CD40L Deficiency Attenuates Diet-Induced Adipose Tissue Inflammation by Impairing Immune Cell Accumulation and Production of Pathogenic IgG-Antibodies

BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease

MTF-1-Mediated Repression of the Zinc Transporter Zip10 Is Alleviated by Zinc Restriction

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy

Peripheral Effects of FAAH Deficiency on Fuel and Energy Homeostasis: Role of Dysregulated Lysine Acetylation

FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/-) mice.FAAH(-/-) mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH(-/-) mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/-) mice. Dysregulated hepatic FAAH(-/-) lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/-) acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH(-/-) mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/-) mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/-) aldolase B. Fed FAAH(-/-) alcohol dehydrogenase (ADH) acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/-) mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models

Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Differential Cerebral Cortex Transcriptomes of Baboon Neonates Consuming Moderate and High Docosahexaenoic Acid Formulas

BACKGROUND: Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels. METHODS AND FINDINGS: Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; “L”, LCPUFA, with 0.33%DHA-0.67%ARA; “L3”, LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner. CONCLUSIONS: These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA

Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations

Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F−, Cl−, Br−, and I−) ions play an important role in many biological phenomena, roles that range from stabilization of biomolecular structure, to influence on biomolecular dynamics, to key physiological influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomolecular structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in solution and the interactions of ions with other molecules. At present, the best force fields for biomolecules employ a simple additive, nonpolarizable, and pairwise potential for atomic interaction. In this work, we describe our efforts to build better models of the monovalent ions within the pairwise Coulombic and 6-12 Lennard-Jones framework, where the models are tuned to balance crystal and solution properties in Ewald simulations with specific choices of well-known water models. Although it has been clearly demonstrated that truly accurate treatments of ions will require inclusion of nonadditivity and polarizability (particularly with the anions) and ultimately even a quantum mechanical treatment, our goal was to simply push the limits of the additive treatments to see if a balanced model could be created. The applied methodology is general and can be extended to other ions and to polarizable force-field models. Our starting point centered on observations from long simulations of biomolecules in salt solution with the AMBER force fields where salt crystals formed well below their solubility limit. The likely cause of the artifact in the AMBER parameters relates to the naive mixing of the Smith and Dang chloride parameters with AMBER-adapted Åqvist cation parameters. To provide a more appropriate balance, we reoptimized the parameters of the Lennard-Jones potential for the ions and specific choices of water models. To validate and optimize the parameters, we calculated hydration free energies of the solvated ions and also lattice energies (LE) and lattice constants (LC) of alkali halide salt crystals. This is the first effort that systematically scans across the Lennard-Jones space (well depth and radius) while balancing ion properties like LE and LC across all pair combinations of the alkali ions and halide ions. The optimization across the entire monovalent series avoids systematic deviations. The ion parameters developed, optimized, and characterized were targeted for use with some of the most commonly used rigid and nonpolarizable water models, specifically TIP3P, TIP4PEW, and SPC/E. In addition to well reproducing the solution and crystal properties, the new ion parameters well reproduce binding energies of the ions to water and the radii of the first hydration shells

Reliability of surface facial electromyography

Data from two studies were used to estimate the reliability of facial EMG when used to index facial mimicry (Study 1) or affective reactions to pictorial stimuli (Study 2). Results for individual muscle sites varied between muscles and depending on data treatment. For difference scores, acceptable internal consistencies were found only for corrugator supercilii, and test-retest reliabilities were low. For contrast measures describing patterns of reactions to stimuli, such as high zygomaticus major combined with low corrugator supercilii, acceptable internal consistencies were found for facial reactions to smiling faces and positive affective reactions to affiliative images (Study 2). Facial reactions to negative emotions (Study 1) and facial reactions to power and somewhat less to achievement imagery (Study 2) showed unsatisfactory internal consistencies. For contrast measures, good temporal stability over 24 months (Study 1) and 15 months (Study 2), respectively, was obtained. In Study 1, the effect of method factors such as mode of presentation was more reliable than the emotion effect. Overall, people's facial reactions to affective stimuli seem to be influenced by a variety of factors other than the emotion-eliciting element per se, which resulted in biased internal consistency estimates. However, the influence of these factors in turn seemed to be stable over time

“Steeping” of Health Expenditure Profiles

If health care expenditure for the elderly grows faster than for younger people, the expenditure profiles become “steeper” – we call that “steeping”. Three instruments for measuring “steeping” are presented: (1) trend of the relation between per capita expenditure of the old and the young; (2) comparing the linear slopes of per capita expenditure in age groups; (3) trend in parameters of nonlinear modelling of expenditure profiles. Using data of the largest German private health insurer over a period of 18 years, “steeping” could be observed by all three methods in most examined insurance plans. There are some hints that steeping also occurs in Germany's social health insurance system. The impact of steeping on the sustainability of the health system is discussed. Consequences for the calculation methods in a capital-funded health insurance system and for the implicit inter-generational contract within a pay-as-you-go financed social health insurance system are analysed. The link between “steeping” and the topic of the “red herring” discussion is elaborated. The Geneva Papers (2006) 31, 581–599. doi:10.1057/palgrave.gpp.2510100

Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca2+ release and apoptosis

Imbalance of signals that control cell survival and death results in pathologies, including cancer and neurodegeneration. Two pathways that are integral to setting the balance between cell survival and cell death are controlled by lipid-activated protein kinase B (PKB)/Akt and Ca2+. PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca2+ stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca2+ release through inositol 1,4,5-trisphosphate receptor (InsP3R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP3Rs, significantly reducing their Ca2+ release activity. Moreover, phosphorylation of InsP3Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca2+ flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKB on InsP3R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKB-mediated abolition of InsP3-induced Ca2+ release may afford tumor cells a survival advantage