ZNT7 binds to CD40 and influences CD154-triggered p38 MAPK activity in B lymphocytes-a possible regulatory mechanism for zinc in immune function.

Zinc deficiency impairs the immune system leading to frequent infections. Although zinc is known to play critical roles in maintaining healthy immune function, the underlying molecular targets are largely unknown. In this study, we demonstrate that zinc is important for the CD154-CD40-mediated activation of downstream signaling pathways in human B lymphocytes. CD40 is a receptor localized on the cell surface of many immune cells, including B lymphocytes. It binds to CD154, a membrane protein expressed on antigen-activated T helper (Th) lymphocytes. This CD154-CD40 interaction leads to B-cell activation. We showed that cellular zinc deficiency impaired the CD154-CD40-mediated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. We also showed that zinc supplemental treatment of B lymphocytes had limited effect on this CD40-mediated p38 MAPK signaling. Most importantly, we demonstrated that the zinc transporter protein zinc transporter 7 (ZNT7) interacted with CD40 using immunoprecipitation analyses. ZNT7 knockdown in B lymphocytes had a negative effect on the cell surface expression of CD40. Consequently, the CD40-mediated p38 MAPK signaling transduction was down-regulated in ZNT7 KD B lymphocytes. Conversely, this p38 MAPK signaling activity was up-regulated by overexpression (OE) of ZNT7 in B lymphocytes. Moreover, we found that ZNT7 knockdown in B lymphocytes constitutively up- and down-regulated the inhibitor of i kappa B kinase and AKT serine/threonine kinase phosphorylation, respectively, which implies the activation of survival signaling in ZNT7 KD B cells. We conclude that CD40 is the target molecule for ZNT7 in regulation of immune function of B lymphocytes.

Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects.

Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with l-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. l-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations ≥25 µM, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 µM. Additionally, l-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase between 2.5- and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. l-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 µM, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted l-C16 carnitine-mediated IL-6 production by >65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the l-C16 carnitine-associated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiological-to-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

A dairy-based high calcium diet improves glucose homeostasis and reduces steatosis in the context of preexisting obesity.

OBJECTIVE: High dietary calcium (Ca) in the context of a dairy food matrix has been shown to reduce obesity development and associated inflammation in diet-induced obese (DIO) rodents. The influence of Ca and dairy on these phenotypes in the context of preexisting obesity is not known. Furthermore, interpretations have been confounded historically by differences in body weight gain among DIO animals fed dairy-based protein or high Ca. DESIGN AND METHODS: Adiposity along with associated metabolic and inflammatory outcomes were measured in DIO mice previously fattened for 12 week on a soy protein-based obesogenic high fat diet (45% energy, 0.5% adequate Ca), then fed one of three high fat diets (n = 29-30/group) for an additional 8 week: control (same as lead-in diet), high-Ca (1.5% Ca), or high-Ca + nonfat dry milk (NFDM). RESULTS AND CONCLUSION: Mice fed high-Ca + NFDM had modestly, but significantly, attenuated weight gain compared to mice fed high-Ca or versus controls (P < 0.001), whereas mice fed high-Ca alone had increased weight gain compared to controls (P < 0.001). Total measured adipose depot weights between groups were similar, as were white adipose tissue inflammation and macrophage infiltration markers (e.g. TNFα, IL-6, CD68 mRNAs). Mice fed high-Ca + NFDM had significantly improved glucose tolerance following a glucose tolerance test, and markedly lower liver triglycerides compared to high-Ca and control groups. Improved metabolic phenotypes in prefattened DIO mice following provision of a diet enriched with dairy-based protein and carbohydrates appeared to be driven by non-Ca components of dairy and were observed despite minimal differences in body weight or adiposity.

Increased lipolysis and altered lipid homeostasis protect γ-synuclein-null mutant mice from diet-induced obesity.

Synucleins are a family of homologous proteins principally known for their involvement in neurodegeneration. γ-Synuclein is highly expressed in human white adipose tissue and increased in obesity. Here we show that γ-synuclein is nutritionally regulated in white adipose tissue whereas its loss partially protects mice from high-fat diet (HFD)-induced obesity and ameliorates some of the associated metabolic complications. Compared with HFD-fed WT mice, HFD-fed γ-synuclein-null mutant mice display increased lipolysis, lipid oxidation, and energy expenditure, and reduced adipocyte hypertrophy. Knockdown of γ-synuclein in adipocytes causes redistribution of the key lipolytic enzyme ATGL to lipid droplets and increases lipolysis. γ-Synuclein-deficient adipocytes also contain fewer SNARE complexes of a type involved in lipid droplet fusion. We hypothesize that γ-synuclein may deliver SNAP-23 to the SNARE complexes under lipogenic conditions. Via these independent but complementary roles, γ-synuclein may coordinately modulate lipid storage by influencing lipolysis and lipid droplet formation. Our data reveal γ-synuclein as a regulator of lipid handling in adipocytes, the function of which is particularly important in conditions of nutrient excess.

A high calcium diet containing nonfat dry milk reduces weight gain and associated adipose tissue inflammation in diet-induced obese mice when compared to high calcium alone.

BACKGROUND: High dietary calcium (Ca) is reported to have anti-obesity and anti-inflammatory properties. Evidence for these properties of dietary Ca in animal models of polygenic obesity have been confounded by the inclusion of dairy food components in experimental diets; thus, effect of Ca per se could not be deciphered. Furthermore, potential anti-inflammatory actions of Ca in vivo could not be dissociated from reduced adiposity. METHODS: We characterized adiposity along with metabolic and inflammatory phenotypes in diet-induced obese (DIO) mice fed 1 of 3 high fat diets (45% energy) for 12 wk: control (n = 29), high-Ca (n = 30), or high-Ca + nonfat dry milk (NFDM) (n = 30). RESULTS: Mice fed high-Ca + NFDM had reduced body weight and adiposity compared to high-Ca mice (P < 0.001). Surprisingly, the high-Ca mice had increased adiposity compared to lower-Ca controls (P < 0.001). Hyperphagia and increased feed efficiency contributed to obesity development in high-Ca mice, in contrast to NFDM mice that displayed significantly reduced weight gain despite higher energy intake compared to controls (P < 0.001). mRNA markers of macrophages (e.g., CD68, CD11d) strongly correlated with body weight in all diet treatment groups, and most treatment differences in WAT inflammatory factor mRNA abundances were lost when controlling for body weight gain as a covariate. CONCLUSIONS: The results indicate that high dietary Ca is not sufficient to dampen obesity-related phenotypes in DIO mice, and in fact exacerbates weight gain and hyperphagia. The data further suggest that putative anti-obesity properties of dairy emanate from food components beyond Ca.

Inflammatory phenotyping identifies CD11d as a gene markedly induced in white adipose tissue in obese rodents and women.

In severe obesity, white adipose tissue (WAT) inflammation and macrophage infiltration are thought to contribute to WAT and whole-body insulin resistance. Specific players involved in triggering and maintaining inflammation (i.e. those regulating adipokine release and WAT macrophage recruitment, retention, or function) remain to be fully elaborated, and the degree to which moderate obesity promotes WAT inflammation remains to be clarified further. Therefore, we characterized adiposity and metabolic phenotypes in adult male C57BL/6J mice fed differing levels of dietary fat (10, 45, and 60% of energy) for 12 wk, concurrent with determinations of WAT inflammation markers and mRNA expression of leukocyte-derived integrins (CD11b, CD11c, CD11d) involved in macrophage extravasation and tissue macrophage homing/retention. As expected, a lard-based, very high-fat diet (60% energy) significantly increased adiposity and glucose intolerance compared with 10% fat-fed controls, coincident with higher retroperitoneal (RP) WAT transcript levels for proinflammatory factors and macrophage markers, including TNFα and CD68 mRNA, which were ~3- and ~15-fold of control levels, respectively (P < 0.001). Mice fed the 45% fat diet had more moderate obesity, less glucose intolerance, and lower WAT macrophage/inflammatory marker mRNA abundances compared with 60% fat-fed mice; TNFα and CD68 mRNA levels were ~2- and ~5-fold of control levels (P < 0.01). Relative WAT expression of CD11d was massively induced by obesity to an extent greater than any other inflammatory marker (to >300-fold of controls in the 45 and 60% fat groups) (P < 0.0001) and this induction was WAT specific. Because we found that CD11d expression also increased in RP-WAT of Zucker obese rats and in the subcutaneous WAT of obese adult women, this appears to be a common feature of obesity. Observed correlations of WAT macrophage transcript marker abundances with body weight in lean to modestly obese mice raises an interesting possibility that the activities of at least some WAT macrophages are closely linked to the normal adipose remodeling that is a requisite for changes in WAT energy storage capacity.

Increased expression of receptors for orexigenic factors in nodose ganglion of diet-induced obese rats.

The vagal afferent pathway is important in short-term regulation of food intake, and decreased activation of this neural pathway with long-term ingestion of a high-fat diet may contribute to hyperphagic weight gain. We tested the hypothesis that expression of genes encoding receptors for orexigenic factors in vagal afferent neurons are increased by long-term ingestion of a high-fat diet, thus supporting orexigenic signals from the gut. Obesity-prone (DIO-P) rats fed a high-fat diet showed increased body weight and hyperleptinemia compared with low-fat diet-fed controls and high-fat diet-induced obesity-resistant (DIO-R) rats. Expression of the type I cannabinoid receptor and growth hormone secretagogue receptor 1a in the nodose ganglia was increased in DIO-P compared with low-fat diet-fed controls or DIO-R rats. Shifts in the balance between orexigenic and anorexigenic signals within the vagal afferent pathway may influence food intake and body weight gain induced by high fat diets.

Molecular Characterization of the Tumor Suppressor Candidate 5 Gene: Regulation by PPARgamma and Identification of TUSC5 Coding Variants in Lean and Obese Humans.

Tumor suppressor candidate 5 (TUSC5) is a gene expressed abundantly in white adipose tissue (WAT), brown adipose tissue (BAT), and peripheral afferent neurons. Strong adipocyte expression and increased expression following peroxisome proliferator activated receptor gamma (PPARgamma) agonist treatment of 3T3-L1 adipocytes suggested a role for Tusc5 in fat cell proliferation and/or metabolism. However, the regulation of Tusc5 in WAT and its potential association with obesity phenotypes remain unclear. We tested the hypothesis that the TUSC5 gene is a bona fide PPARgamma target and evaluated whether its WAT expression or single-nucleotide polymorphisms (SNPs) in the TUSC5 coding region are associated with human obesity. Induction of Tusc5 mRNA levels in 3T3-L1 adipocytes by troglitazone and GW1929 followed a dose-response consistent with these agents' binding affinities for PPARgamma. Chromatin immunoprecipitation (ChIP) experiments confirmed that PPARgamma protein binds a approximately -1.1 kb promotor sequence of murine TUSC5 transiently during 3T3-L1 adipogenesis, concurrent with histone H3 acetylation. No change in Tusc5 mRNA or protein levels was evident in type 2 diabetic patients treated with pioglitazone. Tusc5 expression was not induced appreciably in liver preparations overexpressing PPARs, suggesting that tissue-specific factors regulate PPARgamma responsiveness of the TUSC5 gene. Finally, we observed no differences in Tusc5 WAT expression or prevalence of coding region SNPs in lean versus obese human subjects. These studies firmly establish the murine TUSC5 gene locus as a PPARgamma target, but the significance of Tusc5 in obesity phenotypes or in the pharmacologic actions of PPARgamma agonists in humans remains equivocal.

Gamma-synuclein is an adipocyte-neuron gene coordinately expressed with leptin and increased in human obesity.

Recently, we characterized tumor suppressor candidate 5 (Tusc5) as an adipocyte-neuron PPARgamma target gene. Our objective herein was to identify additional genes that display distinctly high expression in fat and neurons, because such a pattern could signal previously uncharacterized functional pathways shared in these disparate tissues. gamma-Synuclein, a marker of peripheral and select central nervous system neurons, was strongly expressed in white adipose tissue (WAT) and peripheral nervous system ganglia using bioinformatics and quantitative PCR approaches. Gamma-synuclein expression was determined during adipogenesis and in subcutaneous (SC) and visceral adipose tissue (VAT) from obese and nonobese humans. Gamma-synuclein mRNA increased from trace levels in preadipocytes to high levels in mature 3T3-L1 adipocytes and decreased approximately 50% following treatment with the PPARgamma agonist GW1929 (P < 0.01). Because gamma-synuclein limits growth arrest and is implicated in cancer progression in nonadipocytes, we suspected that expression would be increased in situations where WAT plasticity/adipocyte turnover are engaged. Consistent with this postulate, human WAT gamma-synuclein mRNA levels consistently increased in obesity and were higher in SC than in VAT; i.e. they increased approximately 1.7-fold in obese Pima Indian adipocytes (P = 0.003) and approximately 2-fold in SC and VAT of other obese cohorts relative to nonobese subjects. Expression correlated with leptin transcript levels in human SC and VAT (r = 0.887; P < 0.0001; n = 44). Gamma-synuclein protein was observed in rodent and human WAT but not in negative control liver. These results are consistent with the hypothesis that gamma-synuclein plays an important role in adipocyte physiology.

Characterization of Tusc5, an adipocyte gene co-expressed in peripheral neurons.

Tumor suppressor candidate 5 (Tusc5, also termed brain endothelial cell derived gene-1 or BEC-1), a CD225 domain-containing, cold-repressed gene identified during brown adipose tissue (BAT) transcriptome analyses was found to be robustly-expressed in mouse white adipose tissue (WAT) and BAT, with similarly high expression in human adipocytes. Tusc5 mRNA was markedly increased from trace levels in pre-adipocytes to significant levels in developing 3T3-L1 adipocytes, coincident with several mature adipocyte markers (phosphoenolpyruvate carboxykinase 1, GLUT4, adipsin, leptin). The Tusc5 transcript levels were increased by the peroxisome proliferator activated receptor-gamma (PPARgamma) agonist GW1929 (1microg/mL, 18h) by >10-fold (pre-adipocytes) to approximately 1.5-fold (mature adipocytes) versus controls (p<0.0001). Taken together, these results suggest an important role for Tusc5 in maturing adipocytes. Intriguingly, we discovered robust co-expression of the gene in peripheral nerves (primary somatosensory neurons). In light of the marked repression of the gene observed after cold exposure, these findings may point to participation of Tusc5 in shared adipose-nervous system functions linking environmental cues, CNS signals, and WAT-BAT physiology. Characterization of such links is important for clarifying the molecular basis for adipocyte proliferation and could have implications for understanding the biology of metabolic disease-related neuropathies.