Levels of insulin, leptin, and resistin were analyzed with a mouse serum adipokine LINCOplexkit (Linco Research) with the Bio-Plex System (Bio-Rad) according to the manufacturer’s instructions

Levels of insulin, leptin, and resistin were analyzed with a mouse serum adipokine LINCOplexkit (Linco Research) with the Bio-Plex System (Bio-Rad) according to the manufacturer’s instructions. == Glucose and Insulin Tolerance Tests and HOMA. of multiple signaling pathways through its long-form receptor Ob-Rb in leptin-responsive neurons (2,3). Mice deficient in leptin (ob/ob) or functional leptin receptor (db/db) manifest obesity, hyperphagia, and hyperglycemia (4,5). Although leptin resistance is found to be closely associated with obesity (6), the exact molecular defects underlying diminished leptin responsiveness remain poorly defined, largely ascribable to our incomplete understanding of the whole spectrum of Ob-Rb-mediated signaling events in energy homeostasis. Leptin triggers activation of Janus kinase 2 (JAK2) Ginsenoside Rg3 by binding to Ob-Rb (7) and elicits an array of subsequent intracellular tyrosyl-phosphorylation-dependent signaling actions, thought to be mediated through the 3 cytoplasmic tyrosine residues (Tyr985, Tyr1077, and Tyr1138) within mouse Ob-Rb. Phosphorylated Tyr1138is known to recruit signal transducer and activator of transcription 3 (STAT3) and activate the crucial JAK2STAT3 pathway in mediating leptin actions in energy homeostasis (8,9). Phosphorylation at Tyr985has been shown to mediate molecular events involving SH2-containing protein tyrosine phosphatase 2 (SHP2) (10,11), extracellular signal-regulated kinase (ERK) (12,13), and suppressor of cytokine signaling 3 (14,15). However, only recently has an autoinhibitory role been suggested in vivo for Tyr985in thel/lmice harboring a leucine substitution at this site (16). Whereas activation of other downstream signaling molecules, e.g., phosphatidylinositol 3-kinase (PI3K) MGC5370 (17) or STAT5 (18), may also contribute to the metabolic actions by Ob-Rb, the physiological importance of possible tyrosine-independent actions and their functional connections in vivo with tyrosine-dependent mechanisms remain largely unclear. To understand fully the physiological contributions of phosphotyrosine-mediated signaling actions of Ob-Rb, we generated 2 lines of knockin mice by introducing tyrosine-to-phenylalanine substitution mutations simultaneously at all 3 intracellular tyrosine sites or at Tyr1138alone. By comparing withdb/dbmice which are devoid of functional receptor signaling, we assessed Ginsenoside Rg3 the physiological importance of Ob-Rb signaling in the total absence of phosphotyrosine-mediated mechanisms, and actions mediated by Tyr985and Tyr1077but in the absence of Tyr1138-mediated STAT3 cascade, in regulation of energy balance and glucose metabolism. == Results == == Total Loss of Phosphotyrosine Actions of Ob-Rb Does Not Exacerbate the Obesity Phenotype Caused by Loss of STAT3 Ginsenoside Rg3 Action. == To maintain the structural integrity of Ob-Rb receptor, substitution mutations were introduced through homologous gene targeting, replacing with phenylalanine (F) the 3 tyrosine (Y) residues, Tyr985, Tyr1077and Tyr1138(denoted Y123F), or Tyr1138alone (denoted Y3F), within exon 18 of Ob-Rb (Fig. 1AandB). Y123F and Y3F homozygotes were obtained after backcrossing for 6 generations onto the C57BL/6 background, and all subsequent studies were carried out compared with C57BL/6db/dbmice. The wild-type (WT) littermates used were derived from the knockin lines, which showed no differences in body weight (BW), or fed glucose levels compared with the age-matched WT littermates derived from C57BL/6db/+ mice (data not shown), suggesting that little background difference existed metabolically between the two sources of experimental C57BL/6 animals. Quantitative RT-PCR analysis revealed comparable hypothalamic mRNA expression levels of Ob-Rb in Y123F and Y3F mice and in WT littermates, similar to that of the mutant Ob-Rb form indb/dbanimals [supporting information (SI) Fig. S1A]. Hypothalamic expression levels of Ob-Ra, one of the short isoforms of leptin receptor thought to lack signaling functions, were also similar in both knockin lines and WT littermates but dramatically lower than that indb/dbmice because of the genetic mutation of theOb-Rgene (19). Similar to the reported findings in thes/smice (8) with targeted substitution mutation at Tyr1138by serine, replacement by phenylalanine of all 3 tyrosines or Tyr1138within Ob-Rb abolished leptin-stimulated phosphorylation of STAT3 in the hypothalamus (Fig. 1C). Compared withdb/dbanimals, however, both age- and sex-matched Y123F and Y3F mice showed lower obesity levels, greater snoutanus lengths, and interestingly, appreciably higher lean mass (Table 1andFig. S1B). In contrast to Y3F females that exhibited similarly.