Glucose and lipid metabolism in human skeletal muscle

Insulin resistance is characterized by a blunted biological response to insulin, and it is the hallmark of obesity and type 2 diabetes. Since skeletal muscle is among the major metabolic organs in the body, a better understanding of the molecular pathways regulating metabolic responses in muscle will help to develop novel strategies to improve glucose and lipid metabolism. Here, two different models of human skeletal muscle, intact skeletal muscle strips and primary human myotubes, were used to study acute regulation of metabolism. Globular adiponectin modestly increased glucose transport in type 2 diabetic, but not in nondiabetic muscle strips, without any effects on proximal insulin signaling. Insulin sensitizing drug rosiglitazone did not affect glucose transport or proximal insulin signaling events in intact human muscle strips, but it transiently activated AMPK-signaling pathway. Polyphenolic compound resveratrol inhibited fatty acid oxidation, glucose metabolism and insulin signaling in human myotubes. Moreover, resveratrol increased ER stress and directly inhibited AMPK activity. Palmitate impaired glucose metabolism and increased ER stress in primary human myotubes and also in intact skeletal muscle strips from overweight, but not lean men. Thus, the role of ER stress in the pathogenesis of fatty acid induced insulin resistance and T2D warrants further studies.Insuliiniresistenssillä tarkoitetaan heikentynyttä biologista vastetta insuliinille ja se on keskeinen patofysiologinen ilmiö lihavuudessa ja tyypin 2 diabeteksessa. Koska poikkijuovainen luurankolihas on yksi keskeisimmistä aineenvaihduntaa säätelevistä kudoksista, lihaksen aineenvaihduntaa säätelevien molekylääristen reittien tarkempi tuntemus saattaa auttaa kehittämään uusia hoitomuotoja tehostamaan glukoosi- ja rasva-aineenvaihduntaa. Tässä väitöskirjatyössä on käytetty kahta erilaista mallia tutkia ihmisen lihaskudoksen aineenvaihduntaa laboratorio-olosuhteissa: eristettyjä lihassäikeitä sekä primaareja lihassoluviljelmiä. Adiponektiini, joka on rasvakudoksen erittämä insuliiniherkistävä hormoni, lisäsi glukoosin soluunottoa tyypin 2 diabetesta potevilta miehiltä eristetyissä lihassäikeissä. Insuliiniherkistäjänä toimiva lääke, rosiglitatsoni, ei vaikuttanut glukoosin soluunottoon tai insuliinin signaalikuljetukseen eristetyissä lihassäikeissä, mutta se aktivoi energia-aineenvaihdunnassa keskeisen viestinviejän, AMP-aktivoidun proteiinikinaasin, signaalireitin. Resveratroli on esimerkiksi viinirypäleitten sisältämä polyfenolinen yhdiste. Eläimillä ja eläinsoluilla suoritettujen kokeitten perusteella resveratroli on parantanut aineenvaihduntaa ja suojannut tyydyttyneen rasvan aiheuttamalta insuliiniresistenssiltä. Tutkimme resveratrolin vaikutusta ihmisen primaareissa viljellyissä lihassoluissa. Yllättäen resveratroli ei suojannutkaan palmitaatin aiheuttamalta insuliiniresistenssilta, vaan heikensi sekä glukoosi- että rasva-aineenvaihduntaa ja esti insuliinin signaalikuljetusta. Resveratroli lisäsi endoplastisen kalvoston stressiä, ja esti suoraan AMP-aktivoidun proteiinikinaasin aktiivisuutta. Palmitaatti heikensi glukoosiaineenvaihduntaa ja lisäsi endoplastisen kalvoston stressiä viljellyissä lihassoluissa ja myös eristetyissä lihassäikeissä. Endoplastisen kalvoston stressivasteen merkitys tyydyttyneen rasvan aiheuttamassa insuliiniresistenssissä ja tyypin 2 diabeteksessa on tärkeä jatkotutkimuksen aihe

Palmitate and oleate exert differential effects on insulin signalling and glucose uptake in human skeletal muscle cells

Saturated fatty acids are implicated in the development of insulin resistance, whereas unsaturated fatty acids may have a protective effect on metabolism. We tested in primary human myotubes if insulin resistance induced by saturated fatty acid palmitate can be ameliorated by concomitant exposure to unsaturated fatty acid oleate. Primary human myotubes were pretreated with palmitate, oleate or their combination for 12 h. Glucose uptake was determined by intracellular accumulation of [H-3]-2-deoxy-d-glucose, insulin signalling and activation of endoplasmic reticulum (ER) stress by Western blotting, and mitochondrial reactive oxygen species (ROS) production by fluorescent dye MitoSOX. Exposure of primary human myotubes to palmitate impaired insulin-stimulated Akt-Ser(473), AS160 and GSK-3 beta phosphorylation, induced ER stress signalling target PERK and stress kinase JNK 54 kDa isoform. These effects were virtually abolished by concomitant exposure of palmitate-treated myotubes to oleate. However, an exposure to palmitate, oleate or their combination reduced insulin-stimulated glucose uptake. This was associated with increased mitochondrial ROS production in palmitate-treated myotubes co-incubated with oleate, and was alleviated by antioxidants MitoTempo and Tempol. Thus, metabolic and intracellular signalling events diverge in myotubes treated with palmitate and oleate. Exposure of human myotubes to excess fatty acids increases ROS production and induces insulin resistance.Peer reviewe

Angiopoietin-Like 4 Mediates PPAR Delta Effect on Lipoprotein Lipase-Dependent Fatty Acid Uptake but Not on Beta-Oxidation in Myotubes

Peer reviewe

Angptl4 inhibits intracellular LPL activity and co-localizes with intracellular LPL.

<p>(<b>a</b>) Heparin releasable and intracellular LPL activity was measured in C2/LPL myotubes incubated with GW501516 (0.1 µM) for 24 hours, n = 3. (<b>b, c, d</b>) Confocal microscopy of myoblasts transfected with V5 tagged Angptl4 and stained with antibodies against LPL (<b>b</b>) and V5 tag (<b>c</b>). Scale bar: 20 µm. * p<0.05, paired <i>t</i> test.</p

Proposed mechanism regulating LPL activity in the skeletal muscle.

<p>The working hypothesis is that FAs produced locally by LPL-mediated hydrolysis of VLDL and chylomicrons (CM) together with FAs derived from adipose tissue (FA-albumin) can activate PPARδ/RXR heterodimer which in turn upregulates Angptl4 gene expression. Angptl4 inhibits LPL activity mainly at the surface of the sarcolemma where less LPL will be available to be transported at luminal sites via the function of GPIHBP1. LPL inhibition by Angptl4 occurs to a lesser extent also intracellularly. This mechanism may protect the skeletal muscle from lipid overload and insulin resistance but may also contribute to bexarotene induced systemic hypertriglyceridemia.</p

Angptl4 inhibits LPL activity and LPL-dependent fatty acid uptake and mediates PPARδ effect on LPL activity.

<p>(<b>a</b>) Human Angptl4 concentration was evaluated by ELISA in medium from myotubes infected with HSA (human serum albumin)-AAV2 (Control) or hAngptl4-AAV2. (<b>b</b>) Heparin releasable LPL activity was measured in myotubes infected with HSA-AAV2 (Control) or hAngptl4-AAV2, n = 4 for L6 and n = 3 for C2/LPL. LPL activity expressed as 100% represent 0.037 (L6) or 3.38 (C2/LPL) µmol FAs h⁻¹ mg⁻¹. (<b>c</b>) Heparin releasable LPL activity was measured in C2/LPL myotubes exposed to increasing concentration of recombinant hAngptl4. LPL activity expressed as 100% represent 2.58 µmol FAs h⁻¹ mg⁻¹. (<b>d</b>) Myotubes infected with HSA-AAV2 or Angptl4-AAV2 or treated with Orlistat (50 µM) were incubated 16 h with Intralipid or OA-BSA. Oil Red O staining of myotubes was quantified by densitometry, n = 4. (<b>e</b>) Cells transfected with non targeting siRNA (NT-siRNA) or Angptl4 siRNA were incubated with GW501516 for 4 hours and heparin releasable LPL activity was quantified, n = 3. * p<0.05, paired <i>t</i> test (<b>b</b>) or Two-way ANOVA with Bonferroni post-tests (<b>d, e</b>) compared with Control.</p

PPARδ activation increases Angptl4 mRNA and protein levels in human and mouse C2/LPL myotubes.

<p>(<b>a</b>) Human myotubes were incubated for 24 hours in the presence of DMSO (Control) or GW501516 (0.1 µM) and Angptl4 concentration in medium and cell lysate was quantified using ELISA, n = 4. Angptl4 mRNA levels were measured by real time PCR in (<b>b</b>) human and (<b>c</b>) mouse myotubes incubated with DMSO (Control) or GW501516 (0.1 µM) for 24 hours, n = 4. Angptl4 mRNA levels were normalized and analyzed in parallel with human PBGD and mouse HRPT mRNA levels. (<b>d</b>) Secretion of Angptl4 from C2/LPL myotubes was analyzed by Western blot 48 hours after incubation with DMSO (Control) or GW501516 (0.1 µM). Samples from two experiments were loaded on the gel. Right panel shows Ponceau staining of the blotting membrane as a control for protein loading and efficient transfer from the gradient gel to nitrocellulose membrane. *p<0.05, paired <i>t</i> test compared with Control.</p

Regulation of Angptl4 expression and LPL activity by bexarotene in C2/LPL myotubes.

<p>(<b>a</b>) Secretion of Angptl4 from C2/LPL myotubes was analyzed by Western blot 48 hours after incubation with DMSO (Control) or 0.2 µM bexarotene. Samples from two experiments were loaded on the gel. (<b>b</b>) Angptl4 and LPL mRNA levels were measured by real time PCR in C2/LPL myotubes incubated with DMSO (Control) or 0.2 µM bexarotene for 24 hours. Values are expressed relative to mouse 36B4 mRNA levels. (<b>c</b>) C2/LPL myotubes untreated or pre-treated overnight with 1 µM GSK0660, a PPARδ antagonist, were incubated with DMSO (Control) or 0.2 µM bexarotene for 4 hours. Heparin releasable LPL activity was measured and normalized to the protein content. * p<0.05, Two-way ANOVA with Bonferroni post-tests.</p

PPARδ activation by GW501516 inhibits LPL activity and LPL-dependent fatty acid uptake.

<p>(<b>a</b>) Heparin releasable LPL activity was measured in the L6, C2C12 and C2/LPL myotubes after 24 hour incubation of the cells in the presence of GW501516, n = 3. (<b>b</b>) Time dependent inhibition of LPL activity by GW501516 (0.1 µM) was measured in heparin releasable pool from C2/LPL myotubes, n = 3. (<b>c</b>) Oil Red O staining of myotubes was quantified by densitometry in cells incubated with Intralipid or OA-BSA for 5 hours in the presence or absence of GW501516, n = 4. (<b>d</b>) Intracellular triglycerides quantification in cells incubated with Intralipid for 5 hours in the presence or absence of GW501516, n = 3 (<b>e</b>) Fluorescence microscope images of C2/LPL myotubes (highlighted with a line) incubated with Intralipid for 5 hours in the presence or absence of GW501516. Nuclei are stained in blue (DAPI) and lipid droplets in red (Oil Red O). * p<0.05, paired <i>t</i> test (<b>a</b>) or One-way ANOVA with Dunnett's post test (<b>b, c, d</b>) compared with Control.</p

Angptl4 overexpression has no effect on fatty acids oxidation and glucose metabolism in L6 myotubes.

<p>Fatty acid (palmitate) oxidation was evaluated by measuring (<b>a</b>) ¹⁴C-CO₂ or (<b>b</b>) ¹⁴C-acid soluble metabolites (¹⁴C-ASM) production in L6 myotubes 72 hours post-infection with HSA-AAV2 (Control) or Angptl4-AAV2 and compared with cells incubated with DMSO (Control) or GW501516 for 24 hours, n = 3. (<b>c</b>) Palmitate oxidation was evaluated by measuring ³H-H₂O released from myotubes incubated with low doses of GW501516 in the setting of low (HSA-AAV2) and high (Angptl4-AAV2) Angptl4 levels, n = 4. (<b>d</b>) Glucose uptake, (<b>e</b>) glycogen synthesis and (<b>f</b>) glucose oxidation were measured in L6 myotubes 72 hours post-infection with HSA-AAV2 or Angptl4-AAV2 in the absence (Basal) or presence of insulin (100 nM), n = 3. *p<0.05, One-way (<b>a, b</b>) or Two-way (<b>c–f</b>) ANOVA with Bonferroni post-tests.</p