Murine Diet-induced Obesity Research Articles

Noncoding variation near UBE2E2 orchestrates cardiometabolic pathophenotypes through polygenic effectors.

Mechanisms underpinning signals from genome wide association studies remain poorly understood, particularly for non-coding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where non-coding SNVs are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPRi screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro, CRISPR excision of SNV-containing non-coding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes, and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes than homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of non-coding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.

LBODP003 Effects Of 4-weeks’ Treatment With Intraperitoneal Colchicine On Metabolic And Inflammatory Outcomes In Mice Fed A High-fat Diet

Abstract Health-deteriorating effects of obesity include chronic inflammation and insulin resistance (IR). A recent pilot human trial reported reduced IR in colchicine-treated vs. placebo-treated adults with obesity. Thus, we examined anti-inflammatory and metabolic effects of two commonly employed dosing regimens of colchicine, 0.2 mg/kg and 0. 02mg/kg, in a high-fat diet (HFD; 45%) mouse obesity model. We placed male C57BL/6J mice on HFD from ages 8 through 16 weeks. At age 12 weeks, mice were randomized to: high-dose colchicine (CHD 0.2 mg/kg, n=28), low dose colchicine (CLD, 0. 02mg/kg, n=26) or vehicle (V, n=26), injected intraperitoneally (IP) for 4 weeks. Serum CRP was measured by ELISA. Dual-energy X-ray absorptiometry (DXA) scans and fasting IP glucose tolerance (GTT) and insulin-tolerance (ITT) tests were performed at baseline and following treatment. Hepatic tissue was processed for immunoblotting to determine expression of NLRP3 and caspase-1. Changes in pre- vs. post-treatment serum CRP were significantly different in the CHD group (Mean±SD: -0.57±3.1µg/mL) vs. V (+3.64±2.9µg/mL; p<0. 001) and CLD (+0.72±2.3µg/mL) compared to V (p=0. 03). Changes in CRP for CHD compared to CLD did not differ significantly (p=0.47). DXA-measured body composition changes pre-vs. post-treatment for fat mass were significantly reduced for CHD (-0.26±2. 0g) vs. V (+1.24±2.1g; p=0. 04) and CHD vs. CLD (+1.52±2.2g; p=0. 01). Lean body mass changes were not significant between treatments (p=0.69). GTT glucose concentrations were not significantly different between treatments across timepoints (Group x time interaction: p=0.24) and GTT areas under curves (AUC) were not significantly different between treatments (p=0.96). Analysis of ITT glucose measures revealed a significant group x time interaction (p=0. 03) and group effect for CHD vs. V (p=0. 009) as well as significant differences in ITT AUCs (p=0. 03) such that insulin tolerance was significantly worse for CHD: CHD AUC 19803±2662, CLD 18487±3703, V 17436±3138mg/dLx120min. Hepatic NLRP3 fold-change protein expression of treatment groups compared to V was not significant for CHD (p=0.75) or CLD (p=0.21). However, hepatic caspase-1 protein expression fold-change was significantly lower in the CHD (p=0. 008), but not the CLD group (p=0.19). We conclude that 4-weeks of IP colchicine at 0.2mg/kg (CHD) significantly suppressed serum CRP and hepatic caspase-1 protein expression compared to V, indicating CHD reduced inflammation, whereas 0. 02mg/kg colchicine (CLD) did not. However, our data suggest that 4 weeks of the CHD dose may potentially worsen whole-body insulin resistance, even though it induced a reduction in fat mass, suggesting a possible toxicity from chronic high-dose colchicine treatment. Further studies are warranted to elucidate metabolic effects of colchicine administration in murine diet-induced obesity models. Presentation: No date and time listed

HDL Receptor SR-B1 Deficiency Increased Inflammatory Dyslipidemia and Adipocyte Hypertrophy and Attenuated the Hepatic Steatosis in Murine Diet-Induced Obesity

Abstract Objectives We tested whether the scavenger receptor, class B type 1 (SR-B1), a physiologically relevant HDL receptor, modulates the metabolic and inflammatory phenotype of obese mice, accompanied by changes in adipocyte and hepatocyte lipid deposits. Methods Male SR-B1 knock-out (KO) mice and wild-type (WT) littermates were fed for 12 weeks with a high-fat diet (HFD, n = 12 per group) to induce obesity. Animals were euthanized after overnight food deprivation. Blood was obtained and adipose tissue and liver were removed. Plasma or frozen tissues were used for biochemical analyses or assessed by light microscopy and immunohistochemistry. Comparisons between WT and KO mice fed with HFD were performed and differences were considered statistically significant when P < 0.05. Results Compared to obese WT, obese HFD-fed SR-B1 KO mice showed increased plasma total cholesterol (p < 0.0001) and triglycerides (TG) (p < 0.01) as well as tumor necrosis factor-α (TNF-α) (p < 0.0001) levels. Also, these animals exhibited white fat with larger adipocytes (p < 0.0001) and increased macrophage-based crown-like structure formation (p < 0.01), compared to HFD-fed WT mice, revealing a more inflammatory adipose tissue. These changes in adipose tissue were associated with reduced hepatic steatosis: reduced hepatocyte lipid droplet area (p < 0.0001) and decreased liver TG content (p < 0.0001). In addition, obese SR-B1-deficient mice showed reduced hepatic peroxisome proliferator-activated receptor-γ (PPAR-γ) expression, more efficient hepatic fatty acid β-oxidation and increased very-low-density lipoprotein (VLDL)-TG secretion compared to obese HFD-fed WT mice Furthermore, liver fatty acid (FA) composition in obese SR-B1-deficient mice revealed a reduction in monounsaturated (MUFA) (p < 0.01), but an increase in polyunsaturated (PUFA) (p < 0.05), fatty acid content, compared to obese WT mice. Conclusions Our findings demonstrate that SR-B1 expression modulates high fat feeding-associated inflammatory dyslipidemia, adipocyte hypertrophy, and hepatic steatosis; key processes underlying the pathogenesis of highly prevalent chronic diseases, such as obesity and non-alcoholic fatty liver disease (NAFLD). Funding Sources FONDECYT 1,150,399, FONDECYT 1,180,525.

P1939Tumor necrosis factor receptor-associated factor 5 (TRAF-5) deficiency exacerbates diet-induced adipose tissue inflammation and aggravates metabolic syndrome in mice

Abstract Introduction Many clinical and experimental observations have established an association between visceral obesity and chronic adipose tissue inflammation. Potent pro-inflammatory mediators such as TNFα, CD40 and IL-1β are regulated by Tumor necrosis factor (TNF) receptor-associated factors (TRAFs). TRAF5 deficiency accelerates atherogenesis in mice by increasing inflammatory leukocyte recruitment. Since inflammatory cell invasion is also a prerequisite of adipose tissue inflammation, we tested the hypothesis that deficient TRAF5 signaling aggravates adipose tissue inflammation and its metabolic complications in a murine diet-induced obesity (DIO) model. Purpose We aimed to clarify the role of TRAF5 in adipose tissue inflammation and metabolic syndrome. Methods TRAF5−/− mice and gender- and age-matched wild-type (WT) mice consumed a high fat diet (HFD, 45%kcal from fat) or a matched low-fat diet (LFD, 10%kcal from fat) for 18 weeks to induce DIO and adipose tissue inflammation. All mice were then subjected to subsequent analysis, including glucose and insulin tolerance testing, body composition assessment by MRI imaging, flow cytometry, gene expression of different tissues, plasma analysis and histology. Finally, we studied if TRAF5 expression was associated with metabolic syndrome in humans by analyzing plasma and adipocytes samples from 62 patients of the Tumor-Necrosis-Factor Receptor Associated in Cardiovascular Risk Study (TRAFICS). Results TRAF5 expression was significantly attenuated in isolated WT-adipocytes and WT-macrophages after 18 weeks of HFD compared to LFD-fed controls. TRAF5−/− mice on HFD gained significantly more weight compared to TRAF5-competent mice and showed an aggravated metabolic phenotype, including impaired insulin tolerance, hyperinsulinemia and increased fasting glucose plasma levels. The weight gain in TRAF5−/− mice was attributable to a significant increase in adipose tissue and liver weight. Further analysis of the visceral adipose tissue revealed enhanced macrophage accumulation and increased pro-inflammatory CD11c+ subset polarization in HFD-fed TRAF5−/− mice. In line with an increased migratory capacity of inflammatory cells, we observed enhanced peritoneal invasion of leukocytes and subsets in TRAF5−/− mice. Accordingly, TRAF5 deficiency increased inflammatory cytokine expression and ameliorated parameters of insulin sensitivity in adipose tissue. Finally, patients with metabolic syndrome displayed decreased TRAF5 expression in blood and adipocytes compared to humans without metabolic syndrome. Conclusion We show that genetic deficiency of TRAF5 aggravates metabolic syndrome in murine diet-induced obesity. Enhanced accumulation of leukocytes subsets in adipose tissue serves as the likely mechanism. We conclude that TRAF5 signaling properties may favorably affect metabolic disease. Acknowledgement/Funding Forschungskommission Medizinische Fakultät Universität Freiburg, MOTI-VATE Promotionskolleg der Medizinischen Fakultät Freiburg (EKFS)

Fatty acid synthase downregulation contributes to acute lung injury in murine diet-induced obesity

The prevalence of obesity is rising worldwide and obese patients comprise a specific population in the intensive care unit. Acute respiratory distress syndrome (ARDS) incidence is increased in obese patients. Exposure of rodents to hyperoxia mimics many of the features of ARDS. In this report, we demonstrate that high fat diet induced obesity increases the severity of hyperoxic acute lung injury in mice in part by altering fatty acid synthase (FASN) levels in the lung. Obese mice exposed to hyperoxia had significantly reduced survival and increased lung damage. Transcriptomic analysis of lung homogenates identified Fasn as one of the most significantly altered mitochondrial associated genes in mice receiving 60% compared to 10% fat diet. FASN protein levels in the lung of high fat diet mice were lower by immunoblotting and immunohistochemistry. Depletion of FASN in type II alveolar epithelial cells resulted in altered mitochondrial bioenergetics and more severe lung injury with hyperoxic exposure, even upon the administration of a 60% fat diet. This is the first study to show that a high fat diet leads to altered FASN expression in the lung and that both a high fat diet and reduced FASN expression in alveolar epithelial cells promote lung injury.

Purple corn extract induces long-lasting reprogramming and M2 phenotypic switch of adipose tissue macrophages in obese mice

BackgroundObesity is a chronic and systemic inflammatory disorder and an important risk factor for the onset of several chronic syndromes. Adipose tissue (AT) plays a crucial role in the development of obesity, promoting the infiltration and accumulation of leukocytes in the tissue and sustaining adipocyte expansion. Anthocyanins exert a broad range of health benefits, but their effect in improving obesity-related inflammation in vivo has been poorly characterized. We examined the effects of a purple corn cob extract in the context of AT inflammation in a murine diet-induced obesity (DIO) model.MethodsMale C57BL/6J mice were subjected to control diet (CTR + H2O), high fat diet (HF + H2O) or high fat diet plus purple corn extract (HF + RED) for 12 weeks. Blood glucose, AT, and liver gene expression, metabolism, biochemistry, and histology were analysed and flow cytometry was performed on AT leukocytes and Kupffer cells.ResultsRED extract intake resulted in lower MCP-1 mediated recruitment and proliferation of macrophages into crown-like structures in the AT. AT macrophages (ATM) of HF + RED group upregulated M2 markers (ArgI, Fizz1, TGFβ), downregulating inflammatory mediators (TNF-α, IL-6, IL-1β, COX-2) thanks to the suppression of NF-kB signalling. ATM also increased the expression of iron metabolism-related genes (FABP4, Hmox1, Ferroportin, CD163, TfR1, Ceruloplasmin, FtL1, FtH1) associated with a reduction in iron storage and increased turnover. ATM from HF + RED mice did not respond to LPS treatment ex vivo, confirming the long-lasting effects of the treatment on M2 polarization. Adipocytes of HF + RED group improved lipid metabolism and displayed a lower inflammation grade. Liver histology revealed a remarkable reduction of steatosis in the HF + RED group, and Kupffer cell profiling displayed a marked switch towards the M2 phenotype.ConclusionsRED extract attenuated AT inflammation in vivo, with a long-lasting reprogramming of ATM and adipocyte profiles towards the anti-inflammatory phenotype, therefore representing a valuable supplement in the context of obesity-associated disorders.

High dose of linagliptin induces thermogenic beige adipocytes in the subcutaneous white adipose tissue in diet-induced obese C57BL/6 mice.

To verify whether the treatment with linagliptin induces the browning of the subcutaneous WAT (sWAT) and thermogenesis in murine diet-induced obesity (DIO) model. Forty animals were randomly assigned to receive a control diet (C, 10% lipids as energy) or a high-fat diet (HF, 50% lipids as energy) for 10 weeks. Each group was re-divided to begin the 5-week treatment, totalizing four experimental groups: C, C-L (C plus linagliptin, 30 mg/kg body mass; BM), HF, and HF-L (HF plus linagliptin, 30 mg/kg BM). The drug was mixed with diet. HF animals showed overweight, glucose intolerance, and a greater cross-sectional area of adipocytes. The treatment with linagliptin was able to normalize the BM, restore the glucose tolerance and the cross-sectional area of adipocytes. These observations comply with the observation of UCP1-positive multilocular adipocytes in the sWAT of treated animals. Both treated groups (C-L and HF-L) showed high expression of thermogenic and type 2 cytokines genes, which agree with the enhanced body temperature and the lower respiratory exchange ratio, implying enhanced thermogenesis with the use of lipids as fuel. The reduced BM, the enhanced body temperature, and the presence of positive UCP1 beige cells in the sWAT point to the activation of the browning cascade on the sWAT of linagliptin-treated mice, and hence, linagliptin could induce the thermogenic pathway as a pleiotropic effect that can have translational potential.

GW0742 (PPAR-beta agonist) attenuates hepatic endoplasmic reticulum stress by improving hepatic energy metabolism in high-fat diet fed mice.

Endoplasmic reticulum (ER) stress and hepatic steatosis are intertwined with insulin resistance. PPARs are at the crossroads of these pathways. This study aimed to investigate the effects of GW0742 (PPAR-beta agonist) on hepatic energy metabolism and ER stress in a murine diet-induced obesity model. HF diet caused overweight, hyperinsulinemia, hepatic inflammation (increased NF-kB, TNF-alpha, and IL-6 protein expression) and favored hepatic lipogenesis, leading to ER stress, with ultrastructural and molecular alterations, ending up in proapoptotic stimulus. GW0742 rescued the overweight and the glucose tolerance, tackled hepatic inflammation and favored hepatic beta-oxidation over lipogenesis. These results comply with ER ultrastructure improvement, reducing ER stress and apoptosis in treated animals. Our results indicate that the PPAR-beta/delta activation alleviated the ER stress by improving the insulin sensitivity and maximizing the hepatic energy metabolism with a shift towards beta-oxidation. PPAR-beta/delta activation could be an essential tool to avoid the NAFLD progression and other obesity constraints.

Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways

Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-β (TRIF), I-κB, NF-κB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, α1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-κB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-κB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways.

Murine diet-induced obesity remodels cardiac and liver mitochondrial phospholipid acyl chains with differential effects on respiratory enzyme activity

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8–3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6–5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity.

Abstract P1-03-03: Obesity increases the lipid mediator, sphingosine-1-phosphate in the tumor and tumor microenvironment, and promotes tumor progression

Abstract INTRODUCTION: While obesity is an established independent prognostic factor for breast cancer patients, the underlying mechanisms how obesity promotes breast cancer progression are not well understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Our hypothesis is that obesity increases levels of S1P in both tumor and its microenvironment, which promotes obesity-induced inflammation and breast cancer progression. The aim of this study is to test the hypothesis. METHODS: A high fat diet (HFD) was fed C57Bl/6 mice, which is the most commonly used murine diet-induced obesity model. E0771 cells derived from C57Bl/6 mice were implanted into mammary fat pads of mice that were fed with HFD or normal diet (ND) for 12 weeks prior to the implantation. Western blot, immunofluorescent staining, RT-qPCR and LC-ESI-MS/MS assays were used. RESULTS: Mice fed with HFD for 20 weeks developed severe obesity with an almost 2 fold increase of body weight compared with ND fed mice. E0771 mouse breast cancer cells were implanted into mammary fat pad of C57Bl/6 mice, and the mice fed with HFD developed significantly larger tumors within 30 days than those fed with ND. Expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the tumors of HFD fed mice, compared to those fed with ND. Furthermore, immunofluorescent analysis with anti-F4/80 antibody showed that tumors from HFD fed animals recruited significantly more tumor associated macrophages than those from ND fed animals. Expression of SphK1 and S1PR1, but not SphK2, was increased in the tumors from mice fed HFD. Mass spectrometry analysis revealed that while S1P levels in the normal breast mammary fat pad were increased with HFD feeding, S1P levels were even higher in breast tumors. Consistent with increased SphK1 and S1P in tumors, S1P was also significantly increased in the tumor interstitial fluid, which is a component of the tumor microenvironment and bathes cancer cells in the tumor. S1P levels were also increased in the serum of the tumor-implanted animals fed with HFD compared with those fed with ND, while minimal changes in S1P were evident in the serum of non-tumor bearing mice fed with HFD. Furthermore, S1P levels in the lungs of tumor-implanted animals fed with HFD were significantly higher than those fed with ND. CONCLUSION: S1P is increased not only in tumor, but also in tumor microenvironment such as tumor interstitial fluid by obesity. Our results suggest that S1P has a role in obesity-induced inflammation and the cancer progression. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Tsuchida J, Nagahashi M, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Obesity increases the lipid mediator, sphingosine-1-phosphate in the tumor and tumor microenvironment, and promotes tumor progression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-03.

MIR-34A has a role in weight gain, during murine diet-induced obesity

MIR-34A has a role in weight gain, during murine diet-induced obesity

Strong association of serum- and glucocorticoid-regulated kinase 1 with peripheral and adipose tissue inflammation in obesity.

The serum- and glucocorticoid-regulated kinase 1 (SGK1) is an early transcriptional target of glucocorticoids and is activated via insulin. Here we investigate the regulation of SGK1 expression in human obesity, diet-induced murine obesity and human monocytic cell line THP-1 monocytes. SGK1 expression was studied in subcutaneous and omental adipose tissue (AT) of 20 morbidly obese and 20 age- and gender-matched non-obese controls in murine diet-induced obesity and the THP-1 cell line. The regulation of SGK1 by inflammatory signals was tested in THP-1 cells. Murine diet-induced obesity is associated with a significant upregulation of Sgk1 in gonadal AT. Sgk1 expression is highest in the macrophage-rich stromal vascular fraction and lower in adipocytes. In humans, AT SGK1 is predominantly expressed in CD14(+) macrophages and significantly upregulated in omental and subcutaneous AT of obese subjects. SGK1 mRNA expression in both omental and subcutaneous AT correlates with body mass index, circulating leptin and C-reactive protein, and the local expression of inflammatory markers including monocyte chemotactic protein-1 and macrophage inflammatory protein-1α. The expression of SGK1 in THP-1 cells is upregulated by inflammatory signals, such as lipopolysaccharide and tumour necrosis factor-α, as well as during the induction of monocyte-to-macrophage maturation. Our data present the first link between SGK1 and obesity-associated inflammation. SGK1 expression is stimulated in response to inflammatory signals and increased in AT macrophages. The characterisation of SGK1 functions in obesity and immunity may help identify potential therapeutic targets in the treatment of obesity.

PK modulation of haptenylated peptides via non-covalent antibody complexation

We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide–IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide–antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody–hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.

Effect of Chitosan, O-Carboxymethyl Chitosan, and N-[(2-Hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl] Chitosan Chloride on Overweight and Insulin Resistance in a Murine Diet-Induced Obesity

Two water-soluble chitosan derivatives, O-carboxymethyl chitosan (O-CM-chitosan) and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl] chitosan chloride (N-CQ-chitosan), were prepared, and the therapeutic effects of chitosan, O-CM-chitosan, and N-CQ-chitosan on insulin resistance were simultaneously evaluated by rats fed on a high-fat diet. The parameters of high-fat diet-induced rats indicated that chitosan and its two derivatives not only have low cytotoxicity but can control overnutrition by fat and achieve insulin resistance therapy. However, the results in experiment in vivo showed that the therapeutic degree varied by the molecular weight and surface charge of chitosan, O-CM-chitosan, and N-CQ-chitosan. N-CQ-chitosan with a MW of 5 × 10(4) decreased body weight, the ratio of fat to body weight, triglyceride, fasting plasma glucose, fasting plasma insulin, free fatty acid, and leptin by 11, 17, 44, 46, 44, 87, and 64% and increased fecal lipid by 95%, respectively.

Divergent metabolic outcomes arising from targeted manipulation of the gut microbiota in diet-induced obesity

ObjectiveThe gut microbiota is an environmental regulator of fat storage and adiposity. Whether the microbiota represents a realistic therapeutic target for improving metabolic health is unclear. This study explored two...

Rosiglitazone and Metformin Have Opposite Effects on Intestinal Absorption of Oligopeptides via the Proton-Dependent PepT1 Transporter

The intestinal H(+)/peptide cotransporter 1 (PepT1) plays a major role in nitrogen supply to the body by mediating intestinal absorption of di- and tripeptides. Previous studies have reported that in animal models of type 2 diabetes/obesity, PepT1 activity and expression were markedly reduced. This prompted us to investigate the effects of two antidiabetic drugs, rosiglitazone and metformin, on PepT1 activity/expression in a murine diet-induced obesity model. C57BL/6J male mice were fed a high-fat diet (HFD) or a standard chow for 6 weeks and then were treated for 7 days with metformin (250 mg/kg/day) and/or rosiglitazone (8 mg/kg/day). For in vitro studies, Caco-2 enterocyte-like cells were treated for 7 days with metformin (10 mM) and/or rosiglitazone (10 μM). A 7-day rosiglitazone treatment increased PepT1 activity and prevented the 2-fold HFD-induced reduction in PepT1 transport. Metformin alone did not modify PepT1 activity but counteracted rosiglitazone-induced PepT1-mediated transport. As with the in vivo studies, rosiglitazone treatment up-regulated PepT1 transport activity with concomitant induction of S6 ribosomal protein activation in vitro. Furthermore, metformin decreased PepT1 expression (mRNA and protein) and its transport activity. The effect of metformin was linked to a reduction of phosphorylated S6 ribosomal protein (active form) and of phosphorylated 4E-BP1 (inactive form), a translation repressor. These data demonstrate that two antidiabetic drugs exert opposite effects on the PepT1 transport function probably through direct action on enterocytes. In our type 2 diabetes/obesity model, rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist compensated for the HFD-induced PepT1 down-regulation, whereas metformin reversed rosiglitazone activity at the translational level.

Effects of chitosan, O-carboxymethyl chitosan and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl]chitosan chloride on lipid metabolism enzymes and low-density-lipoprotein receptor in a murine diet-induced obesity

O-carboxymethyl chitosan (O-CM-chitosan) and N-[(2-hydroxy-3-N,N-dimethylhexadecyl ammonium)propyl]chitosan chloride (N-CQ-chitosan) were prepared, and the therapeutic effect of them and chitosan in hepatocyte were simultaneously evaluated. The parameters of high-fat diet-induced rats in vivo indicated that chitosan and its two derivatives not only have low cytotoxicity but can alleviate the hepatic fat accumulation. Furthermore, results of the mRNA expression assay showed that chitosan of 10 kDa elevated HMG-CoA, hepatic lipase (HL), lecithin cholesterol acyltransferase (LCAT) and low-density-lipoprotein receptor (LDL-R) by 402%, 177%, 427% and 56%, and N-CQ-chitosan which was synthesized by chitosan of 50 kDa increased HMG-CoA, HL, LCAT and LDL-R by 543%, 162%, 122% and 2% respectively. It was concluded that the mRNA expression of hepatic lipid metabolism enzymes and LDL-R was positively associated with chitosan and its two derivatives, but the therapeutic degree varied by the molecular weight and surface charge of chitosan, O-CM-chitosan and N-CQ-chitosan.

Kinetic Assessment and Therapeutic Modulation of Metabolic and Inflammatory Profiles in Mice on a High-Fat and Cholesterol Diet

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/ 0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed to monitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5 mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6.