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Figure 5.3. Recruitment of Janus kinase-2 upon leptin stimulation

J774.2 macrophages (A) were serum starved either overnight (macrophages) or for 3 hours (adipocytes) prior to stimulation with 2nM leptin, for the indicated times. Tyrosine phosphorylated signalling complexes were immunoprecipitated with anti-phosphotyrosine antibody, py99, and western blotted with anti-JAK-2 antibody.

5.3.2. P I 3-kinase dependence

The majority of insulins' actions involve PI 3-kinase and in adipocytes this is involved in the regulation of HSL. Also leptin has been show n to regulate this enzym e . Therefore w e studied the effect of insulin and leptin on PI 3-kinase recruitm ent and activity in macrophages.

To determ ine if insulin and leptin stim ulated recruitm ent of PI 3-kinase to tyrosine p h o sp h o ry lated signalling com plexes, J774.2 m acro p h ag es w ere stim ulated and im m unoprecipitated w ith anti-phosphotyrosine antibody. The IPs were then subject to SDS PAGE and blotted w ith antibody raised against the p85 regulatory subunit of PI 3-kinase. It can be seen th at 3nM leptin clearly causes recruitm ent of p85 (Figure 5.4, A). This recruitm ent is m axim al (more then 3 fold) at 15 m inutes stimulation and appears to be returning to basal levels w ith longer stimulation. lOnM Insulin also increases recruitm ent of p85 (figure 5.4, B), although to a lesser extent than that seen for 3nM leptin. The m axim al increase in recruitm ent w ith insulin w as approxim ately 70%, observed after 10 m inutes insulin stimulation. It w ould be expected th at increased recruitm ent of PI 3-kinase into tyrosine phosphorylated signalling complexes w ould result in increased PI 3-kinase activity. To confirm this, the stim ulation of m acrophages w ith insulin and leptin were repeated and the lipid kinase activity in the anti- phosphotyrosine IPs w as m easured (figure 5.5). As expected bo th insulin and leptin increased PI 3-kinase activity an d 3nM leptin caused a m uch bigger stim ulation of activity com pared to lOnM insulin. M am mals have three isoforms of the p i 10 catalytic isoform of PI 3-kinase, p i 10a an d p llO p are w id ely

these different isoforms w ere exam ined in J774.2 m acrophages u pon insulin stim ulation using anti-phosphotyrosine IP and w estern blotting w ith antibodies specific for each isoform (figure 5.6). The non-im m unoprecipitated lysate from basal sam ples were also loaded to verify the position of p i 10 on the gel. The p i 10a and p specific antibodies also highlighted b ands of low er m olecular w eight, how ever the bands w hich ru n at the sam e m olecular w eight as the p ro tein detected in the lysate w ere presum ed to be the true llO kD a ban d representing the PI 3-kinase catalytic isoforms. It appears that while there is increased recru itm en t of p i 10 a an d p llO p into tyrosine p h o sp h o ry lated signalling complexes, there is no increase in the am ount of pllOÔ recruitm ent. H o w ev er, the m ajority of p i 10 Ôis a lre a d y asso ciated w ith ty ro sin e phosphorylated protein in basal cells, whereas the majority, if not all, of p i 10a and pllO P isoforms are still present in the lysate after anti-phosphotyrosine IP, in the basal state.

The SH2 containing inositol phosphatase protein (SHIP) is a 5' phosphatase w hich is proposed to be involved in dow nregulating the lipid p roduct of PI 3- kinase, PtdIns(3,4,5)P3, by hydrolysis to P tdlns (3,4)P2 (Dam en et al., 1996; Lioubin et al., 1996). P revious stu d ies have sh o w n th a t g ro w th factor stim ulation causes tyrosine phosphorylation of SHIP. Therefore w e investigated if the same occured in J774.2 cells. It can clearly be seen in the bottom panel of figure 5.6 that SHIP is not tyrosine phosphorylated at the 10 m inute tim epoint used in these experiments.

lipolysis by insulin in adipocytes is dependent on PI 3-kinase activity. Therefore w e d eterm in ed if the effects of in su lin an d lep tin on HSL activ ity in m acrophages req u ired PI 3-kinase activity. This w as done u sin g lOOnM w ortm annin, which at this concentration is a specific inhibitor of PI 3-kinase. Figure 5.7 show s the effect of w ortm annin pretreatm ent on the regulation of HSL by insulin. Experiments carried out in m acrophage lysates (A) and HSL im m unoprecipitates (B) show th at lOOnM w ortm annin had no effect on the dow nregulation of HSL activity by insulin. How ever, the dow nregulation of HSL activity in HSL im m unoprecipitates from adipocytes w as blocked by w ortm annin. Also the upregulation of HSL activity by leptin in m acrophages, was completely blocked by pretreatm ent w ith lOOnM w ortm annin (figure 5.8), clearly indicating th at w o rtm an n in is functional in the hig h ly oxidative environm ent found in m acrophages. This data indicates th at PI 3-kinase is necessary for leptin effects on HSL.

The thiazolidinedione (TZD) insulin-sensitising drugs have been reported to exert effects on insulin signal transduction pathw ays . These d ru g s act as agonists of the transcription factor PPARy and are used w idely in the treatm ent of type 2 diabetes. Troglitazone, pioglitazone and rosiglitazone are three TZD drugs w hich are approved for treatm ent and their effect on insulin signalling w as investigated in m acrophages and adipocytes. A four h o u r treatm ent w ith either pioglitazone, troglitazone or rosiglitazone causes an average 50% increase in basal IRS-1 phosphorylation and also slightly increases p85 recruitm ent in adipocytes (figure 5.9). H ow ever the increase in p85 recruitm ent and IRS-1 p h o sp h o ry latio n in response to insulin is n o t increased fu rth er w ith TZD

m acrophages for four hours did not effect either basal or insulin-stim ulated p85 recruitm ent, how ever, rosiglitazone did appear to increase p85 recruitm ent com pared to basal cells (figure 5.10). The increased insulin signalling in basal cells m ight be expected to alter basal HSL activity, particularly in adipocytes where PI 3-kinase is clearly involved in HSL regulation. To investigate this 3T3 L-1 adipocytes were treated for four hours w ith TZD, or DMSO alone, and HSL activity was m easured. Figure 5.11 shows that, as predicted, basal HSL activity w as reduced by pioglitazone and troglitazone in adipocytes (rosiglitazone was n o t tested). The effect of rosiglitazone w as tested in m acrophages. The experim ent w as only repeated twice and results w ere variable. As a result, although it appears th at rosiglitazone treatm ent is causing a reduction in basal HSL activity by approxim ately the same am ount as acute insulin treatm ent (figure 5.12), the change compared to basal is not statistically significant, due to large errors. Therefore more experiments will be needed to confirm the effect of rosiglitazone on HSL activity in macrophages.

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Figure 5.4. Insulin and leptin stimulate p85 recruitment in macrophages

J774 cells were serum starved overnight prior to stimulation with 3nM leptin (A) or 10 nM insulin (B) before immunoprécipitation with anti-phosphotyrosine antibody and western blotting with anti-p85 antibody, to determ ine how stimulation effected recruitment of PI 3-kinase to tyrosine phosphorylated signalling complexes

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Figure 5.5. Insulin and leptin stimulate phosphatidylinositol 3-kinase activity in macrophages

J774 cells were serum starved overnight prior to stimulation with 10 nM insulin

(shaded bar) or 3nM leptin (filled bar). PI 3-kinase activity was assayed as

described in materials and methods. Phosphorylated lipids were separated by thin layer chromatography and quantitated using a phosphoimager. This is a single experiment which reinforces PI 3-kinase recruitment data in figure 5.4.