Pyruvate dehydrogenase kinase 4 expression is synergistically induced by AMP-activated protein kinase and fatty acids

Organs are flexible as to which substrates they will use to maintain energy homeostasis. Under well-fed conditions, glucose is a preferred substrate for oxidation. During fasting, fatty acid oxidation will become a more important energy source. Glucose oxidation is decreased by fatty acids, a process in which the pyruvate dehydrogenase complex (PDH) and its regulator pyruvate dehydrogenase kinase 4 (PDK4) play important roles. It is currently unknown how energy status influences PDH activity. We show that AMP-activated protein kinase (AMPK) activation by hypoxia and AICAR treatment combined with fatty acid administration synergistically induce PDK4 expression. We provide evidence that AMPK activation modulates ligand-dependent activation of peroxisome proliferator-activated receptor. Finally, we show that this synergistic induction of PDK4 decreases cellular glucose oxidation. In conclusion, AMPK and fatty acids play a direct role in fuel selection in response to cellular energy status in order to spare glucose. S. M. Houten, M. Chegary: These two authors contributed equally to this work. Received 11 July 2008; received after revision 26 January 2009; accepted 02 February 2009     

Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.     

Purification of breast cancer resistance protein ABCG2 and role of arginine-482

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding. Received 10 April 2006; received after revision 22 May 2006; accepted 12 June 2006 A. Pozza and J. M. Perez-Victoria contributed equally to this work     

Cell surface adenylate kinase activity regulates the F1-ATPase/P2Y13-mediated HDL endocytosis pathway on human hepatocytes

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase and nucleoside diphosphokinase activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F₁-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation. Received 13 July 2006; received after revision 29 August 2006; accepted 19 September 2006     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Starch-binding domains in the post-genome era

Starch belongs to the most abundant biopolymers on Earth. As a source of energy, starch is degraded by a large number of various amylolytic enzymes. However, only about 10% of them are capable of binding and degrading raw starch. These enzymes usually possess a distinct sequence-structural module, the so-called starchbinding domain (SBD). In general, all carbohydrate-binding modules (CBMs) have been classified into the CBM families. In this sequence-based classification the individual types of SBDs have been placed into seven CBM families: CBM20, CBM21, CBM25, CBM26, CBM34, CBM41 and CBM45. The family CBM20, known also as a classical C-terminal SBD of microbial amylases, is the most thoroughly studied. The three-dimensional structures have already been determined by X-ray crystallography or nuclear magnetic resonance for SBDs from five CBM families (20, 25, 26, 34 and 41), and the structure of the CBM21 has been modelled. Despite differences among the amino acid sequences, the fold of a distorted β-barrel seems to be conserved together with a similar way of substrate binding (mainly stacking interactions between aromatic residues and glucose rings). SBDs have recently been discovered in many non-amylolytic proteins. These may, for example, have regulatory functions in starch metabolism in plants or glycogen metabolism in mammals. SBDs have also found practical uses. Received 25 May 2006; received after revision 26 June 2006; accepted 3 August 2006     

Monoclonal antibodies to L-asparaginase

Five hybridomas secreting monoclonal antibody to E. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G1 (1 clone), Ig G2 (2 clones) and Ig G3 (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (Kd) ranging between 2.5 X 10(-9) M and 6.3 X 10(-10) M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Endocannabinoids in adipocytes during differentiation and their role in glucose uptake

The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-γ, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake. Received 11 October 2006; received after revision 9 November 2006; accepted 28 November 2006 V.Gasperi and F. Fezza equally contributed to the study.     

Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.     

The SH3-binding domain of Cx43 participates in loop/tail interactions critical for Cx43-hemichannel activity

Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca²⁺ concentration ([Ca²⁺]_i) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca²⁺]_i, which may be involved in pathological hemichannel opening.     

Biological activity of enantiomerically pure forms of insect juvenile hormone I and III inBombyx mori

Summary Biological activity of enantiomerically pure juvenile hormones was assayed by topical application on allatectomizedBombyx fourth instar larvae. JHs tested were (10R)-JH I [methyl (2E,6E,10R,11S)-10,11-epoxy-3,11-dimethyl-7-ethyl-2,6-tridecadienoate], (10S)-JH I [methyl (2E, 6E, 10S, 11R)-10,11-epoxy-3,11-dimethyl-7-ethyl-2,6-tridecadienoate], (10R)-JH III [methyl (2E,6E,10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate] and (10S)-JH III [methyl (2E,6E,10S)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate]. Among these compounds, natural (10R)-JH I was most active and the dose needed to induce 50% larval molting was 0.04 g/larva; it was approximately 12,000 times more active than unnatural (10S)-JH I. Though natural (10R)-JH III showed slight biological activity, it was only one three-thousandth of that of (10R)-JH I. Unnatural (10S)-JH III exhibited no biological activity at the levels assayed.     

Tolerance to cold and glucose homeostasis in adrenal demedullated dogs

Summary The rise in O₂ consumption and in glucose turnover, induced by acute cold exposure is not suppressed by adrenal demedullation in dogs. However, both at neutral and cold ambient temperature, the mean plasma glucose concentrations are higher in normal (N) than in adrenal-demedullated dogs (ADMX). In the cold, the fall in rectal temperature is larger in ADMX than in N dogs.This work was supported by a grant from Université Claude Bernard (U.E.R. Lyon Nord and U.E.R. Biologie Humaine).The authors thank D. Rougier and G. Dallevet for their excellent technical assistance and A. Brillant for secreterial assistance.     

Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells

Akt (PKB) is a critical kinase in cell-survival pathways. Its activity depends on the phosphorylation of Thr308 and Ser473, by PDK1 and mTORC2, respectively. We found that Akt can be further stimulated through phosphorylation of Ser129 by another kinase, CK2. Here we show that phosphorylation of Akt at Ser129 also facilitates its association with Hsp90 chaperone, thus preventing Thr308 dephosphorylation. This is supported by the following observations: (1) phospho-Thr308 decreases when Ser129 is mutated to alanine, (2) this decrease is abolished by cell treatment with okadaic acid (to inactivate PP2A) or geldanamycin (to inactivate Hsp90), (3) phosphorylation of Ser129 neither enhances the activity of PDK1 nor hampers the in vitro activity of PP2A on Thr308, but increases the Hsp90 association to Akt. These data support the view that the antiapoptotic potential of CK2 is at least in part mediated by its ability to maintain Akt in its active form.     

Regulating the human HECT E3 ligases

Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C terminus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibitory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruitment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein–protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways.     

Hsp70 chaperones: Cellular functions and molecular mechanism

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.Received 21 October 2004; received after revision 24 November 2004; accepted 6 December 2004     

Proinsulin C-peptide elicits disaggregation of insulin resulting in enhanced physiological insulin effects

Using surface plasmon resonance (SPR) and electrospray mass spectrometry (ESI-MS), proinsulin C-peptide was found to influence insulin-insulin interactions. In SPR with chip-bound insulin, C-peptide mixed with analyte insulin increased the binding, while alone C-peptide did not. A control peptide with the same residues in random sequence had little effect. In ESI-MS, C-peptide lowered the presence of insulin hexamer. The data suggest that C-peptide promotes insulin disaggregation. Insulin/insulin oligomer μM dissociation constants were determined. Compatible with these findings, type 1 diabetic patients receiving insulin and C-peptide developed 66% more stimulation of glucose metabolism than when given insulin alone. A role of C-peptide in promoting insulin disaggregation may be important physiologically during exocytosis of pancreatic β-cell secretory granulae and pharmacologically at insulin injection sites. It is compatible with the normal co-release of C-peptide and insulin and may contribute to the beneficial effect of C-peptide and insulin replacement in type 1 diabetics. Received 3 May 2006; received after revision 9 June 2006; accepted 12 June 2006 Free Online Access     

Changes in plasma levels of metabolites during long-term fasting in the goose]

In fed geese, plasma levels of glucose and alanine were 1.9 g.l-1 and 560 mumol.l-1, respectively. During a long fast (40 days), plasma glucose and alanine were maintained at a high level (1.5-1.8 g.l-1 and 370-540 mumol.l-1, respectively). Plasma level of acetoacetate was very low (40 mumol.l-1); by contrast, plasma level of beta hydroxybutyrate reached very high values (20 mmol.l-1) after about 20 days of fasting, then it decreased. Plasma levels of lactate and pyruvate decreased along the course of the fast, from 2 500 to 2 000 mumol.l-1 and 220 to 170 mumol.l-1, respectively.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007