Skeletal muscle proteomes reveal downregulation of mitochondrial proteins in transition from prediabetes into type 2 diabetes

Skeletal muscle insulin resistance is a central defect in the pathogenesis of type 2 diabetes (T2D). Here, we analyzed skeletal muscle proteome in 148 vastus lateralis muscle biopsies obtained from men covering all glucose tolerance phenotypes: normal, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and T2D. Skeletal muscle proteome was analyzed by a sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics technique. Our data indicate a downregulation in several proteins involved inmitochondrial electron transport or respiratory chain complex assembly already in IFG and IGT-muscles, with most profound decreases observed in T2D. Additional phosphoproteomic analysis reveals altered phosphorylation in several signaling pathways in IFG, IGT, and T2D muscles, including those regulating glucose metabolic processes, and the structure of muscle cells. These data reveal several alterations present in skeletalmuscle already in prediabetes and highlight impairedmitochondrial energy metabolism in the trajectory from prediabetes into T2D.Peer reviewe

Send in the signals : studies on the mechanisms of insulin resistance in human skeletal muscle

Insulin resistance is a pathophysiological condition characterized by an impaired ability of insulin-sensitive tissues to respond normally to insulin stimulation. Insulin resistance is an early defect in the pathogenesis of type 2 diabetes (T2D), leading to impaired glucose disposal and contributing to elevated plasma glucose concentrations. There are a number of intracellular mechanisms underlying insulin resistance. These range from dysregulated nutrient metabolism to mitochondrial energy imbalance, oxidative stress, and inflammation. These cellular perturbations in turn stem from various etiologies, including obesity or excess fat consumption as well as the use of certain medications. Moreover, there are several gene variants associated with insulin resistance and the increased risk for type 2 diabetes. As skeletal muscle accounts for 80% of glucose disposal under insulin stimulation, it is a primary determinant of whole-body glycemic control. Therefore, the primary skeletal muscle cell cultures established from human muscle biopsies were utilized in this thesis as a study model to examine the impact of fatty acids (I), simvastatin (II), and AKT2 gene variant (III) on glucose metabolism and cell signaling events. Excess dietary saturated fatty acids have been implicated in the development of insulin resistance, whereas unsaturated fatty acids may have a protective effect on metabolism. In study I, we have investigated if insulin resistance induced by saturated fatty acid palmitate can be alleviated by simultaneous exposure to monounsaturated fatty acid oleate. We found that exposure of primary human myotubes to palmitate impaired insulin signaling, increased ER stress, and activated stress kinase JNK. These negative effects were restored by co-incubation with oleate. However, co-exposure to palmitate and oleate impaired insulin action on glucose uptake. This was related to increased mitochondrial ROS production and was ameliorated with antioxidants. Simvastatin use has been associated with an increased risk for T2D. In clinical use, simvastatin is administered as an inactive lipophilic lactone-form, which is then converted to an active acid-form in the body. Statins cause variable degrees of muscular side effects. Especially the lactone-form statins have been observed to be more prone to cause myotoxic effects. In study II, we have investigated if simvastatin affects glucose metabolism and mitochondrial respiration. Moreover, we have investigated if lactone- and acid-forms of simvastatin exert similar effects. We found that lactone- and acid-forms of simvastatin exhibited differential effects on non-oxidative glucose metabolism, as the lactone-form increased, and the acid-form reduced insulin-stimulated glucose storage into glycogen. This suggests impaired insulin sensitivity in response to acid-form simvastatin. Both forms of simvastatin profoundly inhibited glycolysis and mitochondrial energy production. These effects may contribute to the muscular side effects and the risk for type 2 diabetes observed with simvastatin use. Finnish-specific gene variant in insulin signaling target AKT2 has been associated with increased predisposition to type 2 diabetes and in vivo insulin resistance in humans. In study III, we have established primary human muscle cells from 14 AKT2 variant carriers and 14 controls to investigate in vitro the impact of the gene variant on glucose metabolism and cell signaling events. The AKT2 gene variant led to impaired activation of the insulin signaling pathway, which may be a result of a defective binding of the variant AKT2-PH domain to PI(3,4,5)P3. In addition, AKT2 variant myotubes demonstrated reduced insulin-stimulated glycolysis. Global kinase activity profiling (PamGene®) revealed multiple differentially phosphorylated kinase substrates in insulin-stimulated myotubes from variant carriers. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating in intracellular signal transduction, protein translation, and cell cycle events. In conclusion, p.P50T/AKT2 variant myotubes show alterations in several cellular signaling networks in vitro, which may contribute to the increased risk for skeletal muscle insulin resistance and T2D in the carriers of this signaling variant. The understanding of the various mechanisms of insulin resistance in skeletal muscle is far from complete. This thesis provides novel data on the events in cell metabolism and intracellular signaling which may contribute to the development of insulin resistance in human skeletal muscle and predispose to the development of type 2 diabetes.Insuliiniresistenssi on patofysiologinen tila, jolle on tunnusomaista insuliiniherkkien kudosten heikentynyt kyky vastata normaalisti insuliinistimulaatioon. Insuliiniresistenssi on varhainen häiriö tyypin 2 diabeteksen (T2D) patogeneesissä, ja johtaa heikentyneeseen glukoosin soluunottoon ja myötävaikuttaa kohonneeseen plasman glukoosipitoisuuteen. Insuliiniresistenssin taustalla on useita solunsisäisiä mekanismeja, jotka vaihtelevat ravintoaineenvaihdunnan säätelyn häiriöistä epätasapainoon mitokondrioiden energiataloudessa, oksidatiiviseen stressiin ja tulehdukseen. Nämä soluhäiriöt puolestaan johtuvat erilaisista syistä, kuten liikalihavuudesta tai liiallisesta rasvan saannista sekä joidenkin lääkkeiden käytöstä. Lisäksi on tunnistettu useita geenivariantteja, jotka liittyvät insuliiniresistenssiin ja lisääntyneeseen T2D:n riskiin. Koska luurankolihas vastaa 80 % glukoosin käytöstä insuliinistimulaation aikana, on se ensisijainen koko kehon glukoosiaineenvaihdunnan tasapainoa määrittävä tekijä. Siksi hyödynsimme tämän väitöskirjan tutkimusmallina ihmisen lihasbiopsioista johdettuja primaareja lihassoluviljelmiä tutkiaksemme rasvahappojen (I), simvastatiinin (II) ja AKT2-geenivariantin (III) vaikutusta glukoosiaineenvaihduntaan sekä solujen signalointitapahtumiin. Ravinnon liiallisen tyydyttyneiden rasvahappojen määrän on todettu vaikuttavan insuliiniresistenssin kehittymiseen, kun taas tyydyttymättömillä rasvahapoilla voi olla suojaava vaikutus aineenvaihduntaan. Tutkimuksessa I olemme selvittäneet, voidaanko tyydyttyneen rasvahapon, palmitaatin, aiheuttamaa insuliiniresistenssiä lievittää altistamalla erilaistetut lihassolut samanaikaisesti kertatyydyttymättömälle rasvahapolle, oleaatille. Havaitsimme, että lihassolujen altistuminen palmitaatille heikensi insuliinin signaalinsiirtoa, lisäsi ER-stressiä ja aktivoi stressikinaasi JNK:ta. Nämä negatiiviset vaikutukset hävisivät, kun lihassoluja altistettiin samanaikaisesti palmitaatille ja oleaatille. Samanaikainen altistuminen palmitaatille ja oleaatille heikensi kuitenkin insuliinin vaikutusta glukoosin soluunottoon. Tähän liittyi mitokondrioiden lisääntynyt ROS-tuotanto ja sitä voitiin helpottaa antioksidanteilla. Simvastatiinin käyttöön on todettu liittyvän kohonnut T2D:n riski. Kliinisessä käytössä simvastatiinia annetaan inaktiivisena lipofiilisenä laktonimuotona, joka muuttuu kehossa aktiiviseksi happomuodoksi. Statiinit voivat aiheuttaa lihaksissa eriasteisia haitallisia sivuvaikutuksia. Erityisesti laktonimuotoisten statiinien on havaittu olevan taipuvaisempia aiheuttamaan lihastoksisuutta. Tutkimuksessa II olemme selvittäneet, vaikuttaako simvastatiini primaarien lihassolujen glukoosiaineenvaihduntaan ja mitokondriohengitykseen. Lisäksi olemme selvittäneet vaikuttavatko simvastatiinin laktoni- ja happomuodot samankaltaisesti. Havaitsimme eri simvastatiinimuodoilla olevan erilainen vaikutus ei-hapettavaan glukoosiaineenvaihduntaan: laktonimuotoinen simvastatiini lisäsi ja happomuotoinen vähensi insuliinistimuloitua glukoosin varastointia glykogeeniksi. Tämä viittaa siihen, että happomuotoinen simvastatiini heikentää lihasten insuliiniherkkyyttä. Molemmat simvastatiinimuodot kuitenkin heikensivät huomattavasti glykolyysiä ja mitokondrioiden energiantuotantoa. Nämä vaikutukset voivat myötävaikuttaa simvastatiinin käytön yhteydessä havaittuihin lihasperäisiin sivuvaikutuksiin sekä T2D:n riskiin. Suomalainen geenimuunnos insuliinin signaalisiirtoketjun kohdetta koodaavassa AKT2-geenissä on yhdistetty lisääntyneeseen T2D:n riskiin sekä in vivo insuliiniresistenssiin ihmisillä. Tutkimuksessa III olemme luoneet ihmisen primaarit lihassoluviljelmät 14 AKT2-variantin kantajasta ja 14 kontrollihenkilöstä tutkiaksemme geenivariantin vaikutusta glukoosiaineenvaihduntaan ja solujen signalointitapahtumiin in vitro. Havaitsimme, että AKT2-variantin kantajien lihassoluissa insuliinin signaalinsiirto oli heikentynyt, mikä saattaa johtua havaitusta variantti AKT2-PH-domeenin puutteellisesta sitoutumisesta PI(3,4,5)P3:een. Lisäksi osoitimme, että AKT2-variantin lihassoluissa insuliinistimuloitu glykolyysi oli alentunut verrattuna kontrollisoluihin. Globaalin kinaasiaktiivisuuden profilointi, käyttäen PamGene®-menetelmää, paljasti useita erilaisesti fosforyloituja kinaasisubstraatteja AKT2-variantin kantajien insuliinilla stimuloiduissa lihassoluissa. Ylävirran kinaasiaktiivisuuden in silico-analyysi ennusti laaja-alaista toiminnan heikkenemistä kinaaseissa, jotka osallistuvat muun muassa solunsisäiseen signaalinsiirtoon, proteiinien translaatioon sekä solusyklitapahtumiin. Nämä useiden eri signaalinsiirtoverkostojen muutokset variantin kantajien lihassoluissa saattavat myötävaikuttaa p.P50T/AKT2-variantin kantajien suurentuneeseen lihaksen insuliiniresistenssin ja tyypin 2 diabeteksen riskiin. Lihaskudoksen insuliiniresistenssin eri mekanismien ymmärtäminen on vielä puutteellista. Tämä väitöskirjatyö tuottaa uutta tietoa lihassolujen aineenvaihdunnan ja solunsisäisen signaalinsiirron tapahtumista, jotka voivat vaikuttaa insuliiniresistenssin kehittymiselle ihmisen luurankolihaksessa ja altistaa tyypin 2 diabeteksen kehittymiselle

Simvastatin profoundly impairs energy metabolism in primary human muscle cells

Correction: Volume11, Issue: 7 Article Number: x200444 DOI: 10.1530/EC-20-0444e Published: JUL 2022Objectives: Simvastatin use is associated with muscular side effects, and increased risk for type 2 diabetes (T2D). In clinical use, simvastatin is administered in inactive lipophilic lactone-form, which is then converted to active acid-form in the body. Here, we have investigated if lactone- and acid-form simvastatin differentially affect glucose metabolism and mitochondrial respiration in primary human skeletal muscle cells. Methods: Muscle cells were exposed separately to lactone- and acid-form simvastatin for 48 h. After pre-exposure, glucose uptake and glycogen synthesis were measured using radioactive tracers; insulin signalling was detected with Western blotting; and glycolysis, mitochondrial oxygen consumption and ATP production were measured with Seahorse XF(e)96 analyzer. Results: Lactone-form simvastatin increased glucose uptake and glycogen synthesis, whereas acid-form simvastatin did not affect glucose uptake and decreased glycogen synthesis. Phosphorylation of insulin signalling targets Akt substrate 160 kDa (AS160) and glycogen synthase kinase 3 beta (GSK3 beta) was upregulated with lactone-, but not with acid-form simvastatin. Exposure to both forms of simvastatin led to a decrease in glycolysis and glycolytic capacity, as well as to a decrease in mitochondrial respiration and ATP production. Conclusions: These data suggest that lactone- and acid-forms of simvastatin exhibit differential effects on non-oxidative glucose metabolism as lacto ne-form increases and acid-form impairs glucose storage into glycogen, suggesting impaired insulin sensitivity in response to acid-form simvastatin. Both forms profoundly impair oxidative glucose metabolism and energy production in human skeletal muscle cells. These effects may contribute to muscular side effects and risk for T2D observed with simvastatin use.Peer reviewe

Finnish-specific AKT2 gene variant leads to impaired insulin signalling in myotubes

Finnish-specific gene variant p.P50T/AKT2 (minor allele frequency (MAF) = 1.1%) is associated with insulin resistance and increased predisposition to type 2 diabetes. Here, we have investigated in vitro the impact of the gene variant on glucose metabolism and intracellular signalling in human primary skeletal muscle cells, which were established from 14 male p.P50T/AKT2 variant carriers and 14 controls. Insulin-stimulated glucose uptake and glucose incorporation into glycogen were detected with 2-[1,2-H-3]-deoxy-D-glucose and D-[C-14]-glucose, respectively, and the rate of glycolysis was measured with a Seahorse XF(e)96 analyzer. Insulin signalling was investigated with Western blotting. The binding of variant and control AKT2-PH domains to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P-3) was assayed using PIP Strips(TM) Membranes. Protein tyrosine kinase and serine-threonine kinase assays were performed using the PamGene (R) kinome profiling system. Insulin-stimulated glucose uptake and glycogen synthesis in myotubes in vitro were not significantly affected by the genotype. However, the insulin-stimulated glycolytic rate was impaired in variant myotubes. Western blot analysis showed that insulin-stimulated phosphorylation of AKT-Thr(308), AS160-Thr(642) and GSK3 beta-Ser(9) was reduced in variant myotubes compared to controls. The binding of variant AKT2-PH domain to PI(3,4,5)P-3 was reduced as compared to the control protein. PamGene (R) kinome profiling revealed multiple differentially phosphorylated kinase substrates, e.g. calmodulin, between the genotypes. Further in silico upstream kinase analysis predicted a large-scale impairment in activities of kinases participating, for example, in intracellular signal transduction, protein translation and cell cycle events. In conclusion, myotubes from p.P50T/AKT2 variant carriers show multiple signalling alterations which may contribute to predisposition to insulin resistance and T2D in the carriers of this signalling variant.Peer reviewe

A Strategy for Discovery of Endocrine Interactions with Application to Whole-Body Metabolism.

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as beiging. We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions

A Strategy for Discovery of Endocrine Interactions with Application to Whole-Body Metabolism

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions