Regulation of Insulin Secretion II MPB333_Ja2 Glucose stimulated insulin secretion (GSIS) [Ca2+] i V...

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Transcript of Regulation of Insulin Secretion II MPB333_Ja2 Glucose stimulated insulin secretion (GSIS) [Ca2+] i V...

  • 1

    Regulation of Insulin Secretion II

    Cellular Signaling in the Islet of Langerhans

    Richard KP Benninger

    Islet of Langerhans

    -cell -cell -cell

    Endocrine pancreas

    Exocrine pancreas

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

  • 2

    Glucose stimulated insulin secretion (GSIS)

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    Insulin granule exocytosis

    Defects in many genes that underlie GSIS increase risk of diabetes

    Kir6.2, Sur1 (KATP channel subunits) Channel mutations can cause neonatal diabetes SNPs associated with enhanced risk to type 2 diabetes

    GK (Hexokinase 4) Heterozygous mutations cause MODY-2 Homozygous mutations cause neonatal diabetes

    Ins1,Ins2 (Insulin genes) Mutations cause neonatal diabetes

    HNF1- (transcription factor, regulates mitochondria) Mutations cause MODY-3

    ZnT8 (Zn2+ Transporter) SNPs associated with enhanced risk to type 2 diabetes Other SNPs such as TCF7L2 also enhance risk to type 2 diabetes

  • 3

    Many points underlying GSIS are potential therapeutic targets

    Sulfonylureas close KATP channels and trigger insulin secretion: Glibenclamide (Glyburide)

    GLP1R agonists elevate cAMP and enhance insulin secretion and cell proliferation: Exendin-4

    Glucose stimulated insulin secretion (GSIS)

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    Insulin granule exocytosis

  • 4

    GLUT2 and GK

    GLUT2 is a high capacity glucose transporter Intracellular [glucose] Extracellular [glucose]

    GK (Hexokinase 4) phosphorylates glucose Produces glucose-6-phosphate Enters glycolysis and then CAC metabolism Elevates ATP/ADP ratio

    GK is the rate limiting step in GSIS

    KD of GK is ~7mM glucose Rate limiting step for ATP synthesis Compared to

  • 5

    Glucose stimulated insulin secretion (GSIS)

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    Insulin granule exocytosis

    Glucose metabolism leads to membrane depolarization KATP channel closure

    Repeated APs fire following depolarization Regulated by voltage gated K+, Na+, Ca2+ channels

    Electrical activity in the -cell

  • 6

    ATP-sensitive Potassium channel (KATP)

    KATP is a hetero-octomer Kir6.2 and Sur1 4:4

    ATP closes channel MgADP opens channel Channel mutations can shift

    the sensitivity to ATP

    The KATP channel and electrical activity play a key role in islet function

    Expression of ATP-insensitive KATP channels leads to a loss of serum insulin

  • 7

    The KATP channel and electrical activity play a key role in islet function

    Mutations that reduce the ATP sensitivity of KATPchannels cause neonatal diabetes in humans

    The KATP channel and electrical activity play a key role in islet function

    Sulfonylureas are an effective treatment for patients with KATP induced permanent neonatal diabetes

  • 8

    Other ion channels are also important

    Jacobson et al. Cell Metabolism 2007

    Voltage gated K+ channels (Kv) Regulates AP repolarization Kv2.1-/- shows elevated insulin secretion

    Kv2.1+/+

    Kv2.1-/-

    Glucose stimulated insulin secretion (GSIS)

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    Insulin granule exocytosis

  • 9

    Ca2+ and insulin secretion Membrane depolarization activates L-type Ca2+

    channels Influx of Ca2+ into cell Triggers insulin granule exocytosis

    Measuring Ca2+ in cells

    Label cells with Ca2+ sensitive fluorescent dye

    Fura-3 Calcium Response

  • 10

    Measuring Ca2+ in cells

    Image the fluorescence from the Ca2+ dye on a confocal microscope

    Calcium oscillations in the islet

    0 20 40 60 80 100 120

    Fluo

    -4 In

    tens

    ity (n

    orm

    .)

    Time (sec.)

  • 11

    Pulsatile Insulin secretion Oscillations in plasma insulin are found in

    humans, dogs and mice 3-8 minute period

    Pulsatile insulin has been shown to exert a greater glucose lowering action than continuous levels of insulin

    Insulin oscillations are disrupted obese individuals and in patients with type2 diabetes

    Matthews et al. Diabetes 1983Bratuschmarrain et al. Diabetes 1986Porksen et al, Diabetes 2002

    Pulsatile insulin secretion and Ca2+

    Pulsatile insulin levels are related to Ca2+oscillations in isolated islets

    Nunemaker et al. Diabetes 2005

  • 12

    Summary I

    Electrical activity is critical for normal insulin secretion Defects underlie many aspects of diabetes

    Many regulators of electrical activity can be potential therapeutic targets e.g. KATP, KV, CaV, channels, and more

    Oscillations in electrical activity leads to pulsatileinsulin secretion Insulin oscillations have a greater hypoglycemic effect

    and are disrupted in type2 diabetes

    Multi-cellular properties of the islet

    Glucose

    Insulin

    Glucose

    Insulin

    Glucose

    Insulin

    Glucose

    Insulin

    Glucose

    Insulin

    Glucose

    Insulin

    High dynamic range of secretion

    Low dynamic range of secretion

    Time

    Insulin

    Time

    Insulin

    Time

    Insulin

    Time

    InsulinCoordinated

    pulsatile secretionContinuous

    irregular secretion

    Islet transplant

    -cell transplant

  • 13

    Cell-cell communication in the islet

    [Ca2+]i

    Vm

    ATPADP

    KATP

    CaV

    GLUT2

    mitochondria

    GK

    glucose

    glycolysis

    PKA Epac

    cAMP

    ATP AC

    Gs GPCR

    glucagon GLP1

    Gap junction channels

    Receptors for hormones and neurotransmitters

    Cell adhesion molecules

    Islets are vascularizedand innervated

    Cells in the islet express several factors for cell to cell communication

    Role of gap junction in cells Gap junctions are made of 2 connexon hemichannels Each connexon is made of 6 connexin subunits Gap junction channels allow ionic currents, and small

    molecules to pass between cells

  • 14

    Kir6.2[AAA] loss-of-function mutation

    Rocheleau et al. PLoS Biology 2006Koster et al. Cell 2000

    Kir6.2 subunit

    Critical role of gap junction coupling

    Rocheleau et al. PLoS Biology 2006

    Inhibiting gap junction coupling leads to spontaneous Ca2+ bursts in the islet

    Gap junction coupling overcoming the Kir6.2[AAA] induced excitability

  • 15

    Cx36 gap junctions couple -cells

    +/+ +/- -/-0.0

    0.2

    0.4

    0.6

    0.8

    1.0

    1.2

    Cou

    plin

    g co

    nduc

    tanc

    e (n

    S)

    C x-36

    Ravier, et al. (2005) Diabetes Benninger, et al. (2008) BiophysJ

    Ca2+ dynamics in Cx36 knockout

    F/F 0

    0 100 200 300Time (sec)

    Cx36+/+: 100%

    500 600 700 800

    Time (sec)

    T=-5 sec

    T=+5 sec

    T=-5 sec

    T=+5 sec

    Cx36+/-: ~50%

    1200 1300 1400 1500Time (sec)

    T=-5 sec

    T=+5 sec

    Cx36-/-: ~0%

    T=+5 sec

    T=-5 sec

  • 16

    Oscillations in Cx36-/- islets

    Total loss in synchronized [Ca2+]i activity

    Played at x15 normal speed

    Regulation of pulsatile insulin secretion?

    Loss of Cx36 leads to an absence of pulsatile insulin secretion from single isolated islets

    Ravier et al. (2005) Diabetes

  • 17

    GSIS vs gap junctions in intact islets

    +/+ +/- -/-0.0

    0.5

    1.0

    1.5

    Ins.

    con

    tent

    (ug/

    10is

    lets

    )

    0 5 10 15 200

    10

    20

    30

    40

    50

    Insu

    lin s

    ecre

    tion

    (ng/

    10is

    lets

    /60m

    ins.

    )

    Glucose (mM)

    Cx36+/+ Cx36+/- Cx36-/-

    No significant change in GSIS upon a loss of gap junction coupling

    Cx36-/- mice are glucose intolerant

    0 30 60 90 1200

    100

    200

    300

    400

    Plas

    ma

    gluc

    ose

    (mg/

    dl)

    Time (min.)

    Cx36+/+ Cx36-/-

    0 30 60 90 1200

    100

    200

    300

    400

    Pla

    sma

    gluc

    ose

    (mg/

    dl)

    Time (min.)

    Cx36+/+ Cx36-/-

    Male, 16 weeks Female, 16 weeks

    **

    *

    ***** *

  • 18

    Summary II An absence of gap junction coupling leads to

    reduced glucose tolerance Gap junction coupling is necessary to coordinate

    oscillations in electrical activity Necessary for pulsatile insulin secretion from isolated

    islets Gap junction coupling does not play a signific