In vivo screening for secreted factors that enhance glucose tolerance identified interleukin-6. A.

<p>Glucose tolerance tests were performed on a cohort of mice (n = 4 animals per group) undergoing primary screening (1 control group and 3 experimental groups) for genes that modulate the glucose tolerance test. The second group contained the IL-6 ORF in addition to two other factors. ** Indicates p<0.01 using a paired t-test. <b>B.</b> Glucose tolerance tests were performed 48 hours post tail vein injection on 6 mice that received either GLuc (25 µg) and H2B-Cherry (25 µg), or GLuc and IL-6 (25 µg). Mice underwent no fast (right, upper panel, n = 5 animals per group), a 5 hour fast (left, lower panel, n = 5 animals per group) or an overnight fast (right, lower panel, n = 4 animals per group). *Indicates p<0.05 using a paired t-test. ** Indicates p<0.01 using a paired t-test. <b>C.</b> Glucose tolerance tests were performed 7 days post tail vein injection on mice that received either GLuc (25 µg) and H2B-cherry (25 µg), or GLuc and IL-6 (25 µg) (n = 5 animals per group). Animals were fasted for 5 hours prior to performing the GTT. *Indicates p<0.05 using a paired t-test. ** Indicates p<0.01 using a paired t-test.</p

Serum luciferase activity is influenced by both the amount and the complexity of cDNA expression constructs that are transfected into liver cells via hydrostatic tail vein injection. A.

<p>Serum luciferase levels were measured 48 hours after 6-week old ICR mice (n = 5 animals per group) underwent hydrostatic tail vein injection with the indicated amount of the GLuc expression construct. <b>B.</b> Serum luiciferase levels were measured 48 hours after 6-week old ICR mice (n = 5 animals per group) underwent hydrostatic tail vein injection with GLuc (25 µg) and variable amounts (0, 75, 175 or 250 µg) of unrelated pT3 expression constructs. ** Indicates p<0.01 using a paired t-test.</p

Development of an in vivo screening protocol.

<p>A Gateway ready arrayed library of 248 factors (left, top panel) were transferred into the transposable pT3 expression vector and isolated for injection. For each round of injection, a control DNA injection (GLuc (25 µg), SB100 (2 µg), and H2B-Cherry (75 µg)) and 3 experimental DNA injection formulations (GLuc (25 µg), SB100 (2 µg), and 3 secreted factors (25 µg each)) were prepared (right, top panel). Each cohort of 5 mice (aged 6–7 weeks) were injected with a single DNA formulation (left, middle panel). 43 h post-injection, mice were weighed and fasted. At 48 hours, animals underwent an i.p. glucose tolerance test with plasma glucose monitoring at times 0, 37.5 and 60 minutes. Subsequently, serum luciferase levels were determined to exclude mice with unsuccessful tail vein injections (luciferase values <30,000 relative light units). Sample results are illustrated in the left lower panel where the three potential outcomes are observed: improved glucose tolerance (blue formulation), worsened glucose tolerance (green formulation), and no effect (purple formulation).</p

Several IL-6 family members enhance glucose tolerance.

<p>Glucose tolerance tests were performed 2 days post tail vein injection on mice that received either GLuc (25 µg) and H2B-cherry (25 µg), or: <b>A.</b> GLuc and LIF (25 µg) (n = 7 animals per group). <b>B.</b> GLuc and CT-2 (25 µg) (n = 7 animals per group). <b>C.</b> GLuc and OSM (25 µg) (n = 6 animals per group). <b>D.</b> GLuc and CNTF (25 µg) (n = 10 animals per group). All animals were fasted for 5 hours prior to the GTT. * Indicates p<0.05 using a paired t-test. ** Indicates p<0.01 using a paired t-test. <b>E.</b> Glucose tolerance testing was performed on 5-hour fasted animals that were injected i.p. with the indicated recombinant factor (n = 4) at time –30 minutes and then gavaged with a glucose solution (2g/kg) at time 0 minutes. Compared to saline treated animals, glucose values were significantly lower (p<0.04) for all experimentally treated animals at 15, 30, 45, 60 and 90 minutes (except CT-1 at 90 minutes).</p