Distinct metabolic and vascular effects of dietary triglycerides and cholesterol in atherosclerotic and diabetic mouse models.

Cholesterol and triglyceride-rich Western diets are typically associated with an increased occurrence of type 2 diabetes and vascular diseases. This study aimed to assess the relative impact of dietary cholesterol and triglycerides on glucose tolerance, insulin sensitivity, atherosclerotic plaque formation, and endothelial function. C57BL6 wild-type (C57) mice were compared with atherosclerotic LDLr(-/-) ApoB(100/100) (LRKOB100) and atherosclerotic/diabetic IGF-II × LDLr(-/-) ApoB(100/100) (LRKOB100/IGF) mice. Each group was fed either a standard chow diet, a 0.2% cholesterol diet, a high-fat diet (HFD), or a high-fat 0.2% cholesterol diet for 6 mo. The triglyceride-rich HFD increased body weight, glucose intolerance, and insulin resistance but did not alter endothelial function or atherosclerotic plaque formation. Dietary cholesterol, however, increased plaque formation in LRKOB100 and LRKOB100/IGF animals and decreased endothelial function regardless of genotype. However, cholesterol was not associated with an increase of insulin resistance in LRKOB100 and LRKOB100/IGF mice and, unexpectedly, was even found to reduce the insulin-resistant effect of dietary triglycerides in these animals. Our data indicate that dietary triglycerides and cholesterol have distinct metabolic and vascular effects in obese atherogenic mouse models resulting in dissociation between the impairment of glucose homeostasis and the development of atherosclerosis.

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration.

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.

Adiponectin increases LPL activity via RhoA/ROCK-mediated actin remodelling in adult rat cardiomyocytes.

Cardiomyocyte substrate utilization is important in maintaining optimal cardiac function. Adiponectin has been shown to confer cardioprotective effects in part via regulating glucose and fatty acid uptake and oxidation in cardiomyocytes. Here we investigated mechanisms whereby adiponectin mediates a particular metabolic effect by focusing on lipoprotein lipase (LPL), an enzyme that increases free fatty acid availability to the heart by breakdown of chylomicrons and very-low-density lipoproteins in circulation. We used primary adult rat cardiomyocytes and demonstrate that adiponectin increased LPL translocation to the cell surface where it could be released at least partly in its active form, as evidenced by measuring basal and heparin-releasable LPL activity. Furthermore, these effects of adiponectin were mediated via remodeling of the actin cytoskeleton. We quantitatively assessed the filamentous to globular actin ratio and show that increased stress fiber formation, visualized by rhodamine-phalloidin immunofluorescence, in response to adiponectin, is achieved via stimulating Ras homolog gene family A (RhoA) activity, determined using G-LISA RhoA activation assay kit. We also demonstrate that adiponectin induces phosphorylation and inhibition of cofilin, leading to a reduction in actin treadmilling. Increased cofilin phosphorylation and stress fiber formation in response to adiponectin were prevented by inhibition of either RhoA or its downstream kinase Rho-associated protein kinase. Importantly, inhibition of cytoskeletal remodeling prevented adiponectin-stimulated plasma membrane LPL content detected by immunofluorescence and also subsequent LPL activity. In summary, we show that adiponectin mediates actin cytoskeleton remodeling to translocate LPL and allow subsequent activation.

An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart.

Adiponectin promotes cardioprotection by various mechanisms, and this study used primary cardiomyocytes and the isolated working perfused heart to investigate cardiometabolic effects. We show in adult cardiomyocytes that adiponectin increased CD36 translocation and fatty acid uptake as well as insulin-stimulated glucose transport and Akt phosphorylation. Coimmunoprecipitation showed that adiponectin enhanced association of AdipoR1 with APPL1, subsequent binding of APPL1 with AMPKα2, which led to phosphorylation and inhibition of ACC and increased fatty acid oxidation. Using siRNA to effectively knockdown APPL1 in neonatal cardiomyocytes, we demonstrated an essential role for APPL1 in mediating increased fatty acid uptake and oxidation by adiponectin. Importantly, enhanced fatty acid oxidation in conjunction with AMPK and ACC phosphorylation was also observed in the isolated working heart. Despite increasing fatty acid oxidation and myocardial oxygen consumption, adiponectin increased hydraulic work and maintained cardiac efficiency. In summary, the present study documents several beneficial metabolic effects mediated by adiponectin in the heart and provides novel insight into the mechanisms behind these effects, in particular the importance of APPL1.

Differential impact of adipokines derived from primary adipocytes of wild-type versus streptozotocin-induced diabetic rats on glucose and fatty acid metabolism in cardiomyocytes.

The causal relationship between obesity and cardiovascular disease is extensively acknowledged; however, the exact mechanisms linking obesity and heart failure remain unclear. Here, we investigated the influence of adipokines derived from primary adipocytes on glucose and fatty acid uptake and metabolism in isolated primary cardiomyocytes. Either co-culture of these cell types or incubation with adipocyte-conditioned medium significantly increased glucose uptake in cardiomyocytes. When streptozotocin-induced diabetic rats were used as a source of adipocytes, there was a lower ability to elicit glucose uptake in cardiomyocytes which corresponded with lower Akt and AMPK phosphorylation. The profile of glucose metabolism also differed with oxidation being favored upon co-culture with wild-type adipocytes whereas lactate production was strongly induced by adipocytes from diabetic rats. Examination of fatty acid uptake revealed that stimulation only occurred in response to adipokines secreted by wild-type rat adipocytes. Importantly, oxidation of fatty acids by cardiomyocytes was decreased by adipokines derived from diabetic rat adipocytes. Analysis of adipokine profiles in diabetic rat adipocyte-conditioned medium demonstrated the most significant decreases in adiponectin and leptin with increased IL6 expression. Taken together, these data suggest that the profile of adipokines secreted by adipocytes from diabetic rats have a deleterious influence on cardiomyocyte metabolism which may be of relevance in the pathophysiology of heart failure.

Characterization of two components of the 2-naphthoate monooxygenase system from Burkholderia sp. strain JT1500.

2-Naphthoate monooxygenase, a two-protein system, encoded by the nmoA and nmoB genes, was heterologously overexpressed in Escherichia coli. The proteins used for functional characterization were purified to over 90% homogeneity by affinity chromatography. The oxidative component EnmoA (47.9 kDa) lacked substrate catalysis capability on its own, and the reductive component EnmoB (33.4 kDa) and its truncated derivate EnmoB(T) (25 kDa) possessed nearly identical independent flavin reductase activities, c. 130 micromol min(-1) mg(-1) of protein. The inframe fusioned protein EnmoB(T)A (65.2 kDa), containing NmoB(T) and NmoA peptides showed a stable 2-naphthoate monooxygenase activity of 1.2 micromol min(-1) mg(-1) of protein. This is the first report on the purification of a fused form of a two-component flavoprotein monooxygenase. In the specificity experiment, FAD and NADH were shown to be preferred cosubstrates for EnmoB and EnmoB(T). All these data suggest that NmoB(T)A is a two-component flavoprotein monooxygenase, consisting of an oxygenase and a reductase component. NmoA is a member of the class D flavoprotein monooxygenase, and NmoB is an independent NADH:Flavin oxidoreductase.

Coculture with primary visceral rat adipocytes from control but not streptozotocin-induced diabetic animals increases glucose uptake in rat skeletal muscle cells: role of adiponectin.

We developed a coculture system comprising primary rat adipocytes and L6 rat skeletal muscle cells to allow investigation of the effects of physiologically relevant mixtures of adipokines. We observed that coculture, or adipocyte-conditioned media, increased glucose uptake in muscle cells. An adipokine that could potentially mediate this effect is adiponectin, and we demonstrated that small interfering RNA-mediated knockdown of adiponectin receptor-2 in muscle cells reduced the uptake of glucose upon coculture with primary rat adipocytes. Analysis of coculture media by ELISA indicated total adiponectin concentration of up to 1 microg/ml, and Western blotting and gel filtration analysis demonstrated that the adipokine profile was hexamer greater than high molecular weight much greater than trimer. We used the streptozotocin-induced rat model of diabetes and found that high-molecular-weight adiponectin levels decreased in comparison with control animals and this correlated with the fact that diabetic rat-derived primary adipocytes in coculture did not stimulate glucose uptake to the same extent as control adipocytes. Coculture induced phosphorylation of AMP-activated protein kinase (T172) and interestingly also insulin receptor substrate-1 (Y612) and Akt (T308 & S473), which could be attenuated after adiponectin receptor-2-small interfering RNA treatment. In summary, we believe that this coculture system represents an excellent model to study the effects of primary adipocyte-derived adipokine mixtures on skeletal muscle metabolism, and here we have established that in the context of physiologically relevant mixtures of adipokines, adiponectin may be an important determinant of positive cross talk between adipocytes and skeletal muscle.

Globular and full-length forms of adiponectin mediate specific changes in glucose and fatty acid uptake and metabolism in cardiomyocytes.

OBJECTIVE: Our aim was to investigate the regulation of glucose and fatty acid metabolism in cardiomyocytes by the globular (gAd) and full-length (fAd) forms of adiponectin. METHODS: We produced fAd (consisting of high, medium and low molecular weight oligomers) in a mammalian expression system and gAd in bacteria. These were used to treat primary neonatal rat cardiomyocytes (up to 48 h), and we employed 3H- or 14C-labeled substrates to monitor glucose uptake and subsequent metabolism via oxidation, glycogen synthesis or lactate production and fatty acid uptake and oxidation. Enzymatic assay for acetyl CoA carboxylase activity was employed, and protein phosphorylation and expression was determined by immunoblotting cell lysates. The role of adiponectin receptor (AdipoR) isoforms was determined via siRNA-mediated knockdown. RESULTS: There was an initial (1 h) increase in glucose uptake and oxidation in response to gAd or fAd. Fatty acid uptake was stimulated by gAd or fAd, and by 24 h a decrease in acetyl CoA carboxylase activity and elevated fatty acid oxidation were observed. After 48 h increased fatty acid oxidation correlated with decreased glucose oxidation and pyruvate dehydrogenase activity, while glycogen synthesis and lactate production increased. Both gAd and fAd elicited phosphorylation of AMP kinase, insulin receptor substrate-1, Akt and glycogen synthase kinase-3beta. Knockdown of AdipoR1 or AdipoR2 attenuated the effect of both gAd and fAd on fatty acid uptake and oxidation. Only AdipoR1 knockdown prevented the ability of gAd (1 h) to increase glucose uptake and oxidation; however, reducing either AdipoR1 or AdipoR2 expression attenuated the long-term (24 h) effects of gAd. CONCLUSIONS: These results clearly demonstrate that gAd and fAd mediate distinct and time-dependent effects on cardiomyocyte energy metabolism via AdipoR1 and AdipoR2.

[Cloning and expression of a 2-naphthoate monooxygenase gene (nmo) in Burkholderia sp. JT1500].

A 4.8kb DNA fragment from one blue colony of the pLARF1 gene library of Burkholderia sp. JT1500 was subcloned to pUC18, designated as pEK123. The sequence of the inserted 4.8kb DNA' of pEK123 was analyzed and submitted to EMBL nucleotide database, the accession # is AJ566333. The transformants of pEK123 could also become blue in LB agar and sequence analysis showed that three open reading frames and a putative promoter sequence were located in this inserted fragment. Then the 4.4kb insert fragment of pEK123 was double digested with Xba I / Kpn I and EcoR I / Xba I respectively to construct plamsids pXK3 and pEX12. The pXK3 contained only one 1158bp open reading frame (ORF) and pEX12 with other two ORFs. Unlike pEK123, the colonies of pEX12 did not show any blue color even incubated for 72h in LB agar, but the transformants of pXK3 did oxidize indole into indigo. The deduced 43kD protein of 1158bp ORF showed 64% homology of amino acid composition to Ralstonia eutropha HF39 hydroxylase (bec). Results of substrate transformation analysis showed that the transformants of pEK123 was able to catalyze the oxidation of 2-naphthoate but not other key intermediates in 2-naphthoate metabolic pathway. These results confirmed that the product of 1158bp ORF is 2-naphthoate monooxygenase. Though the oxygenase activity of pEK123 is much higher than that of pXK3, SDS-PAGE analysis found no difference between the amount of the band of monooxygenase produced by pXK3 or pEK123, but one more band was found produced by pEK123. According to the difference of substrate analysis between pXK3 and pEK123, it is supposed that the products of two open reading frames up stream of nmo gene had strong influence on the activity of the monooxygenase. Benzoate was oxidized by free-cell extracts of the transformants of pEK123 in the transformation experiment with different aromatic substrates. As the DNA sequence and amino acid sequence of 2-naphthoate monoxygenase (nmo) did no show any homology with the DNA sequence and amino acid sequence of benzoate oxygenases reported, the pathway of benzoate oxidation conducted by nmo is on the investigation.