Mutant Insulin Receptor Research Articles

MON-159 Increased Gluconeogenesis and Lower Glycogenolysis in Patients with Non-Selective Compared Selective Insulin Resistance

Type 2 diabetes is characterized by increased hepatic glucose production and selective insulin resistance that occurs downstream of the insulin receptor. However, the differential pathways and exact molecules implicated in selective insulin signaling are unclear. To elucidate the hepatic mechanistic pathways affected by selective versus non-selective insulin resistance, we compared basal rates of hepatic glucose production and its components, gluconeogenesis and glycogenolysis, in patients with severe non-selective insulin resistance (insulin receptor mutations (INSR)) to patients with selective insulin resistance (lipodystrophy and type 2 diabetes). In 7 patients with INSR, 14 with lipodystrophy and 9 with type 2 diabetes, we measured rates of hepatic glucose production (via [6,6-2H2] glucose) and fractional gluconeogenesis (via 2H2O) after an overnight fast. Absolute rates of gluconeogenesis were calculated as the product of fractional gluconeogenesis and hepatic glucose production, and glycogenolysis were calculated as hepatic glucose production minus gluconeogenesis. In patients with INSR, lipodystrophy and type 2 diabetes, HbA1c (8.2 ± 2.8, 8.4 ± 1.9, 7.4 ± 0.9%) and fasting glucose (120 ± 70, 159 ± 55, 127 ± 32 mg/dl) were comparable (P>0.05). Fasting serum insulin was higher in INSR vs. lipodystrophy and type 2 diabetes (332 ± 355, 48 ± 50, 50 ± 16 μU/ml; P<0.01). Patients with INSR and lipodystrophy had higher hepatic glucose production vs. type 2 diabetes (24.0 ± 4.0, 24.1 ± 6.2, 15.8 ± 4.3 µmol/kgLEAN/min; P<0.01). Fractional gluconeogenesis was higher in INSR vs. lipodystrophy and type 2 diabetes (77.1 ± 9.4, 55.4 ± 11.5, 59.1 ± 5.9%; P<0.01). Absolute gluconeogenesis was highest in INSR, intermediate in lipodystrophy, and lowest in type 2 diabetes (19.1 ± 4.4, 13.5 ± 4.2, 9.5 ± 1.9 µmol/kgLEAN/min; P<0.01). Glycogenolysis was lowest in INSR, highest in lipodystrophy, and intermediate in type 2 diabetes (4.9 ± 2.2 vs. 10.6 ± 4.8, 6.3 ± 2.6 µmol/kgLEAN/min; P<0.01). Higher hepatic glucose production in lipodystrophy vs. type 2 diabetes is consistent with higher hepatic insulin resistance in patients with lipodystrophy, but comparable fractional rates of gluconeogenesis and glycogenolysis confirm similar mechanisms of selective post-receptor insulin resistance. By contrast, the markedly higher contribution of gluconeogenesis to hepatic glucose production in INSR may reflect complete loss of insulin signaling for glycogen accumulation and/or suppression of gluconeogenesis. Further studies should evaluate whether greater substrate and fuel availability for gluconeogenesis, and/or lower hepatic glycogen stores are important determinants of the components of glucose production in patients with non-selective insulin resistance due to INSR mutation.

SAT-293 Novel Presentation Of A Heterozygous INSR Mutation And Markedly Elevated Testosterone Levels In A Pediatric Patient, A Case Report.

Background: Type A Insulin Resistance Syndrome is a rare disorder characterized by severe insulin resistance. Affected females may present with primary amenorrhea, oligomenorrhea, cystic ovaries, hirsutism, and acanthosis nigricans in the absence of obesity. Case: A 14y0m female was seen in endocrinology clinic for the possibility of PCOS due to primary amenorrhea and hirsutism. Mom with history of PCOS. Patient had onset of breast development at age 11, body odor age 12, pubic hair age 12, axillary hair age 12, without menarche or menstrual cramping. She had mild facial acne treated with over the counter facial washes. She noticed hair growth on her face, arms, and stomach since turning 13yo which was gradually getting worse. On physical exam, she was an average sized female with BMI at the 75%. She had a Ferriman Gallwey score of 31, moderate facial acne, acanthosis nigricans, and a deep voice. She had a testosterone of 301ng/dL, free testosterone 39pg/mL, DHEAS 137mcg/dL, androstenedione 475ng/dL, 17-OH-progesterone 127ng/dL, HgA1C 4.8%, and glucose 77mg/dL. Due to the degree of hyperandrogenemia, additional studies were performed to include 12am salivary cortisol <50ng/dL, ACTH stimulation test (not diagnostic for congenital adrenal hyperplasia), OGTT with fasting insulin 131 uIU/mL and 2hour insulin >300uIU/mL, and adiponectin 17mcg/mL. She had normal adrenal ultrasound and MRI. She had pelvic ultrasound with prominent ovarian sizes with numerous follicles. She had genetic testing for an insulin receptor mutation which confirmed suspicions for type A insulin resistance syndrome. INSR molecular analysis revealed heterozygous pathogenic mutation (c.3602G>A; p.Arg1201Gln). She was treated with metformin, drospirenone-ethinyl estradiol, and laser hair removal with improvement in clinical and biochemical features. Conclusion: This case highlights that significantly elevated androgen levels, specifically testosterone, can exceed values much higher than those currently reported or known to be associated with the various INSR mutations causing Type A Insulin Resistance Syndrome. We hope this case will serve to add knowledge and diversity to the variable clinical findings and outcomes, related to Type A Insulin Resistance, especially in the pediatric population.

The Effect of Inulin on Lifespan, Related Gene Expression and Gut Microbiota in InRp5545/TM3 Mutant Drosophila melanogaster: A Preliminary Study.

Inulin is considered an efficient prebiotic and is beneficial for metabolic diseases via promoting intestinal probiotic enrichment and the metabolites of short-chain fatty acids (SCFAs). However, the effect of inulin on patients with InR deficiencies has seldom been reported. In this study, the lifespan, related gene expression, and gut microbiota of InRp5545/TM3 (insulin receptor mutant) Drosophila melanogaster under inulin treatment were investigated. The results showed that the lifespan was extended in only males and not in females. Furthermore, distinctly different patterns of gene expression were found between males and females, especially in the insulin/insulin-like growth factor (IGF)-like signalling (IIS) and target of rapamycin (TOR) pathways. Additionally, as a link between inulin and lifespan responses, the gut microbiota was distinctly separated by gender in both the standard diet group and the inulin treatment group, and the relationship between lifespan and the gut microbiota community was stronger in male flies than in females. This study provides preliminary evidence for the gender-dependent lifespan responses to inulin in insulin signalling-deficient Drosophila. However, controls such as wild-type and TM3 flies, and more InR mutant strains with different genetic backgrounds need to be further investigated to elucidate the mechanisms underlying the phenomenon.

Thyroid Abnormalities in Patients With Extreme Insulin Resistance Syndromes.

Insulin and leptin may increase growth and proliferation of thyroid cells, underlying an association between type 2 diabetes and papillary thyroid cancer (PTC). Patients with extreme insulin resistance due to lipodystrophy or insulin receptor mutations (INSR) are treated with high-dose insulin and recombinant leptin (metreleptin), which may increase the risk of thyroid neoplasia. The aim of this study was to analyze thyroid structural abnormalities in patients with lipodystrophy and INSR mutations and to assess whether insulin, IGF-1, and metreleptin therapy contribute to the thyroid growth and neoplasia in this population. Thyroid ultrasound characteristics were analyzed in 81 patients with lipodystrophy and 11 with INSR (5 homozygous; 6 heterozygous). Sixty patients were taking metreleptin. The prevalence of thyroid nodules in children with extreme insulin resistance (5 of 30, 16.7%) was significantly higher than published prevalence for children (64 of 3202; 2%), with no difference between lipodystrophy and INSR. Body surface area-adjusted thyroid volume was larger in INSR homozygotes vs heterozygotes or lipodystrophy (10.4 ± 5.1, 3.9 ± 1.5, and 6.2 ± 3.4 cm2, respectively. Three patients with lipodystrophy and one INSR heterozygote had PTC. There were no differences in thyroid ultrasound features in patients treated vs not treated with metreleptin. Children with extreme insulin resistance had a high prevalence of thyroid nodules, which were not associated with metreleptin treatment. Patients with homozygous INSR mutation had thyromegaly, which may be a novel phenotypic feature of this disease. Further studies are needed to determine the etiology of thyroid abnormalities in patients with extreme insulin resistance.

Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis.

The insulin‐IGF‐1/DAF‐2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS‐22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS‐22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long‐lived daf‐2 insulin receptor mutants show remarkable low levels of INS‐22::Venus secretion. In contrast, we found that short‐lived mutant worms that lack the oxidation repair enzyme MSRA‐1 show increased levels of INS‐22::Venus secretion, specifically during the reproductive stage. MSRA‐1 is a target of the insulin‐IGF‐1/DAF‐2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra‐1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin‐IGF‐1/DAF‐2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.

Insulin resistance: Review of the underlying molecular mechanisms.

Most human cells utilize glucose as the primary substrate, cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR). IR underpins many metabolic disorders such as type 2 diabetesand metabolic syndrome, impairments in insulin signaling disrupting entry of glucose into the adipocytes, and skeletal muscle cells. Although the exact underlying cause of IR has not been fully elucidated, a number of major mechanisms, including oxidative stress, inflammation, insulin receptor mutations, endoplasmic reticulum stress, and mitochondrial dysfunction have been suggested. In this review, we consider the role these cellular mechanisms play in the development of IR.

Ribosomal protein L10A and signaling pathway

Ribosome: machinery in control of messenger RNA's (mRNAs) and several ribosomal proteins are in the small and large subunit of the ribosome. Various aspects of ribosomal proteins have related to cell growth, cell cycle, and diseases. Ribosomal protein L10A (RpL10A) in shrimp and fruit fly has been demonstrated to play a role in oogenesis. Interestingly, deletion RpL10A gene (RpL10Ab−/−) germ line clone of the fruit fly showed a loss of follicle cells surrounding the egg chamber, but nurse cells appeared normal. This phenotype is reminiscent of insulin receptor mutants (InR−/−). In contrast, over-expression of RpL10A in the eyes of the fruit flies resulted in abnormal ommatidia with a loss of red pigment in the center of the eyes. In this study, the abnormal rearrangements of nuclei and lack of cell membranes in those eyes were demonstrated. Furthermore, the expression of InR gene and the InR protein were extensively increased as determined by real-time PCR and immunohistochemistry, respectively. In addition, some insulin signaling mediators were also detected. The Akt and FOXO proteins were highly phosphorylated in the RpL10A over-expressed mutant. The results revealed that RpL10A induced over-expression of the insulin receptor and consequently activated in insulin signaling pathway which affects cell proliferation and we suggest RpL10A stimulates cell proliferation via the insulin signaling pathway.

Genotype–Phenotype Correlations and Structural Basis of INSR and IGF1R Mutations Causing Severe Insulin/IGF-1 Resistance

Mutations of the insulin receptor (INSR) gene cause severe insulin resistance, resulting in several syndromes with various abnormal phenotypes associated with variable severity of INSR dysfunction. As a member of the insulin receptor family, the IGF-I receptor (IGF1R) shows the same domain structure as INSR containing the fibronectin type III (FnIII) domains where there are an α-β cleavage site and a part of the ligand binding site. Mutations of IGF1R can cause intrauterine and postnatal growth retardation. We compiled 3 mutations of the FnIII-2 and FnIII-3 domains in IGF1R registered in HGMD, ClinVar and UniProt databases as causing growth retardation as well as the SNP with &amp;gt;1% allele frequency in IGF1R from the 1000 Genomes Project data, ESP6500, HGVD and 3.5KJPN, a reference panel of Japanese individuals. We conducted in silico structural analysis of the mutations of the FnIII domains in IGF1R which revealed that the pathogenic mutations were predicted to severely impair stability of the FnIII domains and hydrophobic core formation or affect processing site, while the SNP was predicted not to affect folding of the domains. In addition, IGF-1 proreceptor processing was impaired in cells expressing mutations of FnIII compared to cells expressing WT form. Previously, we performed structural analysis of mutations of the FnIII domains in INSR, which revealed that missense mutations causing the most severe Donohue syndrome were predicted to severely impair hydrophobic core formation and stability of the FnIII domains, while those causing less severe Rabson-Mendenhall syndrome were predicted to lead to localized destabilization and not to affect folding of the FnIII domains. Genotype-structure-phenotype correlations of variants of the FnIII domains in INSR and IGF1R identified in these analyses will enable early diagnosis of pathogenic mutations in INSR and IGF1R and provide a valuable insight into the molecular mechanism of severe insulin or IGF-1 resistance. Disclosure J. Hosoe: None. H. Kadowaki: None. F. Miya: None. M. Takakura: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Novo Nordisk Inc., Daiichi Sankyo Company, Limited, Sanofi, Takeda, Tanabe-Mitsubishi. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp &amp; Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp &amp; Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp &amp; Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

Modeling the Spectrum of Human Insulin Resistance Using Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) represent a unique model to study the genetic components of insulin resistant states in vitro and can be studied in both the undifferentiated and differentiated state. In prior studies we have shown that iPSCs from patients with insulin receptor mutations exhibit altered insulin signaling with reduced insulin-stimulated phosphorylation of IR, IRS, and AKT in iPS-derived myotubes compared to healthy subjects and have reduced insulin stimulation of proliferation and gene expression. We now use iPSCs to characterize the insulin response of type 2 diabetics (T2D) and nondiabetic individuals before and after differentiation to skeletal muscle. The iPSCs from a subgroup of the nondiabetic individuals were further divided into those in the upper and lower quartiles of insulin resistance as determined using steady-state plasma glucose (SSPG) methodology. We find that iPSCs from insulin sensitive controls, insulin resistant (IR) nondiabetics and from patient with type 2 diabetes exhibited similar levels of pluripotency markers and showed similar changes in markers of muscle differentiation through the satellite cell-like and myoblast/myotube stage. This was true for iPSC derived from blood cells and from muscle biopsies. Insulin-induced activation of early signaling steps including phosphorylation of IR, IRS, AKT, ERK and GSK3 showed dose-responsive phosphorylation in the iPSC, as well as myotube stage with significant 10 nM and 100 nM insulin. Importantly, iPSCs differentiated into satellite-like cells displayed insulin resistance at the level of AKT phosphorylation in both the T2D and IR patients (p=0.0134 and p=0.0352). There was also decreased GSK3 phosphorylation in cells from T2D patients (p=0.0325). Thus, iPSC-derived muscle satellite-like cells from T2D and nondiabetic IR individuals retain defects in AKT and GSK3 phosphorylation indicating a level of a genetic or persistent epigenetic signature of insulin resistance in vitro. Disclosure J. Lebastchi: None. T.M. Batista: None. I. Carcamo-Orive: None. A. Krook: None. J.R. Zierath: None. J. Knowles: None. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.

Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging.

Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.

Novel heterozygous mutations of the INSR gene in a familial case of Donohue syndrome

Donohue syndrome (DS), a rare autosomal recessive disease which represents severe insulin resistance, pre- and postnatal growth retardation, hypertrichosis, and dysmorphic features, is caused by mutations in the insulin receptor (INSR) gene. Here, we have reported the clinical, molecular, and biochemical characterizations of a patient with DS. In this article, we have also reported a case with 2 novel INSR mutations and the DS phenotype. Using next-generation sequencing (NGS), we screened 27 known genes involved in inherited maturity-onset diabetes of the young (MODY) and identified compound heterozygous mutations in the INSR gene in the patient with DS, c.62T>G (p.L21R) and c.2563G>T (p.V855F). The positive correlation of these mutations with DS was further validated by Sanger DNA sequencing of his lineal consanguinity, indicating that these pathogenic mutations were inherited maternally and paternally, respectively. Therefore, our finding expanded the number of reported cases of this rare disease and the mutation spectrum of INSR mutation, suggesting that NGS is an accurate, rapid, and cost-effective method for the genetic diagnosis of this rare disease.

Structural Basis and Genotype-Phenotype Correlations of INSR Mutations Causing Severe Insulin Resistance.

The insulin receptor (INSR) gene was analyzed in four patients with severe insulin resistance, revealing five novel mutations and a deletion that removed exon 2. A patient with Donohue syndrome (DS) had a novel p.V657F mutation in the second fibronectin type III domain (FnIII-2), which contains the α-β cleavage site and part of the insulin-binding site. The mutant INSR was expressed in Chinese hamster ovary cells, revealing that it reduced insulin proreceptor processing and impaired activation of downstream signaling cascades. Using online databases, we analyzed 82 INSR missense mutations and demonstrated that mutations causing DS were more frequently located in the FnIII domains than those causing the milder type A insulin resistance (P = 0.016). In silico structural analysis revealed that missense mutations predicted to severely impair hydrophobic core formation and stability of the FnIII domains all caused DS, whereas those predicted to produce localized destabilization and to not affect folding of the FnIII domains all caused the less severe Rabson-Mendenhall syndrome. These results suggest the importance of the FnIII domains, provide insight into the molecular mechanism of severe insulin resistance, will aid early diagnosis, and will provide potential novel targets for treating extreme insulin resistance.

Latent Inflammation and Insulin Resistance in Adipose Tissue.

Obesity is a growing problem in modern society and medicine. It closely associates with metabolic disorders such as type 2 diabetes mellitus (T2DM) and hepatic and cardiovascular diseases such as nonalcoholic fatty liver disease, atherosclerosis, myocarditis, and hypertension. Obesity is often associated with latent inflammation; however, the link between inflammation, obesity, T2DM, and cardiovascular diseases is still poorly understood. Insulin resistance is the earliest feature of metabolic disorders. It mostly develops as a result of dysregulated insulin signaling in insulin-sensitive cells, as compared to inactivating mutations in insulin receptor or signaling proteins that occur relatively rare. Here, we argue that inflammatory signaling provides a link between latent inflammation, obesity, insulin resistance, and metabolic disorders. We further hypothesize that insulin-activated PI3-kinase pathway and inflammatory signaling mediated by several IκB kinases may constitute negative feedback leading to insulin resistance at least in the fat tissue. Finally, we discuss perspectives for anti-inflammatory therapies in treating the metabolic diseases.

Eicosapentaenoic acid ameliorates hyperglycemia in high-fat diet-sensitive diabetes mice in conjunction with restoration of hypoadiponectinemia.

Background/Objective:Eicosapentaenoic acid (EPA) exerts pleiotropic effects on metabolic disorders such as atherosclerosis and dyslipidemia, but its effectiveness in the treatment of type 2 diabetes mellitus remains controversial.Methods:We examined the antidiabetic effect of EPA in insulin receptor mutant (InsrP1195L/+) mice that exhibit high-fat diet (HFD)-dependent hyperglycemia.Results:EPA supplementation was found to alleviate hyperglycemia of InsrP1195L/+ mice fed HFD (InsrP1195L/+/HFD mice), which was accompanied by amelioration of increased gluconeogenesis and impaired insulin signaling, as assessed by glucose-6-phosphatase (G6pc) expression on refeeding and insulin-induced phosphorylation of Akt in the liver, respectively. We found that serum levels of adiponectin, the antidiabetic adipokine, were decreased by HFD along with the body weight gain in InsrP1195L/+ mice but not in wild-type mice, suggesting that InsrP1195L/+ mice are prone to hypoadiponectinemia in response to obesity. Interestingly, the blood glucose levels of InsrP1195L/+ mice were in reverse proportion to their serum adiponectin levels and EPA supplementation ameliorated their hyperglycemia in conjunction with the restoration of hypoadiponectinemia.Conclusions:EPA exerts an antidiabetic effect in InsrP1195L/+/HFD mice, an HFD-sensitive, insulin-resistant animal model, possibly through its action against hypoadiponectinemia.

Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans.

Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB) receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-β ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans.

Unsuppressed lipolysis in adipocytes is linked with enhanced gluconeogenesis and altered bile acid physiology in Insr(P1195L/+) mice fed high-fat-diet.

High-fat diet (HFD) triggers insulin resistance and diabetes mellitus, but their link remains unclear. Characterization of overt hyperglycemia in insulin receptor mutant (InsrP1195L/+) mice exposed to HFD (InsrP1195L/+/HFD mice) revealed increased glucose-6-phosphatase (G6pc) expression in liver and increased gluconeogenesis from glycerol. Lipolysis in white adipose tissues (WAT) and lipolysis-induced blood glucose rise were increased in InsrP1195L/+/HFD mice, while wild-type WAT transplantation ameliorated the hyperglycemia and the increased G6pc expression. We found that the expressions of genes involved in bile acid (BA) metabolism were altered in InsrP1195L/+/HFD liver. Among these, the expression of Cyp7a1, a BA synthesis enzyme, was insulin-dependent and was markedly decreased in InsrP1195L/+/HFD liver. Reduced Cyp7a1 expression in InsrP1195L/+/HFD liver was rescued by WAT transplantation, and the expression of Cyp7a1 was suppressed by glycerol administration in wild-type liver. These findings suggest that unsuppressed lipolysis in adipocytes elicited by HFD feeding is linked with enhanced gluconeogenesis from glycerol and with alterations in BA physiology in InsrP1195L/+/HFD liver.

C. elegans maximum velocity correlates with healthspan and is maintained in worms with an insulin receptor mutation.

Ageing is marked by physical decline. Caenorhabditis elegans is a valuable model for identifying genetic regulatory mechanisms of ageing and longevity. Here we report a simple method to assess C. elegans' maximum physical ability based on the worms' maximum movement velocity. We show maximum velocity declines with age, correlates well with longevity, accurately reports movement ability and, if measured in mid-adulthood, is predictive of maximal lifespan. Contrary to recent findings, we observe that maximum velocity of worm with mutations in daf-2(e1370) insulin/IGF-1 signalling scales with lifespan. Because of increased odorant receptor expression, daf-2(e1370) mutants prefer food over exploration, causing previous on-food motility assays to underestimate movement ability and, thus, worm health. Finally, a disease-burden analysis of published data reveals that the daf-2(e1370) mutation improves quality of life, and therefore combines lifespan extension with various signs of an increased healthspan.

Severe insulin resistance alters metabolism in mesenchymal progenitor cells.

Donohue syndrome (DS) is characterized by severe insulin resistance due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in DS in the undifferentiated state, we generated mesenchymal progenitor cells (MPCs) from induced pluripotent stem cells derived from a 4-week-old female with DS and a healthy newborn male (control). INSR mRNA and protein were significantly reduced in DS MPC (for β-subunit, 64% and 89% reduction, respectively, P < .05), but IGF1R mRNA and protein did not differ vs control. Insulin-stimulated phosphorylation of INSR or the downstream substrates insulin receptor substrate 1 and protein kinase B did not differ, but ERK phosphorylation tended to be reduced in DS (32% decrease, P = .07). By contrast, IGF-1 and insulin-stimulated insulin-like growth factor 1 (IGF-1) receptor phosphorylation were increased in DS (IGF-1, 8.5- vs 4.5-fold increase; INS, 11- vs 6-fold; P < .05). DS MPC tended to have higher oxygen consumption in both the basal state (87% higher, P =.09) and in response to the uncoupler carbonyl cyanide-p-triflouromethoxyphenylhydrazone (2-fold increase, P =.06). Although mitochondrial DNA or mass did not differ, oxidative phosphorylation protein complexes III and V were increased in DS (by 37% and 6%, respectively; P < .05). Extracellular acidification also tended to increase in DS (91% increase, P = .07), with parallel significant increases in lactate secretion (34% higher at 4 h, P < .05). In summary, DS MPC maintain signaling downstream of the INSR, suggesting that IGF-1R signaling may partly compensate for INSR mutations. However, alterations in receptor expression and pathway-specific defects in insulin signaling, even in undifferentiated cells, can alter cellular oxidative metabolism, potentially via transcriptional mechanisms.

Fatty acylated caveolin-2 is a substrate of insulin receptor tyrosine kinase for insulin receptor substrate-1-directed signaling activation

Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated.

Effect of insulin on AKR1C3 expression in female adipose tissue: in-vivo and in-vitro study of adipose androgen generation in polycystic ovary syndrome

BackgroundInsulin resistance and hyperandrogenism are the cardinal features of polycystic ovary syndrome (PCOS). Women with insulin receptor (INSR) mutations develop severe hyperandrogenism secondary to hyperinsulinaemia. We hypothesised that insulin might drive adipose testosterone generation from androstenedione through aldoketoredutase type 3 (AKR1C3) in women with insulin resistance. Here we assessed the effect of insulin on AKR1C3 activity in vivo and in vitro. MethodsTen women with PCOS, ten healthy controls, and three women with INSR mutations underwent dehydroepiandrosterone (DHEA) challenge; serum androgens were measured every 30 min for 4 h after ingestion of 100 mg DHEA. Additionally, paired subcutaneous and omental fat samples were obtained during abdominal surgery from 38 women. AKR1C3 expression was measured by real-time PCR. Serum steroids and cultured cell media androgens were measured with liquid chromatography tandem mass spectrometry. FindingsWomen with PCOS had higher androstenedione concentrations than did controls and women with INSR mutations (p=0·01 and p=0·005, respectively). However, area under the curve for testosterone was higher in women with INSR mutations after DHEA than in women with PCOS and controls (874·2 [SE 242] vs 425 [136] and 375·2 [109], p<0·001 for both). AKR1C3 mRNA expression was significantly higher in subcutaneous than in omental adipose tissue (p=0·004). AKR1C3 expression correlated positively with body-mass index in subcutaneous fat (Spearman correlation=0·51, p=0·006). Insulin significantly increased AKR1C3 expression in differentiated subcutaneous adipocytes (p=0·04). Incubation with insulin significantly increased testosterone generation from androstenedione in cultured subcutaneous cell media compared with controls (p<0·001). InterpretationWe have found in-vivo and in-vitro evidence of modulation of AKR1C3 activity by insulin in PCOS and in women with INSR mutations. Insulin seems to drive adipose androgen generation by increasing AKR1C3 activity in female subcutaneous adipose tissue. Selective AKR1C3 inhibition might offer a novel therapeutic target to reduce androgen burden and improve metabolic phenotype in PCOS. FundingWellcome Trust.