Retinol as a cofactor for PKCδ-mediated impairment of insulin sensitivity in a mouse model of diet-induced obesity.

We previously defined that the mitochondria-localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl-coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet-induced insulin resistance. We crossed mice overexpressing human retinol-binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ(-/-) strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ(+/+)hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ(-/-)hRBP and PKCδ(-/-) mice. Therefore, in addition to the well-defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole-body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.

Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: excimer probe study.

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063µMmin(-1)) and catalytic rate constant kcat (0.42s(-1)) together with a similarity of Km (0.9µM CPD) and KI (2.8µM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.

Two protein kinase C isoforms, δ and ε, regulate energy homeostasis in mitochondria by transmitting opposing signals to the pyruvate dehydrogenase complex.

Energy production in mitochondria is a multistep process that requires coordination of several subsystems. While reversible phosphorylation is emerging as the principal tool, it is still unclear how this signal network senses the workloads of processes as different as fuel procurement, catabolism in the Krebs cycle, and stepwise oxidation of reducing equivalents in the electron transfer chain. We previously proposed that mitochondria use oxidized cytochrome c in concert with retinol to activate protein kinase Cδ, thereby linking a prominent kinase network to the redox balance of the ETC. Here, we show that activation of PKCε in mitochondria also requires retinol as a cofactor, implying a redox-mechanism. Whereas activated PKCδ transmits a stimulatory signal to the pyruvate dehdyrogenase complex (PDHC), PKCε opposes this signal and inhibits the PDHC. Our results suggest that the balance between PKCδ and ε is of paramount importance not only for flux of fuel entering the Krebs cycle but for overall energy homeostasis. We observed that the synthetic retinoid fenretinide substituted for the retinol cofactor function but, on chronic use, distorted this signal balance, leading to predominance of PKCε over PKCδ. The suppression of the PDHC might explain the proapoptotic effect of fenretinide on tumor cells, as well as the diminished adiposity observed in experimental animals and humans. Furthermore, a disturbed balance between PKCδ and PKCε might underlie the injury inflicted on the ischemic myocardium during reperfusion. dehydrogenase complex.

Insights into the molecular mechanisms of the anti-atherogenic actions of flavonoids in normal and obese mice.

Obesity is a major and independent risk factor for cardiovascular disease and it is strongly associated with the development of dyslipidemia, insulin resistance and type 2 diabetes. Flavonoids, a diverse group of polyphenol compounds of plant origin widely distributed in human diet, have been reported to have numerous health benefits, although the mechanisms underlying these effects have remained obscure. We analyzed the effects of chronic dietary supplementation with flavonoids extracted from cranberry (FLS) in normal and obese C57/BL6 mice compared to mice maintained on the same diets lacking FLS. Obese mice supplemented with flavonoids showed an amelioration of insulin resistance and plasma lipid profile, and a reduction of visceral fat mass. We provide evidence that the adiponectin-AMPK pathway is the main mediator of the improvement of these metabolic disorders. In contrast, the reduced plasma atherogenic cholesterol observed in normal mice under FLS seems to be due to a downregulation of the hepatic cholesterol synthesis pathway. Overall, we demonstrate for the first time that the molecular mechanisms underlying the beneficial effects of flavonoids are determined by the metabolic state.

Composition, structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester.

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein-phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.

Serum retinol-binding protein 4 (RBP4) and retinol in a cohort of borderline obese women with and without gestational diabetes.

OBJECTIVES: To evaluate whether serum RBP4 correlates with gestational diabetes mellitus (GDM) in a cohort of borderline obese (BMI>30) pregnant women. DESIGN AND METHODS: Serum RBP4 and retinol were measured in pregnant women with (n=12) and without (n=10) GDM. RESULTS: RBP4, retinol and RBP4:retinol molar ratio were not different between the groups and were not associated with markers of insulin resistance. CONCLUSIONS: GDM is not associated with RBP4 or retinol among borderline obese pregnant women.

Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers.

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation.

Allosteric control of the RNA polymerase by the elongation factor RfaH.

Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that 'fast' RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis-Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 A away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and lambdaQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex.

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids.

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.