Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice

Conjugated linoleic acid (CLA) causes insulin resistance and hepatic steatosis in conjunction with depletion of adipokines in some rodent models. Our objective was to determine whether the maintenance of adipokines, mainly leptin and adiponectin, by either removing CLA from diets or using an adiponectin enhancer, rosiglitazone (ROSI), could attenuate CLA-induced insulin resistance. Male C57BL/6 mice were consecutively fed two experimental diets containing 1.5% CLA mixed isomer for 4 weeks followed by a diet without CLA for 4 weeks. CLA significantly depleted adiponectin but not leptin and was accompanied by hepatic steatosis and insulin resistance. These effects were attenuated after switching mice to the diet without CLA along with restoration of adiponectin. To further elucidate the role of adiponectin in CLA-mediated insulin resistance, ROSI was used in a subsequent study in male ob/ob mice fed either control (CON) or CLA diet. ROSI maintained significantly higher adiponectin levels in CON- and CLA-fed mice and prevented the depletion of epididymal adipose tissue and the development of insulin resistance. In conclusion, we show that insulin resistance induced by CLA may be related more to adiponectin depletion than to leptin and that maintaining adiponectin levels alone either by removing CLA or using ROSI can attenuate these effects.     

Regulation of chemotactic networks by 'atypical' receptors

Directed cell migration is a fundamental component of numerous biological systems and is critical to the pathology of many diseases. Although the importance of secreted chemoattractant factors in providing navigational cues to migrating cells bearing specific chemoattractant receptors is now well-established, how the function of these factors is regulated is not so well understood and may be of key importance to the design of new therapeutics for numerous human diseases. While regulation of migration clearly takes place on a number of different levels, it is becoming clear that so-called 'atypical' receptors play a role in scavenging, or altering the localisation of, chemoattractant molecules such as chemokines and complement components. These receptors do this through binding and/or internalising their chemoattractant ligands without activating signal transduction cascades leading to cell migration. The atypical chemokine receptor family currently comprises the receptors D6, DARC and CCX-CKR. In this review, we discuss the evidence from in vitro and in vivo studies that these receptors play a role in regulating cell migration, and speculate that other orphan receptors may also belong to this family. Furthermore, with the advent of gene therapy on the horizon, the therapeutic potential of these receptors in human disease is also considered.     

Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)

We investigated the evolutionary dynamics of duplicated copies of the granule-bound starch synthase I gene (GBSSI or Waxy) within polyploid Spartina species. Molecular cloning, sequencing, and phylogenetic analyses revealed incongruences between the expected species phylogeny and the inferred gene trees. Some genes within species were more divergent than expected from ploidy level alone, suggesting the existence of paralogous sets of Waxy loci in Spartina. Phylogenetic analyses indicate that this paralogy originated from a duplication that occurred prior to the divergence of Spartina from other Chloridoideae. Gene tree topologies revealed three divergent homoeologous sequences in the hexaploid S. alterniflora that are consistent with the proposal of an allopolyploid origin of the hexaploid clade. Waxy sequences differ in insertion–deletion events in introns, which may be used to diagnose gene copies. Both paralogous and homoeologous coding regions appear to evolving under selective constraints.     

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

Background

Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.

Methods

Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10⁶ cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.

Results

We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.

Conclusion

Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.     

Clinical and molecular characterization of five patients with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inborn error of ketone body metabolism and causes episodic ketoacidosis. We report clinical and molecular analyses of 5 patients with SCOT deficiency. Patients GS07, GS13, and GS14 are homozygotes of S405P, L327P, and R468C, respectively. GS17 and GS18 are compound heterozygotes for S226N and A215V, and V404F and E273X, respectively. These mutations have not been reported previously. Missense mutations were further characterized by transient expression analysis of mutant cDNAs. Among 6 missense mutations, mutants L327P, R468C, and A215V retained some residual activities and their mutant proteins were detected in immunoblot analysis following expression at 37 °C. They were more stable at 30 °C than 37 °C, indicating their temperature sensitive character. The R468C mutant is a distinct temperature sensitive mutant which retained 12% and 51% of wild-type residual activities at 37 and 30 °C, respectively. The S226N mutant protein was detected but retained no residual activity. Effects of missense mutations were predicted from the tertiary structure of the SCOT molecule. Main effects of these mutations were destabilization of SCOT molecules, and some of them also affected catalytic activity. Among 5 patients, GS07 and GS18 had null mutations in both alleles and the other three patients retained some residual SCOT activities. All 5 developed a first severe ketoacidotic crisis with blood gas pH < 7.1, and experienced multiple ketoacidotic decompensations (two of them had seven such episodes). In general, the outcome was good even following multiple ketoacidotic events. Permanent ketosis or ketonuria is considered a pathognomonic feature of SCOT deficiency. However, this condition depends not only on residual activity but also on environmental factors.     

Targeted cancer therapy: What if the driver is just a messenger?

“Shoot the driver” is the paradigm of targeted cancer therapy. However, resistance to targeted inhibitors of signaling pathways is a major problem. In part, the redundancy of signaling networks can bypass targeted inhibitors and thereby reduce their biological effect. In this case, the driver turns out to be one of several potential messengers and is easily replaced. Cocktails of multiple targeted inhibitors are an obvious solution. This is limited, however, by the lack of potent inhibitors and may also produce increased toxicity. Therefore, we explored the direct blockade of a key biological activity downstream from multiple converging oncogenic signals. Specifically, several oncogenic signaling pathways, including AKT, MAPK and PIM kinase signals, converge on the activation of cap-dependent translation. In cancer cells, aberrant activation of cap-dependent translation favors the increased expression of short-lived oncoproteins like c-MYC, MCL1, CYCLIND1 and the PIM kinases. Intriguingly, cancer cells are especially sensitive to even temporary reductions in these proteins. We will discuss our findings concerning translational inhibitor therapy in cancer.Key words: targeted therapy, cancer, lymphoma, translation, eIF4ETargeted cancer therapies are designed to block selected pathways or molecules that are required for tumor cell survival. The most successful examples are inhibitors of the BCR-ABL fusion protein that drive chronic myeloid leukemia (CML). By shutting down the activity of a single molecule driving the growth of CML cells, imatinib and its successors dasatinib and nilotinib can produce complete and sustained remissions as single agents (reviewed in ref. ¹). However, it has proven difficult to translate this success to other cancers. In metastatic melanoma, for example, the BRAF inhibitor vemurafenib produces high response rates in patients whose tumors bear the BRAF-V600E activating mutation (reviewed in ref. ²). Excitement generated by these results is well-deserved, as they have opened a new treatment paradigm in a disease with few available options and a grim prognosis. Unfortunately, however, resistance to the drug generally is seen within a few months. Median progression-free survival resulting from vemurafenib for treatment-naïve metastatic melanoma patients with V600E was about six months,³ compared with imatinib in CML, which kept 93% of patients progression-free at five years.⁴ The success of TKIs in CML is still the exception and not the rule in targeted cancer therapeutics.Resistance to targeted inhibitors is an emerging problem with multiple causes and potential solutions. While mechanisms of resistance to BRAF inhibition in melanoma remain to be elucidated, they are well-described for some targets in other cancers. Drugs against the epidermal growth factor receptor (EGFR) kinase, for example, are bypassed both by activation of downstream mediators, most prominently KRAS, and by signaling through parallel pathways like the MET oncogene (reviewed in ref. ⁵). Resistance mechanisms are varied and complex in some ways, but most boil down to the same idea: evolution has provided multiple routes to the crucial endpoints that allow sustained growth and proliferation of cancer cells. Cocktails of multiple inhibitors have been proposed to prevent or thwart resistance (reviewed in ref. ⁶), and this approach appears highly promising based on some recent preclinical studies. In prostate cancer, for example, combined blockade of androgen receptor signaling and the PI3K/AKT/mTOR pathway showed potent synergy in model systems.⁷ Unfortunately, similar approaches against many other cancers are currently limited by the availability of potent, selective inhibitors and a need to identity which combinations will be effective. Moreover, combined toxicities from simultaneous use of multiple inhibitors will pose limits on the number and intensity of drugs that can be used.An alternative and potentially complementary approach is to directly target the downstream biological processes that are activated by signaling pathways and that cancer cells rely on. Experiences with inhibitors of EGFR, BRAF, and other signaling molecules suggest that most tumors can reliably activate parallel or downstream messengers to thwart efficacy. So inhibiting a signaling intermediate (i.e., a messenger) allows resistance if the biological effects can be achieved via an alternate route. Ultimately, tumor cells don''t depend on the messenger, often a kinase, and, instead, require a downstream biological function. This opens the possibility that targeting the critical effect directly may be an effective cancer therapy and could overcome the problem of redundant messengers.Several inherent properties of signaling pathways are relevant to targeted therapies, their side effects and mechanisms of resistance.⁸ For example, signaling pathways frequently converge on key activities. As explained above, this redundancy can produce resistance to targeted agents. Often redundant pathways are induced via feedback mechanisms, which provide robust signals and can also bypass selective inhibitors. Signals also diverge, however, and seemingly parallel pathways therefore also produce pleiotropic and non-overlapping effects. It is possible not all activities are required by cancer cells. Hence, an upstream block may produce toxicities, in part, by blocking activities that are not strictly required by tumor cells as much as by normal tissues. Finally, unlike metabolic pathways, where a limiting substrate is passed down, signaling cascades amplify signals toward a key activity, and the initial signal or message is both energetically “cheap” and infinitely recurrent. Accordingly, it is conceivable that direct block of the downstream effects provide an alternative or complementary approach to targeting upstream signaling molecules.Multiple oncogenic signals, including the PI3K, MAPK/ERK and PIM kinase pathways converge on the activation of cap-dependent translation, the process by which most capped mRNAs are translated into proteins (reviewed in ref. ⁹). Signaling pathways control the availability of the cap-binding protein eIF4E that is the limiting component of the multimeric translation complex eIF4F, which also includes scaffolds (eIF4G) and RNA helicase activities (eIF4A).^10–12 The complex ultimately mediates loading of mRNAs onto ribosomes. Availability of the eIF4E factor is especially important for mRNAs with long and structured 5′ UTRs. These include, in particular, short-lived cell cycle regulators and oncoproteins. Hence, regulation of eIF4E via upstream signals provides an immediate level of expression control that directly controls levels of proteins, including c-MYC, cyclin D1, BCL2, MCL1 and PIM1.^13–17 Cancer cells require continuous expression of these proteins. For example, even brief loss of MYC expression produces widespread cell death in several cancers but only produces reversible cell cycle arrest in normal tissues.¹⁸ Hence, the increased requirement for the continuous translation of oncoproteins in cancer cells may provide a therapeutic window for inhibitors of capdependent translation.We recently investigated the therapeutic potential of directly blocking capdependent translation in non-Hodgkin lymphoma (NHL).^15,16,19 Rapalogs, inhibitors of mTORC1, the upstream activator of cap-dependent translation, have been extensively studied in NHL in clinical trials (reviewed in ref. ²⁰). However, their activity has, overall, been modest, and our results implicate mTORC1-independent activation of translation by PIM kinases as one mechanism of rapalog resistance in NHLs.¹⁶ The PIM family kinases are active upon expression and do not require activating modifications. They have been known for some time to be able to promote phosphorylation of 4E-BP1 in a manner resistant to rapamycin.^21,22 We now report expression of PIM1 and/or PIM2 in more than 60% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) and more than 75% of mantle cell and small lymphocytic lymphomas. Our study found PIM expression (either PIM1 or PIM2) was associated with worse time to event and overall survival in FL, while another recent report points to PIM2 as a driver of aggressive disease in activated B-cell type DLBCL.²³ In addition, two recent studies of chromosomal translocations mediated by activation-induced deaminase (AID) identified PIM1 as a frequent target.^24,25 In sum, expression of PIM kinases is common in NHLs, may be associated with a more aggressive clinical course and exemplifies how the redundancy of messaging molecules can bypass the clinical activity of selective signaling inhibitors.In experimental systems, we found that direct blockade of cap-dependent translation was highly effective against lymphomas with redundant PI3K/AKT and PIM signals.¹⁶ Briefly, using both a constitutively active mutant 4E-BP1 allele that blocks eIF4E activity and a small molecule inhibitor of the eIF4A helicase, silvestrol, we were able to completely restore rapalog sensitivity in lymphomas engineered to express PIM2 kinase activity. Mechanistically, we found that silvestrol dramatically reduced the translation of critical oncoproteins, including c-MYC, cyclin D1 and MCL1. Interestingly, silvestrol also blocked the translation of both PIM kinases themselves. Moreover, consistent with prior reports, silvestrol treatment at an effective dose was well-tolerated in animals, and we observed no frank toxicity.¹⁹ Hence, blocking cap-dependent translation disrupts upstream signaling molecules, the PIM kinases and also key oncoproteins commonly considered “undruggable” oncoproteins, including c-MYC.Silvestrol worked dramatically better than inhibition of the upstream kinases. Briefly, we tested the SuperGen Inc. PIM kinase inhibitors SGI-1776 and SGI-1773 side by side with silvestrol in a panel of these PIM-expressing human NHL cell lines. Notably, SGI-1776 is the only PIM inhibitor that has entered clinical trials, although these had to be discontinued due to cardiac toxicity of the compound (SuperGen press release, 2010). In any case, silvestrol showed in vitro potency at IC₅₀ of less than 10 nM in all cases, and the PIM kinase inhibitors were 100 to 1,000 times less active. These results highlight some problems associated with the “inhibitor cocktail” approach and indicate a complementary strategy that includes direct blockade of a key biological activity, in this instance, cap-dependent translation of oncoproteins.Silvestrol is not the only means to block cap-dependent translation, and others are reviewed in reference ⁹. These include antisense oligonucleotides against eIF4E and peptide inhibitors of eIF4F complex formation, though neither has entered clinical trials. Silvestrol is the most well-studied of several compounds that emerged from library screens for ability to disrupt the function of the eIF4F subunit eIF4A, an RNA helicase required for its ability to promote mRNA translation. A plant-derived flavagline, silvestrol has shown activity against a variety of tumor types and can be given to mice at high enough concentrations for antitumor activity without major toxicity. The drug shows activity as a single agent against human breast and prostate cancer cell lines in xenograft experiments in nude mice.²⁶ This produced mild transient impairment of hepatic synthetic function but no toxicities producing morbidity or mortality. In genetically defined murine tumor models, silvestrol showed potent synergy with chemotherapy when used against tumors bearing translational activation due to loss of Pten or overexpression of eIf4e.¹⁹ Originally isolated from Aglaia silvestris, silvestrol has a complex structure that has proved difficult to chemically synthesize in quantity. For this reason, the parent compound is not an ideal clinical drug candidate. Efforts are underway by Drs. Pelletier (McGill) and Porco (Boston University) to develop analogs with more efficient synthesis profiles and that retain its biochemical properties. In sum, cap-dependent translation is a promising drug target alternate to mTORC1 and upstream kinase inhibitors.     

Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton

Mapping of genes that play major roles in cotton fiber development is an important step toward their cloning and manipulation, and provides a test of their relationships (if any) to agriculturally-important QTLs. Seven previously identified fiber mutants, four dominant (Li ₁, Li ₂, N ₁ and Fbl) and three recessive (n ₂, sma-4(h _a), and sma-4(fz)), were genetically mapped in six F₂ populations comprising 124 or more plants each. For those mutants previously assigned to chromosomes by using aneuploids or by linkage to other morphological markers, all map locations were concordant except n ₂, which mapped to the homoeolog of the chromosome previously reported. Three mutations with primary effects on fuzz fibers (N ₁, Fbl, n ₂) mapped near the likelihood peaks for QTLs that affected lint fiber productivity in the same populations, perhaps suggesting pleiotropic effects on both fiber types. However, only Li ₁ mapped within the likelihood interval for 191 previously detected lint fiber QTLs discovered in non-mutant crosses, suggesting that these mutations may occur in genes that played early roles in cotton fiber evolution, and for which new allelic variants are quickly eliminated from improved germplasm. A close positional association between sma-4(h _a ), two leaf and stem-borne trichome mutants (t ₁ , t ₂), and a gene previously implicated in fiber development, sucrose synthase, raises questions about the possibility that these genes may be functionally related. Increasing knowledge of the correspondence of the cotton and Arabidopsis genomes provides several avenues by which genetic dissection of cotton fiber development may be accelerated.     

2-Alkoxydihydrocinnamates as PPAR agonists. Activity modulation by the incorporation of phenoxy substituents

Herein we describe a series of potent and selective PPARgamma agonists with moderate PPARalpha affinity and little to no affinity for other nuclear receptors. In vivo studies in a NIDDM animal model (ZDF rat) showed that these compounds are efficacious at low doses in glucose normalization and plasma triglyceride reduction. Compound 1b (LY519818) was selected from our SAR studies to be advanced to clinical evaluation for the treatment of type II diabetes.