Circulating levels of the insulin-sensitizing adipokine, adiponectin, are reduced in obesity, contributing to insulin resistance and the metabolic syndrome, and greatly increasing the risk of type 2 diabetes and cardiovascular disease [3
] (). Increasing adiponectin synthesis pharmacologically might help disrupt this pathogenetic process. Thiazolidinediones are insulin-sensitizing agonistic ligands for the nuclear receptor PPAR-γ that can activate PPAR-γ-RXR-α heterodimers bound to a PPRE in the adiponectin promoter to stimulate adiponectin transcription [8
]. The present study shows that IGFBP-3, hypoxia and TNF-α, three agents that promote insulin resistance in vivo
, inhibit human adiponectin PPRE-dependent promoter activity stimulated by the thiazolidinedione rosiglitazone in mouse fibroblasts.
Fig. 4 Schematic diagram illustrating a possible pathway by which hypoxia, chronic inflammation, and IGFBP-3 inhibit adiponectin transcription. Reduced adiponectin levels contribute to the development of insulin resistance and the metabolic syndrome, increasing (more ...)
Expression of IGFBP-3 in transgenic mice induced hyperglycemia, glucose intolerance and insulin resistance [34
], and IGFBP-3 inhibited insulin-stimulated glucose uptake in 3T3-L1 mouse adipocytes [13
]. Treatment of 3T3-L1 cells with IGFBP-3 also decreased rosiglitazone-stimulated adiponectin protein expression [13
]. IGFBP-3 can bind to the PPAR-γ heterodimer partner, RXR-α, and inhibited transcription stimulated by RAR-RXR-α by disrupting the heterodimer complex [14
]. This led us to postulate that IGFBP-3 also might inhibit rosiglitazone-stimulated adiponectin transcription mediated by PPAR-γ-RXR-α heterodimers. Using immortalized mouse embryo fibroblasts that stably expressed PPAR-γ2 [29
], we confirmed that rosiglitazone stimulated adiponectin-PPRE-dependent promoter activity. Cotransfection with YFP-IGFBP-3 fusion proteins inhibited rosiglitazone-stimulated promoter activity (). Inhibition was a direct effect of IGFBP-3 since similar inhibition was observed using recombinant human IGFBP-3 protein.
To determine whether IGFBP-3 inhibition of adiponectin promoter activity required its binding to RXR-α, we determined whether rosiglitazone-induced adiponectin transcription would be inhibited by an IGFBP-3 mutant that does not bind RXR-α [14
]. We previously reported that the RXR-α-non-binding IGFBP-3 mutant, YFP-HBD-11m-IGFBP-3, retained the ability of wild-type IGFBP-3 to induce apoptosis in human prostate cancer cells, indicating that direct binding of IGFBP-3 to RXR-α was not required for its pro-apoptotic activity [16
]. When mouse embryo fibroblasts were co-transfected with YFP-HBD-11m-IGFBP-3 and a luciferase reporter plasmid containing 3 copies of the human adiponectin PPRE, no inhibition of rosiglitazone-stimulated promoter activity was observed, in contrast to the inhibition observed when wild-type YFP-IGFBP-3 was transfected. The inability of the RXR-α-non-binding IGFBP-3 mutant to inhibit thiazolidinedione-stimulated adiponectin PPRE-dependent promoter activity is consistent with the possibility that direct binding of IGFBP-3 to RXR-α in the RXR-α-PPAR-γ heterodimer might be required for the inhibition of transcription, as described for RAR-RXR-α heterodimers [14
]. Further studies are necessary to determine whether the lack of inhibition is due to the inability of the IGFBP-3 mutant to bind RXR-α, however, since the COOH-terminal region that is mutated in HBD-11m-IGFBP-3 is highly basic and contains a functional nuclear localization signal as well as binding sites for other proteins besides RXR-α [31
]. (During the final stages of preparation of this manuscript, it was reported that wild-type IGFBP-3 (but not an IGFBP-3 mutant analogous to our RXR-α nonbinding mutant) also can bind to PPAR-γ, providing an alternative mechanism by which IGFBP-3 can inhibit adiponectin transcription [38
Two pathophysiologic conditions that develop in obese adipose tissue, hypoxia and chronic inflammation, contribute to the pathogenesis of insulin resistance and the metabolic syndrome [17
] (). Both hypoxia [19
] and the proinflammatory cytokine TNF-α [10
] inhibited adiponectin expression in 3T3-L1 adipocytes and human adipose tissue fragments. It is intriguing that hypoxia [24
] and TNF-α [13
] also induce IGFBP-3 transcription (). This led us to examine whether a hypoxia-mimetic agent, cobalt chloride [21
], and TNF-α could inhibit thiazolidinedione-stimulated adiponectin PPRE-dependent promoter activity in mouse embryo fibroblasts as did IGFBP-3. It previously was shown that hypoxia inhibited adiponectin transcription under basal [19
] and thiazolidinedione-stimulated [20
] conditions, and that TNF-α inhibited transcription under both basal and thiazolidinedione-stimulated conditions [10
]. In these studies, 1.3-3.6 kb adiponectin promoter fragments were used, so that the observed inhibition of thiazolidinedione-stimulated promoter activity could not be specifically attributed to the PPRE in the adiponectin promoter. By contrast, our studies show that both hypoxia and TNF-α inhibited rosiglitazone-stimulated adiponectin promoter activity using a reporter plasmid containing only 3 copies of the PPRE from the human adiponectin promoter, establishing that the PPRE itself is sufficient for the observed inhibition.
In summary, we have shown that IGFBP-3, hypoxia, and the pro-inflammatory cytokine TNF-α inhibit thiazolidinedione-stimulated adiponectin transcription, and may contribute to the pathogenesis of insulin resistance and the metabolic syndrome in patients with abdominal obesity (). Our results suggest that binding of IGFBP-3 to RXR-α in RXR-α-PPAR-γ heterodimers may be required for inhibition, and raise the possibility that the induction of IGFBP-3 by hypoxia and TNF-α may contribute to their ability to inhibit adiponectin transcription and promote insulin resistance.