Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection.

Infection of erythrocytes with Plasmodium species induces clinical malaria. Parasite-specific CD4(+) T cells correlate with lower parasite burdens and severity of human malaria and are needed to control blood-stage infection in mice. However, the characteristics of CD4(+) T cells that determine protection or parasite persistence remain unknown. Here we show that infection of humans with Plasmodium falciparum resulted in higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction. In vivo blockade of the PD-1 ligand PD-L1 and the inhibitory receptor LAG-3 restored CD4(+) T cell function, amplified the number of follicular helper T cells and germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, and proper function can be restored by inhibitory therapies to enhance parasite control.

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer.

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.

A LAG-3-Specific Agonist Antibody for the Treatment of T Cell-Induced Autoimmune Diseases.

T cells chronically stimulated with the same peptide tend to express exhaustion markers such as PD-1 or LAG-3. Deficiencies in the PD-1 and LAG-3 pathways have been linked to the development of autoimmune diseases. IMP761 is a LAG-3-specific humanized agonist Ab with immunosuppressive properties both in vitro and in vivo in an Ag-specific delayed-type hypersensitivity (DTH) model in the cynomolgus macaque (Macaca fascicularis). IMP761 inhibits TCR-mediated NFAT activation and Ag-induced human T cell proliferation and activation. In the DTH model, assessment of T cell infiltration and gene expression profile at the DTH biopsy site corresponds to immunosuppression of an Ag-induced T cell response. IMP761 is the first LAG-3-specific agonist product candidate, acting upstream on activated T cells, the root cause of self-Ag-specific T cell-induced autoimmune diseases.

Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo.

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.

Evodiamine inhibits vasculogenic mimicry in HCT116 cells by suppressing hypoxia-inducible factor 1-alpha-mediated angiogenesis.

Evodiamine (Evo), a quinazoline alkaloid and one of the most typical polycyclic heterocycles, is mainly isolated from Evodia rugulosa. Vasculogenic mimicry (VM) is a newly identified way of angiogenesis during tumor neovascularization, which is prevalent in a variety of highly invasive tumors. The purpose of this study was to investigate the effect and mechanism of Evo on VM in human colorectal cancer (CRC) cells. The number of VM structures was calculated by the three-dimensional culture of human CRC cells. Wound-healing was used to detect the migration of HCT116 cells. Gene expression was detected by reverse transcription-quantitative PCR assay. CD31/PAS staining was used to identify VM. Western blotting and immunofluorescence were used to detect protein levels. The results showed that Evo inhibited the migration of HCT116 cells, as well as the formation of VM. Furthermore, Evo reduced the expression of hypoxia-inducible factor 1-alpha (HIF-1α), VE-cadherin, VEGF, MMP2, and MMP9. In a model of subcutaneous xenotransplantation, Evo also inhibited tumor growth and VM formation. Our study demonstrates that Evo could inhibit VM in CRC cells HCT116 and reduce the expression of HIF-1α, VE-cadherin, VEGF, MMP2, and MMP9.

Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function.

BACKGROUND: Chronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa-associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol-associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear. METHODS: To investigate the impact of alcohol on MAIT cells, mice were treated with binge-on-chronic alcohol for 10 days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol-induced MAIT cell changes: (1) Cecal microbiota from alcohol-fed (AF) mice were adoptive transferred into antibiotic-pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry. RESULTS: Binge-on-chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue-associated MAIT cells was increased in AF mice compared with pair-fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals. CONCLUSION: MAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol-associated dysbiosis. MAIT cell dysfunction may contribute to alcohol-induced innate and adaptive immunity and consequently end-organ pathophysiology.

Butyrophilin3A proteins and Vγ9Vδ2 T cell activation.

Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.

Rikkunshito attenuates induction of epithelial-mesenchymal switch via activation of Sirtuin1 in ovarian cancer cells.

Rikkunshito, a traditional Japanese herbal medicine, improves appetite via activation of gastrointestinal hormone ghrelin pathway. The function of ghrelin is mediated by growth hormone secretagogue receptor (GHSR1a), and ghrelin has been known to possess diverse physiological functions including growth suppression of some cancer cells. Considering that increased ghrelin signaling by Rikkunshito could enhance sirtuin1 (SIRT1) activity in nervous system, we aimed to investigate the effect of Rikkunshito in ovarian cancer cells. Ovarian cancer cell lines were treated with Rikkunshito, and cellular viability, gene expressions and epithelial-mesenchymal transition (EMT) status were investigated. To investigate the involvement of SIRT1 by Rikkunshito in SKOV3 cancer cells, endogenous expression of SIRT1 was depleted using small interfering RNA (siRNA). Treatment with Rikkunshito elevated ghrelin, GHSR1a and SIRT1, while cellular viability was decreased. The treatment of Rikkunshito also inhibited cellular migration and invasion status in a dose-dependent manner, and these effects were translated to the enhanced EMT status, although the role of SIRT1 was not determined. Our study revealed a novel function of Rikkunshito in enhancing EMT status of ovarian cancer cells. Therefore, we would like to propose that Rikkunshito may be used as a novel adjunctive therapy in chemotherapy of ovarian cancer because platinum-based chemotherapy frequently used for the treatment of ovarian cancer inevitably impairs appetite.

Macrophage markers soluble CD163 and soluble mannose receptor are associated with liver injury in patients with paracetamol overdose.

The macrophage activation markers, soluble CD163 (sCD163) and soluble mannose receptor (sMR), are associated with liver disease severity and prognosis. We aimed to investigate macrophage activation reflected by sMR and sCD163 in patients with mild and severe paracetamol (PCM) intoxication and effects of antidote treatment in patients and healthy controls. We measured sMR and sCD163 levels by in-house enzyme-linked immunosorbent assays in two independent prospective cohorts of PCM overdosed patients: 49 patients with early mild PCM overdose from Aarhus University Hospital and 30 patients with severe acute liver injury included at the Royal Infirmary of Edinburgh. Furthermore, we investigated sMR and sCD163 in 14 healthy controls during N-acetylcysteine treatment. Within the mild PCM cohort, patients with elevated alanine transaminase on admission had significantly higher levels of sCD163 compared with patients with normal alanine transaminase (2.92[2.00-5.75] versus 1.29[1.02-1.69] mg/L, p = .009), whereas sMR showed no significant difference. In patients with acute liver injury, both markers were markedly higher compared to the mild PCM cohort (sCD163: 10.73[5.79-14.62] versus 1.34[1.06-1.96], p < .001; sMR: 0.80[0.63-1.14] versus 0.18[0.14-0.25], p < .001). Antidote treatment significantly reduced sCD163 levels in both PCM overdosed patients and healthy controls. In conclusion, macrophage activation assessed by the levels of sMR and sCD163 is associated with the degree of liver injury in patients with PCM intoxication and is ameliorated by antidote treatment, suggesting macrophage involvement in PCM-induced liver injury.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Stress disinhibits microglia via down-regulation of CD200R: A mechanism of neuroinflammatory priming.

Exposure to stressors primes the neuroinflammatory and microglial proinflammatory response to subsequent immune challenges, suggesting that stress might attenuate immunoregulatory mechanisms in the CNS microenvironment. CD200:CD200R is a key immunoregulatory signaling dyad that constrains microglial activation, and disruption of CD200:CD200R signaling primes microglia to subsequent immune challenges. Therefore, the present study examined the mediating role of CD200:CD200R signaling in stress-induced microglial priming. Here, we found that exposure to an acute stressor reduced CD200R expression across sub-regions of the hippocampus, amygdala as well as in isolated hippocampal microglia. A transcriptional suppressor of CD200R, CAAT/Enhancer Binding Proteinß, was induced by stress and inversely associated with CD200R expression. To examine whether disrupted CD200:CD200R signaling plays a mediating role in stress-induced microglial priming, a soluble fragment of CD200 (mCD200Fc) was administered intra-cisterna magna prior to stressor exposure and stress-induced microglia priming assessed ex vivo 24 h later. Treatment with mCD200Fc blocked the stress-induced priming of the microglial pro-inflammatory response. Further, treatment with mCD200R1Fc recapitulated the effects of stress on microglial priming. We previously found that stress increases the alarmin high mobility group box-1 (HMGB1) in hippocampus, and that HMGB1 mediates stress-induced priming of microglia. Thus, we examined whether stress-induced increases in hippocampal HMGB1 are a consequence of disrupted CD200:CD200R signaling. Indeed, treatment with mCD200Fc prior to stress exposure blocked the stress-induced increase in hippocampal HMGB1. The present study suggests that stress exposure disrupts immunoregulatory mechanisms in the brain, which typically constrain the immune response of CNS innate immune cells. This attenuation of immunoregulatory mechanisms may thus permit a primed activation state of microglia to manifest.

Yangxin Tongmai Formula ameliorates impaired glucose tolerance in children with Graves' disease through upregulation of the insulin receptor levels.

Graves' disease (GD) is the leading cause of hyperthyroidism, and the majority of GD patients eventually develop disorders of glucose handling, which further affects their quality of life. Yangxin Tongmai formula (YTF) is modified from a famous formula of traditional Chinese medicine for the treatment of cardiovascular diseases. In this study we investigated the potential effects of YTF in the treatment of pediatric GD patients with impaired glucose tolerance. Forty pediatric GD patients and 20 healthy children were recruited for this clinical study. Based on the glucose tolerance, the GD patients were divided into two groups: 20 patients displayed impaired glucose tolerance, while the other 20 patients displayed normal glucose tolerance. YTF was orally administered for 60 days. YTF administration significantly ameliorated the abnormal glucose tolerance and insulin sensitivity in the GD patients with impaired glucose tolerance. To determine the molecular mechanisms of this observation, the number of plasma insulin receptors was determined by ELISA. Before treatment, the fasting and postprandial levels of the insulin receptor were significantly lower in patients with impaired glucose tolerance compared with those in patients with normal glucose tolerance and healthy children. After YTF treatment, both the fasting and the postprandial circulating insulin receptor levels were upregulated, and close to those in healthy children. Therefore, YTF is a potential effective treatment to enhance glucose handling in GD children with impaired glucose tolerance.

Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.

Overcoming the resistance of pancreatic cancer to immune checkpoint inhibitors.

Immunotherapy has become a new modality of cancer treatment, but has had a limited success in treating PDAC. A combination approach to immunotherapy, using both immune checkpoint inhibitors and immune activating agonists, is needed, as PDAC does not respond to single-agent checkpoint inhibitors. Studies have also supported using vaccine-based therapies to prime the tumor microenvironment of PDAC with effector T-cells. Other therapeutic strategies including epigenetic agents, stroma modulators, radiotherapy, and T-cell transfer therapies may also prime the tumor microenvironment to overcome resistance to immune checkpoint inhibitors.

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

Delayed vasculogenesis and impaired angiogenesis due to altered Ang-2 and VE-cadherin levels in the chick embryo model following exposure to cadmium.

PURPOSE: Cadmium (Cd) causes chick embryo malformation and abnormal extra-embryonic vasculature. This study investigates the effect of Cd on vasculogenesis, quantifies extra-embryonic vascular development following exposure to cadmium acetate (CdAc). METHODS: After 48 or 60 h incubation, chicks were explanted and treated with 50 µl of 50 µM CdAc or equimolar sodium acetate. Embryos were again incubated then re-examined 4, 8, 24 and 48 h later. Gross morphological and histological manifestations were noted. Vasculogenesis was assessed by the development of omphalomesenteric vessels from blood islands. Sinus terminalis (ST), area vasculosa (AV), vessel density and embryo crown-rump length (CRL) were measured. Ang-2 and VE-cadherin mRNA expression was analysed by RT-PCR. RESULTS: Vasculogenesis was delayed on gross and histological examination. ST length, AV area, vessel density and CRL were significantly reduced in the Cd group. Ang-2 was increased 4 h after exposure to Cd, whereas VE-cadherin was reduced. CONCLUSION: Cd exposure inhibits normal development of extra-embryonic vasculature in line with growth retardation of the chick embryo in association with altered expression of Ang-2 and VE-cadherin.

[Progress on anti-tumor molecular mechanisms of dihydroartemisinin].

Artemisinin is an anti-malarial drug with poor water solubility and oral absorption; so a variety of derivatives based on the parent nucleus have been developed. Compared with artemisinin, dihydroartemisinin (DHA) has a stronger anti-malaria activity, and has the advantages of high metabolic rate and better water solubility. Recent studies have discovered that DHA has a good inhibitory effect on tumor cells, which is closely related to the peroxide bridge in its molecular structure. Since tumor cells need more Fe3+ than normal cells, there are a large number of transferrin receptors on the tumor cell membrane. DHA can break the peroxide bridge in the presence of Fe2+, and the free radicals generated can play its lethal effect on tumor cells. In addition, DHA can promote endocytosis of transferrin receptor, and thus prevent cancer cells from taking Fe3+ from microenvironment. This article reviews the anti-tumor molecular mechanism of DHA, including accelerating oxidative damage, inducing apoptosis, inhibiting the growth, proliferation and invasion of tumor cells, reversing tumor multidrug resistance.

[Nebulized budesonide in the treatment of exacerbations of chronic obstructive pulmonary disease: Efficacy, safety, and effects on the serum levels of soluble differentiation molecules].

AIM: To investigate the efficacy and safety of nebulized budesonide and systemic glucocorticosteroids (GCS) (SGCS) in the treatment of an exacerbation of chronic obstructive pulmonary disease (COPD) and their effects on the serum concentration of soluble leukocyte differentiation antigens. MATERIALS AND METHODS: Seventy-eight hospitalized patients with an acute exacerbation of COPD were randomized into two groups: 1) 37 patients took nebulized budesonide 4 mg/day; 2) 41 patients received intravenous prednisolone. The symptoms of COPD, forced expiratory volume in one second (FEV1) and other spirometric indicators, peripheral blood oxygen saturation (SpO2), and adverse events were studied. The serum levels of the soluble adhesion molecules CD50 (sCD50) and CD54 (sCD54) and the lymphocyte activation molecules CD38 (sCD38) and CD25 (sCD25) were investigated by an enzyme immunoassay. RESULTS: There was a significant resolution of the symptoms of COPD, FEV1, and SpO2 in both groups after treatment. The incidence of hyperglycemia episodes was lower in the budesonide group than in the sGCS group. GCSs caused a decrease in the serum level of soluble interleukin-2 receptor (sCD25) in both groups. A prednisolone cycle, unlike a budesonide one, was found to reduce the concentrations of sCD54, sCD50, and sCD38. CONCLUSION: Nebulized budesonide is an effective and safe alternative to SGCS in treating an exacerbation of COPD. Inhaled GCSs, unlike SGCSs, exhibit anti-inflammatory activity, but exert no immunosuppressive activity.

CB1R antagonist increases hepatic insulin clearance in fat-fed dogs likely via upregulation of liver adiponectin receptors.

The improvement of hepatic insulin sensitivity by the cannabinoid receptor 1 (CB1R) antagonist rimonabant (RIM) has been recently been reported to be due to upregulation of adiponectin. Several studies demonstrated that improvement in insulin clearance accompanies the enhancement of hepatic insulin sensitivity. However, the effects of RIM on hepatic insulin clearance (HIC) have not been fully explored. The aim of this study was to explore the molecular mechanism(s) by which RIM affects HIC, specifically to determine whether upregulation of liver adiponectin receptors (ADRs) and other key genes regulated by adiponectin mediate the effects. To induce insulin resistance in skeletal muscle and liver, dogs were fed a hypercaloric high-fat diet (HFD) for 6 wk. Thereafter, while still maintained on a HFD, animals received RIM (HFD+RIM; n = 11) or placebo (HFD+PL; n = 9) for an additional 16 wk. HIC, calculated as the metabolic clearance rate (MCR), was estimated from the euglycemic-hyperinsulinemic clamp. The HFD+PL group showed a decrease in MCR; in contrast, the HFD+RIM group increased MCR. Consistently, the expression of genes involved in HIC, CEACAM-1 and IDE, as well as gene expression of liver ADRs, were increased in the HFD+RIM group, but not in the HFD+PL group. We also found a positive correlation between CEACAM-1 and the insulin-degrading enzyme IDE with ADRs. Interestingly, expression of liver genes regulated by adiponectin and involved in lipid oxidation were increased in the HFD+RIM group. We conclude that in fat-fed dogs RIM enhances HIC, which appears to be linked to an upregulation of the adiponectin pathway.