Insulin resistance and diabetes caused by genetic or diet

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30 800 8 120,Glucose of baseline,Body weight g,Glucose mg dL. Insulin ng ml, tny tny tny tny tny tny tny tny tny tny tny tny 0 15 30 45 60 90 120. Male Female Male Female Time after insulin injection min. of body weight,H E H E H E,Adipose weight,1 0 BAT eWAT iWAT. tny tny tny tny tny tny,H E H E H E,0 0 BAT eWAT iWAT. BAT eWAT iWAT,tny tny tny tny 25,Adiponectin ug ml.
of body weight,of body weight,Leptin ng ml,Lean weight. Fat weight,12 20 42 84 12 20 42 84 tny tny tny tny. Age weeks Age weeks,Aspartate aminotransferase U L. Alanine aminotransferase U L,Liver weight of body weight. 15 60 200 500,Liver triglyceride mg g,H E Oil red O 150.
10 Liver Liver 40,tny tny tny tny,H E Oil red O,Liver Liver. tny tny tny tny tny tny tny tny, Fig 1 The teeny phenotype A Photograph of a male tny homozygote tny tny and WT littermate at 8 wk of age Scale bar 1 cm B D Body weight B. blood glucose C and serum insulin D of 8 wk old mice Glucose and insulin were measured after a 6 h fast E Insulin tolerance test Blood glucose was measured at. indicated times after i p insulin injection in 8 wk old male mice n 3 The baseline blood glucose levels 0 min of tny tny and WT littermates were 626 31 mg dL and. 168 8 mg dL respectively F Representative photographs of BAT eWAT and iWAT from 20 wk old male mice Scale bars 1 cm G Weights of BAT eWAT and. iWAT normalized to body weight in 20 wk old male mice n 3 H M H E staining of sections from different adipose tissues of 20 wk old male mice Scale bars. 30 m N and O Fat weight N and lean weight O normalized to body weight of male mice at indicated ages by MRI n 3 P and Q Serum leptin P and. adiponectin Q in 8 wk old male mice R Representative photographs of liver from 8 wk old male mice Scale bar 1 cm S Liver weight normalized to body weight in. 8 wk old male mice n 3 T W Liver sections of 20 wk old male mice stained with H E T and U and Oil red O V and W Scale bars 30 m X Z Liver triglyceride. X serum ALT Y and AST Z in 8 wk old male mice In B D N Q S and X Z data points represent individual mice P values were determined by Student s t test. Fig 1 H M Magnetic resonance imaging MRI showed that and alanine aminotransferase ALT were increased in the serum. tny tny mice were lean throughout life compared with WT litter of tny mice suggesting liver damage caused by lipid accumulation. mates Fig 1 N and O Presumably as a result of lipodystrophy Fig 1 Y and Z. tny tny mice had reduced adipokines such as leptin and adipo Both male and female tny homozygotes were infertile females. nectin in the serum Fig 1 P and Q In addition 8 wk old tny became pregnant but failed to deliver pups Crosses of hetero. mice had large pallid livers Fig 1 R and S and Oil Red O zygous mice yielded a non Mendelian birth ratio among off. ORO staining showed an abundance of stored lipid Fig 1 T spring Table S1 suggesting prenatal attrition of homozygotes. W possibly a consequence of compromised adipose tissue fat. storage caused by impaired insulin signaling 15 Liver extracts of Tny Phenotypes Are Caused by a Kbtbd2 Mutation Tny was earlier. fasting tny mice showed increased stored triglyceride compared mapped to a premature stop codon in kelch repeat and BTB POZ. Downloaded by guest on July 1 2020, with WT mice Fig 1X Both aspartate aminotransferase AST domain containing 2 Kbtbd2 using body weight as a quantitative. Zhang et al PNAS Published online October 5 2016 E6419. Fig 2 Mutations of Kbtbd2 cause the tny phenotype A Protein domains of WT mouse KBTBD2 Upper and position of tny mutation Lower The tny. mutation is an arginine to premature stop codon substitution at position 121 of the protein B Relative Kbtbd2 mRNA level normalized to Actb mRNA. measured by RT qPCR of mRNA isolated from livers of three male homozygous tny mice or WT littermates C and D Immunoblots of liver C and MEF D. lysates from two male homozygous tny mice and WT littermates E Immunoblots of lysates of 293T cells expressing 3 FLAG tagged full length WT KBTBD2. or KBTBD2tny GFP was coexpressed as a loading control F H Body weight F blood glucose G and serum insulin H values in 6 wk old male homozygous. Kbtbd2 KO Kbtbd2 and WT littermates Glucose and insulin were measured after a 6 h fast I Insulin tolerance test Blood glucose was measured at. indicated times after i p insulin injection in male Kbtbd2 mice n 4 and WT littermates n 3 at 16 wk of age The baseline blood glucose levels 0 min. of Kbtbd2 mice and WT littermates were 728 18 mg dL and 298 28 mg dL respectively In F H data points represent individual mice P values were. determined by Student s t test, trait under a recessive model of inheritance Fig 2A 14 No liver or mouse embryonic fibroblast MEF lysates Fig 2 C and. tny phenotypes were observed in heterozygous mice Fig S1 K D and no full length protein was detected when a tagged ver. and L Although normal levels of Kbtbd2 mRNA were de sion of KBTBD2tny was overexpressed in 293T cells Fig 2E. tected in Kbtbd2tny tny liver Fig 2B expression of endogenous We confirmed that the tny phenotype was caused by Kbtbd2. KBTBD2 was undetectable by immunoblotting in Kbtbd2tny tny mutation in mice homozygous for a second null allele of Kbtbd2. Glucose mg dL,Insulin ng ml,0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8.
Weeks post adipose transplanation Weeks post adipose transplanation. D Kbtbd2 F G H 30,Liver weight of body weight,Body weight g. E Kbtbd2 AT, Fig 3 WT adipose tissue transplantation rescues the tny phenotype Surgery was performed at 4 wk of age and mice received WT adipose tissue AT or PBS n. 4 mice of the indicated genotype per condition A and B Blood glucose A and serum insulin B in mice before 0 wk and at the indicated times after adipose. transplantation C HbA1c in blood of mice 8 wk after transplantation D and E H E staining of sections from liver of Kbtbd2 or Kbtbd2 AT mice at 12 wk. after transplantation Scale bars 30 m F H Liver weight normalized to body weight F representative photograph G or body weight I of mice at 12 wk after. Downloaded by guest on July 1 2020, transplantation Scale bar in G 1 cm Data points represent individual mice C F and H P values were determined by Student s t test. E6420 www pnas org cgi doi 10 1073 pnas 1614467113 Zhang et al. Fig 4 Elevated p85 Expression in KBTBD2 deficient Mice A Protein domains of mouse KBTBD2 and truncated forms for mapping the protein interaction region. B Immunoblot analysis of immunoprecipitates Top and Middle or lysates Bottom of 293T cells expressing HA tagged Cul3 and 3 FLAG tagged full length FL or. truncated KBTBD2 C Immunoblots of lysates of different adipose tissues from two 8 wk old male Kbtbd2 mice or WT littermates D Immunoblots of lysates of. isolated fat cells or SVF from different adipose tissues of 8 wk old WT or Kbtbd2 mice E Immunoblots of lysates of 10 d differentiated MEF derived adipocytes. from two different mice F H Immunoblots of liver F muscle G and brain H lysates from 8 wk old WT or Kbtbd2 mice. generated by CRISPR Cas9 gene targeting Kbtbd2 which Numerous BTB BACK Kelch proteins function as substrate. fully recapitulated all aspects of the tny phenotype Fig 2 F I recognition components of Cul3 based E3 ubiquitin Ub ligase. Taken together these findings indicate that insulin resistance complexes 16 19 Coimmunoprecipitation co IP experiments. diabetes lipodystrophy and fatty liver stem from a homozygous showed that full length KBTBD2 interacted with Cul3 through. null allele of Kbtbd2 in tny mice the KBTBD2 N terminal BTB domain Fig 4 A and B suggesting. that KBTBD2 might fulfill a similar function We hypothesized. Rescue of the tny Phenotype by WT Adipose Tissue Lipodystrophies that putative KBTBD2 specified substrates of the Cul3 Ub ligase. are often associated with insulin resistance diabetes dyslipidemia complex accumulate in Kbtbd2 mutant mice and sought to identify. and hepatic steatosis Thus we tested whether s c implantation of these by semiquantitative mass spectrometry analysis of proteins in. WT adipose tissue into 4 wk old Kbtbd2 mice could rescue the white adipose tissues of WT or Kbtbd2 mice Among the 1 171. hyperglycemia hyperinsulinemia and hepatic steatosis observed proteins identified in all samples Dataset S1 six proteins were. in these mice At 12 wk after surgery transplanted eWAT grafts greater than fivefold more abundant in both eWAT and iWAT. had a healthy vascularized appearance indicating host accep from Kbtbd2 mice relative to WT mice Table S3 p85 a. tance of the tissue Fig S2 Strikingly by 2 wk postsurgery regulatory subunit of PI3K was elevated by 40 and 24 fold in. blood glucose and insulin in transplanted Kbtbd2 mice had Kbtbd2 eWAT and iWAT respectively and we considered it. decreased to concentrations similar to those in WT mice and highly relevant to the tny phenotype because of its key role in. were maintained through 8 wk postsurgery Fig 3 A and B PI3K regulation. HbA1c levels were normal in transplanted Kbtbd2 mice at We verified the specific accumulation of p85 in Kbtbd2. 8 wk postsurgery Fig 3C Moreover hepatic steatosis in adipose tissues in which normal to reduced amounts of the PI3K. Kbtbd2 mice was reversed by adipose transplantation Fig 3 D catalytic subunits p110 and p110 were detected Fig 4C. and E liver weight was similarly restored toward WT levels Fig Within adipose tissues fat cells accumulated greater amounts of. 3F Adipose transplantation also partially rescued the growth p85 than stromal vascular fraction SVF cells Fig 4D Similarly. retardation of Kbtbd2 mice Fig 3 G and H Thus adipose p85 protein accumulated in Kbtbd2 adipocyte like cells differ. tissue expressing functional KBTBD2 is by itself sufficient to entiated from MEFs MEF derived adipocytes Fig 4E We ob. rescue hyperglycemia hyperinsulinemia and hepatic steatosis in served accumulation of p85 in Kbtbd2 liver muscle and brain. Kbtbd2 mice Fig 4 F H Knockdown of KBTBD2 in differentiated human. adipocytes also increased p85 protein levels Fig S4A These data. Elevated p85 Expression in KBTBD2 Deficient Mice The 623 aa KBTBD2 demonstrate that KBTBD2 deficiency results in elevated p85 ex. protein for which no function has been reported previously contains pression in mouse tissues with high expression levels of Kbtbd2. BTB Broad complex Tramtrack and Bric brac and BACK BTB including adipose tissue liver muscle and brain as well as in. and C terminal Kelch domains at its N terminus and four tandem human adipocytes. Kelch motifs at its C terminus Fig 4A similar to other BTB BACK. Kelch proteins 16 The mouse protein is 98 6 identical to human KBTBD2 Interaction with Cul3 and p85 Results in p85 Ubiquitination. KBTBD2 highly similar homologs are found in other vertebrates KBTBD2 interacted with p85 when expressed in 293T cells the. Table S2 but are not present in invertebrate organisms 17 18 p85 inter SH2 domain iSH2 Fig 5 A and B and the KBTBD2. KBTBD2 was observed both in the nucleus and cytoplasm on over C terminal Kelch domains were required for this interaction Figs. expression in 293T cells Fig S3A Kbtbd2 mRNA was detected in a 4A and 5C In vitro GST pull down experiments supported a di. variety of mouse tissues with relatively higher expression in muscle rect interaction between KBTBD2 and p85 Fig 5D Human. Downloaded by guest on July 1 2020, liver brain iBAT heart and eWAT Fig S3B KBTBD2 also interacted with human p85 in differentiated adipocytes. Zhang et al PNAS Published online October 5 2016 E6421. Fig S4B Moreover two step IP demonstrated simultaneous and AKT S473 phosphorylation before and after i p injection. binding of p85 and Cul3 to KBTBD2 to form a three protein of insulin in WT or Kbtbd2 mice In WT iWAT insulin in. complex Fig 5E Coexpression of KBTBD2 p85 Cul3 and Ub duced tyrosine phosphorylation of IRS 1 and formation of a. in 293T cells resulted in p85 polyubiquitination which was re complex containing IRS 1 p110 and p85 Fig 6A In con. duced in the absence of Cul3 or KBTBD2 Fig 5F The BTB trast IRS 1 was associated with large amounts of p85 in. domain is critical for the recruitment of Cul3 Ub ligase complexes Kbtbd2 iWAT independent of insulin stimulation and p110. because KBTBD2 C failed to mediate p85 ubiquitination Fig bound minimally to IRS 1 either before or after insulin stimu. 5F These polyubiquitin chains were K48 linked Fig 5 F H lation Fig 6A Moreover phosphorylation of AKT S473 in. which serves as a degradation signal for the proteasome These creased in WT iWAT after insulin stimulation but this response. data suggest that KBTBD2 recruits p85 to Cul3 Ub ligase was diminished in Kbtbd2 iWAT Fig 6A We also observed. complexes for K48 linked ubiquitination leading to proteasome increased levels of insulin independent tyrosine phosphorylation. mediated degradation of p85 on IRS 1 in Kbtbd2 iWAT likely a result of elevated endog. enous insulin in the mice Fig 6A These data suggest that el. Impaired PI3K Signaling due to Elevated p85 in Kbtbd2 Mice evated p85 expression in Kbtbd2 adipose tissue results in. Based on its striking rescue of metabolic phenotypes we fo impaired PI3K signaling These signaling defects are likely adi. cused on adipose tissue to determine the effect of increased p85 pocyte intrinsic given that Kbtbd2 MEF derived adipocytes. expression on insulin signaling We examined Kbtbd2 adipose and human adipocytes in which KBTBD2 was knocked down. tissue in vivo for the association between PI3K subunits and IRS 1 also exhibited impaired AKT S473 phosphorylation in response. Insulin resistance and diabetes caused by genetic or diet induced KBTBD2 deficiency in mice Zhao Zhanga Emre Turera Xiaohong Lia Xiaoming Zhana Mihwa Choia Miao Tanga Amanda Pressa Steven R Smithb c Adeline Divouxb c Eva Marie Y Morescoa and Bruce Beutlera 1 aCenter for the Genetics of Host Defense University of Texas Southwestern Medical Center Dallas TX 75390 bTranslational

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