Gut-derived GIP activates central Rap1 to impair neural leptin sensitivity during overnutrition.

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

Luciferase Reporter Mice for In Vivo Monitoring and Ex Vivo Assessment of Hypothalamic Signaling of Socs3 Expression.

Suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of actions of cytokines and the metabolic hormone leptin. In the hypothalamus, SOCS3 is induced in response to several conditions such as inflammation and high-fat diet feeding, modulates cellular signaling of cytokines and leptin, and mediates the effects of these biological conditions. However, signaling mechanisms controlling hypothalamic Socs3 expression remains to be fully established. To facilitate the identification of molecular pathways of Socs3 induction, we generated a real-time gene expression reporter mouse of Socs3 (Socs3-Luc mice). We successfully detected a remarkable increase in luciferase activity in various tissues of Socs3-Luc mice in response to a peripheral injection of lipopolysaccharide, a potent inducer of inflammation, reflecting expression levels of endogenous Socs3 mRNA. Using ex vivo hypothalamic explants of Socs3-Luc mice, we demonstrate that hypothalamic luciferase activity was significantly elevated in slices stimulated with known inducers of Socs3 such as proinflammatory cytokines IL-6, IL-1ß, and TNF-α, lipopolysaccharide, and cAMP-inducing agent forskolin. Using the ex vivo model, we found glycogen synthase kinase-3 (GSK3)ß-specific inhibitors to be potent inducers of Socs3. Furthermore, pharmacological inhibitors of ß-catenin, a downstream mediator of GSK3ß signaling, reduced Socs3 luciferase activity ex vivo. Finally, hypothalamic inhibition of GSK3ß hindered leptin-induced phosphorylation of signal transducers and activators of transcription 3 in hypothalamic explants. These results suggest that the Socs3-luciferase mouse is useful for in vivo monitoring of Socs3 gene expression and for ex vivo slice-based screening to identify signaling pathways that control Socs3 in the hypothalamus.

A cell death assay for assessing the mitochondrial targeting of proteins.

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.

Neuronal Rap1 Regulates Energy Balance, Glucose Homeostasis, and Leptin Actions.

The CNS contributes to obesity and metabolic disease; however, the underlying neurobiological pathways remain to be fully established. Here, we show that the small GTPase Rap1 is expressed in multiple hypothalamic nuclei that control whole-body metabolism and is activated in high-fat diet (HFD)-induced obesity. Genetic ablation of CNS Rap1 protects mice from dietary obesity, glucose imbalance, and insulin resistance in the periphery and from HFD-induced neuropathological changes in the hypothalamus, including diminished cellular leptin sensitivity and increased endoplasmic reticulum (ER) stress and inflammation. Furthermore, pharmacological inhibition of CNS Rap1 signaling normalizes hypothalamic ER stress and inflammation, improves cellular leptin sensitivity, and reduces body weight in mice with dietary obesity. We also demonstrate that Rap1 mediates leptin resistance via interplay with ER stress. Thus, neuronal Rap1 critically regulates leptin sensitivity and mediates HFD-induced obesity and hypothalamic pathology and may represent a potential therapeutic target for obesity treatment.

Inhibition of acetyl-CoA carboxylases by soraphen A prevents lipid accumulation and adipocyte differentiation in 3T3-L1 cells.

Acetyl-CoA carboxylases (ACC) 1 and 2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA and depend on biotin as a coenzyme. ACC1 localizes in the cytoplasm and produces malonyl-CoA for fatty acid (FA) synthesis. ACC2 localizes in the outer mitochondrial membrane and produces malonyl-CoA that inhibits FA import into mitochondria for subsequent oxidation. We hypothesized that ACCs are checkpoints in adipocyte differentiation and tested this hypothesis using the ACC1 and ACC2 inhibitor soraphen A (SA) in murine 3T3-L1 preadipocytes. When 3T3-L1 cells were treated with 100nM SA for 8 days after induction of differentiation, the expression of PPARγ mRNA and FABP4 mRNA decreased by 40% and 50%, respectively, compared with solvent controls; the decrease in gene expression was accompanied by a decrease in FABP4 protein expression and associated with a decrease in lipid droplet accumulation. The rate of FA oxidation was 300% greater in SA-treated cells compared with vehicle controls. Treatment with exogenous palmitate restored PPARγ and FABP4 mRNA expression and FABP4 protein expression in SA-treated cells. In contrast, SA did not alter lipid accumulation if treatment was initiated on day eight after induction of differentiation. We conclude that loss of ACC1-dependent FA synthesis and loss of ACC2-dependent inhibition of FA oxidation prevent lipid accumulation in adipocytes and inhibit early stages of adipocyte differentiation.

Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster.

Holocarboxylase synthetase (HLCS) is the sole protein-biotin ligase in the human proteome. HLCS has key regulatory functions in intermediary metabolism, including fatty acid metabolism, and in gene repression through epigenetic mechanisms. The objective of this study was to identify food-borne inhibitors of HLCS that alter HLCS-dependent pathways in metabolism and gene regulation. When libraries of extracts from natural products and chemically pure compounds were screened for HLCS inhibitor activity, resveratrol compounds in grape materials caused an HLCS inhibition of >98% in vitro. The potency of these compounds was piceatannol>resveratrol>piceid. Grape-borne compounds other than resveratrol metabolites also contributed toward HLCS inhibition, e.g., p-coumaric acid and cyanidin chloride. HLCS inhibitors had meaningful effects on body fat mass. When Drosophila melanogaster brummer mutants, which are genetically predisposed to storing excess amounts of lipids, were fed diets enriched with grape leaf extracts and piceid, body fat mass decreased by more than 30% in males and females. However, Drosophila responded to inhibitor treatment with an increase in the expression of HLCS, which elicited an increase in the abundance of biotinylated carboxylases in vivo. We conclude that mechanisms other than inhibition of HLCS cause body fat loss in flies. We propose that the primary candidate is the inhibition of the insulin receptor/Akt signaling pathway.

ß-Keto and ß-hydroxyphosphonate analogs of biotin-5'-AMP are inhibitors of holocarboxylase synthetase.

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (ß-ketoP) and hydroxyphosphonate (ß-hydroxyP) analogs of biotin-5'-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 µM and 203.7 µM. By comparison, an IC50 value of 7 µM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 µM and 17.3 µM, respectively, are consistent with the IC50 results, and close to the Ki obtained for biotinol-5'-AMP (7 µM). The ß-ketoP and ß-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5'-AMP inhibited HLCS by a mixed mechanism.

Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism.

The role of holocarboxylase synthetase (HLCS) in catalyzing the covalent binding of biotin to the five biotin-dependent carboxylases in humans is well established, as are the essential roles of these carboxylases in the metabolism of fatty acids, the catabolism of leucine, and gluconeogenesis. This review examines recent discoveries regarding the roles of HLCS in assembling a multiprotein gene repression complex in chromatin. In addition, emerging evidence suggests that the number of biotinylated proteins is far larger than previously assumed and includes members of the heat-shock superfamily of proteins and proteins coded by the ENO1 gene. Evidence is presented linking biotinylation of heat-shock proteins HSP60 and HSP72 with redox biology and immune function, respectively, and biotinylation of the two ENO1 gene products MBP-1 and ENO1 with tumor suppression and glycolysis, respectively.

Biotin requirements for DNA damage prevention.

Biotin serves as a covalently bound coenzyme in five human carboxylases; biotin is also attached to histones H2A, H3, and H4, although the abundance of biotinylated histones is low. Biotinylation of both carboxylases and histones is catalyzed by holocarboxylase synthetase. Human biotin requirements are unknown. Recommendations for adequate intake of biotin are based on the typical intake of biotin in an apparently healthy population, which is only a crude estimate of the true intake due to analytical problems. Importantly, intake recommendations do not take into account possible effects of biotin deficiency on impairing genome stability. Recent studies suggest that biotin deficiency causes de-repression of long terminal repeats, thereby causing genome instability. While it was originally proposed that these effects are caused by loss of biotinylated histones, more recent evidence suggests a more immediate role of holocarboxylase synthetase in forming multiprotein complexes in chromatin that are important for gene repression. Holocarboxylase synthetase appears to interact physically with the methyl-CpG-binding domain protein 2 and, perhaps, histone methyl transferases, thereby creating epigenetic synergies between biotinylation and methylation events. These observations might offer a mechanistic explanation for some of the birth defects seen in biotin-deficient animal models.

Different sources of omega-3 polyunsaturated fatty acids affects apparent digestibility, tissue deposition, and tissue oxidative stability in growing female rats.

BACKGROUND: Numerous health benefits associated with increased omega-3 polyunsaturated fatty acid (n-3 PUFA) consumption has lead to an increasing variety of available n-3 PUFA sources. However, sources differ in the type, amount, and structural form of the n-3 PUFAs. Therefore, the objective of this study was to determine the effect of different sources of ω-3 PUFAs on digestibility, tissue deposition, eicosanoid metabolism, and oxidative stability. METHODS: Female Sprague-Dawley rats (age 28 d) were randomly assigned (n = 10/group) to be fed a high fat 12% (wt) diet consisting of either corn oil (CO) or n-3 PUFA rich flaxseed (FO), krill (KO), menhaden (MO), salmon (SO) or tuna (TO) oil for 8 weeks. Rats were individually housed in metabolic cages to determine fatty acid digestibility. Diet and tissue fatty acid composition was analyzed by gas chromatography and lipid classes using thin layer chromatography. Eicosanoid metabolism was determined by measuring urinary metabolites of 2-series prostaglandins (PGs) and thromoboxanes (TXBs) using enzyme immunoassays. Oxidative stability was assessed by measuring thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC) using colorimetric assays. Gene expression of antioxidant defense enzymes was determined by real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Rats fed KO had significantly lower DHA digestibility and brain DHA incorporation than SO and TO-fed rats. Of the n-3 PUFA sources, rats fed SO and TO had the highest n-3 PUFAs digestibility and in turn, tissue accretion. Higher tissue n-3 LC-PUFAs had no significant effect on 2-series PG and TXB metabolites. Despite higher tissue n-3 LC-PUFA deposition, there was no increase in oxidation susceptibility indicated by no significant increase in TBARS or decrease in TAC and gene expression of antioxidant defense enzymes, in SO or TO-fed rats. CONCLUSIONS: On the basis that the optimal n-3 PUFA sources should provide high digestibility and efficient tissue incorporation with the least tissue lipid peroxidation, TO and SO appeared to be the most beneficial of the n-3 PUFAs sources evaluated in this study.

The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events.

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to histones. Biotinylated histones are gene repression marks and are particularly enriched in long terminal repeats, telomeres, and other repeat regions. The effects of HLCS in gene regulation are mediated by its physical interactions with chromatin proteins such as histone H3, DNMT1, MeCP2, and EHMT-1. It appears that histone biotinylation depends on prior methylation of cytosines. De-repression of long terminal repeats in biotin- or HLCS-deficient cell cultures and organisms is associated with genome instability.