Leptin receptor action and mechanisms of leptin resistance.

The adipose tissue-derived hormone leptin regulates energy balance and neuroendocrine function. Resistance to the appetite-suppressing effects of leptin is associated with common forms of obesity. Here, we review the mechanisms by which leptin activates intracellular signals and the roles that these signals play in leptin action in vivo. Furthermore, we discuss potential mechanisms of leptin resistance, specifically focusing on data regarding the neuroanatomical locus of leptin resistance and potential mechanisms by which expression of the suppressor of cytokine signaling-3 may impair leptin action.

Acute stimulation of glucose uptake by leptin in l6 muscle cells.

The adipocyte hormone, leptin, acts via the central nervous system to modulate glucose metabolism by skeletal muscle, but the direct effects of leptin on glucose metabolism by skeletal muscle are unclear. In this study, we have examined effects of leptin on glucose uptake by cultured L6 muscle cells assessed with the non-metabolised glucose analogue 2-deoxy-D-glucose. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of RNA showed that L6 muscle cells express a short isoform of the leptin receptor (ObRa), but not the long isoform (ObRb). In the absence of added insulin, incubation of L6 muscle cells with murine leptin (10( -11)-10( -8) M) for 10 min and 1 h increased glucose uptake by 15 % - 23 %. This effect of leptin was lost by 4 h. Leptin (10( -10) - 10( -9) M) initially (after 10 min) suppressed insulin-stimulated glucose uptake by 14 - 16 %, but had no effect in the longer term. Leptin-stimulated glucose uptake was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not by the janus kinase-2 (JAK-2) inhibitor tyrphostin AG490. The results suggest that leptin can act directly on L6 muscle cellsvia a short leptin receptor isoform to acutely stimulate basal (but not insulin-stimulated) glucose uptake via a PI3K-dependent pathway.

Sibutramine metabolites increase glucose transport by cultured rat muscle cells.

BACKGROUND: The anti-obesity agent sibutramine, a serotonin and noradrenaline reuptake inhibitor (SNRI), has been shown to reduce insulin resistance and improve glycaemic control in obese-diabetic ob/ob mice and overweight type 2 diabetic patients. OBJECTIVE: To investigate whether sibutramine or its metabolites act directly on muscle cells to improve glucose uptake and insulin action. DESIGN: Uptake of the non-metabolized glucose analogue 2-deoxyglucose was measured in cultured L6 rat muscle cells after incubation with sibutramine, its two pharmacologically active metabolites and related agents. RESULTS: Sibutramine itself (10(-8)-10(-6) M) did not significantly affect 2-deoxyglucose uptake during incubations up to 72 h. The primary amine metabolite M2 (10(-7) and 10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 12% and 34%) after 24 h incubation. These effects of M2 were lost by 72 h incubation. However, the secondary amine metabolite M1 (10(-6) M) increased basal and insulin-stimulated 2-deoxyglucose uptake (by 50%) after 72 h incubation, although M1 was ineffective after 24 h. M2 stimulated 2-deoxyglucose uptake in the presence of LY-294,002 (an inhibitor of phosphatidylinositol 3-kinase) but the effect of M2 was inhibited by cytochalasin B, which acutely blocks glucose transporters. Incubations with serotoninergic, noradrenergic and dopaminergic agents, or agents known to stimulate release or inhibit reuptake of these substances in nervous tissues indicated that the sibutramine metabolites were not affecting 2-deoxyglucose uptake via mechanisms associated with their SNRI properties. CONCLUSIONS: Sibutramine metabolites can improve insulin-sensitive 2-deoxyglucose uptake by cultured muscle cells independently of SNRI effects.

Divergent roles of SHP-2 in ERK activation by leptin receptors.

The protein tyrosine phosphatase SHP-2 has been proposed to serve as a regulator of leptin signaling, but its specific roles are not fully examined. To directly investigate the role of SHP-2, we employed dominant negative strategies in transfected cells. We show that a catalytically inactive mutant of SHP-2 blocks leptin-stimulated ERK phosphorylation by the long leptin receptor, ObRb. SHP-2, lacking two C-terminal tyrosine residues, partially inhibits ERK phosphorylation. We find similar effects of the SHP-2 mutants after examining stimulation of an ERK-dependent egr-1 promoter-construct by leptin. We also demonstrate ERK phosphorylation and egr-1 mRNA expression in the hypothalamus by leptin. Analysis of signaling by ObRb lacking intracellular tyrosine residues or by the short leptin receptor, ObRa, enabled us to conclude that two pathways are critical for ERK activation. One pathway does not require the intracellular domain of ObRb, whereas the other pathway requires tyrosine residue 985 of ObRb. The phosphatase activity of SHP-2 is required for both pathways, whereas activation of ERK via Tyr-985 of ObRb also requires tyrosine phosphorylation of SHP-2. SHP-2 is thus a positive regulator of ERK by leptin receptors, and both the adaptor function and the phosphatase activity of SHP-2 are critical for this regulation.

SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985.

During leptin signaling, each of the phosphorylated tyrosine residues on the long form of the leptin receptor (LRb) mediates distinct signals. Phosphorylated Tyr(1138) binds STAT3 to mediate its tyrosine phosphorylation and transcriptional activation, while phosphorylated Tyr(985) binds the tyrosine phosphatase SHP-2 and reportedly mediates both activation of ERK kinases and inhibition of LRb-mediated STAT3 activation. We show here that although mutation of Tyr(985) does not alter STAT3 signaling by erythropoietin receptor-LRb (ELR) chimeras in transfected 293 cells at short times of stimulation, this mutation enhances STAT3 signaling at longer times of stimulation (>6 h). These data suggest that Tyr(985) may mediate feedback inhibition of LRb signaling by an LRb-induced LRb inhibitor, such as SOCS3. Indeed, SOCS3 binds specifically to phosphorylated Tyr(985) of LRb, and SOCS3 fails to inhibit transcription by ELR following mutation of Tyr(985), suggesting that SOCS3 inhibits LRb signaling by binding to phosphorylated Tyr(985). Additionally, overexpression of SOCS3, but not SHP-2, impairs ELR signaling, and the overexpression of SHP-2 blunts SOCS3-mediated inhibition of ELR signaling. Thus, our data suggest that in addition to mediating SHP-2 binding and ERK activation during acute stimulation, Tyr(985) of LRb mediates feedback inhibition of LRb signaling by binding to LRb-induced SOCS3.

Insulin-like effect of pinitol.

D-pinitol (3-O-methyl-chiroinositol), an active principle of the traditional antidiabetic plant Bougainvillea spectabilis, is claimed to exert insulin-like effects. This study investigates the effect of D-pinitol on glucose homeostasis in animal models of diabetes, and on glucose transport by cultured muscle cells. Plasma glucose concentrations were measured in normal, obese-diabetic (ob/ob) and streptozotocin (STZ)-diabetic mice after oral (p.o.) and intraperitoneal (i.p.) administration of D-pinitol. Glucose transport was measured in L6 rat muscle cells by 2-deoxyglucose (2DG) uptake. In STZ-diabetic mice, 100 mg kg(-1) p.o. D-pinitol acutely decreased the hyperglycaemia (by 22% at 6 h). A similar decrease in plasma glucose (by 21%) was observed after 100 mg kg(-1) i.p. D-pinitol. Insulin concentrations and the rate of insulin-induced (1 unit kg(-1) actrapid i.p.) glucose disappearance were not altered by 100 mg kg(-1) p.o. D-pinitol. Chronic administration of D-pinitol (100 mg kg(-1) i.p. twice daily for 11 days) to STZ-diabetic mice maintained a reduction in plasma glucose concentrations from about 14 to 10 mmol l(-1). In normal non-diabetic and severely insulin resistant ob/ob mice, 100 mg kg(-1) p.o. D-pinitol did not significantly affect plasma glucose or insulin during acute studies. Incubation of L6 muscle cells with D-pinitol (10(-3) M) increased basal 2DG uptake by 41% after 10 min and by 34% after 4 h. The effect of D-pinitol was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. D-pinitol did not increase insulin-stimulated 2DG uptake by L6 cells. The data support the view that D-pinitol can exert an insulin-like effect to improve glycaemic control in hypoinsulinaemic STZ-diabetic mice. D-pinitol may act via a post-receptor pathway of insulin action affecting glucose uptake.

Activation of downstream signals by the long form of the leptin receptor.

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.