The FASEB Journal • FJ Express
Hepatic leptin signaling in obesity Georg Brabant,*,1 Gu¨nter Mu¨ller,† Ru¨diger Horn,* Christian Anderwald,㥋 Michael Roden,‡ and Heike Nave§ *Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; †Aventis Pharma, Frankfurt, Germany; ‡Medical Department, Hanusch Hospital, Vienna, Austria; §Department of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany; and 㛳Department of Internal Medicine III, University of Vienna, Austria To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2846fje; doi: 10.1096/fj.04-2846fje SPECIFIC AIMS Leptin released by adipose tissue exerts well known and profound effects on glucose metabolism. Numerous observations support an insulin-sensitizing action of leptin both via central hypothalamic actions and through peripheral mechanisms including direct hepatic effects. In diet induced obesity (DIO), a model of human obesity, hepatic insulin signaling is impaired but the links between leptin and insulin signaling pathways are incompletely defined. The aim of the present study was to evaluate the hepatic alterations of leptin signaling due to DIO and their effects on insulin cross-signaling in the liver. PRINCIPAL FINDINGS 1. Decreased leptin receptor expression in the liver of obese animals In lean animals, in situ hybridization with a probe recognizing Ob-R isoforms a– c and Ob-Rf revealed strong leptin receptor expression. Immunoblot analysis of the samples identified a predominance of the short isoforms of the receptor. After a bolus of exogenous human recombinant leptin, Ob-R mRNA expression was significantly reduced after 45 min but normalized 5 h later (Fig. 1A, B). DIO induced a significantly higher body weight compared with controls and increased circulating total leptin levels by ⬃4-fold. DIO rats did not express leptin receptor transcripts at any time point (Fig. 1B) and the expression of short isoforms in immunoblots was greatly reduced (Fig. 2). 2. Insulin signaling is impaired and the cross-talk between leptin and insulin signaling is differentially regulated by the nutritional status To further test the functional significance of these findings, we dynamically evaluated leptin receptor dependent intracellular signaling under in vivo stimulation with an exogenous leptin bolus. Under basal 1048
Figure 1. A) Quantification of mean density of in situ hybridizations in lean and diet-induced obese rats (n⫽6 in each group; *Pⱕ0.0001). B) In situ hybridization of Ob-R mRNA in cryostat sections of livers of lean (top panels) and dietinduced obese (bottom panels) rats, before (first row), 45 min (second row), and 6 h (third row) after an intravenous leptin infusion (⫻200).
conditions JAK-2pY protein expression was comparable in lean and DIO rats (Fig. 2). In DIO rats, in vivo stimulation with hrec-leptin did not alter Ob-R protein levels 45 min or 6 h later, but induced an ⬃5-fold JAK-2pY increase in lean animals after 45 min. Basal levels of IRS-1pY, IRS-1p85, IRS-2pY, and IRS-2p85 levels in DIO rats averaged at ⬃25/20% of lean controls. The decline of IRS-1pY after ad lib feeding was completely inhibited by hrec-leptin stimulation at 45 min and 6 h in lean animals, whereas in DIO rats basal 1
Correspondence: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neubergstr. 1, Hannover 30625, Germany. E-mail:
[email protected] 0892-6638/05/0019-1048 © FASEB
Figure 2. Ob-Rs, JAK-2, IRS-1/2, PKB, GSK-3, and AMPK␣ were immunoprecipitated with appropriate antibodies from liver extracts of lean (triangles) or DIO rats (squares, circles) which had been pretreated with hrecleptin or saline for 45 min or 6 h or left untreated (bars). Collected and washed immunoprecipitates were analyzed by immunoblot for the amount of Ob-Rs, pY (JAK-2, IRS-1/2), p85 (IRS-1/2), pT308 (PKB), pS9 (GSK-3), and AMPK␣ (AMPK␣) or assayed for AMPK activity as indicated. Representative autoradiograms of the immunoblots are shown in the insets. Quantitative data are given in means corrected for the amount of immunoprecipitated protein actually applied onto the gel (as revealed by homologous immunoblot analysis [data not shown]) and for blot-to-blot variation (as revealed by parallel analysis of a constant amount of corresponding marker proteins on each gel). PEPCK activity was measured with total liver extracts using [14C]NaHO3 fixation assay. *P ⬍0.05, leptin vs. saline.
levels, remained constant and leptin treatment had no effects including IRS-2pY and IRS-2p85. Phosphorylation of AKT/PKBp(T308) was clearly diminished in DIO as compared with lean controls and did not change under leptin stimulation. In normal weight animals, AKT/PKBp(T308) continuously decreased HEPATIC LEPTIN SIGNALING IN OBESITY
over time whereas PKBpT levels were significantly stimulated after leptin application. Lean animals showed higher basal GSK-3(S9) phosphorylation but lower PEPCK activity levels. Leptin stimulated GSK-3 phosphorylation acutely (45 min) followed by a decrease below the saline controls after 6 h. No change with time 1049
and no leptin effect was seen in DIO states, but basal PEPCK activity was significantly higher in DIO animals. Finally, we tested the effects on AMPK ␣-subunit. Minokoshi and co-workers previously demonstrated in muscle a time-dependent activation of AMPK by leptin whereas Jakobsen and co-workers described positive effects of AMPK on IRS-1 phosphorylation, which may explain the interaction between the leptin and insulin signaling pathways at the molecular level. Protein expression of AMPK ␣-subunit, which was significantly reduced in DIO compared with lean rats, possibly reflecting the leptin-resistant state of the former (Fig. 2). Only in lean animals was a small but significant increase shown 45 min after leptin. This increase was not significant in DIO rats, mostly due to slightly elevated basal AMPK activity in comparison to lean rats (Fig. 2). Figure 3. Schematic diagram of the leptin-to-insulin cross-talk in lean and obese subjects.
CONCLUSIONS AND SIGNIFICANCE DIO is associated with distinct alterations in leptin signaling and with the development of resistance to the central feedback action of leptin. Here we show a 4-fold increase in circulating total leptin levels along with a significant decrease of Ob-R (Ob-Rs) expression in the liver under basal conditions as measured by in situ hybridization and immunoblot analysis. These results were supported by a completely abolished postreceptor signaling in DIO rats. JAK will in turn lead to STAT3 and 5b phosphorylation, a process known to be decreased under chronic leptin stimulation. The recently demonstrated changes in leptin binding to the liver and in circulating leptin levels further support the observed alterations in leptin receptor expression, indicating that DIO renders the liver insensitive to leptin The altered leptin binding in DIO changed downstream signaling and its putative cross-talk to the insulin signaling pathways. Under basal conditions as well as under leptin stimulation, we reproduced the inhibitory effects of leptin on hepatic gluconeogenesis in lean rats. Interaction between leptin and insulin dependent signaling has been described on several levels. Recent data presented by Lam suggest a direct effect of leptin on insulin receptor degradation. Huang et al., studying leptin effects as well under a 90 min liver perfusion, indicated that leptin has additional effects on PI3K, an effect attenuated in DIO. These alternative pathways may, despite any dynamic effect on IRS-1p85 or IRS-2p85, explain the acute leptin action on serine phosphorylation of PKB/AKT and GSK-3. All these results are detectable in lean animals only. Because both effects fit the assumption that leptin attenuates hepatic glucose production and insulin resistance under normal conditions of lean animals, leptin adds to the inhibitory effects of insulin on gluconeogenesis. This effect is completely lost in obese animals, which suggests an important physiological dysregulation of leptin in DIO.
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Vol. 19
June 2005
Total AMPK activity was comparable in both groups. There was a slight stimulation of AMPK total activity in the lean 45 min after a bolus of leptin, whereas in DIO rats this effect did not reach significance. Our findings are at variance to the action of leptin in muscle. B. Kahn and co-workers recently suggested a major stimulatory effect of leptin on AMPK activity in muscle and hypothalamus, indicating a potential role of leptin on AMPK-dependent fatty acid oxidation. Our data, however, confirm recent results of Huang et al. when considering total AMPK activity in the liver. In summary, hepatic leptin resistance with an attenuated cross-talk between insulin and leptin signaling in obesity may be an important cofactor in the development of hepatic insulin resistance and increased glucose production in DIO. The increase in AMPK activity was not accompanied by elevation in the energy charge (AMP/ATP ratio; data not shown), arguing for AMPindependent stimulation of AMPK as has been described previously for AMPK up-regulation by leptin in muscle and liver. The apparent correlation among increased JAK-2 tyrosine phosphorylation, AMPK activation, IRS-1 tyrosine phosphorylation, PKB phosphorylation at T308, and GSK-3 phosphorylation at S9 in the liver of lean rats upon short-term challenge with leptin and the complete lack of corresponding leptin effects in DIO rats allows us to suggest the following: 1) involvement of JAK-2 in AMP-independent leptin regulation of AMPK; 2) positive cross-talk of AMPK to PKB and GSK-3 of the insulin signaling cascade; 3) operation of IRS-1 as point of convergence for positive interference between leptin and insulin signaling, possibly via AMPK-mediated serine phosphorylation leading to IRS-1pY-dependent but IRS-1p85-independent downstream signaling; and 4) desensitization of the leptin-to-insulin signaling cross-talk in DIO rats at the level of JAK-2, which may be based on hyperleptinemiainduced down-regulation of the liver-specific leptin receptor Ob-Rs (Fig. 3).
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BRABANT ET AL.
