Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity.

BACKGROUND: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear. METHODS: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery. RESULTS: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis. CONCLUSION: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

The Effect of FGF21 and Its Genetic Variants on Food and Drug Cravings, Adipokines and Metabolic Traits.

Fibroblast growth factor 21 (FGF21) is a regulator of addictive behavior. Increasing evidence suggests an impact of FGF21 on eating behavior, food and drug cravings and on other adipokines like insulin-like growth factor 1 (IGF-1) or adiponectin. We investigated the association of serum FGF21 and genetic variants with aspects of food and drug craving and obesity related metabolic parameters including serum adipokine levels. Standardized questionnaires, blood samples and anthropometric data of the Sorbs cohort (n = 1046) were analyzed using SPSS. For genetic analyses, the FGF21-locus ±10 kb was genotyped and analyzed using PLINK. Validation was conducted in a second independent cohort (n = 704). FGF21 was significantly associated with alcohol and coffee consumption, smoking and eating behavior (disinhibition). We confirmed correlations of FGF21 serum levels with IGF-1, adiponectin, pro-enkephalin, adipocyte fatty-acid-binding protein, chemerin and progranulin. FGF21 genetic variants were associated with anthropometric and metabolic parameters, adipokines, food and drug craving while strongest evidence was seen with low-density lipoprotein cholesterol (LDL-C). We highlight the potential role of FGF21 in food and drug cravings and provide new insights regarding the link of FGF21 with other adipokines as well as with metabolic traits, in particular those related to lipid metabolism (LDL-C).

Structural Studies on the Inhibitory Binding Mode of Aromatic Coumarinic Esters to Human Kallikrein-Related Peptidase 7.

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.

Membrane Phospholipids and Polyphosphates as Cofactors and Binding Molecules of SERPINA12 (vaspin).

Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (KD: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in ß-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.

Kallikrein-related peptidase 14 is the second KLK protease targeted by the serpin vaspin.

Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multi-specific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.

Vaspin suppresses cytokine-induced inflammation in 3T3-L1 adipocytes via inhibition of NFκB pathway.

Vaspin expression is increased in white adipose tissue (WAT) of diet-induced obese mice and rats and is supposed to compensate HFD-induced inflammatory processes and insulin resistance in adipose tissue by counteracting pro-inflammatory gene expression in obesity. Multiple studies have also demonstrated strong anti-inflammatory effects in vascular and skin cells. Here, we used vaspin treated 3T3-L1 murine adipocytes as well as 3T3-L1 cells with stable vaspin expression to investigate the effect of exogenous and endogenous vaspin on inflammatory processes and insulin signaling in adipocytes. Our stably transfected cells secreted significant amounts of vaspin which was in the physiological range of ∼0.5 ng/ml in cell supernatants. Adipocyte differentiation was not affected by vaspin as expression of adipogenic marker genes as well as lipid accumulation after full differentiation was similar to control cells. We found that IL-1ß induced expression and secretion of pro-inflammatory cytokines, such as IL-6, MCP1 and TNFα was significantly blunted in vaspin expressing 3T3-L1 cells. Treatment of 3T3-L1 cells with exogenous vaspin resulted in reduced cytokine-induced activation of the intracellular and pro-inflammatory NFκB signaling cascades (IKKα/ß, IκB and NFκB). Moreover, endogenous vaspin positively affected insulin signaling by increasing insulin-stimulated phosphorylation of the key mediator protein kinase B (AKT). Together, we demonstrate anti-inflammatory effects of vaspin in 3T3-L1 adipocytes as well as increased insulin signaling by endogenous expression or exogenous treatment. The results provide evidence for potent anti-inflammatory action of vaspin not only in vascular cells but also in adipose tissue.

Anti-Inflammatory Action of Keratinocyte-Derived Vaspin: Relevance for the Pathogenesis of Psoriasis.

Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1ß, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation. In conclusion, vaspin expression by maturing KCs modulates cutaneous immune responses and may be involved in the pathogenesis of psoriasis.

Extensive weight loss reveals distinct gene expression changes in human subcutaneous and visceral adipose tissue.

Weight loss has been shown to significantly improve Adipose tissue (AT) function, however changes in AT gene expression profiles particularly in visceral AT (VAT) have not been systematically studied. Here, we tested the hypothesis that extensive weight loss in response to bariatric surgery (BS) causes AT gene expression changes, which may affect energy and lipid metabolism, inflammation and secretory function of AT. We assessed gene expression changes by whole genome expression chips in AT samples obtained from six morbidly obese individuals, who underwent a two step BS strategy with sleeve gastrectomy as initial and a Roux-en-Y gastric bypass as second step surgery after 12 ± 2 months. Global gene expression differences in VAT and subcutaneous (S)AT were analyzed through the use of genome-scale metabolic model (GEM) for adipocytes. Significantly altered gene expressions were PCR-validated in 16 individuals, which also underwent a two-step surgery intervention. We found increased expression of cell death-inducing DFFA-like effector a (CIDEA), involved in formation of lipid droplets in both fat depots in response to significant weight loss. We observed that expression of the genes associated with metabolic reactions involved in NAD+, glutathione and branched chain amino acid metabolism are significantly increased in AT depots after surgery-induced weight loss.

Liver-restricted Repin1 deficiency improves whole-body insulin sensitivity, alters lipid metabolism, and causes secondary changes in adipose tissue in mice.

Replication initiator 1 (Repin1) is a zinc finger protein highly expressed in liver and adipose tissue and maps within a quantitative trait locus (QTL) for body weight and triglyceride (TG) levels in the rat. The QTL has further been supported as a susceptibility locus for dyslipidemia and related metabolic disorders in congenic and subcongenic rat strains. Here, we elucidated the role of Repin1 in lipid metabolism in vivo. We generated a liver-specific Repin1 knockout mouse (LRep1(-/-)) and systematically characterized the consequences of Repin1 deficiency in the liver on body weight, glucose and lipid metabolism, liver lipid patterns, and protein/mRNA expression. Hyperinsulinemic-euglycemic clamp studies revealed significantly improved whole-body insulin sensitivity in LRep1(-/-) mice, which may be due to significantly lower TG content in the liver. Repin1 deficiency causes significant changes in potential downstream target molecules including Cd36, Pparγ, Glut2 protein, Akt phosphorylation, and lipocalin2, Vamp4, and Snap23 mRNA expression. Mice with hepatic deletion of Repin1 display secondary changes in adipose tissue function, which may be mediated by altered hepatic expression of lipocalin2 or chemerin. Our findings indicate that Repin1 plays a role in insulin sensitivity and lipid metabolism by regulating key genes of glucose and lipid metabolism.

Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization.

The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far. Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling.

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.

Localization of novel adiponectin receptor constructs.

Adiponectin is one of the most abundant fat-derived hormones involved in a multitude of metabolism pathways. The receptors AdipoR1 and AdipoR2 of this metabolically active protein have been identified recently. AdipoR1 and AdipoR2 are most abundantly expressed in the skeletal muscle and in the liver, respectively. It has been postulated that although they both consist of seven transmembrane helices, they are distinct from other G protein-coupled receptors (GPCRs). We cloned both receptors as fusion proteins with enhanced yellow fluorescent protein (YFP) to determine their localization and orientation in the cell membrane. By confocal microscopy and immune staining we demonstrated that both receptor-YFP-fusion proteins are integral membrane proteins with the predicted topology--an intracellular N-terminus and an extracellular C-terminus. In parallel, comparative experiments were performed with the NPY Y2-receptor, a classical rhodopsin-like GPCR.