Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

A convenient synthesis of 3-keto bile acids by selective oxidation of bile acids with silver carbonate-Celite.

A number of 3-keto bile acids were synthesized by the selective oxidation of bile acid methyl esters with silver carbonate-Celite in refluxing toluene. The pure 3-keto bile acids were isolated simply by filtering the reaction mixture and concentrating the filtrate. The relation of the bile acid structure to the oxidation rate is also discussed.     

Spiropentaneacetic acid as a specific inhibitor of medium-chain acyl-CoA dehydrogenase

To study the structure-activity relationship between pentanoic acid analogues and the inhibition of fatty acid oxidation, a number of 4-pentenoic and methylenecyclopropaneacetic acid derivatives were prepared. All compounds inhibited palmitoylcarnitine oxidation in rat liver mitochondria, with 50% inhibition occurring at a concentration between 6 and 100 microM. However, only methylenecyclopropaneacetic acid (MCPA) and spiropentaneacetic acid (SPA) showed in vivo inhibitory activity in rats as indicated by the occurrence of dicarboxylic aciduria. Rats treated with SPA excreted metabolites derived only from fatty acid oxidation whereas MCPA-treated rats also excreted metabolites derived from branch-chained amino acid and lysine metabolism. SPA is a specific inhibitor of fatty acid oxidation without affecting amino acid metabolism. The site of inhibition is medium-chain acyl-CoA dehydrogenase (MCAD). In contrast, MCPA inhibited both MCAD and short-chain acyl-CoA dehydrogenase with a stronger inhibition toward the latter. The inhibition of fatty acid oxidation by both inhibitors was partially reversible by glycine or l-carnitine. Since SPA does not form a ring-opened nucleophile such as that proposed for MCPA in the inhibition of FAD prosthetic group in acyl-CoA dehydrogenases, we propose that the irreversible inhibition by SPA occurs by a tight complex without forming a covalent bond to the isoalloxazine ring in FAD.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway

About a decade ago calcitroic acid was isolated as a major side chain cleaved water-soluble metabolite of 1,25-dihydroxyvitamin D3 [Esvelt, R. P., Schnoes, H. K., & Decula, H. F. (1979) Biochemistry 18, 3977]. Presently, calcitroic acid is being considered as the major excretory form of 1,25-dihydroxyvitamin D3. However, the exact site or sites of calcitroic acid production and the possible side chain modified intermediary metabolites that may be formed during the conversion of 1,25-dihydroxyvitamin D3 into calcitroic acid are not fully understood. In the mean time there have been many advances in our understanding of the side-chain metabolism of 1,25-dihydroxyvitamin D3. It is now well established that both the kidney and the intestine metabolize 1,25-dihydroxyvitamin D3 through the C-24 oxidation pathway according to the following steps: 1,25-dihydroxyvitamin D3----1,24,25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3-----1,23,25-trihydroxy-24-oxovitamin D3. Recently, we identified 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-23 alcohol) as a major side chain cleaved lipid-soluble metabolite of 1,25-dihydroxyvitamin D3 and further extended the aforementioned C-24 oxidation pathway in the kidney by demonstrating 1,23,25-trihydroxy-24-oxovitamin D3 as the precursor of C-23 alcohol [Reddy, G. S., Tserng, K. Y., Thomas, B. R., Dayal, R., & Norman, A. W. (1987) Biochemistry 26, 324]. In this present study, we investigated the metabolic fate of 1,25-dihydroxyvitamin D3 (3 X 10(-10) M) in the perfused rat kidney and identified calcitroic acid as the major water-soluble metabolite of 1,25-dihydroxyvitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and identification of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, a new metabolite of 1,25-dihydroxyvitamin D3 produced in rat kidney

A new metabolite of vitamin D3 was produced in vitro by perfusing rat kidneys with 1,25-dihydroxyvitamin D3 (4 X 10(-6) M). It was isolated and purified from the lipid extract of the kidney perfusate by high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, chemical derivatization, and chemical synthesis, the new metabolite was identified as 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Along with the new metabolite, three other previously identified metabolites, namely, 1,24,25-trihydroxyvitamin D3, 1,25-dihydroxy-24-oxovitamin D3, and 1,23,25-trihydroxy-24-oxovitamin D3, were also isolated. The new metabolite was also formed when 1,23,25-trihydroxy-24-oxovitamin D3 was used as the substrate. Thus, the new metabolite fits into the following metabolic pathway: 1,25-dihydroxyvitamin D3----1,24(R),25-trihydroxyvitamin D3----1,25-dihydroxy-24-oxovitamin D3----1,23,25-trihydroxy-24-oxovitamin D3----1,23-dihydroxy-24,25,26,27-tetranorvitamin D3. Further, we used 1 alpha,25-dihydroxy[1 beta-3H]vitamin D3 in the kidney perfusion system and demonstrated 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 as the major further metabolite of 1,25-dihydroxyvitamin D3, circulating in the final perfusate when kidneys were perfused with 1,25-dihydroxyvitamin D3 (6 X 10(-10) M) for 4 h. The biological activity of 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 (C-3 alcohol) and its metabolic relationship to 1-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D3 (calcitroic acid or C-23 acid), the other previously identified side-chain cleavage metabolite of 1,25-dihydroxyvitamin D3, are unknown and are presently undergoing investigation.     

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development.Excitatory synaptic transmission occurs primarily at dendritic spines, small protrusions that extend from dendritic shafts. Emerging studies have shown that dendritic spines are dynamic structures which undergo changes in size, shape and number during development, and remain plastic in adult brain.¹ Regulation of spine morphology has been implicated to associate with changes of synaptic strength.² For example, enlargement and shrinkage of spines was reported to associate with certain forms of synaptic plasticity, i.e., long-term potentiation and long time depression, respectively.³ Thus, understanding the molecular mechanisms underlying the regulation of spine morphogenesis would provide insights into synapse development and plasticity. Receptor tyrosine kinases (RTKs) such as the Ephs are known to play critical roles in regulating spine morphogenesis. Eph receptors are comprised of 14 members, which are classified into EphAs and EphBs according to their sequence homology and ligand binding specificity. With a few exceptions, EphAs typically bind to A-type ligands, whereas EphBs bind to B-type ligands. During development of the central nervous system (CNS), ephrin-Eph interactions exert repulsive/attractive signaling, leading to regulation of axon guidance, topographic mapping and neural patterning.⁴ Activated Ephs trigger intracellular signaling cascades, which subsequently lead to remodeling of actin cytoskeleton through tyrosine phosphorylation of its target proteins or interaction with various cytoplasmic signaling proteins. Intriguingly, emerging studies have revealed novel functions of Ephs in synapse formation and synaptic plasticity.⁵ Specific Ephs expressed in dendritic spines of adult brain are implicated in regulating spine morphogenesis, i.e., EphBs promote spine formation and maturation, while EphA4 induces spine retraction.^6,7In the adult hippocampus, EphA4 is localized to the dendritic spines.^7,8 Activation of EphA4 at the astrocyte-neuron contacts, triggered by astrocytic ephrin-A3, leads to spine retraction and results in a reduction of spine density.⁷ It has been well established that actin cytoskeletal rearrangement is critical for spine morphogenesis, and is controlled by a tight regulation of Rho GTPases including Rac1/Cdc42 and RhoA. Antagonistic regulation of Rac1/Cdc42 and RhoA has been observed to precede changes in spine morphogenesis, i.e., activation of Rac1/Cdc42 and inhibition of RhoA is involved in spine formation, and vice versa in spine retraction.⁹ Rho GTPases function as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. The activation status of GTPase is regulated by an antagonistic action of guanine-nucleotide exchange factors (GEFs) which enhance the exchange of bound GDP for GTP, and GTPase-activating proteins (GAPs) which increase the intrinsic rate of hydrolysis of bound GTP.¹⁰ Previous studies have implicated that Rho GTPases provides a direct link between Eph and actin cytoskeleton in diverse cellular processes including spine morphogenesis.¹¹ In particular, EphBs regulate spine morphology by modulating the activity of Rho GTPases, thereby leading to rearrangement of actin networks.^12–14 Although EphA4 activation results in spine shrinkage, the molecular mechanisms that underlie the action of EphA4 at dendritic spines remain largely unclear.Work from our laboratory recently demonstrated a critical role of cyclin-dependent kinase 5 (Cdk5) in mediating the action of EphA4 in spine morphogenesis through regulation of RhoA GTPase.¹⁵ Cdk5 is a proline-directed serine/threonine kinase initially identified to be a key regulator of neuronal differentiation, and has been implicated in actin dynamics through regulating the activity of Pak1, a Rac effector, during growth cone collapse and neurite outgrowth.¹⁶ We found that EphA4 stimulation by ephrin-A ligand enhances Cdk5 activity through phosphorylation of Cdk5 at Tyr15. More importantly, we demonstrated that ephexin1, a Rho GEF, is phosphorylated by Cdk5 in vivo. Ephexin1 was reported to transduce signals from activated EphA4 to RhoA, resulting in growth cone collapse during axon guidance.^17,18 Interestingly, we found that ephexin1 is highly expressed at the post-synaptic densities (PSDs) of adult brains.¹⁵ Loss of ephexin1 in cultured hippocampal neurons or in vivo perturbs the ability of ephrin-A to induce EphA4-dependent spine retraction. The loss of ephexin1 function in spine morphology can be rescued by reexpression of wild-type ephexin1, but not by expression of its phosphorylation-deficient mutant. Our findings therefore provide important evidence that phosphorylation of ephexin1 by Cdk5 is required for the EphA4-dependent spine retraction.Molecular mechanisms underlying the action of Cdk5/ephexin1 on actin networks in EphA4-mediated spine retraction is just beginning to be unraveled. It was reported that activation of EphA4-signaling induces tyrosine phosphorylation of ephexin1 through Src family kinases (SFKs), and promotes its exchange activity towards RhoA.¹⁷ Interestingly, mutation of the Cdk5 phosphorylation sites of ephexin1 attenuates the Src-dependent tyrosine phosphorylation of ephexin1 at Tyr⁸⁷ upon EphA4 activation. These findings suggest that Cdk5 is the “priming” kinase for ephexin1. We propose that EphA4 activation by ephrin-A ligand increases Cdk5 activity, leading to phosphorylation and priming of ephexin1 for the subsequent phosphorylation of ephexin1 by Src kinase at Tyr⁸⁷, resulting in an increase of its exchange activity towards RhoA. Thus, regulation of Cdk5 activity might indirectly control the phosphorylation of ephexin1 by Src. It is tempting to speculate that phosphorylation of ephexin1 by Cdk5 at the amino-terminal region leads to a conformational change of protein, thus facilitating the access of Tyr⁸⁷ site on ephexin1 to Src kinase. Whereas accumulating evidence have pointed to a pivotal role of various GEFs including Tiam1, intersectin and kalirin in regulating spine morphogenesis, the involvement of GAPs is not clear. For example, oligophrenin-1, a Rho GAP, is implicated in maintaining the spine length through repressing RhoA activity.¹⁹ Thus, it is conceivable that a specific GAP is involved in EphA4-dependent spine retraction. Recently, we found that α2-chimaerin, a Rac GAP, regulates EphA4-dependent signaling in hippocampal neurons (Shi and Ip, unpublished observations). Taken into consideration that α2-chimaerin is enriched in the PSDs, α2-chimaerin is a likely candidate that cooperates with ephexin1 during EphA4-dependent spine retraction.In addition to stimulation of the RTK signaling cascade following EphA4 receptor activation, clustering of EphA4 signaling complex is required for eliciting maximal EphA4 function.²⁰ It is tempting to speculate that Cdk5 also regulates the formation of EphA4-containing clusters in neurons. Indeed, Cdk5^-/- neurons show reduced size of EphA4 clusters upon ephrin-A treatment, suggesting that Cdk5 regulates the recruitment of downstream signaling proteins to activate EphA4. Moreover, since ephrinA-EphA4 interaction stimulates the activity of Cdk5 at synaptic contacts, it is possible that Cdk5 might play additional roles at the post-synaptic regions through phosphorylation of its substrates. For example, PSD-95, the major scaffold protein in the PSDs, and NMDA receptor subunit NR2A are both substrates for Cdk5. Interestingly, phosphorylation of these proteins by Cdk5 has been implicated in regulating the clustering of neurotransmitter receptors as well as synaptic transmission.^21,22 Consistent with these observations, spatial distribution of neurotransmitter receptors at neuromuscular synapses is altered and abnormal neurotransmission is observed in Cdk5^-/- mice.²³ Thus, further analysis to delineate the precise roles of Cdk5 in EphA4-dependent synapse development, including regulation of neurotransmitter receptor clustering, is required.Recently, Cdk5 was shown to regulate dendritic spine density and shape through controlling the phosphorylation status of Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE-1), a critical component of actin cytoskeletal network.²⁴ In particular, phosphorylation of WAVE-1 by Cdk5 prevents actin from Arp2/3 complex-dependent polymerization and leads to a loss of dendritic spines at basal state, while reduced Cdk5-dependent phosphorylation of WAVE-1 through cAMP-dependent dephosphorylation leads to an enhanced actin polymerization and increased number of spines. It is interesting to note that phosphorylation of ephexin1 and WAVE-1 by Cdk5 both results in a reduction of spine density. Whether a concerted phosphorylation of these proteins at synapses by Cdk5 plays a role in synaptic plasticity awaits further studies. Precise regulation of Cdk5 activity is unequivocally important to maintain its proper functions at synaptic contacts. Activation of Cdk5 is mainly dependent on its binding to two neuronal-specific activators, p35 or p39, and its activity can be enhanced upon phosphorylation at Tyr¹⁵.While the signals that lie upstream of Cdk5 have barely begun to be unraveled, Cdk5 has been demonstrated to be a key downstream regulator of signaling pathways activated by extracellular cues such as neuregulin, BDNF and semaphorin. To the best of our knowledge, ephrin-EphA4 signaling is the first extracellular cue that has been identified to phosphorylate Cdk5 and promote its activity at CNS synapses.^15,25 Since BDNF-TrkB and semaphorin3A-fyn signaling have also been implicated in synapse/ spine development, it is of importance to examine whether Cdk5 is the downstream integrator of these signaling events at synapses during spine morphogenesis.^26,27Although accumulating evidence highlights a role of Cdk5 in spatial learning and synaptic plasticity, the molecular mechanisms underlying the action of Cdk5 are largely unclear.^28,29 With the recent findings that reveal the critical involvement of Cdk5 in the regulation of Rho GTPases to affect spine morphology, it can be anticipated that precise regulation of actin dynamics by Cdk5 at synapses will be an important mechanism underlying synaptic plasticity in the adult brain.? Open in a separate windowFigure 1Phosphorylation of actin regulators by Cdk5 during dendritic spine morphogenesis. (A) In striatal and hippocampal neurons, phosphorylation of WAVE-1 by Cdk5 at basal condition prevents WAVE-1-mediated actin polymerization and leads to a loss of dendritic spines. However, activation of cyclic AMP-dependent signaling by neurotransmitter such as dopamine, reduces the Cdk5-dependent phosphorylation of WAVE-1 in these neurons. Dephosphorylation of WAVE-1 promotes actin polymerization and results in an increased number of mature dendritic spines. (B) In mature hippocampal neurons, activation of EphA4 by ephrin-A increases Cdk5-dependent of ephexin1. The phosphorylation of ephexin1 by Cdk5 facilitates its EphA4-stimulated GEF activity towards RhoA activation and leads to spine retraction.     

Effects of chronic activation of peroxisome proliferator-activated receptor-alpha or high-fat feeding in a rat infarct model of heart failure

Intracardiac accumulation of lipid and related intermediates (e.g., ceramide) is associated with cardiac dysfunction and may contribute to the progression of heart failure (HF). Overexpression of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) increases intramyocellular ceramide and left ventricular (LV) dysfunction. We tested the hypothesis that activation of fatty acid metabolism with fat feeding or a PPARalpha agonist increases myocardial triglyceride and/or ceramide and exacerbates LV dysfunction in HF. Rats with infarct-induced HF (n = 38) or sham-operated rats (n = 10) were either untreated (INF, n = 10), fed a high-fat diet (45% kcal fat, INF + Fat, n = 15), or fed the PPARalpha agonist fenofibrate (150 mg.kg(-1).day(-1), INF + Feno, n = 13) for 12 wk. LV ejection fraction was significantly reduced with HF (49 +/- 6%) compared with sham operated (86 +/- 2%) with no significant differences in ejection fraction (or other functional or hemodynamic measures) among the three infarcted groups. Treatment with the PPARalpha agonist resulted in LV hypertrophy (24% increase in LV/body mass ratio) and induced mRNAs encoding for PPARalpha-regulated genes, as well as protein expression and activity of medium chain acyl-CoA dehydrogenase (compared with INF and INF + Fat groups). Myocardial ceramide content was elevated in the INF group compared with sham-operated rats, with no further change in the INF + Fat or INF + Feno groups. Myocardial triglyceride was unaffected by infarction but increased in the INF + Fat group. In conclusion, LV dysfunction and dilation are not worsened despite upregulation of the fatty acid metabolic pathway and LV hypertrophy or accumulation of myocardial triglyceride in the rat infarct model of HF.