Skeletal Muscle: from Basic Physiology to Exercise and Diabetes Nicolas J

Nicolas J. Pillon 09/04/2019

Molecular Physiology Master Course

Skeletal muscle: From basic physiology to exercise and diabetes Nicolas J. Pillon

www.nicopillon.com [email protected] www.nicopillon.com

Interactive teaching using Poll Everywhere How to join?

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Who are these people?

. Fredrick Banting (1891 –1941) Canadian Nobel laureate with John Macleod for the discovery of insulin and its therapeutic potential.

. Otto Fritz Meyerhof (1884 –1951) German Nobel laureate with Archibald Vivian Hill or his discovery of the fixed relationship between the consumption of oxygen and the metabolism of lactic acid in the muscle (glycolysis).

. Charles Best (1899 –1978) Canadian co-discoverer of insulin with James Collip, Frederick Banting and John Macleod. Best and Collip were ignored by the Nobel committee and did not co-share the Prize for the discovery of insulin.

. August Krogh (1874 –1949) Danish Nobel laureate for the discovery of the mechanism of regulation of the capillaries and oxygen flow in skeletal muscle.

Outline of the course

. Introduction - skeletal muscle structure and basic physiology . Metabolic signalling in skeletal muscle  Insulin signalling and glucose metabolism  Fatty acid signalling and metabolism  Metabolic flexibility . Skeletal muscle dysfunction in metabolic diseases . Lifestyle and pharmacological interventions

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Skeletal muscle STRUCTURE AND PHYSIOLOGY

Skeletal Muscle from the encyclopaedia Britannica

“Contractile tissue found in animals, the function of which is to produce motion.”

• Skeletal muscle is the most common and widely distributed muscle tissue in the body, making up around 40% of the body’s total mass • Skeletal muscles are attached to bones by tendons, and they produce all the movements of body parts in relation to each other. • Unlike smooth muscle and cardiac muscle, skeletal muscle is under voluntary control. • Similar to cardiac muscle, however, skeletal muscle is striated; its long, thin, multinucleated fibres are crossed with a regular pattern of fine red and white lines, giving the muscle a distinctive appearance. • Skeletal muscle fibres are bound together by connective tissue and communicate with nerves and blood vessels.

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Skeletal muscle organ

Skeletal muscle cell: fiber or myotube

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Skeletal muscle contractile unit: myofibril

M-band Z-disk M-band Z-disk M-band ↓ ↓ ↓ ↓ ↓

Thin filament (actin)

Thick filament (myosin)

Lipid droplet

Mitochondria

Skeletal muscle structure

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Three ways to generate ATP in skeletal muscle

50 meters: 5–6 seconds 400 meters: 50–60 seconds 1500 meters: 5–6 minutes

Phosphagen system = immediate energy source Anaerobic cellular respiration = short-term energy source Aerobic cellular respiration = long-term energy source

1500-meter track

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The phosphagen system provides an immediate supply of ATP

ATPase Myokinase Creatine kinase ATP  ADP + Pi 2 ADP  ATP + AMP ADP + CP  ATP + Creatine

ATP ADP ADP ADP Creatine P

ADP

ATP AMP ATP Pi Creatine

ATP already present in skeletal tissue Myokinase transfers one phosphate Creatine phosphate (CP) can supply hydrolyzed by (myosin) ATPase into from adenosine diphosphate (ADP) to ATP in skeletal muscle only. Creatine

ADP and Pi (phosphate ion). another ADP to yield ATP and AMP. kinase transfers one phosphate from creatine phosphate to ADP to yield Initial energy source good for about 5 Generates an additional few secs of creatine and ATP. sec maximal exertion. energy. Provides additional 10 to 15 seconds of energy. Process reversed during rest.

Cellular respiration

Sugars Fatty acids

Glycolysis Beta-oxidation

Anaerobic Aerobic Acetyl-CoA

Lactate CO2 + H2O ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP ATP

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Metabolic signalling in skeletal muscle Glucose uptake and metabolism

1 Glucose Uptake

2 Glycogenesis

3 Glycolysis 1

2 3

Storage Energy

Glycogen Glycolysis

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Glucose transport

The glucose transporter family members

20 Augustin R. The protein family of glucose transport facilitators: It's not only about glucose after all. IUBMB Life. 2010 May;62(5):315-33. Nicolas J. Pillon 09/04/2019

Class 1 tissue distribution: RNA and protein

GLUT1 GLUT2 GLUT3 GLUT14 GLUT4 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

21 https://www.proteinatlas.org

Class 2 tissue distribution: RNA and protein

GLUT5 GLUT7 GLUT9 GLUT11 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

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Class 3 tissue distribution: RNA and protein

GLUT6 GLUT8 GLUT10 GLUT12 GLUT13 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

Glucose transporter deficient mouse models

Gene Major phenotype

Embryonic lethality SLC2A1 SLC2A1−/− embryonic lethal, SLC2A1+/− resemble human GLUT1 deficiency syndrome

SLC2A2−/− develop symptoms of T2D, early neonatal death SLC2A2 Normal hepatic glucose output, normal intestinal glucose and fructose absorption, glucosuria

SLC2A3−/− embryonic lethal, SLC2A3+/− slight alterations in behaviour, no metabolic phenotype SLC2A3 SLC2A3−/− embryonic lethal SLC2A3+/− features of autism spectrum disorders, male mice with adult‐onset adiposity & insulin resistance SLC2A4−/− growth‐retarded, cardiomegaly, normoglycemic; SLC2A4+/− diabetes, male mice with adult‐onset adiposity and insulin resistance

SLC2A4−/− hyperglycemic, glucose intolerant and insulin resistant, subset develops diabetes SLC2A4 SLC2A4−/− hyperglycemic, glucose intoleranct and insulin resistant, subset develops diabetes, insulin resistance in liver and muscle

SLC2A4−/− modest (compensated) cardiac hypertrophy

24 Augustin R. The protein family of glucose transport facilitators: It's not only about glucose after all. IUBMB Life. 2010 May;62(5):315-33. Nicolas J. Pillon 09/04/2019

GLUT4 translocation to the plasma membrane

Myc epitope extracellular

Intracellular

L6 myoblasts transfected with GFP-GLUT4-myc were stained without permeabilization with anti- myc antibody to detect surface GLUT4. The GFP (green) signal represents all GLUT4 while the myc signal (red) represents plasma membrane-inserted GLUT4.

25 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611.

GLUT4 is Highly Compartmentalized and Undergoes Continuous Cycling to and from the Plasma Membrane

26 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611. Nicolas J. Pillon 09/04/2019

Membrane Fusion by SNAREs and Their Regulators

27 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611.

INSULIN-DEPENDENT GLUCOSE UPTAKE

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Canonical and Novel Signals in Insulin-Stimulated GLUT4 Translocation

29 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611.

Rac1 is activates cortical actin filament branching

30 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611. Nicolas J. Pillon 09/04/2019

CONTRACTION-DEPENDENT GLUCOSE UPTAKE

Mechanisms of exercise-regulated glucose uptake

32 Sylow L, Kleinert M, Richter EA, Jensen TE. Exercise-stimulated glucose uptake - regulation and implications for glycaemic control. Nat Rev Endocrinol. 2017 Mar;13(3):133-148. Nicolas J. Pillon 09/04/2019

Mechanisms of exercise-regulated glucose uptake

33 Sylow L, Kleinert M, Richter EA, Jensen TE. Exercise-stimulated glucose uptake - regulation and implications for glycaemic control. Nat Rev Endocrinol. 2017 Mar;13(3):133-148.

Storage Glycogenesis

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Healthy vs unhealthy storage in muscle

Glucose Fatty acids Glucose Fatty acids

Storage compartments Storage compartments

Glycogen Triacylglycerol Glycogen Triacylglycerol

Pyruvate Acyl-CoA Pyruvate Acyl-CoA

Acetyl-CoA Acetyl-CoA

TCA cycle TCA cycle

Glycogen

Glycogenin

Glucose

36 McArdle et al. (2006). Exercise physiology: energy, nutrition, and human performance (6th ed.). Lippincott Williams & Wilkins. p. 12. ISBN 978-0-7817-4990-9. Nicolas J. Pillon 09/04/2019

Insulin-dependent glycogenesis

37 Bouskila et al. Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle. Cell Metab. 2010 Nov 3;12(5):456-66.

Insulin-dependent glycogenesis

38 https://themedicalbiochemistrypage.org/glycogen.php Nicolas J. Pillon 09/04/2019

Energy Glycolysis

From glucose to pyruvate: 2 ATP + 2 NADH

D-Glucose 2 × Pyruvate + +2[NAD] + 2 [NADH] + +2[ADP] +2H +2[Pi] +2[ATP] +2H2O

40 http://www.simplinotes.com/glycolysis/ Nicolas J. Pillon 09/04/2019

Preparatory phase Cost: 2 ATP

Make: 2 ADP

The “lysis” step of glycolysis

41 https://www.slideshare.net/BarahJafari/chapter14-160419080639

From glucose to pyruvate Cost: 2 NAD+ 2 Pi 4 ADP

Make: 2 NADH + 2 H+ 4 ATP 2 H2O

42 https://www.slideshare.net/BarahJafari/chapter14-160419080639 Nicolas J. Pillon 09/04/2019

Fates of pyruvate (in mammals)

Glucose

1. Lactic acid fermentation: after vigorous exercise, [O2] in muscles is low (hypoxia) NADH cannot be Pyruvate reoxidized to NAD+ for glycolysis so aerobic pyruvate is reduced to lactate hypoxic or conditions Alanine accepting electrons from NADH anaerobic Leucine conditions CO2 Valine 2. Citric acid cycle: pyruvate is Isoleucine oxidized and decarboxylated to Acetyl-CoA release CO2. The electrons that are Building blocks for moving go through the electron amino acid and fatty Lactate acid synthesis transport chain in the mitochondria 4CO2 + 4H2O and are used to make ATP. Fermentation to lactate Transport to the Fatty acids for example in vigorously mitochondria and 3. Anabolism: synthesis of amino contracting muscle complete oxidation acids and fatty acids in the TCA cycle

Metabolic signalling in skeletal muscle Glucose uptake and metabolism

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45 www.genecards.com / www.proteinatlas.org / www.biogps.org

Glucose transport Questions

. What is the main mechanism regulating GLUT4-dependent glucose uptake? . What are the two major stimuli regulating glucose uptake in muscle?

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Glucose transport Questions

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Glucose transport Questions

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Glucose storage - Glycogenesis Questions

. Around which protein are the glycogen polymers organized? . On which carbons are the glucose molecules polymerizing? . On which carbon is the glycogen polymer branching? . Draw the insulin signalling pathway leading to glycogen synthesis and the negative feedback loops regulating glycogen synthase activity

Glucose utilization - Glycolysis Questions

. How many reactions are in glycolysis? . How are defined the two “phases” of glycolysis? . What is the ultimate product of glycolysis? . Why is glycolysis called “lysis”? . What is the energy yield of glycolysis? . What are the 3 possible fates of pyruvate in muscle?

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Metabolic signalling in skeletal muscle Fatty acid uptake and metabolism

The many roles of fatty acids

54 Knobloch. The Role of Lipid Metabolism for Neural Stem Cell Regulation. Brain Plast. 2017 Nov 9;3(1):61-71 Nicolas J. Pillon 09/04/2019

1 Lipid Uptake

2 Lipogenesis

3 Oxidative phosphorylation

2 3

Storage Energy

Triglycerides Oxidative phosphorylation

Lipid transport

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From the extracellular space to the mitochondrial matrix

Very Long Chain Long Chain Medium/Short Chain Fatty Acids Fatty Acids Fatty Acids

FATP FATP Cytoplasm

Acyl-CoA Acyl-CoA

CPT1 Acyl CoA CPT2 Beta oxidation

Acyl-CoA

Peroxisome TCA Beta Cycle Mitochondrial oxidation Acetyl matrix CoA

Fatty acid transporters

Name Nomenclature Systematic nomenclature Endogenous substrates

Fatty acid transport protein 1 FATP1 SLC27A1 arachidonic acid > palmitic acid > oleic acid > butyric acid

Fatty acid transport protein 2 FATP2 SLC27A2

Fatty acid transport protein 3 FATP3 SLC27A3

Fatty acid transport protein 4 FATP4 SLC27A4 palmitic acid > oleic acid > butyric acid, γ-linolenic acid > arachidonic acid

Fatty acid transport protein 5 FATP5 SLC27A5

Fatty acid transport protein 6 FATP6 SLC27A6 palmitic acid > oleic acid > γ-linolenic acid > octanoic acid

Fatty acid translocase CD36 CD36

Caveolins CAV1, CAV2, CAV3

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Tissue distribution: RNA and protein

FATP1 FATP2 FATP3 FATP4 FATP5 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

Tissue distribution: RNA and protein

FATP6 CD36 CAV1 CAV2 CAV3 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

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Storage Lipogenesis

Healthy vs unhealthy storage in muscle

Glucose Fatty acids Glucose Fatty acids

Storage compartments Storage compartments

Glycogen Triacylglycerol Glycogen Triacylglycerol

Pyruvate Acyl-CoA Pyruvate Acyl-CoA

Acetyl-CoA Acetyl-CoA

TCA cycle TCA cycle

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Lipid droplets are located close to the mitochondrial network

Mitochondria were stained using an antibody against the translocase of the outer mitochondrial membrane-20 (TOMM20), labelled with Alexa Fluor 555.

Lipid droplets have been stained green using BODIPY 493/503.

63 Daemen et al. The effect of diet and exercise on lipid droplet dynamics in human muscle tissue. Journal of Experimental Biology 2018 221: jeb167015

Lipid droplets are located close to the mitochondrial network

SM: sarcomere SSR: subsarcolemmal region Lipid droplets Mitochondria

64 Bosma M. Lipid droplet dynamics in skeletal muscle. Exp Cell Res. 2016 Jan 15;340(2):180-6. Nicolas J. Pillon 09/04/2019

Lipogenesis

Energy Beta-oxidation Citric acid cycle Krebs cycle Tricarboxylic acid (TCA) cycle

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Beta-oxidation

O ׀׀

H3C-C-SCoA Acetyl CoA FAD O ׀׀ (RR-CH-2)-CH-CH-CH-C-SCoA-C-SCoA Acyl CoA 3-ketoacyl CoA 2 2 2 2 dehydrogenase thiolase AcylAcyl CoA CoA

SCoA FADH2

O O O ׀׀ ׀׀ ׀׀

R-C-CH2-C-SCoA R-CH=CH-C-SCoA 3-ketoacyl CoA 2-trans-enoylCoA

H O NADH + H+ 2 OH O 3-hydroxy acyl Enoyl CoA hydratase ׀׀ | CoA dehydrogenase R-CH-CH2-C-SCoA L-3-hydroxyl acyl CoA NAD+

Glucose and fatty acid metabolism converge on acetyl-CoA

Glucose Fatty acid

Glycolysis

Acyl-CoA

Pyruvate

Beta-oxidation Pyruvate dehydrogenase Acetyl-CoA

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Krebs Cycle

69 http://tamaraclark.com/project/tca-cycle/

Respiration

70 de Goede et a. Circadian rhythms in mitochondrial respiration. J Mol Endocrinol. 2018 Apr; 60(3): R115–R130. Nicolas J. Pillon 09/04/2019

Metabolic signalling in skeletal muscle Fatty acid uptake and metabolism

The Seven Learning Styles • Visual (spatial):You prefer using pictures, images, and spatial understanding. • Aural (auditory-musical): You prefer using sound and music. • Verbal (linguistic): You prefer using words, both in speech and writing. • Physical (kinesthetic): You prefer using your body, hands and sense of touch. • Logical (mathematical): You prefer using logic, reasoning and systems. •Social(interpersonal): You prefer to learn in groups or with other people. • Solitary (intrapersonal): You prefer to work alone and use self-study.

71 https://www.learning-styles-online.com/overview/

72 www.genecards.com / www.proteinatlas.org / www.biogps.org Nicolas J. Pillon 09/04/2019

Fatty acid transport Questions

. How many membranes do fatty acids have to cross before they can be used for energy? . Which transporters are responsible for each crossing?

Fatty acid transport Questions

Very Long Chain Long Chain Medium/Short Chain Fatty Acids Fatty Acids Fatty Acids

Cytoplasm

Peroxisome

Mitochondrial matrix

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Fatty acid transport Questions

Very Long Chain Long Chain Medium/Short Chain Fatty Acids Fatty Acids Fatty Acids

FATP FATP Cytoplasm

Acyl-CoA Acyl-CoA

CPT1 Acyl CoA CPT2 Beta oxidation

Acyl-CoA

Peroxisome TCA Beta Cycle Mitochondrial oxidation Acetyl matrix CoA

Fatty acid storage Questions

. What is the main form of lipid storage in skeletal muscle? . Where are lipid stored in skeletal muscle cells? . How are glycolysis and lipogenesis connected?

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Fatty acid metabolism Questions

. Of how many steps is beta-oxidation constituted? . How many carbons are removed from fatty acids at each cycle? . What is the end product of beta-oxidation? . How much energy does beta-oxidation produce?

Glucose and Fatty Acid Transport, Storage and Metabolism Summary

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Nutrient uptake and metabolism

79 https://www.diapedia.org/metabolism-and-hormones/5105765817/metabolic-pathways

80 Kono et al. Pathway projector: web-based zoomable pathway browser using KEGG atlas and Google Maps API. PLoS One. 2009 Nov 11;4(11):e7710. Nicolas J. Pillon 09/04/2019

Substrate Preference and Metabolic Flexibility

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Fuel exchange during fasting

Meal X ↓ Glucose uptake ↓ Glycogenogenesis ↓ Lipogenesis ↑ Neoglucogenesis ↑ Lipolysis

FA Glc XGlc Glc XFA X

↓ Glucose (Glc)

Glc FA Glc Glucagon

β‐cells

Intramyocellular Glycogen triglycerides

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Fuel exchange after a meal

Meal ↑ Glucose uptake ↑ Glycogenogenesis ↑ Lipogenesis ↓ Neoglucogenesis ↓ Lipolysis

Glc X Glc Glc FA FA

↑ Glucose ↑ Fatty Acids ↓ Glucose ↓ Fatty Acids (Glc) (FA) (Glc) (FA)

Glc FA Glc Insulin

β‐cells

Glycogen Intramyocellular (75-80%) triglycerides

Sensing of Fuel and Energy Status AMP-activated Protein Kinase (AMPK)

• Activated when intracellular ATP levels decrease • Inhibits synthesis of fatty acids and activates lipid uptake • Stimulates glucose uptake

Regulatory

β1-2

α1-2 γ1-3

Catalytic Sensor

Hardie DG. AMP-activated protein kinase: maintaining energy homeostasis at the cellular and whole-body levels. Annu Rev Nutr. 2014;34:31-55. 84 Hardie DG. AMPK-sensing energy while talking to other signaling pathways. Cell Metab. 2014 Dec 2;20(6):939-52. Nicolas J. Pillon 09/04/2019

Sensing of Fuel and Energy Status AMP-activated Protein Kinase (AMPK)

AMPK activation • increased uptake of glucose (↑ GLUT4) • increased oxidation of glucose (↑ HKII) • Inhibition of glycogen synthesis (↓GS) • increased uptake of fatty acids (↑ FAT/CD36) • Increased fatty acid oxidation (↓ ACC)

Metabolic changes known to be induced by AMPK in muscle, including stimulation of glucose and fatty acid uptake, fatty acid oxidation, and mitochondrial biogenesis, and inhibition of glycogen synthesis and, via inhibition of TOR, hypertrophy Question marks indicate that the direct target for AMPK responsible for the observed downstream effect is not known. The effect on fatty acid uptake has to date only been observed in cardiac muscle.

85 Hardie DG, Sakamoto K. AMPK: a key sensor of fuel and energy status in skeletal muscle. Physiology (Bethesda). 2006 Feb;21:48-60.

Modulation of substrate preference Mechanism of reciprocal inhibition of glucose and fatty acid oxidation

Mechanism of reciprocal inhibition of glucose and fatty acid oxidation. When glucose uptake and consumption increases, fatty acid oxidation is suppressed by malonyl-CoA’s allosteric inhibition of CPT-1, and increased pyruvate from glycolysis inhibits PDK, which stimulates glucose oxidation (yellow lines). CPT-1 inhibition increases the concentration of LCFA-CoAs, which then are used for triglyceride synthesis and stored (pink arrow). Vice versa, when fatty acid oxidation is high, glucose uptake, glycolysis, and pyruvate oxidation are decreased (red lines) because rising levels of acetyl-CoA and NADH impede PDH activity. Additionally, increased citrate levels inhibit GLUT4 and PFK-1. PFK-1 inhibition results in increased glucose-6- phosphate concentrations that inhibit HK. A decrease in pyruvate oxidation enables pyruvate to be used as either a gluconeogenic precursor or, in energetically demanding tissues, a substrate for PC, which produces oxaloacetate that is used as anaplerotic substrate (purple arrows). During caloric restriction, the rise in AMP/ATP activates AMPK, which inhibits ACC, stimulating fatty acid uptake by the mitochondria via CPT-1. ACL, ATP-citrate lyase; CACT, carnitine acylcarnitine translocase; CTP, citrate transport protein; CYTO, cytosol; FAS, fatty acid synthase; LCFA, long-chain fatty acid; MITO, mitochondria; MPC, mitochondrial pyruvate carrier; PC, pyruvate carboxylase. Green arrows indicate stimulatory reactions.

From: Metabolic Flexibility as an Adaptation to Energy Resources and Requirements in Health and Disease. Endocr Rev. 2018;39(4):489-517. doi:10.1210/er.2017-00211 Endocr Rev | Copyright © 2018 Endocrine Society

86 Hue & Taegtmeyer. The Randle cycle revisited: a new head for an old hat. Am J Physiol Endocrinol Metab. 2009 Sep;297(3):E578-91 Nicolas J. Pillon 09/04/2019

Modulation of substrate preference The Randle cycle: inhibition of fat oxidation by glucose

87 Hue & Taegtmeyer. The Randle cycle revisited: a new head for an old hat. Am J Physiol Endocrinol Metab. 2009 Sep;297(3):E578-91

Modulation of substrate preference The Randle cycle: inhibition of glucose oxidation by fat

88 Hue & Taegtmeyer. The Randle cycle revisited: a new head for an old hat. Am J Physiol Endocrinol Metab. 2009 Sep;297(3):E578-91 Nicolas J. Pillon 09/04/2019

Metabolic flexibility The capacity for an organism to adapt fuel oxidation to fuel availability

The primary purpose of the substrate shift is to move from catabolic to anabolic processes. Lipids

Insulin is a major driver of this shift. Catabolism Anabolism Energy is stored after nutrient intake (glycogen, TG). Stored energy is restituted during fasting.

Inappropriate storage leads to ectopic lipid Carbohydrates accumulation in non-adipose tissues (lipotoxicity), that contributes to T2D and its complications.

Measuring Energy Sources in vivo Calorimetry

Cage Oxygen in (VO2i) Oxygen out (VO2o) 2 or 2 2 Chamber Carbon Dioxide in (VCO ) Carbon dioxide out (VCO ) 2i 2o 22 2

2 2

Promethion Cages Maastrich Instruments BV Dual Cosmed Desktop Metabolic System Sable Systems International Chamber Whole Body Calorimeter for Indirect Calorimetry with Mask

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Measuring Energy Sources in vivo Respiratory Exchange Ratio = Respiratory Quotient

2 The source of energy dictates the amount of O2 required to oxidize the carbons and hydrogens into CO2 2

Carbohydrate (Glucose)

C6H12O6 + 6O2 → 6CO2 + 6H2O + 38ATP

RER = 6.CO2 ÷6.O2 =1

Fat (Palmitate)

C16H32O2 + 23O2 → 16CO2 + 16H2O + 129ATP

RER = 16.CO2 ÷ 23.O2 =0.7

91 Emily K. Sims et al. Am J Physiol Endocrinol Metab. 2013 Dec;305(12):E1495-511.

Measuring Energy Sources in vivo Respiratory Exchange Ratio = Respiratory Quotient

Breakfast A Breakfast B

1.0 1.0

0.9 0.9

0.8 0.8 RER RER

0.7 0.7

0.6 0.6 23456789101112 23456789101112 Time Time

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Substrate Preference and Metabolic Flexibility • What is the main sensor of energy status in muscle cells? What metabolic effects does its activation trigger? • What is the main mechanism of the inhibition of glucose oxidation by fatty acids? • What is the main mechanism of the inhibition of fatty acid oxidation by glucose? • What is the definition of metabolic flexibility? • How can substrate preference measured?

Metabolic Flexibility and the Metabolic Syndrome

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The metabolic syndrome

Abdominal obesity

Insulin resistance

HDL- Plasma cholesterol triglycerides H<40mg/dL >150mg/dL F<50mg/dL Metabolic Type 2 diabetes Syndrome

Cardio-vascular diseases

Fasted Hypertension >130/85mmHg hyperglycemia >110mg/dL

Body Mass Index

HEIGHT Weight in kilograms 1.80 BMI Height in meters WEIGHT 110 BMI 32.4

< 18.5 kg/m² underweight 18.5 – 24.9 « normal » > 25 kg/m² overweight > 30 kg/m² obese > 40 kg/m² Morbidly obese Nicolas J. Pillon 09/04/2019

Body Mass Index evolution

3 million years

1.5 millions years

200,000 years

40 years… Nicolas J. Pillon 09/04/2019

Worldwide diabetes in women 1980-2014

https://www.lshtm.ac.uk/newsevents/expert-opinion/visualising-slow-march-chronic-disease-apocalypse

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The complexity of metabolic disorders

Inactivity Epigenetics

Psychology Genetics

Type 2 Exercise Ageing Circadian Diabetes Rhythms Metabolic Control Nutrition Sleeplessness

Inflammation Skeletal Air pollution Muscle

Sociocultural factors Oxidative Stress

Fuel exchange after a meal

Meal ↑ Glucose uptake ↑ Glycogenogenesis ↑ Lipogenesis ↓ Neoglucogenesis ↓ Lipolysis

Glc X Glc Glc FA FA

↑ Glucose ↑ Fatty Acids ↓ Glucose ↓ Fatty Acids (Glc) (FA) (Glc) (FA)

Glc FA Glc Insulin

β‐cells

Glycogen Intramyocellular (75-80%) triglycerides

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Progression to type 2 diabetes Oral glucose tolerance test

Healthy Prediabetes T2D 4 160

3 120

2 80

1 40 Blood glucose (g/L) Blood Insulin (μU/mL)

0 0 0 123 0 123 Time (h) Time (h) Meal Meal Nicolas J. Pillon 09/04/2019

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Skeletal muscle plays a major role in regulating whole body insulin sensitivity

7 Splanchnic ) 6 ‐1 Insulin 5 Adipose sensitivity/signaling x min 4 ‐1 Glucose Plasma glucose levels 3 Muscle utilization/storage Glucose Utilization

(mg x kg 2 Fuel oxidation 1 Brain 0 Control T2DM

“Regardless of whether or not insulin resistance in muscle can cause type 2 diabetes, it is likely that insulin resistance is part of an overall metabolic inflexibility”

DeFronzo RA, Tripathy D. Skeletal muscle insulin resistance is the primary defect in type 2 diabetes. Diabetes Care. 2009 Nov;32 Suppl 2:S157-63. Goodpaster BH, Sparks LM. Metabolic Flexibility in Health and Disease. Cell Metab. 2017 May 2;25(5):1027-1036. Nicolas J. Pillon 09/04/2019

Metabolic flexibility The capacity for an organism to adapt fuel oxidation to fuel availability

The ability to switch from one substrate to another is crucial to maintain whole body energy balance and a healthy storage of energy.

107 Goodpaster BH, Sparks LM. Metabolic Flexibility in Health and Disease. Cell Metab. 2017 May 2;25(5):1027-1036.

Metabolic flexibility is impaired in individuals with type 2 diabetes

● Metabolically flexible ○ Metabolically inflexible (T2D)

Galgani, Moro & Ravussin. Metabolic flexibility and insulin resistance. Am J Physiol Endocrinol Metab. 2008 Nov;295(5):E1009-17. Apostolopoulou et al. Metabolic flexibility and oxidative capacity associate with insulin sensitivity in individuals with type 2 diabetes. Diabetologia. 2016 Oct;59(10):2203-7. Nicolas J. Pillon 09/04/2019

Modulation of substrate preference Physical activity

The degree to which each fuel acts as the primary or secondary source of energy and the efficiency with which energy is utilized depends on the prior nutrition and the intensity and duration of the exercise. Short intense exercise  carbohydrate. Prolonged moderate exercise  fat

Carbohydrate oxidation increases with intensity Lipid oxidation increases with duration

Carbohydrate

Fat Fat

Watt et al. Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise. J Appl Physiol (1985). 2003 Jul;95(1):314-21 109 Venables et al. Determinants of fat oxidation during exercise in healthy men and women. J Appl Physiol (1985). 2005 Jan;98(1):160-7. Epub 2004 Aug 27

Metabolic flexibility is enhanced in athletes

Glucose uptake Fatty acid oxidation

Trained subjects more effectively decrease glucose uptake and increase fatty acid oxidation than untrained individuals.  Healthy trained muscle can rapidly switch between fuels

light bars: control dark bars: co-infusion of intralipid

Dube et al. Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance. Am. J. Physiol. Endocrinol. Metab. 307, E1117–E1124. Nicolas J. Pillon 09/04/2019

Metabolic Flexibility and the Metabolic Syndrome • How is BMI calculated? What are its advantages and limitations? • Which organ is responsible for the majority of post-prandial glucose uptake? • Why is metabolic flexibility important in the context of metabolic diseases? • What type of fuel does skeletal muscle use at low intensity, long duration exercise? At high intensity, short duration exercise? Why?

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Skeletal muscle dysfunction in metabolic diseases Molecular mechanisms

www.nicopillon.com Nicolas J. Pillon 09/04/2019

Insulin Resistance, Molecular Mechanisms LIPOTOXICITY

Lipid classes and metabolites linked with insulin resistance

114 Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023. Nicolas J. Pillon 09/04/2019

Lipotoxicity Intramyocellular triglycerides

TAGs can be considered as a reservoir for FFA storage, on similar principles to the larger-scale role played by adipose tissue.

Inappropriate TAG accumulation is indicative of a disturbance at some level in lipid metabolism pathways.

Intramyocellular TAG accumulation have both been observed in the absence and presence of IR.

Intracellular TAG synthesis and storage is a highly dynamic process, suggested to have a protective role by warding against accumulation of more lipotoxic species in muscle. Enhanced channelling of FA substrate to TAG can reduce lipotoxic pressure despite greater cellular lipid content

Moro C, Bajpeyi S, Smith SR. Determinants of intramyocellular triglyceride turnover: implications for insulin sensitivity. Am J Physiol Endocrinol Metab. 2008 Feb;294(2):E203-13. 115 Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023.

Lipotoxicity Intramyocellular triglycerides

TAGs can be considered as a reservoir for FFA storage, on similar principles to the larger-scale role played by adipose tissue.

Inappropriate TAG accumulation is indicative of a disturbance at some level in lipid metabolism pathways.

Intramyocellular TAG accumulation have both been observed in the absence and presence of IR.

116 Moro C, Bajpeyi S, Smith SR. Determinants of intramyocellular triglyceride turnover: implications for insulin sensitivity. Am J Physiol Endocrinol Metab. 2008 Feb;294(2):E203-13. Nicolas J. Pillon 09/04/2019

Lipotoxicity LCFA-CoAs and acylcarnitines

Long-chain fatty acyl-CoA (LCA-CoA), the initial active intermediate formed during FA metabolism, has also been implicated in the development of IR. Studies have demonstrated negative correlations between LCA-CoA intracellular accumulation and insulin action in the skeletal muscle of HFD-fed rodents (Oakes et al. 1997a,b, Ellis et al. 2000, Wright et al. 2011), with acute lipid infusion-based studies in rodents and humans recapitulating these associations (Tsintzas et al. 2007, Hoy et al. 2009). Impairment of insulin signalling and glucose metabolism in this context is theorised to arise, in part, via LCA-CoA interactions with proteins including protein kinase C (Færgeman & Knudsen 1997), glycogen synthase (Wititsuwannakul & Kim 1977), glucokinase (Tippet & Neet 1982a,b), hexokinase (Thompson & Cooney 2000), as well as LCA-CoA-induced alterations in gene transcription (Hertz et al. 1998). LCA-CoA can also impact insulin sensitivity through flow-on effects on FA metabolism and synthesis of other deleterious lipids (Fig. 1). The influence of LCA-CoAs on insulin action is speculated to better reflect acute changes in tissue lipid metabolism than other lipid molecules that arise through more chronic exposure to FA excess (Ellis et al. 2000).

Acylcarnitines are generated during an early stage of mitochondrial FAO, converted from LCA- CoAs and carnitine via carnitine palmitoyltransferase 1 (CPT1) at the outer mitochondrial membrane before reconversion to constituent parts via CPT2 in the mitochondrial matrix. Accumulation of this lipid intermediate can often therefore serve as a measure of incomplete FAO (Van Hove et al. 1993, Koves et al. 2008, Mihalik et al. 2010, Aguer et al. 2013) and thereby also of mitochondrial energy substrate overload. Given the association of diminished FAO with IR, it is perhaps unsurprising that long-term acylcarnitine accumulation in plasma and skeletal muscle has also been presented as a feature of the condition (Ukropcova et al. 2005, Mihalik et al. 2010, Wolf et al. 2013, Aguer et al. 2015, Xiang et al. 2017). Pharmacological reduction of acylcarnitine content in a mouse model of IR has been reported to recover insulin sensitivity and reduce blood glucose levels (Liepinsh et al. 2016). An accompanying study, which heightened acylcarnitine content in mice through both acute and long-term administration of palmitoylcarnitine, saw the consequential induction of muscle-specific IR (Liepinsh et al. 2017). Regulation of metabolic flexibility, production of reactive oxygen species and inhibition of insulin signalling have all been suggested as mechanisms linking acylcarnitines with IR (Muoio et al. 2012, Aguer et al. 2015, Liepinsh et al. 2017), although it is still unclear to what extent acylcarnitines have a primary role in the induction of IR.

117 Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023.

Lipotoxicity Diacylglycerols

Correlations between DAG accumulation and impaired insulin action in various tissues and decreases in DAG content are associated with protection from IR

In muscle, DAGs activate the novel PKCθ and PKCδ isoforms.

PKC prevents insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and promotes inhibitory serine phosphorylation

Ablation of PKCθ protects mice from lipid- induced defects in muscle insulin signalling and glucose transport.

Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023. 118 Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. Diabetes. 2006 Dec;55 Suppl 2:S9-S15. Nicolas J. Pillon 09/04/2019

Lipotoxicity Ceramides

• Ceramide accumulation correlates with development of IR • Exercise and/or weight loss interventions reduce ceramide content in skeletal muscle • Pharmacological and genetic interventions to reduce (whole-body) ceramide content improve insulin

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Lipotoxicity Ceramides

Ceramides can be generated by hydrolysis of plasma membrane sphingomyelin or by de- novo synthesis from long-chain saturated FAs [29]. Numerous studies have implicated ceramides in the development of impaired insulin-stimulated muscle glucose uptake and indicate that these lipid species act downstream of IRS1 to reduce muscle glucose uptake [48,49*]. Ceramides can impair insulin signalling in muscle by reducing Akt activation [50,51]. Possible explanations are the ceramide-mediated activation of protein phosphatases that dephosphorylate Akt, and the atypical PKC[zeta]-mediated phosphorylation of the pleckstrin homology domain of Akt, precluding Akt migration to the plasma membrane to become activated [50,52,53]. However, the moderate reductions observed in Akt activity may not be the cause of insulin resistance of muscle glucose uptake, since GLUT4 translocation can proceed with as little as 20% of Akt activation [54]. This suggests that targets other than Akt may contribute to the ceramide-induced insulin resistance. In this regard, the cell-permeating short chain C2-ceramide inhibits insulin- dependent Rac activation and its consequent actin remodelling in cultured L6 myotubes, which is essential for GLUT4 translocation [55]. Interestingly, muscle ceramide levels are only elevated when muscle is exposed to saturated FA (palmitate) and not unsaturated FA (linoleate), and inhibition of ceramide biosynthesis via serine palmitoyl transferase with myriocin prevents palmitate-induced insulin-resistant glucose uptake, but not the insulin resistance caused by linoleate [48]. Compellingly, treatment of Zucker diabetic fatty rats with myriocin prevented the onset of diabetes, correlating with lower ceramide levels in muscle [48]. Similarly, in L6 myotubes the palmitate-induced reduction in insulin-stimulated Akt and glucose uptake was relieved by myriocin, which also reduced ceramide levels. Surprisingly, longer treatment with myriosin diverted palmitate towards DAG synthesis and reduced IRS1-directed insulin signalling [49*]. Insulin resistance could also be relieved upon incubating human muscle cells with palmitate along with the unsaturated FA oleate, which redirected palmitate away from ceramides to IMTG storage [56]. These results suggest that palmitate generates insulin resistance primarily via ceramides, provided its metabolism is not diverted to DAG or IMTG. Whether ceramides directly influence insulin sensitivity in human skeletal muscle in vivo remains unclear [57-59].

In summary, ceramide build-up may be a contributor to insulin resistance as a result of a diet high in saturated FAs and further studies should clarify the influence of specific FAs on muscle ceramide content and insulin resistance and their mode of action.

120 Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023. Nicolas J. Pillon 09/04/2019

Metabolic dysfunction in skeletal muscle: Lipotoxicity • What are the most deleterious lipid species for insulin signalling? • What is the primary target of ceramides to impair insulin signalling? • What are the primary mediators of the deleterious effects of DAGs?

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Insulin Resistance, Molecular Mechanisms INFLAMMATION Nicolas J. Pillon 09/04/2019

“Inflammation” Exercise Tissue Wounds damage Auto- Virus immunity

Bacteria Metabolic stress

Inflammation “to set on fire”

Further damage Recruitment of immune cells Resolution (loss of function) to the site of infection / damage Repair

Inflammatory signals occur inside all cells Macrophages are professional inflammatory cells

Cell-autonomous inflammation Professional inflammatory cells (within the same cell) (monocytes and macrophages) NFkB pathway Monocytes

IKK Complex (migrate from blood to tissues)

phosphorylation IB degradation Macrophages Transcription factor to Cytokine gene nucleus expression (pro- or anti-inflammatory)

124 Nicolas J. Pillon 09/04/2019

Crosstalk between skeletal muscle and immune cells

Acute muscle injury, mechanical stress (e.g. exercise, muscular dystrophies), endotoxins and saturated fat signal through different pathways, activating the transcription factors NF-κB and/or c-Jun/AP-1 leading to expression and secretion of muscle factors (e.g. chemokines and non-protein mediators, see Table 1) (a,d). These compounds are responsible for the recruitment of immune cells from the circulation (neutrophils, monocytes) into the muscle (b). Once in the tissue, the infiltrating and resident immune cells produce additional factors that reciprocally affect the muscle (c). This leads to the acute inflammatory response necessary for pathogen clearance and tissue repair (green arrow). If this crosstalk is deregulated, chronic inflammation ensues (red arrow) and leads to pathological complications such as fibrosis and possibly impaired insulin action (insulin resistance).

125 Pillon NJ, Bilan PJ, Fink LN, Klip A. Cross-talk between skeletal muscle and immune cells. Am J Physiol Endocrinol Metab. 2013 Mar 1;304(5):E453-65.

Inflammatory mediators released by skeletal muscle

Produced by Conditions Electrical pulse stimulation Muscle cells Lipids Lactate Macrophages N/A Sepsis (LPS) Muscle tissue Obesity/Diabetes Exercise Muscle cells Electrical pulse stimulation Cell damage ATP LPS Macrophages Chemotaxis Muscle tissue Exercise Muscle repair Muscle cells Palmitate exposure Eicosanoids Macrophages LPS Muscle tissue Exercise Electrical pulse stimulation Muscle cells Palmitate LPS, TNFα, IL-1β IL-6 LPS Macrophages Palmitate Exercise Muscle tissue High fat feeding LPS Muscle cells Palmitate Differentiation LPS TNFα Macrophages Palmitate Exercise Muscle tissue Myopathies High fat feeding LPS, TNFα, IL-1β Muscle cells Palmitate CCL2 Macrophages LPS Acute injury, Endotoxemia Muscle tissue Myopathies, High fat feeding 126 Nicolas J. Pillon 09/04/2019

Molecular mechanisms of inflammation-induced insulin resistance

Catrysse L, van Loo G. Inflammation and the Metabolic Syndrome: The Tissue-Specific Functions of NF-κB. Trends Cell Biol. 2017 Jun;27(6):417-429. 127 Tilg H, Moschen AR. Insulin resistance, inflammation, and non-alcoholic fatty liver disease. Trends Endocrinol Metab. 2008 Dec;19(10):371-9.

Metabolic dysfunction in skeletal muscle: Inflammation • What are the two “types” of inflammatory responses? • What molecules are the main mediators of inflammatory responses? Which ones have been associated with skeletal muscle insulin resistance? • What are the two main inflammatory signalling pathways impacting on insulin sensitivity. What are their main targets?

128 Nicolas J. Pillon 09/04/2019

Molecular Physiology Master Course

Skeletal muscle: From basic physiology to exercise and diabetes Nicolas J. Pillon

www.nicopillon.com [email protected] www.nicopillon.com

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133 www.genecards.com / www.proteinatlas.org / www.biogps.org

GLUTs in skeletal muscle: RNA

134 Nicolas J. Pillon 09/04/2019

Class 1 tissue distribution: RNA and protein

135 https://www.proteinatlas.org

Class 2 tissue distribution: RNA and protein

GLUT5 GLUT7 GLUT9 GLUT11 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

136 Nicolas J. Pillon 09/04/2019

Class 3 tissue distribution: RNA and protein

137

Glucose metabolism Questions

. What is the main mechanism regulating GLUT4-dependent glucose uptake? . What are the two major stimuli regulating glucose uptake in muscle? . How many reactions are in glycolysis? . How are defined the two “phases” of glycolysis? . What is the ultimate product of glycolysis? . Why is glycolysis called “lysis”? . What is the energy yield of glycolysis? . What are the 3 possible fates of pyruvate in muscle?

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Insulin signalling

140 Jaldin-Fincati et al. Update on GLUT4 Vesicle Traffic: A Cornerstone of Insulin Action. Trends Endocrinol Metab. 2017 Aug;28(8):597-611. Nicolas J. Pillon 09/04/2019

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Exercise-regulated glucose uptake

142 Sylow L, Kleinert M, Richter EA, Jensen TE. Exercise-stimulated glucose uptake - regulation and implications for glycaemic control. Nat Rev Endocrinol. 2017 Mar;13(3):133-148. Nicolas J. Pillon 09/04/2019

143 www.genecards.com / www.proteinatlas.org / www.biogps.org

Tissue distribution: RNA and protein

FATP1 FATP2 FATP3 FATP4 FATP5 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

144 Nicolas J. Pillon 09/04/2019

Tissue distribution: RNA and protein

FATP6 CD36 CAV1 CAV2 CAV3 RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression RNA expression Protein expression

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Fatty acid transport Questions

Very Long Chain Long Chain Medium/Short Chain Fatty Acids Fatty Acids Fatty Acids

FATP FATP Cytoplasm

Acyl-CoA Acyl-CoA

CPT1 Acyl CoA CPT2 Beta oxidation

Acyl-CoA

Peroxisome TCA Beta Cycle Mitochondrial oxidation Acetyl matrix CoA

146 Nicolas J. Pillon 09/04/2019

Fatty acid metabolism Questions

. What is the main form of lipid storage in skeletal muscle? . Where are lipid stored in skeletal muscle cells? . How are glycolysis and lipogenesis connected? . Of how many steps is beta-oxidation constituted? . How many carbons are removed from fatty acids at each cycle? . What is the end product of beta-oxidation? . How much energy does beta-oxidation produce?

147

148 Nicolas J. Pillon 09/04/2019

Sensing of Fuel and Energy Status AMP-activated Protein Kinase (AMPK)

149 Hardie DG, Sakamoto K. AMPK: a key sensor of fuel and energy status in skeletal muscle. Physiology (Bethesda). 2006 Feb;21:48-60.

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Measuring Energy Sources in vivo Respiratory Exchange Ratio = Respiratory Quotient

Breakfast A Breakfast B 2 2

1.0 1.0

0.9 0.9

0.8 0.8 RER RER

0.7 0.7

0.6 0.6 23456789101112 23456789101112 Time Time

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Modulation of substrate preference Physical activity

Carbohydrate oxidation increases with intensity Lipid oxidation increases with duration

Carbohydrate

Fat Fat

Watt et al. Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise. J Appl Physiol (1985). 2003 Jul;95(1):314-21 154 Venables et al. Determinants of fat oxidation during exercise in healthy men and women. J Appl Physiol (1985). 2005 Jan;98(1):160-7. Epub 2004 Aug 27 Nicolas J. Pillon 09/04/2019

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156 Nicolas J. Pillon 09/04/2019

Progression to type 2 diabetes Oral glucose tolerance test

Healthy Prediabetes T2D 4 160

3 120

2 80

1 40 Blood glucose (g/L) Blood Insulin (μU/mL)

0 0 0 123 0 123 Time (h) Time (h) Meal Meal

158 Nicolas J. Pillon 09/04/2019

Lipid classes and metabolites linked with insulin resistance

159 Metcalfe LK, Smith GC, Turner N. Defining lipid mediators of insulin resistance - controversies and challenges. J Mol Endocrinol. 2018 Aug 1. pii: JME-18-0023.

Lifestyle and pharmacological interventions

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Literature review PHARMACOLOGICAL INTERVENTIONS

162 Nicolas J. Pillon 09/04/2019

Diabetes medication

Class Target organ How They Work

Liver Biguanides Decreases glucose released from liver. Adipose tissue? (Metformin) Increases peripheral insulin sensitivity? Skeletal muscle?

Stimulates the pancreas to release more insulin, both right after Sulfonylureas/Glinides Pancreas a meal and then over several hours

Liver Thiazolidinediones Adipose tissue Increases insulin sensitivity through PPARγ activation. Skeletal muscle?

DPP-4 Inhibitors Pancreas Improves insulin secretion and reduces glucagon after a meal.

Alpha-glucosidase Inhibitors Intestine Slows the absorption of carbohydrates.

Liver Insulin Adipose tissue Increases glucose uptake by peripheral tissues. Skeletal muscle

SGLT2 inhibitors Kidney Increases glucose excretion in urine.

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Literature review LIFESTYLE INTERVENTIONS Nicolas J. Pillon 09/04/2019

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Dietary measures and weight loss

. Low Fat, Low Calorie

. High Protein

. Low Glycemic Index Diet

. Moderate Fat Diets (Mediterranean Diet)

. Newer Dietary Approaches

. Bariatric surgery

166 Shai I, Schwarzfuchs D, Henkin Y, et al.Weight loss with a low-carbohydrate, Mediterranean or low-fat diet. N Engl J Med. 2008;359(3):229-241 Nicolas J. Pillon 09/04/2019

Cognitive-Behavioral Strategies and Mindfulness-Based Eating Awareness Therapies

http://spectrum.diabetesjournals.org/content/30/3/171

http://spectrum.diabetesjournals.org/content/30/4/244

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Physical activity Nicolas J. Pillon 09/04/2019

Prevalence of physical inactivity Moderate intensity ≤150 min/week, ages 18+, males 2010

World Health Organization facts Physical Inactivity

40 r2=0.113 p<0.0001 . Insufficient physical activity is 1 of the 10 leading risk factors for death 30 worldwide.

. Insufficient physical activity is a key risk factor for noncommunicable 20 diseases such as cardiovascular diseases, cancer and diabetes. . Physical activity has significant health benefits and contributes to prevent 10

noncommunicable diseases . Diabetes of Prevalence 0 . Globally, 1 in 4 adults is not active enough. 0 20406080 Inactivity . More than 80% of the world's adolescent population is insufficiently physically active. 60 r2=0.235 . Policies to address insufficient physical activity are operational in 56% of p<0.0001 WHO Member States. 40 . WHO Member States have agreed to reduce insufficient physical activity by 10% by 2025. 20

*Diabetes: Fasting Blood Glucose ≥ 7 mM Prevalence of Obesity *Obesity: Body mass index >30 kg/m2 0 *Inactivity: Moderate physical activity ≤150 min/week 0 20406080 Inactivity

Source: WHO, processed by Nicolas J. Pillon, unpublished Nicolas J. Pillon 09/04/2019

Is exercise really beneficial? Cumulative survival

Men with type 2 diabetes completed a maximal exercise test. Three fitness categories were established (low-, moderate-, and high-fit) based on peak METs achieved. Subjects were followed for all-cause mortality for 7.3 ± 4.7 years.

CONCLUSION. Exercise capacity is a strong predictor of all-cause mortality.

BUT people were classified based on MET, not randomized to undergo training…

Kokkinos. Exercise Capacity and All-Cause Mortality in African American and Caucasian Men With Type 2 Diabetes. Diabetes Care 2009 Apr; 32(4): 623-628.

Is exercise really beneficial? Lifestyle intervention

BMI>24, impaired glucose tolerance (prediabetes). Randomly assigned to one of three interventions: standard lifestyle recommendations plus metformin (Glucophage) at a dose of 850 mg twice daily, standard lifestyle recommendations plus placebo twice daily, or an intensive program of lifestyle modification. Lifestyle intervention was healthy low-calorie, low-fat diet and physical activity of moderate intensity, such as brisk walking, for at least 150 minutes per week.

CONCLUSIONS. Lifestyle changes and treatment with metformin both reduced the incidence of diabetes in persons at high risk. The lifestyle intervention was more effective than metformin.

BUT the goal was at least a 7 percent weight loss with a drastic diet in addition to exercise…

Diabetes Prevention Program Research Group. Reduction in the Incidence of Type 2 Diabetes with Lifestyle Intervention. N Engl J Med 2002; 346:393-403 Nicolas J. Pillon 09/04/2019

Is exercise really beneficial? A Randomized Clinical Trial

Type 2 diabetes diagnosed less than 10 years, BMI of 25 to 40, taking 2 or fewer glucose- lowering medications. Participants were randomized in permuted blocks of 3 and 6, stratified by sex, to either the lifestyle group or the standard care group. Intensive lifestyle intervention consisted of 5 to 6 weekly aerobic sessions (duration 30-60 minutes), of which 2 to 3 sessions were combined with resistance training.

Outcome: change in HbA1c.

CONCLUSIONS. Among adults with type 2 diabetes, a lifestyle intervention compared with standard care resulted in a change in glycemic control that did not reach the criterion for equivalence, but was in a direction consistent with benefit.

BUT no significance and a clear classical effect of adherence…

Johansen et al. Effect of an Intensive Lifestyle Intervention on Glycemic Control in Patients With Type 2 Diabetes. JAMA. 2017 Aug 15;318(7):637-646.

Is exercise really beneficial? Meta-analyses

PubMed, Embase and Ovid databases were searched for Electronic database searches were performed in AMED, MEDLINE, SPORTDiscus, prospective studies and randomized trials up to 2nd of March 2015. CINAHL, EMBASE, and Web of Science Core Collections from earliest record to Eighty-one studies were included. Outcome: risk of developing T2D. December 2014. Oucome: improved insulin sensitivity (Clamp or glucose tolerance tests).

CONCLUSION. All types of physical activity are associated with a statistically significant reduction in the risk of type 2 diabetes and increased insulin sensitivity. BUT Meta-analyses are inherently biased…

Aune et al. Physical activity and the risk of type 2 diabetes: a systematic review and dose-response meta-analysis. Eur J Epidemiol. 2015 Jul;30(7):529-42. Way et al. The Effect of Regular Exercise on Insulin Sensitivity in Type 2 Diabetes Mellitus. Diabetes Metab J. 2016 Aug;40(4):253-71. Nicolas J. Pillon 09/04/2019

Exercise benefits Glucose uptake

Three main sites/processes can be regulated: 1) glucose delivery 2) glucose transport 3) glucose metabolism

Richter & Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013 Jul;93(3):993-1017. 175 Wahren J , Felig P , Hagenfeldt L. Physical exercise and fuel homeostasis in diabetes mellitus. Diabetologia 14: 213–222, 1978.

GLUT4 and hexokinase II determine skeletal muscle glucose uptake during exercise

At rest, overexpression of GLUT4 leads to increased glucose uptake independently of HKII expression. During exercise, HKII overexpression leads to increased glucose uptake at normal and increased levels of GLUT4 expression. Furthermore, GLUT4 overexpression does not in itself lead to increased glucose uptake during exercise.

176 Richter & Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013 Jul;93(3):993-1017. Nicolas J. Pillon 09/04/2019

GLUT4 levels in human skeletal muscle fiber types before and after exercise training

The differences in GLUT4 expression between muscle fiber types are much smaller in humans than rodents.

In type I fibers, GLUT4 is increased 20–30%

This is not observed in all muscles, with relatively little difference between fiber types observed in soleus and triceps muscles. This could reflect differences in habitual activity levels.

177 Richter & Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013 Jul;93(3):993-1017.

molecular signaling involved in contraction-induced GLUT4 gene activation.

178 Richter & Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev. 2013 Jul;93(3):993-1017. Nicolas J. Pillon 09/04/2019

Benefits of Exercise

• Manage blood sugar and insulin levels • Body weight control • Reduced risk of heart diseases • Improve your mental health • Helps thinking and learning • Maintain muscle mass during ageing • Reduce your risk of falls • Reduce your risk of some cancers • Improve sleep • Improve sexual health

Is exercise really beneficial? Position Statement of the American Diabetes Association

Aerobic Exercise Benefits • Substantially lower cardiovascular and overall mortality risks in both type 1 and type 2 diabetes • In individuals with type 2 diabetes, reduces A1C, triglycerides, blood pressure, and insulin resistance • High-intensity interval training (HIIT) promotes rapid enhancement of skeletal muscle oxidative capacity, insulin sensitivity, and glycemic control in adults with type 2 diabetes

Resistance Exercise Benefits • Improvements in muscle mass, body composition, strength, physical function, mental health, bone mineral density, insulin sensitivity, blood pressure, lipid profiles, and cardiovascular health • Improvements in glycemic control, insulin resistance, fat mass, blood pressure, strength, and lean body mass in individuals with T2D

Recommendations • Daily exercise, or at least not allowing more than 2 days to elapse between exercise sessions, is recommended to enhance insulin action. • Adults with type 2 diabetes should ideally perform both aerobic and resistance exercise training for optimal glycemic and health outcomes. • Children and adolescents with type 2 diabetes should be encouraged to meet the same physical activity goals set for youth in general. • Structured lifestyle interventions that include at least 150 min/week of physical activity and dietary changes resulting in weight loss of 5%–7% are recommended to prevent or delay the onset of type 2 diabetes in populations at high risk and with prediabetes.

Colberg et al. Physical Activity/Exercise and Diabetes: A Position Statement of the American Diabetes Association. Diabetes Care. 2016 Nov;39(11):2065-2079. Nicolas J. Pillon 09/04/2019

Take home message: Exercise is an excellent therapeutic tool

• A 150 min of moderate-to-vigorous physical activity accumulated per week can reduce the risk of most major chronic diseases by 25–50%.

• A 15 min of moderate-to-vigorous physical activity per day (or 75 min/week) is associated with a ∼15% relative mortality risk reduction.

Benefits increase with the dose: a little bit is better than nothing and the more the better.

DocMikeEvans – 23½ hours https://www.youtube.com/watch?v=aUaInS6HIGo

Thornton et al. Physical Activity Prescription: A Critical Opportunity to Address a Modifiable Risk Factor for the Prevention and Management of Chronic Disease: A Position Statement by the Canadian Academy of Sport and Exercise Medicine. Clin J Sport Med. 2016 Jul;26(4):259-65.

Molecular Physiology Master Course

Skeletal muscle: From basic physiology to exercise and diabetes Nicolas J. Pillon

www.nicopillon.com [email protected] www.nicopillon.com Nicolas J. Pillon 09/04/2019