Advertisement

Loss of postprandial insulin clearance control by Insulin-degrading enzyme drives dysmetabolism traits

  • Author Footnotes
    1 These authors contributed equally to these work.
    Diego O. Borges
    Footnotes
    1 These authors contributed equally to these work.
    Affiliations
    Centro de Estudos de Doenças Crónicas (CEDOC), NOVA Medical School-FCM, Universidade Nova de Lisboa, Lisboa, Portugal

    Molecular Biosciences PhD Program, Instituto de Tecnologia Química e Biológica António Xavier - ITQB NOVA, Universidade Nova de Lisboa, Oeiras, Portugal
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to these work.
    Rita S. Patarrão
    Footnotes
    1 These authors contributed equally to these work.
    Affiliations
    Centro de Estudos de Doenças Crónicas (CEDOC), NOVA Medical School-FCM, Universidade Nova de Lisboa, Lisboa, Portugal

    Instituto Gulbenkian de Ciência, Oeiras, Portugal
    Search for articles by this author
  • Rogério T. Ribeiro
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal

    Departamento de Ciências Médicas, Instituto de Biomedicina - iBiMED, Universidade de Aveiro, Aveiro, Portugal
    Search for articles by this author
  • Rita Machado de Oliveira
    Affiliations
    Centro de Estudos de Doenças Crónicas (CEDOC), NOVA Medical School-FCM, Universidade Nova de Lisboa, Lisboa, Portugal
    Search for articles by this author
  • Nádia Duarte
    Affiliations
    Instituto Gulbenkian de Ciência, Oeiras, Portugal
    Search for articles by this author
  • Getachew Debas Belew
    Affiliations
    Center for Neurosciences and Cell Biology, University of Coimbra, Portugal
    Search for articles by this author
  • Madalena Martins
    Affiliations
    Instituto Gulbenkian de Ciência, Oeiras, Portugal
    Search for articles by this author
  • Rita Andrade
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • João Costa
    Affiliations
    Instituto Gulbenkian de Ciência, Oeiras, Portugal
    Search for articles by this author
  • Isabel Correia
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • José Manuel Boavida
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • Rui Duarte
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • Luís Gardete-Correia
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • José Luís Medina
    Affiliations
    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal
    Search for articles by this author
  • João F. Raposo
    Affiliations
    Centro de Estudos de Doenças Crónicas (CEDOC), NOVA Medical School-FCM, Universidade Nova de Lisboa, Lisboa, Portugal

    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • John G. Jones
    Affiliations
    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal

    Center for Neurosciences and Cell Biology, University of Coimbra, Portugal
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to these work.
    Carlos Penha-Gonçalves
    Footnotes
    1 These authors contributed equally to these work.
    Affiliations
    Instituto Gulbenkian de Ciência, Oeiras, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to these work.
    M. Paula Macedo
    Correspondence
    Corresponding author at: CEDOC, NOVA Medical School (NMS/FCM), Edifício CEDOC II, Rua Câmara Pestana, N° 6, 1150-082 Lisboa, Portugal.
    Footnotes
    1 These authors contributed equally to these work.
    Affiliations
    Centro de Estudos de Doenças Crónicas (CEDOC), NOVA Medical School-FCM, Universidade Nova de Lisboa, Lisboa, Portugal

    Sociedade Portuguesa de Diabetologia, Lisboa, Portugal

    APDP-Diabetes Portugal Education and Research Center (APDP-ERC), Lisboa, Portugal

    Departamento de Ciências Médicas, Instituto de Biomedicina - iBiMED, Universidade de Aveiro, Aveiro, Portugal
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to these work.
Published:February 22, 2021DOI:https://doi.org/10.1016/j.metabol.2021.154735

      Highlights

      • IDE polymorphisms strongly associate with postprandial IC in NGT men.
      • IDE polymorphisms association with IC is weaken in women and prediabetes.
      • Liver-specific IDE KO display reduced postprandial IC.
      • Liver-specific IDE KO display glucose intolerance correlated with reduced glut2.
      • Liver-specific IDE KO exacerbates hepatic steatosis induced by high fat diet.

      Abstract

      Systemic insulin availability is determined by a balance between beta-cell secretion capacity and insulin clearance (IC). Insulin-degrading enzyme (IDE) is involved in the intracellular mechanisms underlying IC. The liver is a major player in IC control yet the role of hepatic IDE in glucose and lipid homeostasis remains unexplored.
      We hypothesized that IDE governs postprandial IC and hepatic IDE dysfunction amplifies dysmetabolic responses and prediabetes traits such as hepatic steatosis.
      In a European/Portuguese population-based cohort, IDE SNPs were strongly associated with postprandial IC in normoglycemic men but to a considerably lesser extent in women or in subjects with prediabetes. Liver-specific knockout-mice (LS-IDE KO) under normal chow diet (NCD), showed reduced postprandial IC with glucose intolerance and under high fat diet (HFD) were more susceptible to hepatic steatosis than control mice. This suggests that regulation of IC by IDE contributes to liver metabolic resilience. In agreement, LS-IDE KO hepatocytes revealed reduction of Glut2 expression levels with consequent impairment of glucose uptake and upregulation of CD36, a major hepatic free fatty acid transporter.
      Together these findings provide strong evidence that dysfunctional IC due to abnormal IDE regulation directly impairs postprandial hepatic glucose disposal and increases susceptibility to dysmetabolic conditions in the setting of Western diet/lifestyle.

      Graphical abstract

      Keywords

      1. Introduction

      Development of type 2 diabetes and comorbidities, such as metabolic associated fatty liver disease (MAFLD), is propelled by interactions of genetic and environmental factors [
      • Eslam M.
      • Newsome P.N.
      • Sarin S.K.
      • Anstee Q.M.
      • Targher G.
      • Romero-Gomez M.
      • et al.
      A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.
      ]. While type 2 diabetes genetic association studies identified many risk loci for diabetes-related traits, the functional validation of such candidates is still ongoing. Polymorphisms in the insulin-degrading enzyme (IDE) genomic region were associated with increased risk of type 2 diabetes development [
      • Groves C.J.
      • Wiltshire S.
      • Smedley D.
      • Owen K.R.
      • Frayling T.M.
      • Walker M.
      • et al.
      Association and haplotype analysis of the insulin-degrading enzyme (IDE) gene, a strong positional and biological candidate for type 2 diabetes susceptibility.
      ,
      • Karamohamed S.
      • Demissie S.
      • Volcjak J.
      • Liu C.
      • Heard-Costa N.
      • Liu J.
      • et al.
      Polymorphisms in the insulin-degrading enzyme gene are associated with type 2 diabetes in men from the NHLBI Framingham Heart Study.
      ,
      • Kwak S.H.
      • Cho Y.M.
      • Moon M.K.
      • Kim J.H.
      • Park B.L.
      • Cheong H.S.
      • et al.
      Association of polymorphisms in the insulin-degrading enzyme gene with type 2 diabetes in the Korean population.
      ,
      • Grarup N.
      • Rose C.S.
      • Andersson E.A.
      • Andersen G.
      • Nielsen A.L.
      • Albrechtsen A.
      • et al.
      Studies of association of variants near the HHEX, CDKN2A/B, and IGF2BP2 genes with type 2 diabetes and impaired insulin release in 10,705 Danish subjects: validation and extension of genome-wide association studies.
      ,
      • Dimas A.S.
      • Lagou V.
      • Barker A.
      • Knowles J.W.
      • Magi R.
      • Hivert M.F.
      • et al.
      Impact of type 2 diabetes susceptibility variants on quantitative glycemic traits reveals mechanistic heterogeneity.
      ]. IDE is the main protease to degrade insulin and is therefore a key element in the control of systemic insulin levels, via insulin clearance (IC) [
      • Farris W.
      • Mansourian S.
      • Chang Y.
      • Lindsley L.
      • Eckman E.A.
      • Frosch M.P.
      • et al.
      Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.
      ].
      The magnitude of IC varies across human populations [
      • Pina A.F.
      • Patarrao R.S.
      • Ribeiro R.T.
      • Penha-Goncalves C.
      • Raposo J.F.
      • Gardete-Correia L.
      • et al.
      Metabolic footprint, towards understanding type 2 diabetes beyond glycemia.
      ]. It was shown to be reduced in some ethnic groups such as African-Americans [
      • Osei K.
      • Schuster D.P.
      Ethnic differences in secretion, sensitivity, and hepatic extraction of insulin in black and white Americans.
      ] and Hispanics [
      • Haffner S.M.
      • Stern M.P.
      • Watanabe R.M.
      • Bergman R.N.
      Relationship of insulin clearance and secretion to insulin sensitivity in non-diabetic Mexican Americans.
      ] that coincidently have higher genetic predisposition for insulin resistance and type 2 diabetes [
      • Lee C.C.
      • Haffner S.M.
      • Wagenknecht L.E.
      • Lorenzo C.
      • Norris J.M.
      • Bergman R.N.
      • et al.
      Insulin clearance and the incidence of type 2 diabetes in Hispanics and African Americans: the IRAS Family Study.
      ]. Remarkably, when hepatic versus extrahepatic IC were evaluated in African-American and European-American women, hepatic IC was already lower even in youth for the African-Americans [
      • Piccinini F.
      • Polidori D.C.
      • Gower B.A.
      • Bergman R.N.
      Hepatic but not extrahepatic insulin clearance is lower in African American than in European American women.
      ,
      • Piccinini F.
      • Polidori D.C.
      • Gower B.A.
      • Fernandez J.R.
      • Bergman R.N.
      Dissection of hepatic versus extra-hepatic insulin clearance: ethnic differences in childhood.
      ]. Raising the question of whether IDE genetic variance impacts insulin homeostasis and particularly hepatic IC in the postprandial phase.
      Beyond genetic regulation, IDE activity can also be directly modified by both metabolites and pharmacological agents [
      • Cordes C.M.
      • Bennett R.G.
      • Siford G.L.
      • Hamel F.G.
      Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation.
      ,
      • Pivovarova O.
      • Gogebakan O.
      • Pfeiffer A.F.
      • Rudovich N.
      Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells.
      ,
      • Wei X.
      • Ke B.
      • Zhao Z.
      • Ye X.
      • Gao Z.
      • Ye J.
      Regulation of insulin degrading enzyme activity by obesity-associated factors and pioglitazone in liver of diet-induced obese mice.
      ,
      • Martins F.O.
      • Delgado T.C.
      • Viegas J.
      • Gaspar J.M.
      • Scott D.K.
      • O’Doherty R.M.
      • et al.
      Mechanisms by which the thiazolidinedione troglitazone protects against sucrose-induced hepatic fat accumulation and hyperinsulinaemia.
      ]. Several studies in mice and humans showed that IDE function is important for both insulin secretion and degradation [
      • Wei X.
      • Ke B.
      • Zhao Z.
      • Ye X.
      • Gao Z.
      • Ye J.
      Regulation of insulin degrading enzyme activity by obesity-associated factors and pioglitazone in liver of diet-induced obese mice.
      ,
      • Martins F.O.
      • Delgado T.C.
      • Viegas J.
      • Gaspar J.M.
      • Scott D.K.
      • O’Doherty R.M.
      • et al.
      Mechanisms by which the thiazolidinedione troglitazone protects against sucrose-induced hepatic fat accumulation and hyperinsulinaemia.
      ,
      • Fosam A.
      • Sikder S.
      • Abel B.S.
      • Tella S.H.
      • Walter M.F.
      • Mari A.
      • et al.
      Reduced insulin clearance and insulin-degrading enzyme activity contribute to hyperinsulinemia in African Americans.
      ,
      • Steneberg P.
      • Bernardo L.
      • Edfalk S.
      • Lundberg L.
      • Backlund F.
      • Ostenson C.G.
      • et al.
      The type 2 diabetes-associated gene ide is required for insulin secretion and suppression of alpha-synuclein levels in beta-cells.
      ]. Recently, liver IDE activity, but not protein expression, was associated with reduced IC when comparing Afro-Americans with non-Hispanic whites [
      • Fosam A.
      • Sikder S.
      • Abel B.S.
      • Tella S.H.
      • Walter M.F.
      • Mari A.
      • et al.
      Reduced insulin clearance and insulin-degrading enzyme activity contribute to hyperinsulinemia in African Americans.
      ]. The liver is indeed the main site of IC, and while the role of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in insulin receptor isoform A internalization is well established, that of IDE is less well characterized [
      • Poy M.N.
      • Yang Y.
      • Rezaei K.
      • Fernstrom M.A.
      • Lee A.D.
      • Kido Y.
      • et al.
      CEACAM1 regulates insulin clearance in liver.
      ,
      • Najjar S.M.
      • Perdomo G.
      Hepatic insulin clearance: mechanism and physiology.
      ]. In vitro studies showed that IDE activity is downregulated in hyperglycemia, however there is lack of in vivo studies exploring the role of IDE in hepatic-IC. Indeed, animals with global knockout (KO) of IDE presented clinical features of prediabetes such as reduced insulin degradation, hyperinsulinemia and glucose intolerance [
      • Farris W.
      • Mansourian S.
      • Chang Y.
      • Lindsley L.
      • Eckman E.A.
      • Frosch M.P.
      • et al.
      Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.
      ,
      • Abdul-Hay S.O.
      • Kang D.
      • McBride M.
      • Li L.
      • Zhao J.
      • Leissring M.A.
      Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance.
      ]. Nevertheless, the molecular mechanisms that explain IDE involvement remain speculative.
      To uncover IDE role in the regulation of postprandial insulin levels, we investigated Ide genomic region aiming to unveil IDE association with postprandial IC. Furthermore, we tested the hypothesis that IDE controls postprandial IC, and that hepatic IDE dysfunction caused by either, genetically and/or dietary factors, is associated with impairment of glucose and lipid homeostasis.

      2. Materials and methods

      2.1 Human studies

      2.1.1 Ethics statement

      All subjects were volunteers and provided written informed consent for participation in this study. Ethics Committee of Associação Protectora dos Diabéticos de Portugal (APDP) and from the Instituto Gulbenkian de Ciência (IGC) gave permition to conduct this study. The study protocol adhered to the Declaration of Helsinki and was approved by the Autoridade Nacional de Protecção de Dados (permit nr.3228/2013).

      2.1.2 Subjects

      The study population comprises the participants of a diabetes prevalence study performed in Portugal (PREVADIAB2), as a follow-up of PREVADIAB1 [
      • Pina A.F.
      • Patarrao R.S.
      • Ribeiro R.T.
      • Penha-Goncalves C.
      • Raposo J.F.
      • Gardete-Correia L.
      • et al.
      Metabolic footprint, towards understanding type 2 diabetes beyond glycemia.
      ,
      • Gardete-Correia L.
      • Boavida J.M.
      • Raposo J.F.
      • Mesquita A.C.
      • Fona C.
      • Carvalho R.
      • et al.
      First diabetes prevalence study in Portugal: PREVADIAB study.
      ]. All subjects with prediabetes or normal glucose metabolism were contacted in PREVADIAB1, in a selection of sampling sites to maintain national representativeness. For each participant medical history was assessed, BMI was recorded and routine blood tests and oral glucose tolerance test (OGTT) were performed.

      2.1.3 Inclusion criteria

      The diabetes status of each participant was determined using the WHO criteria for increased risk of diabetes and prediabetes (WHO 2016, Global Report on Diabetes). Participants fulfilling the criteria for diabetes were excluded from this study. Participants fulfilling the criteria for impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) were classified as prediabetic subjects (n = 233). The remaining subjects were classified as normal glucose tolerant (NGT, n = 736, Supplementary Fig. 1).

      2.1.4 Biochemical measurements

      Participants underwent a standardized 75 g OGTT. To determine plasma glucose, insulin and C-peptide, venous blood (12 h fasting) was drawn at baseline (0 min), 30 and 120 min of the OGTT. For biochemical and genetic analysis, the ratio between C-peptide area under the curve (AUC) and insulin AUC along the OGTT estimates IC AUC. The AUC during the OGTT was estimated according to the trapezoid method. Missing phenotypic data was <99%. Individuals with missing data were not included. NGT and prediabetic subjects were compared using Mann-Whitney test in GraphPad Prism 7.0. Results were listed as mean with the 95% confidence interval (CI) or represented in violin plots with probability density, median, interquartile range and 95% CI.

      2.1.5 Genotyping

      Genomic DNA was extracted from whole blood (Chemagen Magnetic Beads) and quantified using PicoGreen reagents (Invitrogen). A total of 46 single nucleotide polymorphisms (SNPs) covering the IDE region in chromosome 10 (92.45–92.71 Mb) were genotyped using the Sequenom iPlex assay (San Diego, USA) and the Sequenom MassArray K2 platform at the Genomics Unit of the Instituto Gulbenkian de Ciência. Genotyping calling quality was controlled using two samples with known genotypes. Genotype calling were performed blinded to affection status. SNPs deviating from Hardy-Weinberg equilibrium (HWE) were excluded (P < 0.05). Final dataset under analysis comprised 969 subjects with non-missing genotyping rate above 95%, genotyped for 36 SNPs that passed the exclusion criteria (minor allele frequency < 8% or call rate < 99%) (Supplementary Table 1). Linkage disequilibrium (LD) map was generated by Haploview 4.2 software (Supplementary Fig. 2).

      2.1.6 Genetic association analysis

      Quantitative trait association analysis was performed with PLINK software package; nominal P-values for 36 IDE SNPs were obtained for allelic and genotypic association under the additive model using age and BMI (Body Mass Index) as covariates. Empirical P values were obtained with max(T) permutation procedure implemented in PLINK package with 1,000,000 label permutations. Since the tested metabolic traits (IC AUC 0–120 min) are not independent no correction was applied. Data storage and interfacing with PLINK package used the BC|Gene platform (version 3.6–036).

      2.2 Mouse studies

      2.2.1 Animals generation

      Animals were ordered from European Mouse Mutant Archive (EMMA) ID: 05902. Mice were in a C57Bl/6 N background presenting in one of Ide alleles a Frt-flanked cassette blocking the expression of the gene. Animals were housed at the rodent animal facility from Instituto Gulbenkian de Ciência (Oeiras, Portugal). After generation of animals presenting both alleles with the cassette (Full IDE KO), further backcross with a Flippase knock in driver (The Jackson Laboratory stock: 009086) restored the function of the enzyme, leaving exon 3 flanked with lox-P (floxed) sites. Finally, an Albumin-Cre driver (The Jackson Laboratory stock: 003574) was used for deletion of exon 3 only in hepatocytes. Animals used for phenotypic characterization were all generated by a breeding pair where Ide allele was floxed and just one breeder presented Albumin-Cre+/+, generating LS-IDE KO (Idefloxed|Albuminn-Cre+) and littermates' controls (Idefloxed|Abumin-Cre). All procedures followed ARRIVE guidelines and the European laws (Directive, 2010/63/EU) that rule the use of animals in research. Animals were genotyped to confirm liver specific IDE knockout (LS-IDE KO) 4 weeks after weaning by DNA extraction from the tip of the tail.

      2.2.2 Diet protocol

      Animals were maintained under NCD (SDS Standard-RM3A) or HFD (58Kcal% Lipid - Research Diets D12331) from 6 to 18 weeks old. Blood glucose and weight was measured weekly and at the end of the protocol an OGTT was performed as previously described [
      • Borges D.O.
      • Meneses M.J.
      • Dias T.R.
      • Martins F.O.
      • Oliveira P.F.
      • Alves M.G.
      • et al.
      Data on metabolic profile of insulin-degrading enzyme knockout mice.
      ].

      2.2.3 Biochemical measurements

      Glucose was measured from venous tail blood with a glucometer (Bayer-Contour-Next-Portugal). Plasma was obtained and stored at −80 °C. Plasma C-peptide and insulin levels were measured with Mouse C-peptide ELISA and ultra-sensitive mouse Insulin ELISA kit respectively (Crystal Chem, USA). Plasma triglycerides and total cholesterol levels were measured with enzymatic-colorimetric assays (Spinreact, Spain). Non-esterified free fatty acids were measured according to manufacturer instructions (Wako, Fujifilm-Japan).

      2.2.4 Postprandial IC and liver IDE activity

      C-peptide and insulin AUC during the OGTT were calculated for estimation of postprandial IC. AUC was calculated between OGTT timepoints with the trapezoid method. Insulin-stimulated glucose clearance was estimated by glucose AUC(0120min) to insulin AUC(0120min). Liver IDE activity was measured with SensoLyte® 520 IDE activity assay kit (AnaSpec-USA).

      2.2.5 Liver steatosis and lipid content

      Liver steatosis was evaluated by a pathologist in formalin-fixed paraffin-embedded liver sections after hematoxylin-eosin staining. Lipid extraction was performed in liver samples based on methyl-tert-butyl ether protocol as described [
      • Silva J.C.P.
      • Marques C.
      • Martins F.O.
      • Viegas I.
      • Tavares L.
      • Macedo M.P.
      • et al.
      Determining contributions of exogenous glucose and fructose to de novo fatty acid and glycerol synthesis in liver and adipose tissue.
      ]. Triglycerides and total cholesterol levels were measured, after lipid extraction, as described for plasma and normalized by liver weight.

      2.2.6 Gene expression analysis

      Liver, skeletal muscle, epidydimal white adipose tissue and brown adipose tissue RNA was extracted with Trizol (Thermo Fisher Scientific-USA). Real time PCR was performed using SYBR green master mix [
      • Schmittgen T.D.
      • Livak K.J.
      Analyzing real-time PCR data by the comparative C(T) method.
      ] (Supplementary Table 2).

      2.2.7 Western blot analysis

      Liver samples were homogenized with sonication in presence of lysis buffer plus protease inhibitors cocktail. Protein quantification was done with Pierce™ BCA Protein assay (Thermo Fisher Scientific-USA). Protein was loaded on SDS-PAGE gels and transferred to PVDF membranes. ECL Prime western blotting detection was used in a ChemiDoc imaging system (BioRad-USA) (Supplementary Table 3).

      2.2.8 Primary hepatocytes isolation

      Primary hepatocytes were isolated from LS-IDE KO and controls at 10–12 weeks old. The isolation protocol was adapted from Zhande et al. [
      • Zhande R.
      • Zhang W.
      • Zheng Y.
      • Pendleton E.
      • Li Y.
      • Polakiewicz R.D.
      • et al.
      Dephosphorylation by default, a potential mechanism for regulation of insulin receptor substrate-1/2, Akt, and ERK1/2.
      ].

      2.2.9 Glucose uptake assay

      Hepatocytes from LS-IDE KO and control animals after 3 h starvation in glucose-free DMEM were incubated in presence or absence of 20 uM of a fluorescent glucose analogue (2-NBDG: 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglycose) for 1 h. Dye uptake was stopped by ice-cold PBS, and cells were resuspended in cold HBSS without phenol red. Fluorescence was measured with a VICTOR3 plate reader. An MTT assay was performed as previously described [
      • Mosmann T.
      Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
      ] and 2-NBDG fluorescence was normalize by viability.

      2.2.10 Statistical analysis

      The t-test was used for two groups comparison and One-way ANOVA for more than two groups comparison with Tukey post-test.

      3. Results

      3.1 IDE genetic variation significantly controls postprandial IC in humans

      We studied the genetic control of postprandial IC by the IDE gene in a population-based cohort of 969 Portuguese subjects that took an OGTT mirroring the absorptive state (Supplementary Fig. 1). The OGTT revealed that 736 subjects were NGT while 233 showed abnormal post-glycemic control and fell in the prediabetes spectrum showing increase glucose, insulin and C-peptide (Fig. 1A–C ; Table 1) and reduced postprandial IC during OGTT (Fig. 1D). Using 36 SNPs covering the IDE gene region (Supplementary Table 1 and Supplementary Fig. 2) we found that postprandial IC quantitatively associated with alleles of several SNPs within the IDE gene (Fig. 1E) with a peak in rs4646957 (P = 3.47 × 10−5). Although the SNPs with highest association to postprandial IC were not in strong linkage disequilibrium (Supplementary Fig. 2) a linked genetic effect is not excluded. When comparing males and females, we found IDE SNPs strongly controlled postprandial IC (0–120 min) in NGT males (rs35831196, P = 8.82 × 10−5) but had clear lower control in NGT females as measured by allelic association (Fig. 1F and G) or under the genotypic additive model (Supplementary Table 1). This uncovered that control of postprandial IC by IDE is under a strong gender effect.
      Fig. 1
      Fig. 1Insulin clearance area under the curve (AUC) during the Oral Glucose Tolerance Test (OGTT) and quantitative trait locus (QTL) analysis for SNPs in the IDE gene region in normoglycemic (NGT) and prediabetic subjects. The area under the curve (AUC) of glucose (A), insulin (B), C-peptide (C) and insulin clearance 0–120 min (D) in response to the OGTT was calculated for each subject that completed the OGTT. The scatter dot plot represents the distributions in 736 NGT (white circles) and 233 prediabetic (black circles) subjects. Plot of quantitative trait locus (QTL) analysis for 36 SNPs in the IDE gene region, testing for association with insulin clearance AUC 0–120 min (E). Separated analyses are shown for 736 normoglycemic (NGT, white circles) and 233 prediabetic (black circles) subjects. Results represent the nominal -log10 (P-value) for the additive model adjusted for age and BMI. SNPs position in Chromosome 10 is represented in Megabases. *P<0.05, unpaired t-test Mann-Whitney. Insulin clearance in response to OGTT is controlled by the IDE gene region in normoglycemic males, but not in females nor in prediabetic individuals (males and females). Plots of quantitative trait locus (QTL) analysis for 36 SNPs in the IDE gene region, testing for association with insulin clearance AUC 0–120 min in NGT males and females (F) and insulin clearance AUC 0–120 min in prediabetic males and females (G). Separated analyses for insulin clearance AUC 0–120 min are shown for 292 normoglycemic males (NGT males, blue open squares), 444 normoglycemic females (NGT females, orange open triangles), 93 prediabetic males (Prediabetes males, blue close squares) and 140 prediabetic females (Prediabetes females, orange close triangles) subjects. Results represent the nominal -log10 (P-value) for the additive model adjusted for age and BMI. SNPs position in Chromosome 10 is represented in Megabases. Violin plots represent genotypic effects of rs4646957 (H), rs35831196 (I) and rs11187025 (J) on insulin clearance AUC 0–120 min during OGTT in normoglycemic males and prediabetic males subjects (light blue, homozygotes NGT males' allele; dark blue, homozygotes Prediabetes males' allele). Unpaired t-test Mann-Whitney (**P < 0.01, *P < 0.05). The plots represent the probability density, the median, the interquartile range and the 95% confidence interval of the phenotype distributions per genotype class in 292 NGT males and 93 Prediabetes males' subjects.
      Table 1Glucose and insulin metabolism parameters of NGT and prediabetes subjects in the PREVADIAB2 cohort.
      NGTPrediabetes
      n736233
      Gender M/F (%)292/444 (39.7/60.3)93/140 (39.9/60.1)
      Age (years)58.5±13.3
      P < 0.1E-04.
      65.0±10.5
      P < 0.1E-04.
      Fasting glucose (mg/dl)89.1±8.9
      P < 0.1E-04.
      99.7±12.7
      P < 0.1E-04.
      2 h-OGTT glucose (mg/dl)99.1±21.8
      P < 0.1E-04.
      150.6±25.9
      P < 0.1E-04.
      Glucose AUC 0–120 min14,544±2147
      P < 0.1E-04.
      18,762±1960
      P < 0.1E-04.
      Insulin AUC 0–120 min2522±2016
      P < 0.1E-04.
      5101±3790
      P < 0.1E-04.
      C-peptide AUC 0–120 min578.4±238.1
      P < 0.1E-04.
      826.8±297
      P < 0.1E-04.
      Insulin Clearance AUC 0–120 min0.1799±0.063
      P < 0.01.
      0.1670±0.060
      P < 0.01.
      Mann-Whitney test. Values are mean ± SD.
      a P < 0.1E-04.
      b P < 0.01.
      Strikingly, the strong association observed in NGT males was flattened in subjects with prediabetes, suggesting that hyperglycemic states alter the genetic control of IC by IDE (Fig. 1F and G). Minor alleles of rs4646957, rs35831196 and rs11187025 were found to increase postprandial IC in NGT males (Supplementary Table 4) but in prediabetic individuals these alleles showed to confer lower postprandial IC (Supplementary Table 5) (Fig. 1H–J). Together these data indicate that IDE genetically governs postprandial IC mainly in normoglycemic males and suggest that hyperglycemic states abrogate this mechanism of adaptation to the postprandial state.

      3.2 Hepatic IDE deletion impairs postprandial insulin and glucose clearance in lean mice

      As postprandial IC is highly determined by the hepatic capacity to extract insulin from portal vein blood, we evaluated IC and its consequences in a LS-IDE KO mouse. Because in human studies, IDE allelic association was stronger in males, we used male mice. Hepatic Ide mRNA levels in IDE-LS KO mice were decreased by ~90% compared to control; no alterations in other organs were detected (Supplementary Fig. 3A). A residual presence of IDE protein was observed in hepatic tissue of LS-IDE KO mice (Fig. 2A ), explained by the Albumin-Cre driver and the consequent expression of IDE in non-hepatocyte cells. However, in isolated primary hepatocytes, there was absence of IDE protein expression (Fig. 2B). While LS-IDE depleted animals experienced higher levels of insulin along OGTT (Fig. 2D); C-peptide levels, to estimate insulin secretion, were not altered. The increment in peripheral insulinemia in the face of unmodified insulin secretion in the LS-IDE KO animals is explained by impaired IC (Fig. 2E). Additionally, using the ratio of glucose AUC(0120min) to insulin AUC(0120min), an estimative of overall insulin-stimulated glucose clearance, we observed that LS-IDE KO present an impairment in glucose clearance (Fig. 2F).
      Fig. 2
      Fig. 2Characterization of control and liver-specific (LS)-IDE knockout (KO) littermates' mouse. A – Comparison of liver IDE protein expression between genotypes and diet regimens. B – Comparison of IDE protein levels in primary hepatocytes isolated from controls and LS-IDE KO. C – Comparison of liver IDE activity between genotypes and diet regiments. D – Insulin and C-peptide excursions during an oral glucose tolerance test (OGTT) in controls and LS-IDE KO littermates under NCD. E – Estimation of postprandial hepatic insulin clearance calculated by the ratio of the area under the curve (AUC) from c-peptide and insulin excursions during the OGTT for controls and LS-IDE KO littermates under NCD. F – Estimation of postprandial glucose clearance capacity with the ratio of AUC from glucose excursions to AUC from insulin excursions during OGTT for controls and LS-IDE KO under NCD. G - Insulin and C-peptide excursions during an oral glucose tolerance test (OGTT) in controls and LS-IDE KO littermates under HFD. H – Estimation of postprandial hepatic insulin clearance calculated by the ratio of the area under the curve (AUC) from c-peptide and insulin excursions during the OGTT for NCD LS-IDE KO and control mice. I – Estimation of postprandial glucose clearance capacity with the ratio of AUC from glucose excursions to AUC from insulin excursions during OGTT for controls and LS-IDE KO under HFD. *t-Test used for two groups comparison and One-way ANOVA used for more than two groups comparison. Statistical results between NCD and HFD LS-IDE KO not shown due to the lack of same genotype or diet regimen.

      3.3 Diet-induced obesity impairs postprandial IC through deterioration of IDE function

      Hypercaloric diets such as high-fat diets (HFD) are widely used in animal models of diabetes onset, and effectively recapitulate prediabetes. To evaluate diet-induced obesity on genetically impaired IC background, we studied LS-IDE KO under HFD. As observed in lean LS-IDE KO, there was a reduction of Ide mRNA and protein was observed under HFD (Supplementary Figs. 3A and 2A). Moreover, a sizable decrease of IDE activity, attributable to HFD, was also observed in LS-IDE KO mice. Interestingly, IDE enzyme activity in control mice under HFD was substantially decreased, compared to NCD (Fig. 2C). Yet, no differences in insulin and C-peptide excursions were detected (Fig. 2G). Thus, postprandial IC was unchanged while glucose clearance was not significantly different (Fig. 2H–I). We propose that HFD-driven reduction of liver IDE activity, as exemplified by the data from control animals in Fig. 2C (gray bars, NCD vs HFD), masked the effects of LS-IDE KO per se. Given that HFD caused a reduction in IDE activity comparable to the one observed in LS-IDE KO mice fed with NCD, the greater insulin excursions observed for HFD over NCD mice is explained by two effects. First, an undoubted increase in glucose-stimulated insulin secretion, as evidenced by elevated C-peptide levels (Fig. 2G). Second, and no less important, the 50% reduction in postprandial IC (Fig. 2H). HFD feeding also resulted in impaired glucose clearance, reinforcing the importance of functional hepatic IDE and postprandial IC in glucose homeostasis (Fig. 2I).

      3.4 LS-IDE KO increases glucose excursions and impairs hepatic glucose uptake

      NCD LS-IDE KO presented impaired glucose tolerance when compared to NCD controls (Fig. 3A ). As expected, control animals under HFD presented an increment in glucose AUC. However, in LS-IDE KO mice fed HFD, a pronounced increment in glucose excursions above and beyond that of the same mice fed on NCD was observed, suggesting a synergistic effect of HFD and IDE genetic deletion (Fig. 3A). Having in mind that liver can account for a substantial glucose consumption in postprandial state, we sought to determine if IDE deletion can impact hepatic glucose uptake, contributing to the observed glucose intolerance. We evaluated the main glucose transporter in the liver, GLUT2, and observed a blunted expression of its mRNA in both NCD and HFD fed LS-IDE KO, when compared to NCD control (Fig. 3B). Concomitantly, a reduction in Glut2 protein levels was observed (Supplementary Fig. 3B). Nevertheless, this down-regulation of Glut2 is unlikely to fully explain the observed substantial increment in glucose excursions in HFD LS-IDE KO. In any case, net hepatic glucose balance is controlled via reciprocal regulation of glucokinase (GCK) and glucose-6-phosphatase (G6Pase) actions with GCK being the rate-limiting for net hepatic glucose uptake. Interestingly, we observed that GCK (Gck) gene expression in NCD of controls vs LS-IDE KO upon a t-test (p = 0,024) resulting in a decrease of 66,4%. Consistently, the GCK in HFD LS-IDE KO was significantly reduced when compared to HFD control (p = 0,013) corresponding to a percentage decrease of 58,8% (Fig. 3D). The decreased GCK actions and glucose uptake by the loss of liver IDE activity even without any diet regimen is consistent with the observed glucose intolerance. Under HFD, hyperinsulinemia increases GCK levels as observed in control animals resulting in an additional divergence in GCK capacity from that of LS-IDE KO. G6Pase (G6P) and phosphoenolpyruvate carboxylase mRNA expression were unchanged (Fig. 3C and E) indicating no effects of liver IDE deletion on gluconeogenesis and net glucose production. Finally, L-pyruvate kinase mRNA levels were higher in LS-IDE KO fed HFD compared to their NCD-fed littermates while no such differences were seen for HFD and NCD-control mice (Fig. 3F). This pattern was also observed for the levels of carbohydrate response element-binding protein (ChREBP), a master regulator of L-pyruvate kinase (Supplementary Fig. 3C).
      Fig. 3
      Fig. 3Evaluation of glucose tolerance and liver glucose metabolism in controls and liver-specific (LS)-IDE KO littermates' mouse. A – Glucose excursions during an oral glucose tolerance test (OGTT – left panel) and respective area under the curve (AUC – right panel) calculated from glucose excursions under normal chow diet (NCD) and high fat diet (HFD). B – Comparison of Glut2 mRNA expression between genotypes and diet regimens. C – Comparison of G6PC (glucose 6-phosphatase) mRNA expression between genotypes and diet regimens. D – Comparison of Gck (glucokinase) mRNA expression between genotypes and diet regimens. E – Comparison of PEPCK (Phosphoenolpyruvate caboxykinase) mRNA expression between genotypes and diet regimens. F – Comparison of L-PK (Liver-pyruvate kinase) mRNA expression between genotypes and diet regimens G – Glut2, IDE and calnexin protein levels in primary hepatocytes isolated from control and LS-IDE KO littermates (left panel) and fluorescence of a glucose analog (2-NBDG) reflecting glucose internalization in primary hepatocytes from control and LS-IDE KO mice (right panel). *t-Test used for two groups comparison and One-way ANOVA used for more than two groups. Statistical results between NCD and HFD LS-IDE KO not shown due to the lack of same genotype or diet regimen.
      To confirm if net hepatic glucose uptake was impaired, we isolated hepatocytes from control and LS-IDE KO animals (Fig. 3G). Primary hepatocytes replicated the downregulation of Glut2 protein levels (Fig. 3G-left and middle panel). Moreover, we observed that hepatocytes from LS-IDE KO mice showed a significant reduction (37%) in glucose internalization (Fig. 3G-right panel). These results suggest that higher insulin levels might result in a desensitization of the insulin-Glut2 axis - via downregulation of Glut2 expression. The impaired hepatic glucose uptake observed in the LS-IDE KO can be explained by the decreased Glut2 with the cooperation of the reduced GCK mRNA contributing to an exacerbated glucose intolerance as observed in HFD LS-IDE KO.

      3.5 LS-IDE deletion increases lipid deposition through CD36 up-regulation

      Given that a reduction in hepatic glucose uptake might have an impact on uptake and utilization of lipid substrates, we looked at the impact of IDE deletion on lipid metabolism. As expected, serum triglycerides and cholesterol were significantly higher in HFD controls when compared to NCD controls (Fig. 4A and B ). In response to IDE deletion, HFD LS-IDE KO, and a combination of both, liver triglyceride levels increased in a stepwise fashion (Fig. 4D). Liver triglycerides were significantly higher in HFD LS-IDE KO compared to both their NCD littermates and HFD controls. Serum and hepatic cholesterol levels were less responsive with the only significant difference being an increase in serum levels for HFD-control compared to NCD-control mice (Fig. 4B and E). HFD promoted the presence of microsteatosis in control animals. Importantly, for the LS-IDE KO mice fed HFD, hepatic lipid droplets were markedly larger in size, when compared to their HFD-control littermates (Fig. 4C).
      Fig. 4
      Fig. 4Evaluation of lipid metabolism in control and liver-specific (LS)-IDE KO littermates' mouse. A – Comparison of serum triglycerides levels between genotypes and diet regimens. B – Comparison of serum total cholesterol between genotypes and diet regimens. C – Hematoxylin and eosin staining of formalin-fixed paraffin-embedded liver slices from controls and LS-IDE KO under normal chow diet (NCD) and high fat diet (HFD). D – Comparison of liver triglycerides content between genotypes and diet regimens. E – Comparison of liver total cholesterol content between genotypes and diet regimens. F – Rate of free fatty acid synthesis by in vivo measurement of de novo lipogenesis in livers of controls and LS-IDE KO under HFD. G – Comparison of liver ACC (acetyl-coenzyme A carboxylase) between genotypes and diet regimens. H – Comparison of liver Fasn (fatty acid synthase) between genotypes and diet regimens. I – Comparison of liver Elovl2 (elongation of very long chain fatty acids protein 2) mRNA expression between genotypes and diet regimens. J – Comparison of liver DGAT2 (diacylglycerol O-acyltransferase 2) mRNA expression between genotypes and diet regimens. K – Comparison of liver SCD1 (stearoyl-coenzyme A desaturase-1) mRNA expression between genotypes and diet regimens. L – Comparison of serum free fatty acids levels 30 min after oral glucose administration between genotypes and diet regimens. M – Comparison of liver CPT1b (carnitine palmitoyltranferase 1b) mRNA expression between genotypes and diet regimens. N – Comparison of liver CD36 mRNA expression between genotypes and diet regimens. O –CD36, IDE and calnexin protein levels in primary hepatocytes isolated from control and LS-IDE KO littermates. *t-Test used for two groups comparison and One-way ANOVA used for more than two groups. Statistical results between NCD and HFD LS-IDE KO not shown due to the lack of same genotype or diet regimen.
      Such increase in hepatic triglyceride content, led us to evaluate gene expression of hepatic lipogenesis transcription factors and enzymes. An increase in SREBP2 mRNA expression was observed in HFD LS-IDE KO (Supplementary Fig. 3D). However, SREBP1a and SREBP1c gene expression were unchanged between genotypes and diets (Supplementary Fig. 3E–F). SREBP2 was associated with lipid homeostasis, regulating cholesterol synthesis and de novo lipogenesis (DNL). Indeed, ACC gene expression was upregulated mostly by HFD (Fig. 4G). FASn mRNA expression was also upregulated by HFD with a tendency to be increased in HFD LS-IDE KO compared to HFD controls (Fig. 4H). Similarly, Elovl2 mRNA was upregulated in HFD LS-IDE KO (Fig. 4I). Unexpectedly, SCD1 - whose expression is usually highly correlated with that of ELOVL2 - presented no changes between groups (Fig. 4J). We evaluated in vivo fractional DNL rates for triglyceride fatty acids and no alterations were observed in the overnight rate of newly synthesized fatty acids (Fig. 4F). In the crossroad between newly synthesized lipids and external lipid output, DGAT2 is a key enzyme in the final step for triglyceride synthesis, but its mRNA levels did not change between genotypes and diet regimens (Fig. 4K).
      Given that DNL rates were not altered by IDE deletion, the observed changes in hepatic triglyceride levels reflect alterations in the balance between hepatic fatty acid inflow and oxidation. Regarding fatty acid oxidation, HFD resulted in a significant decrease in CPT1b mRNA expression that is concordant with the higher liver triglyceride content of HFD over NCD mice (Fig. 4M). As expected, control HFD animals presented higher levels of circulating FFA 30 min after oral glucose administration, a typical feature of diet-induced insulin resistance (Fig. 4L). However, this effect was not observed for HFD LS-IDE KO. HFD LS-IDE KO showed lower plasma levels of FFA when compared to control HFD (Supplementary Fig. 3G). This could be due to a reduction in adipose tissue lipolysis, and/or increased hepatic FFA uptake via CD36. CD36 mRNA expression tended to be increased in LS-IDE KO mice, independently of the diet (Fig. 4N). CD36 protein levels were also increase in primary hepatocytes isolated from LS-IDE KO, corroborating the idea of increased FFA uptake (Fig. 4O). Therefore, in the absence of alterations in DNL, the increased hepatic triglyceride content is most likely associated to increased FFA uptake, consistent with decreased FFA in the postprandial.

      4. Discussion

      In the onset of glucose intolerance and prediabetes, the development of insulin resistance accompanied by hyperinsulinemia is a very well characterized observation. Acknowledging that 50–70% of newly-secreted insulin is immediately cleared and degraded by the liver before it has the opportunity to enter the general circulation, hepatic IDE activity is a strong controller of systemic insulin levels [
      • Farris W.
      • Mansourian S.
      • Chang Y.
      • Lindsley L.
      • Eckman E.A.
      • Frosch M.P.
      • et al.
      Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.
      ,
      • Wei X.
      • Ke B.
      • Zhao Z.
      • Ye X.
      • Gao Z.
      • Ye J.
      Regulation of insulin degrading enzyme activity by obesity-associated factors and pioglitazone in liver of diet-induced obese mice.
      ,
      • Fosam A.
      • Sikder S.
      • Abel B.S.
      • Tella S.H.
      • Walter M.F.
      • Mari A.
      • et al.
      Reduced insulin clearance and insulin-degrading enzyme activity contribute to hyperinsulinemia in African Americans.
      ]. Therefore, hepatic IDE impairments may also be a strong driver of hyperinsulinemia. We provide robust genetic evidence that IDE controls IC in men and mice and regulates postprandial glucose excursion. Moreover, this genetic control is abrogated under metabolic disturbances, such as early stages of type 2 diabetes development in humans (prediabetes) and other sequelae of prediabetes such as hepatic steatosis, in mice.
      While several studies have found genetic associations between IDE polymorphisms and plasma insulin levels [
      • Batool A.
      • Jahan N.
      • Sun Y.
      • Hanif A.
      • Xue H.
      Genetic association of IDE, POU2F1, PON1, IL1alpha and IL1beta with type 2 diabetes in Pakistani population.
      ,
      • Gu H.F.
      • Efendic S.
      • Nordman S.
      • Ostenson C.G.
      • Brismar K.
      • Brookes A.J.
      • et al.
      Quantitative trait loci near the insulin-degrading enzyme (IDE) gene contribute to variation in plasma insulin levels.
      ,
      • Guo X.
      • Cui J.
      • Jones M.R.
      • Haritunians T.
      • Xiang A.H.
      • Chen Y.D.
      • et al.
      Insulin clearance: confirmation as a highly heritable trait, and genome-wide linkage analysis.
      ,
      • Rudovich N.
      • Pivovarova O.
      • Fisher E.
      • Fischer-Rosinsky A.
      • Spranger J.
      • Mohlig M.
      • et al.
      Polymorphisms within insulin-degrading enzyme (IDE) gene determine insulin metabolism and risk of type 2 diabetes.
      ], their relationship with postprandial IC is inadequately explored. We found that SNPs within the IDE gene region, that represent natural human genetic variation, exert a significant quantitative control of postprandial IC in normoglycemic individuals and importantly, this was abrogated in individuals with prediabetes. This genetic effect was more prominent in males compared to females indicating that gender influences the IDE-mediated regulation of IC. Likewise, IDE SNPs control glycemic traits in males of the Framingham Heart Study [
      • Karamohamed S.
      • Demissie S.
      • Volcjak J.
      • Liu C.
      • Heard-Costa N.
      • Liu J.
      • et al.
      Polymorphisms in the insulin-degrading enzyme gene are associated with type 2 diabetes in men from the NHLBI Framingham Heart Study.
      ,
      • Gu H.F.
      • Efendic S.
      • Nordman S.
      • Ostenson C.G.
      • Brismar K.
      • Brookes A.J.
      • et al.
      Quantitative trait loci near the insulin-degrading enzyme (IDE) gene contribute to variation in plasma insulin levels.
      ]. Several studies showed linkage and association of the IDE gene with glycemic imbalances, diabetes clinical biomarkers, and type 2 diabetes [
      • Pivovarova O.
      • Nikiforova V.J.
      • Pfeiffer A.F.
      • Rudovich N.
      The influence of genetic variations in HHEX gene on insulin metabolism in the German MESYBEPO cohort.
      ,
      • Wu Y.
      • Li H.
      • Loos R.J.
      • Yu Z.
      • Ye X.
      • Chen L.
      • et al.
      Common variants in CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, and HHEX/IDE genes are associated with type 2 diabetes and impaired fasting glucose in a Chinese Han population.
      ], strongly suggesting that polymorphic IDE variants are involved in the loss of insulin-mediated glycemic control. Our human and mouse data converge towards the paradigm that impaired IDE-mediated hepatic IC is central to explain IDE polymorphisms association with diabetogenic traits.
      Glucose intolerance, a feature of prediabetes, was observed in the majority of IDE KO mice studies [
      • Farris W.
      • Mansourian S.
      • Chang Y.
      • Lindsley L.
      • Eckman E.A.
      • Frosch M.P.
      • et al.
      Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.
      ,
      • Steneberg P.
      • Bernardo L.
      • Edfalk S.
      • Lundberg L.
      • Backlund F.
      • Ostenson C.G.
      • et al.
      The type 2 diabetes-associated gene ide is required for insulin secretion and suppression of alpha-synuclein levels in beta-cells.
      ,
      • Abdul-Hay S.O.
      • Kang D.
      • McBride M.
      • Li L.
      • Zhao J.
      • Leissring M.A.
      Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance.
      ,
      • Villa-Perez P.
      • Merino B.
      • Fernandez-Diaz C.M.
      • Cidad P.
      • Lobaton C.D.
      • Moreno A.
      • et al.
      Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.
      ]. Herein, mice with liver-specific-IDE ablation, had impaired glycemic control, demonstrating a clear relationship between impaired hepatic IC and postprandial hyperinsulinemia. Exposure to insulin leads to downregulation of GLUT2 in hepatocytes [
      • Postic C.
      • Burcelin R.
      • Rencurel F.
      • Pegorier J.P.
      • Loizeau M.
      • Girard J.
      • et al.
      Evidence for a transient inhibitory effect of insulin on GLUT2 expression in the liver: studies in vivo and in vitro.
      ], accordingly, we observed downregulation of Glut2 mRNA and protein expression in the liver of LS-IDE KO animals with a concomitant postprandial hyperinsulinemia. Importantly, we determined that hepatocytes derived from this LS-IDE-ablated animals had a reduced capacity to uptake glucose. Burcelin et al. experiments in liver-specific GLUT2 KO mice observed a decrease in glucokinase expression in the liver advocating a cooperative interplay between the two proteins and thus suggesting a joined mechanism that results in a reduced capacity to uptake glucose [
      • Burcelin R.
      • del Carmen Munoz M.
      • Guillam M.T.
      • Thorens B.
      Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.
      ]. Given that a substantial amount of absorbed glucose is taken up by the liver [
      • Ferrannini E.
      • Bjorkman O.
      • Reichard Jr., G.A.
      • Pilo A.
      • Olsson M.
      • Wahren J.
      • et al.
      The disposal of an oral glucose load in healthy subjects. A quantitative study.
      ,
      • Mari A.
      • Wahren J.
      • DeFronzo R.A.
      • Ferrannini E.
      Glucose absorption and production following oral glucose: comparison of compartmental and arteriovenous-difference methods.
      ,
      • Fernandes A.B.
      • Patarrao R.S.
      • Videira P.A.
      • Macedo M.P.
      Understanding postprandial glucose clearance by peripheral organs: the role of the hepatic parasympathetic system.
      ], our data suggested that the hyperinsulinemic state, secondary to decreased hepatic IC, is directly impairing a key component of postprandial glucose disposal, finally disentangling IDE KO effect in glucose tolerance.
      In human studies is unclear whether the effects of IDE polymorphisms are exerted at the level of gene expression and/or protein function. Recently, it was shown in humans that variations in IC are more closely linked with differences in IDE activity rather than protein expression [
      • Fosam A.
      • Sikder S.
      • Abel B.S.
      • Tella S.H.
      • Walter M.F.
      • Mari A.
      • et al.
      Reduced insulin clearance and insulin-degrading enzyme activity contribute to hyperinsulinemia in African Americans.
      ], similar to our findings in HFD mice. IDE activity possibly regulated by hepatic nitric oxide (NO), whose levels raise as a consequence of diet induced obesity [
      • Sousa-Lima I.
      • Fernandes A.B.
      • Patarrao R.S.
      • Kim Y.B.
      • Macedo M.P.
      S-nitrosoglutathione reverts dietary sucrose-induced insulin resistance.
      ,
      • Dominguez-Vias G.
      • Segarra A.B.
      • Ramirez-Sanchez M.
      • Prieto I.
      The role of high fat diets and liver peptidase activity in the development of obesity and insulin resistance in Wistar rats.
      ], conceivably via S-nitrosylation of its cysteine residues [
      • Cordes C.M.
      • Bennett R.G.
      • Siford G.L.
      • Hamel F.G.
      Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation.
      ,
      • Ralat L.A.
      • Ren M.
      • Schilling A.B.
      • Tang W.J.
      Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.
      ,
      • Neant-Fery M.
      • Garcia-Ordonez R.D.
      • Logan T.P.
      • Selkoe D.J.
      • Li L.
      • Reinstatler L.
      • et al.
      Molecular basis for the thiol sensitivity of insulin-degrading enzyme.
      ]. In fact, S-nitrosylation impairs IDE activity in brain tissue of subjects with Alzheimer's disease [
      • Akhtar M.W.
      • Sanz-Blasco S.
      • Dolatabadi N.
      • Parker J.
      • Chon K.
      • Lee M.S.
      • et al.
      Elevated glucose and oligomeric beta-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.
      ]. Additionally, lower hepatic NO levels and higher liver IDE activity were described as mechanisms leading to improved IC in rats exposed to high-sucrose and treated with thiazolidinediones with parallel decrease in triglycerides [
      • Martins F.O.
      • Delgado T.C.
      • Viegas J.
      • Gaspar J.M.
      • Scott D.K.
      • O’Doherty R.M.
      • et al.
      Mechanisms by which the thiazolidinedione troglitazone protects against sucrose-induced hepatic fat accumulation and hyperinsulinaemia.
      ]. In line with these observations, mice under HFD showed impaired IDE activity and increased IDE S-nitrosylation (unpublished data) strongly suggesting that this mechanism underlies impaired insulin degradation and leads to hyperinsulinemia, insulin resistance and glucose intolerance.
      Interestingly, development of MAFLD highly prevalent in prediabetes was associated with impairment of IC [
      • Ortiz-Lopez C.
      • Lomonaco R.
      • Orsak B.
      • Finch J.
      • Chang Z.
      • Kochunov V.G.
      • et al.
      Prevalence of prediabetes and diabetes and metabolic profile of patients with nonalcoholic fatty liver disease (NAFLD).
      ,
      • Bril F.
      • Lomonaco R.
      • Orsak B.
      • Ortiz-Lopez C.
      • Webb A.
      • Tio F.
      • et al.
      Relationship between disease severity, hyperinsulinemia, and impaired insulin clearance in patients with nonalcoholic steatohepatitis.
      ] and in our mouse model of liver-specific IDE ablation, we observed an increase in hepatic triglyceride accumulation. Surprisingly, DNL appeared not to be involved in this scenario. We searched for evidence of possible mechanisms mediating hepatic lipid imbalance that could account for this observation. We found that expression of CD36, a major transporter in FFA uptake, was increased both in liver and primary hepatocytes of LS-IDE KO (Fig. 4N and O). In agreement, postprandial FFA excursion were decrease suggesting an enhanced liver FFA uptake capacity in the LS-IDE-KO mice. Even though gene expression data has limitations in functional analysis, our results converge to the notion that increased hepatic triglycerides in the LS-IDE-KO mice result from enhanced FFA uptake through CD36. Accordingly, Steneberg et al. [
      • Steneberg P.
      • Sykaras A.G.
      • Backlund F.
      • Straseviciene J.
      • Soderstrom I.
      • Edlund H.
      Hyperinsulinemia enhances hepatic expression of the fatty acid transporter Cd36 and provokes hepatosteatosis and hepatic insulin resistance.
      ] also showed that hyperinsulinemia triggered an increase in hepatic CD36 expression and thus the development of hepatosteatosis. Therefore, the effects of IDE dysfunction on insulin degradation might impinge on MAFLD development.
      Our observations of increased insulin excursions in LS-IDE ablated mice during an OGTT can be explained by two different mechanisms; impaired insulin internalization and/or insulin degradation. On one hand, internalization might be diminished due to the described ablation of CEACAM1 phosphorylation in LS-IDE KO mice [
      • Villa-Perez P.
      • Merino B.
      • Fernandez-Diaz C.M.
      • Cidad P.
      • Lobaton C.D.
      • Moreno A.
      • et al.
      Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.
      ]. On the other hand, after insulin/insulin-receptor complex internalization, degradation can be impaired due to decreased IDE activity. In agreement with IDE activity role on insulin degradation, Shah et al. [
      • Shah N.
      • Zhang S.
      • Harada S.
      • Smith R.M.
      • Jarett L.
      Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.
      ] elegantly showed that impairment of IDE activity, resulted in increased insulin levels both in cytoplasm and nucleus of hepatocytes. Nevertheless, it should be noted that liver's inefficiency to shut down insulin actions can lead to both, GLUT2 down-regulation as shown by Postic et al. [
      • Postic C.
      • Burcelin R.
      • Rencurel F.
      • Pegorier J.P.
      • Loizeau M.
      • Girard J.
      • et al.
      Evidence for a transient inhibitory effect of insulin on GLUT2 expression in the liver: studies in vivo and in vitro.
      ] and up-regulation of CD36 [
      • Steneberg P.
      • Sykaras A.G.
      • Backlund F.
      • Straseviciene J.
      • Soderstrom I.
      • Edlund H.
      Hyperinsulinemia enhances hepatic expression of the fatty acid transporter Cd36 and provokes hepatosteatosis and hepatic insulin resistance.
      ] as observed in the LS-IDE KO mice. Overall, suggesting that modulation of insulin degradation is important for hepatic glucose and lipid homeostasis.
      Herein, we demonstrated for the first time that postprandial IC is impaired by the deletion of hepatic IDE expression, as well as, when under HFD by a decrease in IDE activity. While another group did not observe an hepatic IDE regulatory effect on postprandial IC, this apparent discrepancy can easily be explained by differential experimental assessments and procedures [
      • Villa-Perez P.
      • Merino B.
      • Fernandez-Diaz C.M.
      • Cidad P.
      • Lobaton C.D.
      • Moreno A.
      • et al.
      Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.
      ,
      • Merino B.
      • Fernandez-Diaz C.M.
      • Parrado-Fernandez C.
      • Gonzalez-Casimiro C.M.
      • Postigo-Casado T.
      • Lobaton C.D.
      • et al.
      Hepatic insulin-degrading enzyme regulates glucose and insulin homeostasis in diet-induced obese mice.
      ]. We evaluate IC not only in fasting but also during an OGTT and we observed a reduction of IC during the OGTT in animals lacking liver IDE (Fig. 2E), which was not evaluated in previous publications. Crucially, the genetic control of IDE is also strongly associated with postprandial IC in our human data (Fig. 1). Noteworthy, Merino et al. did not measure IDE activity and exclusively assessed LS-IDE KO under HFD in the latest manuscript; therefore, by lacking the control setting and the measurements along the OGTT. We found that the effect of genetic ablation of hepatic IDE on postprandial IC is lost under HFD, which can be attributed to the pronounced decrease in IDE activity resulting in a suppressed IC both in controls and LS-IDE KO under a high-fat diet (Fig. 2C). These results are highly consistent with those of Merino et al., who also found no effects of deleting hepatic IDE expression on IC in HFD mice. In further agreement with our study, mice with deleted hepatic expression of IDE had poorer glucose tolerance compared to controls [
      • Villa-Perez P.
      • Merino B.
      • Fernandez-Diaz C.M.
      • Cidad P.
      • Lobaton C.D.
      • Moreno A.
      • et al.
      Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.
      ]. More importantly, when we performed a functional assay for glucose uptake using primary cultures of LS-IDE KO, we observed a significant reduction in glucose internalization relative to control mice (Fig. 3I). We observed that the genetic deletion of IDE in the liver resulted in decreased expression of GLUT2 and diminished glucokinase mRNA levels. In their paper they observed an increase in GLUT2, however this increment was just assessed under HFD which can be masked by the diet effect. Our study performs an in depth study of lipid and glucose related pathways, expanding the knowledge of how IDE mechanistically regulates IC both in physiological and pathological settings.
      In conclusion, we unveiled a key role for liver IDE in regulating hepatic IC and consequent control of postprandial glucose and insulin levels. This action is influenced by IDE genetic variants that are compromised in a dysmetabolic setting. Evidence from mouse models demonstrate that glucose intolerance and hepatic steatosis are due to impaired hepatic IDE function. Therefore, we conclude that IDE polymorphisms governing postprandial IC are likely to be affected by epigenetic modifications induced by hypercaloric diets, leading to an impaired capacity to fine-tune postprandial insulin levels.

      Acknowledgments

      The authors thank the study participants for commitment and loyalty. We are thankful to Inês Sousa-Lima for critical reading of the manuscript and to Maria João Meneses for help with figure execution.

      Funding

      This research was funded by “ Fundação para a Ciência e a Tecnologia ” - FCT to Diego O. Borges ( LISBOA-01-0145-FEDER-024325 ) and received a full mobility doctorate award by the program FELLOW-MUNDUS supported by the Erasmus Mundus Action 2 Programme of the European Union; M. Paula Macedo ( PTDC/BIM-MET/2115/2014 , PTDC/DTP-EPI/0207/2012 and iNOVA4Health ( UIDB/Multi/04462/2020 )); by the European Commission H2020 Marie Skłodowska-Curie Actions (grant agreements no. 734719 ), by the Portuguese Diabetology Society and a grant from the Portuguese Directorate General Health . The mouse studies were developed with the support of the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and Foundation for Science and Technology (Portugal).

      CRediT authorship contribution statement

      Conceptualization and supervision of PREVADIAB2 study, JFR, MPM, RD, LGC, JMB; PREVADIAB2 administration, RTR and RSP. Conceptualization and Methodology, MPM, CPG, DOB, RSP, ND and RMO; Formal analysis, DOB and RSP. Discussion, MPM, CPG, DOB, RMO and RSP. Writing-original draft preparation, MPM, CPG, RSP, DOB, RMO. Writing-review and editing, MPM, CPG, RSP, DOB, RMO. All authors have read and agreed to the published version of the manuscript. Funding acquisition, MPM. All authors critically edited the manuscript and approved the final version. MPM and CPG are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

      Declaration of competing interest

      No potential conflicts of interest relevant to this article were reported.

      Appendix A. Supplementary data

      References

        • Eslam M.
        • Newsome P.N.
        • Sarin S.K.
        • Anstee Q.M.
        • Targher G.
        • Romero-Gomez M.
        • et al.
        A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.
        J Hepatol. 2020; 73: 202-209
        • Groves C.J.
        • Wiltshire S.
        • Smedley D.
        • Owen K.R.
        • Frayling T.M.
        • Walker M.
        • et al.
        Association and haplotype analysis of the insulin-degrading enzyme (IDE) gene, a strong positional and biological candidate for type 2 diabetes susceptibility.
        Diabetes. 2003; 52: 1300-1305
        • Karamohamed S.
        • Demissie S.
        • Volcjak J.
        • Liu C.
        • Heard-Costa N.
        • Liu J.
        • et al.
        Polymorphisms in the insulin-degrading enzyme gene are associated with type 2 diabetes in men from the NHLBI Framingham Heart Study.
        Diabetes. 2003; 52: 1562-1567
        • Kwak S.H.
        • Cho Y.M.
        • Moon M.K.
        • Kim J.H.
        • Park B.L.
        • Cheong H.S.
        • et al.
        Association of polymorphisms in the insulin-degrading enzyme gene with type 2 diabetes in the Korean population.
        Diabetes Res Clin Pract. 2008; 79: 284-290
        • Grarup N.
        • Rose C.S.
        • Andersson E.A.
        • Andersen G.
        • Nielsen A.L.
        • Albrechtsen A.
        • et al.
        Studies of association of variants near the HHEX, CDKN2A/B, and IGF2BP2 genes with type 2 diabetes and impaired insulin release in 10,705 Danish subjects: validation and extension of genome-wide association studies.
        Diabetes. 2007; 56: 3105-3111
        • Dimas A.S.
        • Lagou V.
        • Barker A.
        • Knowles J.W.
        • Magi R.
        • Hivert M.F.
        • et al.
        Impact of type 2 diabetes susceptibility variants on quantitative glycemic traits reveals mechanistic heterogeneity.
        Diabetes. 2014; 63: 2158-2171
        • Farris W.
        • Mansourian S.
        • Chang Y.
        • Lindsley L.
        • Eckman E.A.
        • Frosch M.P.
        • et al.
        Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo.
        Proc Natl Acad Sci U S A. 2003; 100: 4162-4167
        • Pina A.F.
        • Patarrao R.S.
        • Ribeiro R.T.
        • Penha-Goncalves C.
        • Raposo J.F.
        • Gardete-Correia L.
        • et al.
        Metabolic footprint, towards understanding type 2 diabetes beyond glycemia.
        J Clin Med. 2020; 9
        • Osei K.
        • Schuster D.P.
        Ethnic differences in secretion, sensitivity, and hepatic extraction of insulin in black and white Americans.
        Diabet Med. 1994; 11: 755-762
        • Haffner S.M.
        • Stern M.P.
        • Watanabe R.M.
        • Bergman R.N.
        Relationship of insulin clearance and secretion to insulin sensitivity in non-diabetic Mexican Americans.
        Eur J Clin Investig. 1992; 22: 147-153
        • Lee C.C.
        • Haffner S.M.
        • Wagenknecht L.E.
        • Lorenzo C.
        • Norris J.M.
        • Bergman R.N.
        • et al.
        Insulin clearance and the incidence of type 2 diabetes in Hispanics and African Americans: the IRAS Family Study.
        Diabetes Care. 2013; 36: 901-907
        • Piccinini F.
        • Polidori D.C.
        • Gower B.A.
        • Bergman R.N.
        Hepatic but not extrahepatic insulin clearance is lower in African American than in European American women.
        Diabetes. 2017; 66: 2564-2570
        • Piccinini F.
        • Polidori D.C.
        • Gower B.A.
        • Fernandez J.R.
        • Bergman R.N.
        Dissection of hepatic versus extra-hepatic insulin clearance: ethnic differences in childhood.
        Diabetes Obes Metab. 2018; 20: 2869-2875
        • Cordes C.M.
        • Bennett R.G.
        • Siford G.L.
        • Hamel F.G.
        Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation.
        Biochem Pharmacol. 2009; 77: 1064-1073
        • Pivovarova O.
        • Gogebakan O.
        • Pfeiffer A.F.
        • Rudovich N.
        Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells.
        Diabetologia. 2009; 52: 1656-1664
        • Wei X.
        • Ke B.
        • Zhao Z.
        • Ye X.
        • Gao Z.
        • Ye J.
        Regulation of insulin degrading enzyme activity by obesity-associated factors and pioglitazone in liver of diet-induced obese mice.
        PLoS One. 2014; 9e95399
        • Martins F.O.
        • Delgado T.C.
        • Viegas J.
        • Gaspar J.M.
        • Scott D.K.
        • O’Doherty R.M.
        • et al.
        Mechanisms by which the thiazolidinedione troglitazone protects against sucrose-induced hepatic fat accumulation and hyperinsulinaemia.
        Br J Pharmacol. 2016; 173: 267-278
        • Fosam A.
        • Sikder S.
        • Abel B.S.
        • Tella S.H.
        • Walter M.F.
        • Mari A.
        • et al.
        Reduced insulin clearance and insulin-degrading enzyme activity contribute to hyperinsulinemia in African Americans.
        J Clin Endocrinol Metab. 2020; 105: e1835-e1846
        • Steneberg P.
        • Bernardo L.
        • Edfalk S.
        • Lundberg L.
        • Backlund F.
        • Ostenson C.G.
        • et al.
        The type 2 diabetes-associated gene ide is required for insulin secretion and suppression of alpha-synuclein levels in beta-cells.
        Diabetes. 2013; 62: 2004-2014
        • Poy M.N.
        • Yang Y.
        • Rezaei K.
        • Fernstrom M.A.
        • Lee A.D.
        • Kido Y.
        • et al.
        CEACAM1 regulates insulin clearance in liver.
        Nat Genet. 2002; 30: 270-276
        • Najjar S.M.
        • Perdomo G.
        Hepatic insulin clearance: mechanism and physiology.
        Physiology (Bethesda). 2019; 34: 198-215
        • Abdul-Hay S.O.
        • Kang D.
        • McBride M.
        • Li L.
        • Zhao J.
        • Leissring M.A.
        Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance.
        PLoS One. 2011; 6e20818
        • Gardete-Correia L.
        • Boavida J.M.
        • Raposo J.F.
        • Mesquita A.C.
        • Fona C.
        • Carvalho R.
        • et al.
        First diabetes prevalence study in Portugal: PREVADIAB study.
        Diabet Med. 2010; 27: 879-881
        • Borges D.O.
        • Meneses M.J.
        • Dias T.R.
        • Martins F.O.
        • Oliveira P.F.
        • Alves M.G.
        • et al.
        Data on metabolic profile of insulin-degrading enzyme knockout mice.
        Data Brief. 2019; 25: 104023
        • Silva J.C.P.
        • Marques C.
        • Martins F.O.
        • Viegas I.
        • Tavares L.
        • Macedo M.P.
        • et al.
        Determining contributions of exogenous glucose and fructose to de novo fatty acid and glycerol synthesis in liver and adipose tissue.
        Metab Eng. 2019; 56: 69-76
        • Schmittgen T.D.
        • Livak K.J.
        Analyzing real-time PCR data by the comparative C(T) method.
        Nat Protoc. 2008; 3: 1101-1108
        • Zhande R.
        • Zhang W.
        • Zheng Y.
        • Pendleton E.
        • Li Y.
        • Polakiewicz R.D.
        • et al.
        Dephosphorylation by default, a potential mechanism for regulation of insulin receptor substrate-1/2, Akt, and ERK1/2.
        J Biol Chem. 2006; 281: 39071-39080
        • Mosmann T.
        Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
        J Immunol Methods. 1983; 65: 55-63
        • Batool A.
        • Jahan N.
        • Sun Y.
        • Hanif A.
        • Xue H.
        Genetic association of IDE, POU2F1, PON1, IL1alpha and IL1beta with type 2 diabetes in Pakistani population.
        Mol Biol Rep. 2014; 41: 3063-3069
        • Gu H.F.
        • Efendic S.
        • Nordman S.
        • Ostenson C.G.
        • Brismar K.
        • Brookes A.J.
        • et al.
        Quantitative trait loci near the insulin-degrading enzyme (IDE) gene contribute to variation in plasma insulin levels.
        Diabetes. 2004; 53: 2137-2142
        • Guo X.
        • Cui J.
        • Jones M.R.
        • Haritunians T.
        • Xiang A.H.
        • Chen Y.D.
        • et al.
        Insulin clearance: confirmation as a highly heritable trait, and genome-wide linkage analysis.
        Diabetologia. 2012; 55: 2183-2192
        • Rudovich N.
        • Pivovarova O.
        • Fisher E.
        • Fischer-Rosinsky A.
        • Spranger J.
        • Mohlig M.
        • et al.
        Polymorphisms within insulin-degrading enzyme (IDE) gene determine insulin metabolism and risk of type 2 diabetes.
        J Mol Med. 2009; 87: 1145-1151
        • Pivovarova O.
        • Nikiforova V.J.
        • Pfeiffer A.F.
        • Rudovich N.
        The influence of genetic variations in HHEX gene on insulin metabolism in the German MESYBEPO cohort.
        Diabetes Metab Res Rev. 2009; 25: 156-162
        • Wu Y.
        • Li H.
        • Loos R.J.
        • Yu Z.
        • Ye X.
        • Chen L.
        • et al.
        Common variants in CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, and HHEX/IDE genes are associated with type 2 diabetes and impaired fasting glucose in a Chinese Han population.
        Diabetes. 2008; 57: 2834-2842
        • Villa-Perez P.
        • Merino B.
        • Fernandez-Diaz C.M.
        • Cidad P.
        • Lobaton C.D.
        • Moreno A.
        • et al.
        Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.
        Metabolism. 2018; 88: 1-11
        • Postic C.
        • Burcelin R.
        • Rencurel F.
        • Pegorier J.P.
        • Loizeau M.
        • Girard J.
        • et al.
        Evidence for a transient inhibitory effect of insulin on GLUT2 expression in the liver: studies in vivo and in vitro.
        Biochem J. 1993; 293: 119-124
        • Burcelin R.
        • del Carmen Munoz M.
        • Guillam M.T.
        • Thorens B.
        Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.
        J Biol Chem. 2000; 275: 10930-10936
        • Ferrannini E.
        • Bjorkman O.
        • Reichard Jr., G.A.
        • Pilo A.
        • Olsson M.
        • Wahren J.
        • et al.
        The disposal of an oral glucose load in healthy subjects. A quantitative study.
        Diabetes. 1985; 34: 580-588
        • Mari A.
        • Wahren J.
        • DeFronzo R.A.
        • Ferrannini E.
        Glucose absorption and production following oral glucose: comparison of compartmental and arteriovenous-difference methods.
        Metabolism. 1994; 43: 1419-1425
        • Fernandes A.B.
        • Patarrao R.S.
        • Videira P.A.
        • Macedo M.P.
        Understanding postprandial glucose clearance by peripheral organs: the role of the hepatic parasympathetic system.
        J Neuroendocrinol. 2011; 23: 1288-1295
        • Sousa-Lima I.
        • Fernandes A.B.
        • Patarrao R.S.
        • Kim Y.B.
        • Macedo M.P.
        S-nitrosoglutathione reverts dietary sucrose-induced insulin resistance.
        Antioxidants (Basel). 2020; 9
        • Dominguez-Vias G.
        • Segarra A.B.
        • Ramirez-Sanchez M.
        • Prieto I.
        The role of high fat diets and liver peptidase activity in the development of obesity and insulin resistance in Wistar rats.
        Nutrients. 2020; 12: 636
        • Ralat L.A.
        • Ren M.
        • Schilling A.B.
        • Tang W.J.
        Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.
        J Biol Chem. 2009; 284: 34005-34018
        • Neant-Fery M.
        • Garcia-Ordonez R.D.
        • Logan T.P.
        • Selkoe D.J.
        • Li L.
        • Reinstatler L.
        • et al.
        Molecular basis for the thiol sensitivity of insulin-degrading enzyme.
        Proc Natl Acad Sci U S A. 2008; 105: 9582-9587
        • Akhtar M.W.
        • Sanz-Blasco S.
        • Dolatabadi N.
        • Parker J.
        • Chon K.
        • Lee M.S.
        • et al.
        Elevated glucose and oligomeric beta-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.
        Nat Commun. 2016; 7: 10242
        • Ortiz-Lopez C.
        • Lomonaco R.
        • Orsak B.
        • Finch J.
        • Chang Z.
        • Kochunov V.G.
        • et al.
        Prevalence of prediabetes and diabetes and metabolic profile of patients with nonalcoholic fatty liver disease (NAFLD).
        Diabetes Care. 2012; 35: 873-878
        • Bril F.
        • Lomonaco R.
        • Orsak B.
        • Ortiz-Lopez C.
        • Webb A.
        • Tio F.
        • et al.
        Relationship between disease severity, hyperinsulinemia, and impaired insulin clearance in patients with nonalcoholic steatohepatitis.
        Hepatology. 2014; 59: 2178-2187
        • Steneberg P.
        • Sykaras A.G.
        • Backlund F.
        • Straseviciene J.
        • Soderstrom I.
        • Edlund H.
        Hyperinsulinemia enhances hepatic expression of the fatty acid transporter Cd36 and provokes hepatosteatosis and hepatic insulin resistance.
        J Biol Chem. 2015; 290: 19034-19043
        • Shah N.
        • Zhang S.
        • Harada S.
        • Smith R.M.
        • Jarett L.
        Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.
        Endocrinology. 1995; 136: 2825-2835
        • Merino B.
        • Fernandez-Diaz C.M.
        • Parrado-Fernandez C.
        • Gonzalez-Casimiro C.M.
        • Postigo-Casado T.
        • Lobaton C.D.
        • et al.
        Hepatic insulin-degrading enzyme regulates glucose and insulin homeostasis in diet-induced obese mice.
        Metabolism. 2020; 113: 154352