Energy restriction enhances therapeutic efficacy of the PPARgamma agonist, rosiglitazone, through regulation of visceral fat gene expression.

AIM: Consumption of a palatable diet can induce hyperphagia, leading to weight gain (dietary obesity) and insulin resistance in rats. Thiazolidinediones (TZDs) can also induce hyperphagia in rats but conversely have an insulin-sensitizing effect. The aim of this study was to investigate whether preventing TZD-induced hyperphagia (i.e. energy restriction) in dietary obese (DIO) rats would enhance the insulin-sensitizing effects of treatment at a therapeutic dose; and, within this paradigm, to produce an original survey of candidate TZD-gene targets in the clinically relevant visceral white adipose tissue (WAT) depot. METHODS: DIO rats that were either freely fed or energy restricted (i.e. pair-fed to the level of untreated controls) were treated with rosiglitazone maleate (RSG; 3 mg/kg/day) for 2 weeks, the restricted group controlling for treatment-induced hyperphagia and weight gain. The outcome measures were circulating concentrations of various biochemical markers of insulin resistance, and gene expression was measured in epididymal WAT. RESULTS: In both freely fed and pair-fed groups, compared to untreated DIO controls, RSG reduced plasma levels of insulin (-29% and -43%; p < 0.05 and p < 0.001, respectively), free fatty acids (FFAs; -45% and -48%; p < 0.01 and p < 0.001, respectively) and triglycerides (TGs; -63% and -72%; both p < 0.001), reflected in improved insulin sensitivity, as measured by homeostasis model assessment (-29% and -43%; p < 0.01 and p < 0.0001). RSG also increased the expression of the fatty acid transport/synthesis genes, fatty acid transport protein (2.4-3.2-fold), epidermal fatty acid-binding protein (FABP; 1.7-2.0-fold), heart FABP (25-29-fold) and fatty acid synthase (2.3-2.9-fold; all p < 0.05) in both groups. Adipocyte FABP was also increased by RSG treatment, but only in combination with energy restriction (1.52-fold; p < 0.05) as was hexokinase II expression (p < 0.001). In contrast, the drug had no effect on expression of several genes associated with lipolysis. Although obesity-induced hyperleptinaemia was normalized only in the energy-restricted group, leptin messenger RNA (mRNA) expression was reduced in both treated groups (all p < 0.01). Resistin and tumour necrosis factor-alpha expression was also reduced, though in the latter case, only with energy restriction (p < 0.05). Other adipokines were unaffected by RSG treatment. CONCLUSION: Our results clearly show that energy restriction enhances the therapeutic efficacy of TZDs and suggest that this occurs, at least in part, through a modulatory effect on gene expression in visceral WAT. These findings improve our understanding of the underlying mechanistic basis for the clinical usefulness of dietary restriction as an adjunct to TZD therapy in type 2 diabetes.

Leptin receptor long-form signalling in a human liver cell line.

Expression of the long form of the leptin receptor was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting in the human liver cell line WRL68. Leptin (50-200 nM) significantly increased tyrosine phosphorylation of STAT cytoplasmic transcription factors STAT3 and STAT5b in a dose-dependent manner and produced a gel-shift with STAT3- and STAT5-specific oligonucleotides. WRL68 cells therefore provide the first human in vitro hepatocyte system in which to study leptin receptor-mediated signalling and to elucidate the role of leptin in liver.

Fatty acids inhibit leptin signalling in BRIN-BD11 insulinoma cells.

The effect of treatment with a 0.03% fatty acid (FA) cocktail on leptin-receptor-mediated STAT (signal transducers and activators of transcription) activation in the rat insulinoma cell line BRIN-BD11 was investigated. Leptin (10 nM) stimulated the tyrosine phosphorylation of STAT3 and STAT5b. Acute treatment with FAs prevented leptin-stimulated STAT3 tyrosine phosphorylation and significantly raised basal STAT5 phosphorylation. A chronic treatment (5 days) of BRIN-BD11 cells with FAs similarly attenuated leptin-stimulated STAT tyrosine phosphorylation. Chronic FA treatment also attenuated prolactin-stimulated STAT5b tyrosine phosphorylation but not interleukin-6-stimulated STAT3 tyrosine phosphorylation, suggesting that the effect is receptor/ligand specific. TaqMan analysis of gene expression following chronic FA treatment showed neither a decrease in the amount of leptin receptor (Ob-R) mRNA, nor an increase in the negative regulators of STAT signalling, SOCS3 (suppressors of cytokine signalling) or cytokine inducible sequence (CIS). These data demonstrate that FAs modulate leptin and prolactin signalling in beta-cells, implying that high levels of circulating FAs present in obese individuals affect the action of selective cytokines in beta-cell function.

Heterologous desensitization of bombesin- and vasopressin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

Bombesin- and vasopressin-stimulated phospholipase D (PLD) activities are rapidly desensitized in 3T3 cells, in addition both agonists are subject to heterologous desensitization. Binding studies showed that homologous desensitization was partly a result of loss of cell surface receptors, whilst heterologous desensitization was independent of receptor changes. Pretreatment with either agonist reduced subsequent GTP gamma S-stimulated PLD activity by 50% whereas a pretreatment with GTP gamma S did not attenuate the response, suggesting that the G-protein or downstream effector systems were affected by receptor activation resulting in desensitization. The desensitization of receptor-stimulated PLD activation provides support for the phospholipase functioning in a key signalling pathway.

The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

Rapid desensitization and resensitization of bombesin-stimulated phospholipase D activity in Swiss 3T3 cells.

The kinetics of bombesin-stimulated phospholipase D (PLD) activity were examined in Swiss 3T3 fibroblasts. The stimulated activity was found to rapidly desensitize, being completely absent after 40 s. This activity then quickly, but incompletely, resensitized, with PLD being detectable after a 4.5 min wash of the desensitized cells and 75-80% of the activity being recovered after 10 min. The desensitization was dose-dependent; however, the half-maximal stimulatory concentration of bombesin was an order of magnitude lower than that required for bombesin-stimulated second messenger generation and the KD for bombesin receptor binding. This suggested that desensitization was stimulated by a 'downstream' effect, but experiments have ruled out changes in protein kinase C activity and Ca2+ concentration. Binding experiments suggested that part of the desensitization is due to receptor internalization, and the requirement for an extracellular agonist for resensitization implies that receptor recycling plays a role. Over an extended time course, cycles of desensitization and resensitization of bombesin-stimulated PLD activity were apparent which may be relevant to mitogenic signalling. These studies add further evidence for a second messenger pathway of PLD activation, and the disparity between the kinetics of diacylglycerol generation and PLD activation supports the possibility that phosphatidic acid may have a messenger role in stimulated cells.

The regulation of phospholipase D activity and its role in sn-1,2-diradylglycerol formation in bombesin- and phorbol 12-myristate 13-acetate-stimulated Swiss 3T3 cells.

Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) to quiescent Swiss 3T3 cells resulted in a sustained increase in sn-1,2-diradylglycerol (DG) mass and [3H]DG in [3H]palmitate-labelled cells where phosphatidylcholine was the major labelled phospholipid. This occurred in the absence of inositol phosphate accumulation. In [3H]palmitate-labelled cells both bombesin and PMA stimulated the formation of phosphatidylbutanol ([3H]PtdBut) in the presence of 0.3% (v/v) butan-1-ol. The kinetics of [3H]PtdBut formation were consistent with phospholipase D (PLD) activation preceding sustained DG formation. The inclusion of butan-1-ol inhibited 70% of PMA-stimulated DG formation but only 30% of the bombesin response. The ability of bombesin and PMA to stimulate the accumulation of [3H]PtdBut was completely abolished in Swiss 3T3 cells which had been pre-treated with 400 nM-PMA for 48 h to down-regulate protein kinase C activity. PMA-stimulated [3H]PtdBut formation was inhibited by 90% by the protein kinase C inhibitor Ro-31-8220 (10 microM), but bombesin-stimulated PtdBut accumulation was inhibited by at most 50% by the same concentration of inhibitor. Cyclic AMP-elevating agents, i.e. forskolin, dibutyryl cyclic AMP and isobutylmethylxanthine, did not inhibit bombesin stimulation of PLD activity. Bombesin-stimulated PLD activity was inhibited by 50% by buffering of the extracellular Ca2+ concentration to 150 nM, but combination of this treatment with Ro-31-8220 addition was less than additive. Ionophore A23187 alone was able to stimulate PLD activity, but this response was inhibited 50% by Ro-31-8220. Thapsigargin was unable to stimulate PLD activity and had no modulatory effect upon bombesin-stimulated PLD activity at any agonist concentration. The results are discussed in terms of the role of PLD in DG generation and the regulation of PLD activity both by bombesin and by PMA.