptor antagonists (100 nmol/L), as indicated. CB1R, CB2R, and TRPV1 antagonists rimonabant (R), AM630, and iodoresiniferatoxin (IRTX), respectively, were added prior to stimulation with high glucose and anandamide. Plots indicate the mean of 3–9 replicates for each dog. (<b>B</b>) When tested alone, none of the antagonist drugs had effect on islet viability compared with vehicle (n = 3). Total magnification: 100X. (<b>C</b>) Islets were continuously perifused with glucose 3 mmol/L and challenged with 15 mmol/L glucose (15G) from t = 0, as shown with the horizontal bar, in presence (n = 3) or absence (n = 3) of anandamide (10 nmol/L). Anandamide perifusion started concomitant with 15G, as indicated by the arrow. Plots of perifusion experiments represent means; bars represent S.E.M. P value represent the difference in overall profile between treatment and control during the second phase (t = 9–36 min). Analysis was performed using mixed-model linear regression to account for repeated measures.</p