Beta Cell ERKonomics. Functions of ERK1/2 in Pancreatic Beta Cells


Uploaded by nihvcast on 31.05.2012

Transcript:
>> GOOD AFTERNOON, EVERYONE.
WELCOME TO YOU IN THE MASUR
AUDITORIUM AND ALL OF THOSE
LISTENING BY VIDEOCONFERENCE AND
I HAVE ALREADY ASSURED OUR
SPEAKER THAT THE NUMBER OF
PEOPLE WHO WILL SOAK UP THE
SCIENCE THAT SHE PRESENTS IS
CONSIDERABLY GREATER THAN THE
PEOPLE SHE CAN SEE IN FRONT OF
US.
THANK YOU FOR THOSE OF YOU WHO
TURNED OUT IN PERSON BECAUSE
THAT'S ALWAYS A LITTLE BIT MORE
INTERACTIVE THAT WAY.
I'M DELIGHTED TO INTRODUCE OUR
SPEAKER, MELANIE COBB WHO IS THE
PROFESSOR OF PHARMACOLOGY AT UT
SOUTHWESTERN MEDICAL CENTER AND
HOLDS THE JANE AND BILL
BROWNING, JR. ENDOWED CHAIR IN
MEDICAL SCIENCE.
IF YOU'RE WATCHING THE CALENDAR,
SHE WAS SCHEDULED TO SPEAK TO US
ON APRIL 4, AND IF YOU WEREN'T
PAYING ATTENTION TO WHAT ELSE
HAPPENED THAT DAY, YOU MIGHT
WONDER WHAT HAPPENED TO THAT
PLAN.
WELL, IT WAS NOT EXACTLY IN HER
PLAN NOT TO GET ON A PLANE THAT
DAY AND IT WASN'T IN OUR PLAN TO
NOT HAVELER ARRIVE.
IT HAD SOMETHING TO DO WITH A
TORNADO THAT PLOWED THROUGH
DALES AND DISRUPTED THE
DALLAS-FORT WORTH AIRPORT FOR
QUITE A FEW DAYS.
SO WE WERE VERY PLEASED THAT
DESPITE THAT PARTICULAR
UNFORTUNATE OCCURRENCE, MELANIE
WAS WILLING TO RESCHEDULE AND
COME BACK AND SPEAK TO US TODAY,
MAY 30.
WE ARRANGED A THUNDERSTORM LAST
NIGHT, WHICH DELAYED HER PLANE
FROM LANDING FOR TWO HOURS.
SO, YES, THE TRADITION
CONTINUES.
HOPEFULLY, THE FLIGHT HOME WILL
BE UNEVENTFUL.
DR. COBB OBTAINED HER
UNDERGRADUATE DEGREE AT THE
UNIVERSITY OF CHICAGO, Ph.D.
IN BIOLOGICAL CHEMISTRY AT WASH
U AFTER POSTDOCTORAL TRAINING AT
MOUNT SINAI AND A THREE-YEAR
PERIOD AS A RESEARCH ASSOCIATE
AT ALBERT EINSTEIN.
SHE LANDED A FACULTY POSITION AT
UT SOUTHWESTERN AND ESSENTIALLY
BEEN THERE SINCE THAT TIME FOR
MOST OF THE REMARKABLE CAREER
SHE HAS CARRIED OUT, STUDYING A
VARIETY OF ISSUES, PARTICULARLY
WELL-KNOWN FOR HER WORK ON MAP
KINASES AT UT SOUTHWESTERN.
FOR A TIME SHE WAS THE DEAN OF
THE SOUTHWESTERN GRADUATE SCHOOL
AND NOW IS PROFESSOR AND HOLDER
OF ENDOWED CHAIR.
FOR ALL OF US WHO HAVE STRUGGLED
TRYING TO UNDERSTAND MAP KINASES
AND MAP KINASES AND MAP KINASES
AND KINASES AND I DON'T KNOW HOW
MANY KS WE ARE UP TO, IT'S A
RELIEF TO HAVE SOMEBODY EXPLAIN
THIS STUFF.
NOT ONLY DOE WE HAVE A
DISTINGUISHED SPEAKER MEMBER OF
THE NATIONAL ACADEMY TO LEAD US
THROUGH THIS, BUT SHE MADE
SEMINOLE CONTRIBUTIONS TO THIS
WHOLE FIELD, PARTICULARLY IN THE
ORIGINAL IDENTIFICATION OF ERK1
AND ERK2 AND MANY OTHER STEPS
THAT FOLLOWED NOW BEING APPLIED
AS SHE WILL TELL US ABOUT TODAY
IN TERMS OF THEIR FUNCTION IN
THE PANCREATIC BETA CELL, MOST
OBVIOUS IMPLICATIONS FOR
DIABETES, A DISEASE WHICH IS A
VIRTUAL EPIDEMIC PROPORTIONS
RIGHT NOW IN OUR COUNTRY AND
INCREASINGLY SO IN THE REST OF
THE WORLD.
SO IT IS A GREAT PLEASURE TO
HAVE HER HERE TODAY.
PLEASE JOIN ME IN WELCOMING
PROFESSOR MELANIE COBB.
[ APPLAUSE ]
>> THANK YOU VERY MUCH FOR THE
INTRODUCTION AND THANK YOU FOR
INVITING ME TWICE.
I'M GLAD I ACTUALLY MADE IT ONE
OF THOSE TIMES.
OKAY, SO JUST BRIEFLY TODAY I'M
GOING TO TELL YOU ALL STORIES
ABOUT PANCREATIC BETA CELLS
AFTER A LITTLE BIT OF BACKGROUND
ON ERK1 AND 2.
SO, I THINK IT'S BECOME
INCREASINGLY CLEAR THAT ERK1 AND
2 ARE INVOLVED IN ALL KINDS OF
PROCESSES IN CELLS AND IN FACT,
I CAN THINK OF FEW EVENTS THAT
DON'T HAVE SOME INPUT FROM THESE
PARTICULAR MAP KINASES.
THEY ARE KEY IN PROLIFERATION
AND CELL LINEAGE COMMITMENT AND
MAINTENANCE OF DIFFERENTIATED
FUNCTIONS AND THAT'S WHAT I'M
MOST INTERESTED IN AND WHY WE
LOOKED A LOT AT PANCREATIC BETA
CELLS.
THEY ARE MISREGULATED IN ALL
KINDS OF DISEASE SYNDROMES,
PARTICULARLY CANCER DUE TO
ACTIVATION OF UPSTREAM
ACTIVATORS OF THE PATHS WAY BY
MUTATION AND A NUMBER OF
CANCERS.
THERE ARE A RANGE OF
DEVELOPMENTAL SYNDROMES THAT END
UP IMPACTING THE ACTIVITY STATE
OF ERK1 AND 2.
SOME OF THESE ARE AMONG THE MOST
COMMON DEVELOPMENTAL SYNDROMES
IN PEOPLE, AND THERE ARE ALSO
NEGATIVE REGULATORS, TYROSINE
KINASE SIGNALING THAT ARE
INVOLVED IN CAUSING ERK
IMPACTIVE AND DEVELOPMENTAL
ISSUES.
SO, A NUMBER OF YEARS AGO
THROUGH COLLABORATIONS WITH
BETSY SMITH AND ONE OF HER
POSTDOCS AND STUDENTS WHO MIGHT
BE HERE AS A FELLOW, WE SOLVED
THE CRYSTAL STRUCTURES OF ERK2
AND THE UNPHOSPHORYLATED STATE
HERE AND IN THE PHOSPHORYLATED
STATE.
IT LOOKS LIKE A TYPICAL PROTEIN
KINASE, ONE OF THE 9s THINGS
IS IT IS A RELATIVELY SMALL
PROTEIN SO WE COULD SEE THE
WHOLE PROTEIN.
IT'S 41 KILLADALTONS, IT HAS A
SMALLER END TERMINAL DOMAIN AND
A LARGER C TERMINAL DOMAIN AND
ATP BINDS AT THE INTERIOR OF THE
INTERFACE BETWEEN THE DOMAINS.
IT'S ACTIVATED BY TWO
PHOSPHORYLATIONS AND IN THE CASE
OF ERK2 THAT ACTIVATION IS
REALLY PROFOUND CAUSING ABOUT A
50,000-FOLD INCREASE IN P CAT
AND THE TWO PHOSPHORYLATIONS ARE
IN THIS ACTIVATION LOOP REGION
HERE AND THEY CAUSE STRUCTURAL
REARRANGEMENTS IN THE PROTEIN TO
CAUSE THIS ACTIVITY INCREASE.
A COUPLE OF POINTS I'LL COME
BACK TO LATER A LITTLE BIT ABOUT
THE MAP KINASE INSERT.
THESE PURPLE REGIONS ARE REGIONS
THAT ARE NOT CONTAINED IN THE
CORE KINASE DOMAIN.
THEY ARE INSERTS AND MAP KINASES
IN PARTICULAR.
THIS INSERT HERE AND WINDING UP
THE BACK OF THE PROTEIN, WE HAVE
A C TERMINAL REGION THAT HAS AN
IMPORTANT 310 HELIX HERE AND
HELIX AT THE TOP OF THE
MOLECULE.
IN TERMS OF THE DETECTION OF
ACTIVITY WHILE WE USE FOR MANY
YEARS IN THE COMPLEX KINASE
ASSAY, NOW JUST ABOUT EVERYBODY
USES ANTIBODIES TO THE
PHOSPHORYLATED FORMS OF ERK1 AND
2.
A COMB OF REASONS THOSE ARE SO
GOOD, THE TWO
FORFOES -- PHOSPHORYLATIONS
SITES ARE SEPARATED BY A SINGLE
RESIDUE AND THE SECOND IS, THESE
ARE ABUNDANT PROTEINS AS WE
THINK OF SIGNALING PROTEINS
SOMETIMES AS PERHAPS SOMEWHAT
RARE BUT THESE MAY BE ON THE
ORDER OF A COUPLE OF 100 NANA
MOLOR OR MICROMOLAR IN CERTAIN
CELLS.
SO THEY ARE EASY TO MEASURE AND
DETECT.
AND SO, I'M GOING TO TALK ABOUT
THIS PATHWAY AND I JUST FOR THE
SAKE OF FULL DISCLOSURE WANT TO
POINT OUT THAT ERK2 FITS HERE IN
THIS EXAMPLE OF THE SIGNALING
NETWORK AND IT'S VERY EASY TO
FOCUS ON ONE LITTLE PATHWAY AND
LOSE SITE OF THE BIG PICTURE,
WHICH IS ALL OF THESE
INTERCONNECTIONS AND SO, MORE
AND MORE WE ARE TRYING TO
ACKNOWLEDGE THESE
INTERCONNECTIONS AND FIGURE OUT
HOW THEY REALLY CONTRIBUTE TO
WHAT ALL HAPPENED WHEN THIS
PATHWAY IS TURNED ON.
OKAY.
SO MUCH OF WHAT I'M GOING SAY
WILL FOCUS ON SPECIFICITY IN
DIFFERENT WAYS AND THE FIRST
THING I WANT TO POINT SOUGHT
ALTHOUGH THIS CASCADE CAN BE
ACTIVATED BY ALL KINDS OF
LIGANDS, ALMOST EVERY SINGLE
AGENT THAT EFFECTS CELL FUNCTION
WILL CHANGE THE ACTIVITY OF ERK1
AND 2.
THERE IS ONE STEP WHERE
INCREDIBLE SPECIFICITY IS
GENERATED IN MAP KINASE PATH
EXPWAIS I HAVE 3 HERE.
THIS IS THE MAP KINASE STEP AND
JUST TO CLARIFY, THIS IS THE MAP
TWO KINASE STEP OR MAP KK.
AND IT IS AT THIS STEP THAT WE
HAVE INCREDIBLE SPECIFICITY AND
THIS JUST SHOWS HERE
PHOSPHORYLATION AND ACTIVATION
OF IN THIS CASE, PHOSPHORYLATION
OF ERK2 BY THE MEK AND ITS
PATHWAY COMPARED TO THE MAP
2Ks AND THE MAP 38 PATHWAY.
THERE IS MORE OR LESS ABSOLUTE
SPECIFICITY DIFFERENCE.
WHILE LOTS OF THINGS TURN ON
THIS PATHWAY, THEY HAVE TO WORK
THROUGH THIS PARTICULAR CASCADE
TO DO IT.
SO I MENTION THE THAT MANY
AGENTS CONTROL THIS PATHWAY AND
CAN CHANGE ITS ACTIVITY.
ALMOST A DIZZYING NUMBER AND
IT'S SOMETIMES HARD TO GET A
GRIP ON HOW DIFFERENT ACTIVATING
MECHANISMS CAN LEAD TO DIFFERENT
OUTPUTS.
AND WHAT CAN YOU SEE HERE IS OLD
DATA B-20 YEARS FOLD A WHOLE
BUNCH OF DIFFERENT LABORATORIES
WE WORKED ON THIS PROBLEM.
ACTIVATION OF ERK1 AND 2 AND
PC12 CELLS BY TWO DIFFERENT
LIGANDS.
EGF IS A RAPID INCREASE IN
ACTIVITY THAT IS TRANSIENT AND
NGF, NERVE GROWTH FACTOR THAT
CAN BE DETECTED THE NEXT DAY.
SO THE DIFFERENCES IN LIGANDS
ACTIVATION MECHANISM HAS A BIG
IMPACT ON THE OUTPUT OF THE
PATHWAY SO IT'S THIS PATHWAY
THAT IS ASSOCIATED PRIMARILY
WITH PROLIFERATION OF PC12 AND
NGF WITH NURRITE EXTENSION.
SO PHOSPHATASES HAVE BEEN
IMPLICATED AS ONE MECHANISM FOR
CONTROLLING THIS TYPE OF
LIGAND-DEPENDENT KINETIC
SPECIFICITY.
AND THIS WILL COME UP AGAIN WHEN
WE GET TO BETA CELLS IN JUST A
MINUTE.
SO ANOTHER IMPORTANT FACTOR IN
DETERMINING WHAT THESE ENZYMES
DO, THEY CAN BE ACTIVATED BY A
NUMBER OF LIGANDS BUT THEY DON'T
ALWAYS LOCALIZE IN ACTIVE FORM
AT THE SAME PLACE.
AND I'LL SHOW A COUPLE MORE
SLIDES ABOUT THAT BUT FIRST I
WANT TO POINT OUT THERE ARE
LARGE NUMBER OF SCAFFOLDING
PROTEINS THAT DETECT THESE
ENZYMES AND LOCALIZE THEM IN
PARTICULAR PLACES DEPENDING ON
WHICH LIGANDS HAVE ACTIVATED
THEM.
SO FOR EXAMPLE, ONES THAT WERE
PARTICULARLY INTERESTED IN ARE
THESE NUCLEAR POOR PROTEINS AND
3 OR 4 OF THESE ARE KNOWN TO BE
VERY TIGHT BINDERS FOR MAP
KINASES.
THERE ARE SOME PROTEINS LIKE
EEA15, WHICH KEEPS ERK2 OUT OF
THE NUCLEUS.
THERE ARE SCAFFOLDS THAT
LOCALIZE TO THE CYTOSKELETON AND
VARIOUS ORGANELLES, FOR EXAMPLE,
AND ONES THAT HAVE VERY DISTINCT
CELL TYPE DEPENDENT FUNCTIONS
SUCH AS K -- KSR PROTEINS.
SCAFFOLDING HAS A LOT TO DO WITH
IT.
THIS MAYBE ONE OF THE
LEAST-UNDERSTOOD ASPECTS OF HOW
THESE PROTEINS ARE CONTROLLED.
ABOUT LOCATION.
WE STARTED LOOKING AT NUCLEAR
LOCALIZATION ALONG A NUMBER OF
YEARS AGO, QUITE A LONG TIME
AGO.
AND WHAT WE FOUND WAS THAT IF WE
USED RECOMBINANT ERK2 AND PERM
BRIDES CELLS, WE COULD SEE THAT
THE PROTEIN LOCALIZED IN THE
NUCLEUS WHETHER OR NOT WE ADDED
AN ENERGY SOURCE OR CYTOSOLIC
TRANSPORT FACTORS.
AND HERE SAY STUDY WITH GFP ERK2
AND ALSO LOCALIZES TO THE
NUCLEUS.
IN THIS CASE, WE COMPARED TO
NUCLEAR LOCALIZATION IN
CONSTITUTION STUDIES TO NLSVSA
IMPORT SUBSTRATE USED FREQUENTLY
TO LOOK AT BETA ONE DEPENDENT
IMPORT.
AND YOU CAN SEE HERE THAT IMPORT
OF NLSBSA REQUIRES TRANSPORT
FACTORS AND REQUIRES AN ENERGY
SOURCE AND GETS INTO THE NUCLEUS
EQUALLY PRETTY MUCH REGARDLESS
OF ADDITIONAL FACTORS ADDED.
AND IMPORT CARRIERS COMPETE WITH
ERK2 FOR ENTRY BECAUSE ERK2
BINDS TO A SIMILAR LOCATION ON
NUCLEAR POOR PROTEINS AS THE
IMPORT FACTORS.
HERE, FOR EXAMPLE, IS A
SCHEMATIC MODEL OF NUCLEAR POOR
PROTEINS ON THE NUCLEAR POOR AND
SOME OF THESE PROTEINS CONTAIN A
WHOLE STRING OF FX-FG TYPE OF
MOTIFS.
THESE WERE SHOWN BY A LAB, IN
PARTICULAR ERK1 AND 2 AND SELECT
ACTIVITY AND SO ERK CAN BIND TO
THESE FX AND FG MOTIFS COMMON ON
THESE NUCLEAR POOR PROTEINS AND
YOU CAN SEE HERE JUST A
PULL-DOWN ASSAY SHOWING BINDING
OF ERK2 TO THE C TERMINAL DOMAIN
OF NUCLEAR PORE 163.
SO 358 NUCLEAR PORE PROTEIN 358
AND PPR HAVE ALSO BEEN SHOWN TO
BIND AND I BELIEVE THERE ARE TWO
OR THREE OTHERS THAT HAVE ALSO
BEEN SHOWN TO BIND.
SO ERK2 PROBABLY SPENDS A LOT OF
TIME AROUND THE NUCLEAR PORE.
INTERESTINGLY, MOST OF THE
CHARACTERIZATION OF THE
LOCALIZATION OF ERK2 WAS DONE IN
TRANSFORMED CELLS, THE STANDARD
CELL LINES THAT PEOPLE USE IN
THE LABORATORY LIKE RACK 52
CELLS.
IF WE USE ERK2, YOU CAN SEE IN
THE UNSTIMULATED STATE, THERE
MIGHT BE A SIGNAL FROM ONE CELL.
BUT IF YOU LOOK AT CELLS
STIMULATED WITH EGF ORESTER FOR
10 MINUTES, THAT MUCH OF THE
PROTEIN IS LOCALIZED IN THE
NUCLEUS ALTHOUGH YOU CAN SEE
SOME LOCALIZATION AROUND THE
CELL PERIPHERY IN THE CASE OF
EGF.
ON THE OTHER HAND, IF YOU LOOK
AT UNTRANSFORMED CELLS, CELLS
THAT WERE INSTEAD IMMORTALIZED
BUT NOT TRANSFORMED, LIKE HUMAN
FORESKIN FIBROBLASTS, AND A LOT
OF OTHER CELLS THAT HAVE BEEN
IMMOTOR A.ALIDES LIKE THIS, YOU
SEE SOMETHING SOMEWHAT
DIFFERENT.
STIMULATION WITH EGF DOESN'T
CAUSE THAT PRO MOUNTAINED
LOCALIZATION WHEREAS STIMULATION
WITHESTER DOES.
SO IN NONTRANSFORMED CELLS WHICH
HAVEN'T BEEN THE MODELS WE USE
VERY MUCH BUT BY-AND-LARGE IN
NON-TRANSFORMED CELLS, THE
LOCALIZATION OF THESE ENZYMES
CAN BE QUITE DIFFERENT DEPENDING
ON ACTIVATING LIGANDS.
SO, THAT'S ALL THE BACKGROUND.
THE REST OF WHAT I WANT TO TELL
YOU FOCUSES ON THE ROLE OF THESE
ENZYMES EONE AND TWO AND
PANCREATIC BETA CELLS --
ERK1 AND 2 -- ONE OF THE REASONS
WE WERE INTERESTED IN THEIR
ACTIONS AND BETA CELLS IS THAT
WE FOUND EARLY ON THAT THEY WERE
IMPORTANT FOR REGULATING INSULIN
GENE TRANSCRIPTION AND I'LL HAVE
A GOOD BIT MORE TO SAY ABOUT
THAT.
ONE OF THE FASCINATING THINGS
ABOUT BETA CELLS THAT WE HAVE
LEARNED IS THAT ERK1 AND 2 ARE
ACTIVATED BY ALL OF THE SIGNALS
THAT STIMULATE INSULIN SECRETION
AND INHIBITED BY AGENTS THAT
PREVENT SECRETION.
SO THEY ARE REALLY WONDERFUL
MARKERS STIMULATION BETA CELLS
TO PRODUCTIVE INSULIN RELEASE.
I DON'T THINK THEY ARE DIRECTLY
INVOLVED IN SECRETION OR IF THEY
ARE, IT MIGHT BE A MORE MINOR
ROLE THAN SOME OF THE OTHER
ROLES, NEVERTHELESS, THEY ARE
WONDERFUL AS A READ OUT OF
STIMULATORY AGENTS ON BETA
CELLS.
SO WE USED IT AND WE LEARNED A
LOT ABOUT HOW BETA CELLS WORK
USING THIS MODEL.
SO JUST TO SHOW SOME PICTURES OF
BETA CELLS ON ONE OF MY POINTS
HERE IS THAT THIS IS A
COLORED-IN MODEL WITH ALPHA
CELLS IN RED AND BETA CELLS IN
GREEN.
AND YOU CAN SEE HERE AS THE
MOUSE ISLET FROM A NORMAL MOUSE
AND HERE IS A HUMAN ISLET FROM A
RELATIVELY NORMAL HUMAN.
AND ONE OF THE THINGS THAT YOU
SEE IS THE RATIOS OF BETA CELLS
ARE QUITE DIFFERENT AND THE
WHYED THAT ALL OF THE ALPHA
CELLS ARE LOCATED ON THE OUTSIDE
OF THE ISLET DOESN'T REALLY HOLD
UP WHEN YOU LOOK AT NORMAL HUMAN
ISLETS SO MUCH.
AND SO, WHILE WE USE HUMAN
ISLETS FOR ALL THE STUDIES WE
CAN TO LOOK AT SIGNALING, WE TRY
TO REPRODUCE EVERYTHING IN HUMAN
ISLETS, WE HAVE USED BETA CELL
LINES A GOOD BIT.
I THINK THE POINT HERE IS THAT
MICE MIGHT NOT BE THE PERFECT
MODEL TO STUDY CONTROL OF
PANCREATIC BETA CELL FUNCTION.
IT'S JUST ARCHITECTURE ISSUE
ALONE IS IMPORTANT.
SO, I'LL SHOW THIS SLIDE TWICE.
SO THE THINGS I WANT TO REALLY
POINT OUT IS THAT ALL OF THESE
AGENTS THAT ACTIVATE INSULIN
SECRETION, AMINO ACIDS, GLUCOSE,
ACH, GLC1, ALL ACTIVATE ERK1 AND
2 AND DO IT THROUGH CALCIUM
INFLUX AND THEY ALL REQUIRE
CALCIUM DEPENDENT FASFOE TAZE TO
BE ABLE TO ACTIVATE ERK1 AND 2.
AGENTS SUCH AS EPINEPHRINE WHICH
ACTS THROUGH THE ALPHA 2 ADD
NERGIC RECEPTOR AND TURNS ON GI
AND GI BLOCKS INSULIN SECRETION
AND BLOCKS ERK1 TWO ACTIVATION
BY ALL OF THESE AGENTS.
SO ALL OF THESE NUTRIENT SENSING
EVENTS DEPEND ON CALCIUM NEURON.
AGENTS THAT ACTIVATE ERK1 AND 2
THROUGH MORE CONVENTIONAL
TYROSINE KINASE LIGAND-TYPE
MECHANISMS, THEY DON'T REQUIRE
IT.
THEY CAN ACTIVATE ERK1 AND 2
WHETHER CALCIUM IS IN ITS
PRESENCE OR INHIBITED.
SOMEHOW THERE IS INSULATION
BETWEEN THE GLUCOSE NUTRIENT
SENSING PATHWAYS AND THE TYPICAL
PATHWAYS THAT WE THINK OF AS
REGULATORS OF ERK1 AND 2 AND
MOST OTHER CELL TYPES.
SO, FIRST SOME EVIDENCE FOR WHAT
I'M TELLING YOU ABOUT THIS
NUTRIENT SENSING INSULIN DEMAND
SELECT ACTIVITY.
SO HERE IS THE COMPARISON OF
ERK1 AND 2 ACTIVATION.
HERE IS TOTAL AMOUNT OF ERK IN
THE LIE PAYS OF THESE CELLS AND
THEN A MEASURE OF ACTIVITY USING
ABILITIES TO PHOSPHOERK1 AND 2.
YOU CAN SEE 11 OR 3 MILL I MOLAR
GLUCOSE WHICH IS LOW.
11 IS PRETTY HIGH, GLUCOSE
CONCENTRATION.
ERK1 AND 2 SHOWS ACTIVITY
ALTHOUGH IT IS INCREASED AT 11
RELATIVE TO 3.
IF WE ADD A CYCLIC AMP
INCREASER, A MORE OR LESS MIMIC
OF GLP1, WE GET A POTENTIATION
OF ACTIVATION.
WHEN WE LOOKED AT THIS
CONCENTRATION IN MORE DETAIL, WE
SEE ERK1 AND 2 ARE ACTSIVATED
MAXIMALLY BY ABOUT 8 OR 9 MILL I
MOLAR GLUCOSE.
AND RESTING CONCENTRATION, WE
HAVE NOW BEEN USING ABOUT 4 OR 4
1/2 MILL MOLAR GLUCOSE INSTEAD
OF THE STANDARD FOR A NORMAL
GLUCOSE TOLLERRANCE
CONCENTRATION -- YOU CAN SEE
HERE, WE STILL SEE ACTIVATION AT
3.
SO I GUESS THE IMPORTANT POINT
IS THAT EONE AND TWO ARE
ACTIVATED IN BETA CELLS UNDER
NORMAL CONDITIONS.
THE AMOUNT OF ACTIVITY CHANGES
BUT IT GOES UP AND DOWN OVER A
FAIRLY SMALL RANGE.
ONE REASON WHY WE CAN'T DO ALL
OF THE EXPERIMENTS WE HAVE DONE
IN ISLETS IS THE FOLLOWING.
HERE IS WHAT WE SEE IF WE LOOK
AT ALPHA CELLS AND BOTH ARE
CULTURED CELL LINES.
IF WE LOOK AT ALPHA CELLS, YOU
SEE ERK1 AND 2 ARE LOW AT HIGH
GLUCOSE AND HIGH AT LOW GLUCOSE.
SO, JUST THE OPPOSITE OF WHAT
HAPPENS IN BETA CELLS.
SO ALPHA CELLS WORK AN OPPOSITE
WAY.
SO IF WE LOOK AT WHOLE ISLETS,
YOU CAN IMAGINE THAT THE SIGNAL
THAT WE WOULD SEE FROM BETA
CELLS WILL BE MITIGATED SOMEWHAT
REDUCED A LITTLE BIT BY THE OR
MAYBE A LOT, BY THE CONTRIBUTION
FROM ALPHA CELLS.
AND HERE YOU CAN SEE AN
ACTIVATION VERY NICE ACTIVATION
BY INSULIN IN ALPHA CELLS.
OKAY.
SO ALPHA AND BETA CELLS DON'T
WORK THE SAY SAME WAY AND IF WE
WANT TO UNDERSTAND THE
SIGNALING, WE ARE SOMEWHAT
RESTRICTED TO USING BETA CELL
PREPARATIONS OR ISOLATED BETA
CELLS FROM IMPACT ISLETS WHICH
WE HAVE DONE ON NUMEROUS
OCCASIONS.
SO, THE ISSUE OF NUTRIENTS AND
SECRETED ACTIVATING ERK1 TWO BY
MECHANISMS DIFFERENT FROM GROWTH
FACTORS.
HERE IS EVIDENCE TO SUPPORT
THAT.
AND IT'S IMPORTANT TO POINT OUT
THAT SQ506 IS AN INHIBITOR OF
CALCIUM NEURONS AND BASICALLY
THIS IS THE MOLECULE THAT WILL
PREVENT ERK ACTIVATION AND THIS
WILL DAMPEN CALLS NEWSPAPER
ACTIVITY.
HERE IS GLUCOSE ACTIVATION.
IF WE ADD CALLS NEURIN ACTIVITY
TO THAT, WE SEE A SIGNIFICANT
SEE EXPRESSION OF ACTIVATION BY
GLUCOSE.
IF YOU LOOK AT GLP1, YOU SEE
SOMETHING SIMILAR, STIMULATION
OF ERK1 AND 2 WITH GLP1 AND A
BLOCKADE WITH CALLS IN NEURIN
INHIBITION.
AND THIS IS VERY CLEAR.
THIS IS TRUE WITH ALL THE AGENTS
THAT ENHANCE INSULIN SECRETION,
ALL THE NUTRIENTS AND HORMONES.
OKAY.
GROWTH FACTORS THAT ACTIVATE
ERKGON TWO AND ANY CELL YOU LOOK
AT.
HERE YOU CAN SEE FORMA LESTER
BLOCKING.
NO REQUIREMENT FOR CALLS NEURIN
TO GET THE PATHWAY TURNED ON AND
HERE IS EGF.
NICE ACTIVATION OF ERK1 AND 2
BUT NO BLOCKADE BY CALLS NEURIN.
SO NUTRIENTS IN GROWTH FACTORS
ACTIVATE BY MECHANISMS EVEN
THOUGH WE PRESUME THEY USE ALL
THE SAME KINASE CASCADE.
EVERYONE THINKS ABOUT
CONTROLLING THE PATHWAY.
WHAT ABOUT SECRETION INHIBITORS?
I MENTIONED BEFORE THAT
EPINEPHRINE BLOCKS INSULIN
SECRETION FROM BETA CELLS AND
THAT HAPPENS THROUGH ALPHA 2 AB
NERGIC RECEPTORS AND HERE IS
ACTIVATION OF ERK1 AND 2 BY
GLUCOSE.
IF WE INCLUDE AN ALPHA 2
SELECTIVE AGONIST, WE BLOCK
ACTIVATION OF ERK1 AND 2 AND IF
WE USE EPINEPHRINE ITSELF, WE
ALSO BLOCK ACTIVATION OF ERK1
AND 2.
IF WE LOOK AT A HELIX CELL WHERE
PEOPLE HAVE CHARACTERIZED HOW
THESE PATHWAYS WORK, YOU CAN SEE
THAT CONTROL CONDITIONS, THERE
IS NO ACTIVITY DEDUCTIBLE.
IF WE ADD EPINEPHRINE, WE CAN
ACTIVATE ERK1 AND 2 AND TURN IT
ON.
IF WE ADD THE UK, SELECTIVE
ALPHA TWO ADD NERGIC AGONIST, WE
CAN ALSO ACTIVATE ERK1 AND 2.
SO TURNING ON GI IN AN AVERAGE
CELL THAT IS USED FOR THIS KIND
OF EXPERIMENT WILL ACTIVATE ERK1
AND 2 AND BETA CELLS THAT
COMPLETELY TURNS IT OFF.
AND AGAIN, THE GROWTH FACTOR
INSULATION COMPONENT OF THIS
ARGUMENT, YOU CAN SEE HERE THAT
GLUCOSE AGAIN IS BLOCKED BITE
ALPHA TWO ABNER GICK AGONIST BUT
THERE IS NO EFFECT ON THAT DRUG
AND ACTIVATION OF THE PATHWAY.
SO JUST PUTTING THAT ALL
TOGETHER, WE HAVE ALL THESE
NUTRIENT SENSING MECHANISMS, ALL
OF THESE MECHANISMS TELLING THE
CELL WHERE INSULIN NEEDS TO BE
SECRETED AND THEY ALL REQUIRE
CALLS NEURIN IN ORDER TO TURN ON
THIS PATHWAY.
AND THIS WILL INHIBIT INSULIN
SECRETION, BLOCKS ERK1 TWO
ACTIVATION.
SO, I WANT -- ONE OF THE
QUESTIONS THAT REALLY PUZZLED US
FOR A LONG PERIOD OF TIME WAS
HOW THE PHOSPHOTAZE CALLS
NEURINES IS REQUIRED TO TURN ON
THIS KINASE CASCADE.
WHAT IS IT DOING?
I'LL SHOW YOU EVIDENCE THAT IT
IS WORKING BY DEPHOSPHORYLATING
THE MAP KKK OR MAP 3K UP STREAM
OF THIS CASCADE.
AND ALL OF THIS WORK WAS DONE BY
A FORMER GRADUATE
STUDENT,LINGLING.
AND THE FIRST THING SHE HAD TO
DO WAS FIGURE OUT WHAT THE MAP
3K WAS UP STREAM OF THE PATHWAY.
THERE ARE MULTIPLE RAV ISOFORMS.
RAV IS CONSIDERED TO BE THE MAB
3K UPSTREAM OF EONE AND TWO.
SHE ASSAYED ALL THREE OF THESE.
SHE COULDN'T MEASURE ANY
ACTIVITY FROM ARA.
AND HERE IS WHAT SHE GOT WITH
CRAF.
YOU CAN SEE BASICALLY NO
ACTIVITY DETECTED.
JUST TO SHOW YOU SHE COULD
REALLY DO THE ASSAY, HERE IS A
PMA STIMULATED HELA CELL WITH
THE SAME TYPE OF COCK POLICE
DEPARTMENT ASSAY PHOSPHORYLATING
THE KINASE SUBSTRATE HERE --
ON THE OTHER HAND,
IMMUNOPRECIPITATED B RAV,
ACTIVATION BY GLUCOSE AND
REDUCTION IN ACTIVITY BY CALLS
IMMUNE.
ONE OFONE OF THE -- CALLS I NEURON.
THIS IS THE EXPECTATION WAS THAT
CRAF WOULD BE THE ACTIVATOR.
IT'S THE MOST COMMONLY
CONSIDERED ACTIVATOR IN A
TYPICAL CELL FOR THE ERK1 AND
TWO PATHWAY AND SO WHATLINGLING
DID WAS TO IMMUNOPREIMSIPITATE
CRAF AND SHOW SHE COULD BRING
DOWN THE PROTEIN BY BLOTTING IT
AND THEN ALSO BLOT WITH AN
ANTIBODY TO PHOSPHOSENIOR 338, A
KEY ACTIVATING PHOSPHORYLATION
SITE ON THE PROTEIN.
SO PHOSPHORYLATION IS THE SITE
AND WOULD INDICATE IT HAD BEEN
ACTIVATED BUT IF ANYTHING, THE
CONTENT DECREASED FOLLOWING
EXPOSE TOWER GLUCOSE.
SO, ANOTHER EXPERIMENT HERE SHE
DID A TIME COURSE OF STIMULATION
WITH GLUCOSE, BRAFIP KINASE
AGAIN AND TREATED WITH CALLS
NEURON -- CYCLOSPORIN A.
SHE COULD REDUCE THAT BRAF
KINASE ACTIVITY.
SO THAT IS THE INCREASED
STRAIGHTFORWARD AND THEN SHE DID
ANOTHER EXPERIMENT BASED ON A
LOT OF WORK FROM A NUMBER OF
LABORATORIES INITIALLY FROM
JOE'S LAB BUT A HUGE NUMBER OF
GROUPS HAVE REALLY PRESENTED
INCREDIBLY VALUABLE INFORMATION
THAT SHOWS HOW COMPLICATED THIS
KINASE PATHWAY CAN BE.
AND THAT INVOLVED THE IDEA THAT
THESE ISOFORMS CAN DIMER ICE,
HETERODIMER ICE AND THE PARTNERS
IN THE HETERODIMER COMBINATIONS
BOTH POSITIVELY AND NEGATIVELY
INFLUENCE EACH OTHER'S
ACTIVITIES.
SO JUST TOASTER IF THAT WAS
GOING TO IN BETA CELLS, WHAT SHE
DID WAS IPd CRA.
AND BLOTTED FOR BRAF.
HERE IS HER CRAF BLOT.
IT WAS PRESENT IN UNTREATED
CELLS OR CELL STIMULATED WITH
GLUCOSE AND HERE IS HER BRAF
BLOT.
IT DID NOT COPRECIPITATION
WITH -- COPRECIPITATE BUT IT DID
IN THE STIMULATED CELLS.
SO WE HAVE THE ISSUE OF
COMBINATIONS OF THESE MOLECULES
BEING PRESENT IN SOME OF THE
IMMUNOPRECIPITATION AND
OBVIOUSLY EFFECTING EACH OTHER'S
ACTIVITY.
TO ANSWER THAT QUESTION WHAT SHE
DID WAS TO USE RNAI IN BETA
CELLS AND THIS IS NOT SUCH AN
EASY PROSPECT IN BETA CELLS
BECAUSE JUST ABOUT THE BEST
KNOCK DOWN THAT IS WE HAVE
ACHIEVED IN THESE CELLS WAS
ABOUT 50%.
HERE WHAT SHE DID WAS KNOCKED
DOWN BRA.
AND YOU CAN SEE ERK ACTIVATION
IS ATTENUATED.
THIS IS HER BRAF BLOT.
THIS IS THE REDUCTION FROM THIS
TO THIS.
AND HERE IS JUST A PLOT OF THE
ACTIVITY.
HERE IS CRA.
AND THIS ONE WAS A SURPRISE.
SHE STIMULATED AND WHEN SHE
KNOCKED IT DOWN, SHE ACTUALLY
GOT AN INCREASE IN ERK1 TWO
ACTIVITY.
SO SHORT-TERM WHAT THAT SUGGESTS
IS THAT CONSISTENT AT LEAST WITH
OTHER DATA IN THE LITERATURE
THAT CRAF COULD SUPPRESS THE
ACTIVITY OF BRAF IN A RELATIVELY
RAPID ASSAY.
INTERESTINGLY, WHEN SHE LOOKED
AT A COUPLE OF HOURS OF
STIMULATION WITH GLUCOSE, SHE
FOUND THE OPPOSITE.
IN THIS CASE, CRAF WAS
ACTIVATING AND FORCING MORE
ACTIVITY THROUGH BRAF.
SO, A COMPLEX TIME DEPENDENT
RESPONSE OF THESE KINASES TO
ACTIVATION BY NUTRIENTS.
SO, HOW WAS CALLS NEURNIN THIS
SMITH WHAT WAS IT DOING?
SHE FIGURED OUT THAT CALLS
NEURIN ESSENTIALLY
DEPHOSPHORYLATES A NEGATIVE
FEEDBACK SITE ON BRA.
AND THAT SITE NEEDS TO BE
DEPHOSPHORYLATED IN ORDER TO
ALLOW CONTINUED ACTIVATION OF
BRAF.
IF THE SITE PHOSPHORYLATED AND
THAT SITE ISN'T REVERSED
BASICALLY, BRAF WILL BE TURNED
OFF AND ERK CAN'T BE ACTIVATED
ANY FURTHER.
SO A COUPLE OF DIFFERENT
EXPERIMENTS TO PUT THAT IDEA
TOGETHER SOME OF WHICH DEPENDED
ON DETERMINATION OF BRAF
PHOSPHORYLATION SITES BY DEBBIE
MORSON'S LAB A COUPLE OF YEARS
AGO.
SO, HERE YOU CAN SEE SHE
STIMULATED WITH GLUCOSE AND HERE
SHE IS ASSAYING BRAF ACTIVITY
USING PHOSPHORYLATION OF KINASE
METH ONE AGAIN AND THERE IS THE
INCREASE IN ACTIVITY.
AND SHE BLOTS CALLS I NEURIN AND
THE ACTIVITY IS DECREASED.
WE KNOW BRAF IS SENSITIVE STEP.
IF SHE BLOCKED ERK ACTIVITY, SHE
DOES NOT GET THE -- SHE STILL
CAN MEASURE BRAF ACTIVITY, AND
IF SHE INHIBITS CALLS NEURIN AND
BLOCKS ERK ACTIVITY, WITH THE
MEC INHIBITOR, SHE DOESN'T
SUPPRESS BRAF ACTIVITY ANY
LONGER.
BY BLOCKING ERK SHE IS
PREVENTING THE FEEDBACK
INHIBITION.
AND HERE YOU CAN READ THE RED
LINES IF YOU COULDN'T FOLLOW
WHAT I SAID BECAUSE IT'S FAIRLY
CONFUSING.
SO, SHE IDENTIFIED A SITE THAT
CEMENTED TO CARRY MOST OF THE
INHIBITORY ACTIVITY AND THAT WAS
401.
SO SHE THEN EXPRESSED BRAF IN
CELLS AND COMPARED THE WILDTYPE
PROTEIN TO THE ALANIN MUTATION
AND SO HERE YOU SEE STIMULATION
BY GLUCOSE AND DEPRESSION OF
ACTIVITY BY BLOCKING CALLS
NEURIN WITH P401BRAF, SHE FOUND
A STIMULATION OF ACTIVITY HIGHER
BASAL ACTIVITY TO BEGIN WITH,
AND THERE WAS NO SUPPRESSION BY
INHIBITING CALLS NEURIN
PRESUMABLY BECAUSE THIS SITE CAN
NO LONGER BE PHOSPHORYLATED STOW
DOESN'T NEED TO BE
DEPHOSPHORYLATED.
SO ALL TOGETHER THAT GIVES US A
MODEL WHICH IS CALCIUM INFLUX
ENHANCES CALLS NEURIN ACTIVITY
AND IT'S IMPORTANT FUNCTION IS
TO DEPHOSPHORYLATE BRAF SO IT
CAN CONTINUE TO BE LEAD TO
ACTIVATION OF ERK1 AND 2 BY
NUTRIENTS.
SO, NEXT, A FEW SLIDES ON WHAT
WE HAVE DONE TO LOOK AT ERK1 AND
2 AND REGULATION OF REGULATION
OF GENE TRANSCRIPTION AND WE
WORKED ON THIS FOR A LONG TIME
BUT A LOT OF THE WORK WAS DONE
BY A FORMER GRADUATE STUDENT AND
THEN MUCH OF IT HAS BEEN DONE BY
A FORMER POSTDOC.
AND THIS WAS THE KEY EXPERIMENT
THAT MICHAEL DID THAT REALLY
MADE IT IMPORTANT, EVERY DETAIL
OF HOW WE DID THESE EXPERIMENTS
BECAME IMPORTANT AND SO, WHAT HE
SHOWED HERE, IT WAS WELL-KNOWN
THAT GLUCOSE COULD SUPPRESS GENE
TRANSCRIPTION BUT WE DIDN'T KNOW
ANYTHING ABOUT THE TIME COURSE.
SO WHAT HE DID WAS TO SHOW THAT
GLUCOSE ACTIVATED THE INSULIN
GENE PROMOTOR ON THE SHORT-TERM.
THIS IS TWO AND 6 HOURS.
BY 12 HOURS, HE FOUND A
SUPPRESSION OF THAT STIMULATION
AND BY 24 HOURS, AND THEREAFTER,
HE FOUND SUPPRESSION BELOW BASIL
TRANSCRIPTION LEFT --
LEVEL.
SO THAT WASN'T TWO SURPRISING.
THE SURPRISING PART IS WE
BLOCKED ERK1 AND 2 ACTIVITY, WE
REDUCED THE GENE TRANSCRIPTION
IN RESPONSE TO GLUCOSE SO
THE -- SO WE WERE BASICALLY
CLOSE TO BASAL LEVELS AT THIS
POINT.
BUT IF WE BLOCKED ERK1 AND 2, HE
RESTORED THE SUPPRESSION CAUSED
BY HIGH GLUCOSE AT LONG TIME.
SO WE ARE LOOKING AT A BIPHASIC
EFFECT OF GLUCOSE A MULTIPHASIC
EFFECT OF ERK PHOSPHORYLATION.
WE BLOCK OUR ACTIVITY EARLY AND
PREVENT A STIMULATORY EFFECT ON
THE PROMOTOR, WE BLOCK ERK
ACTIVITY LATE, WE PREVENT THE
INHIBITORY EFFECT OF GLUCOSE BY
THE INSULIN GENE PROMOTER.
SO THIS BECAME REALLY A KEY TO
HOW WE DID MANY OF THESE
EXPERIMENTS THAT COME NEXT.
SO WE ARE REALLY BASICALLY
COMPARING RAPID OR SOMEWHAT
ACUTE STIMULATION WITH GLUCOSE
TO A MORE CHRONIC LONG-TERM
STIMULATION.
AND THIS JUST SHOWS THE FIRST
EXPERIMENT THAT WE DID IN
COLLABORATION WITH MIKE GERMAN
AND STEVE GRIFFIN AT UCSF.
IF THE CAT REPORTER AS A N-THIS
CASE IT WAS IN FRESHLY ISOLATED
MOUSE ISLETS AND HERE YOU COULD
SEE RELATIVELY LOW PROMOTOR
ACTIVITY WHETHER ERK IS ADDED
AND TRANSFECTED.
WHAT HE STIMULATED WAS 16 MILL
MOLAR GLUCOSE.
YOU CAN SEE INCREASE IN ACTIVITY
AND WHEN HE BLOCKED ERK1 AND 22
WITH A MEC INHIBITOR AND
INCLUDING KINASE, PRETTY MUCH
POTENTLY BLOCKED ACTIVITY FROM
THIS PROMOTOR IN THE ISLET.
AND SO, OVER A NUMBER OF YEARS,
WE AND MANY OTHER PEOPLE
IDENTIFIED SUBSTRATES THAT ARE
TRANSCRIPTION FACTORS THAT WORK
ON THE INSULIN GENE PROMOTOR AND
THE IMPORTANT FACTORS THAT HAVE
BEEN IDENTIFIED ARE NEUROD1, PDX
AND MOP A.
INTERESTINGLY, TWO OF THESE,
PARTICULARLY NEUROD1 AND TO SOME
EXTENT PDX ONE, ARE IMPORTANT
POINTS OF SUBSTRATES AND THEN IN
FACT, NEUROD1 REQUIRES A
PHOSPHORYLATION IN BETA CELLS TO
FUNCTION WELL.
ERK ALSO PHOSPHORYLATES P300
THAT'S BEEN SHOWN IN THE
LITERATURE.
IT PHOSPHORYLATES THE COMMON
GENERAL EBOX DIMER PARTNER FOR
NORMAL D1, E47 AND
PHOSPHORYLATES CEBT BETA WHICH
IS AN INHIBITORY REGULATOR OF
INSULIN GENE TRANSCRIPTION AND
THAT HAS BEEN SHOWN FOR A NUMBER
OF YEARS FOR EXAMPLE BY WORK
FROM HABNER'S LAB AND IT CAN
PHOSPHORYLATE THE C GENE.
WE TRIED TO FIGURE OUT HOW ALL
OF THESE FACTORS INNER
PLATE -- INTERPLAY WITH ERK
SIGNALING TO REGULATE THE
PROMOTOR.
ONE OF THE IMPORTANT THINGS THAT
IS A NONISSUE IF YOU'RE WORKING
ON ISLETS FROM ANIMALS BUT IT'S
A SIGNIFICANT ISSUE IF YOU'RE
LOOKING AT CULTURED ISLET CELL
LINES, THE MAINTENANCE
CONDITIONS FOR THESE CELLS
GENERALLY THE CELLS ARE
MAINTAINED AT VERY HIGH GLUCOSE
CONCENTRATIONS.
WE WONDERED WHY THAT WAS AND SO
MICHAEL LOWERED THE
CONCENTRATION TO WHAT WOULD BE
CONSIDERED A SOMEWHAT MORE
NORMAL GLUCOSE CONCENTRATION AND
WHAT HE FOUND WAS AFTER A COUPLE
OF DAYS, HE LOST EXPRESSION OF
MOP A AND HE KEPT THE CELLS IN
VERY HIGH GLUCOSE.
INHIBITING ERK ACTIVITY WITH THE
MEC INHIBITOR DOESN'T DO
ANYTHING TO THIS.
SO THE GLUCOSE CONCENTRATION
ITSELF HAS A BIG IMPACT EVEN ON
A RELATIVELY SHORT-TERM OF
EXPRESSION OF MAFA.
WE KNOW THAT GENE EXPRESSION
GOES UP WITH LOW GLUCOSE.
YOU CAN SEE HERE AND AGAIN, BY
HABNER AND A NUMBER OF OTHER
GROUPS HAVE SHOWN THAT CP BETA
IS INDUCED WHEN CELLS ARE
EXPOSED TO VERY HIGH GLUCOSE FOR
A LONG PERIOD OF TIME.
SO YOU CAN SEE HERE 48 HOURS ON
HIGH GLUCOSE IS ENOUGH TO INDUCE
THE BAIT A SO IT'S IMPORTANT TO
TREAT THESE CELLS ON A
PARTICULAR WAY SO YOU KNOW WHAT
THE STATE OF THE RELEVANT
FACTORS IS THAT WILL BE
CONTROLLING PROMOTOR ACTIVITY.
OKAY.
SO IN THIS CASE, WHAT WE DID WAS
TO LOOK AT WHAT HAPPENED TO
FACTORS THAT WE KNOW ARE
IMPORTANT, MAFA, NEUROD1 AND
PDX1 AND CPD BETA.
THESE ARE PREIMSIPITATION ASSAYS
SO YOU CAN SEE BANDS.
AND THESE ARE CELLS TREATED FOR
30 MINUTES WITH GLUCOSE AND
COMPARED TO THE CONTROL, YOU CAN
SEE INCREASES IN BINDING OF MAFA
AND NEUROD AND PDX TO THE
NEUROGENE PROMOTOR.
IF WE USE THE MEC INHIBITOR OR
CALLS NEURIN INHIBITOR WE BLOCK
THOSISH ACTIONS.
IT'S JUST A SUBSTANTIAL
REDUCTION.
RAP MICE IN, INHIBITOR OF MTOR
C1 DOESN'T DO ANYTHING.
IF WE DEPOLARIZE WITH POTASSIUM,
WE SEE BINDING OF MAFA AND BOTH
ARE BLOW BLOCKED BY INHIBITING
ERK 1 AND 2 ACTIVITY.
IF WE LOOK AT THOSE INCUBATED
FOR A COUPLE DAYS WITH HIGH
GLUCOSE WITHOUT REMOVAL OF THAT
GLUCOSE, WHAT WE SEE HERE IS
THAT DROPPING THE GLUCOSE FOR 30
MINUTES AND THEN ADDING 11 MILLI
MOLAR GLUCOSE, NO BINDING OF
MAFA, NEUROD1 OR PDX.
BUT WE SEE BINDING OF CEP BETA
CONCENTRATION, AND IT'S HIGHER
IN 11 MILL I MOLAR GLUCOSE.
INTERESTINGLY, IF WE TREAT THESE
SAME CELLS WITH POTASSIUM
DEPOLARIZATION, WE CAN STILL SEE
BINDING OF MAF AND THIS IS JUST
THAT THE BETA IS PREVENTING
THESE OTHER FACTORS.
SO, ONE OF THE OBSERVATIONS THAT
WE MADE THAT GOTTEN US
PARTICULARLY INTERESTED IN
STUDYING THIS IN MORE DETAIL IS
WHAT HAPPENS WITH THE SIGNALING
MOLECULES THEMSELVES HERE WE DID
CHROMATIN IMMUNOPRECIPITATION ON
HUMAN ISLETS WITH ANTIBODIES TO
ERK1 AND 2, ANTIBODIES TO
DOWNSTREAM SUBSTRATE RISK TWO
WHICH CAN PHOSPHORYLATE HISTONE
TAIL AND CALLS NEURIN AND THE
PHOSPHOTAZE.
YOU CAN SEE WE CAN SEE IRK ONE
AND 2 ARE ASSOCIATED WITH THE
INSULIN GENE PROMOTOR UNDER
STIMULATORY CONTINUES AND WE CAN
SEE HERE WITH GLP1.
IF WE ADD FK INTERACTION IS
REDUCED.
WE CAN SEE THAT UNDER A NUMBER
OF DIFFERENT CONDITIONS AND WITH
OTHER PROMOTORS SO WE ARE NOW
EXPLORING THE DENSITY OF
INTERACTIONS OF ERK1 AND 2 WITH
CHROMATIN AND BETA CELLS.
JUST FOR COMPARISON, AGAIN MY
POINT ABOUT LIGANDS, IF I
HAVEN'T MADE THAT STRONGLY
ENOUGH YET, HERE AGAIN WE HAVE
CELLS COMPARING GLUCOSE EG.
AND NGF AS ACTIVATORS OF THESE
CELLS AND LOOKING AT ACTIVATED
ERK1 AND 2 BY IMMUNOBLOTTING.
IF WE BLOCK MEF, WE CAN LIP LIMB
NAT THE SIGNALS AND REDUCE THEM
SUBSTANTIALLY.
HERE WE ARE LOOKING AT JUST
CHROMATIN IMMUNOPRECIPITATION
AND YOU CAN SEE THAT WE ARE
COMPARING THE INSULIN AND THE
PHOS PROMOTORS AND GLUCOSE AND
EGF DON'T ACTIVATE VERY MUCH AND
BETA CELLS BUT NGF DOES AND YOU
CAN SEE A BIG DIFFERENCE IN
INTERACTIONS AND IF WE ADD TO A
126 WHICH BLOCKS ACTIVITY, YOU
CAN SEE WE CAN DAMPEN GLUCOSE
DEPENDENT INTERACTIONS
CONSISTENT WITH THE CHIPS I WAS
SHOWING BEFORE.
WE DON'T REALLY INHIBIT EGF
SIGNALING IN THESE CELLS AND YOU
CAN STILL SEE A ACTIVITY AND YOU
CAN SEE REDUCTION IN BOTH TYPES
OF INTERACTIONS IN RESPONSE TO
NGF.
SO DIFFERENT LIGANDS DO
DIFFERENT THINGS AND THAT
INCLUDES DIFFERENT DNA
ASSOCIATIONS WITH ACTIVATED
MOLECULES DOWNSTREAM OF THOSE
LIGANDS.
SO ONE OF THE THINGS THAT WE
NOTICED WAS ERK1 AND 2 HAD AN
IMPACT ON HISTONE ACETYLATION
AND BETA CELLS AND THIS IS TO
SHOW YOU NOT SO MUCH ABOUT THE
HISTONE MODIFICATIONS THEMSELVES
BUT ABOUT THE GLUCOSE DEPENDENCE
OF THESE INTERACTIONS.
SO HERE IF WE LOOK UNDER NORMAL
CONDITIONS, YOU CAN SEE
STIMULATORY GLUCOSE AND INCREASE
IN ACETYLATED H3 AND H4 AND
INCREASE IN BINDING OF P300 TO
THE INSULIN GENE PROMOTOR.
IF WE BLOCK ERK ERK1 TWO
ACTIVITY, ALL OF THESE ARE
REDUCED.
IF WE LOOK AT CELLS THAT HAVE
BEEN EXPOSED TO HIGH GLUCOSE FOR
A COUPLE OF DAYS, THE PATTERN IS
QUITE DIFFERENT.
YOU CAN SEE HERE THAT UNDER LOW
GLUCOSE CONDITIONS WE CAN SEE
ASSOCIATION OF P300 AND
ACETYLATION OF HISTONES AND
BLOCKING ERK1 AND 2 AT HIGH
GLUCOSE ENHANCES THEIR
ASSOCIATION RELATIVE TO GLUCOSE
ALONE.
AND THIS IS BASICALLY A
COMPARABLE TYPE OF EXPERIMENT.
HERE WE ARE LOOKING AT CYTOKINE
ACTION AND I'LL GET BACK TO THAT
IN A FEW MINUTES.
BUT THE COMBINATION OF HIGH
GLUCOSE IL AND BETA CYTOKINE,
CAUSES BASICALLY AN INHIBITION
OF ACETYLATION OF HISTONES AND
BINDING OF P300 AND BINDING OF
POLYMERASE TWO TO THE INSULIN
GENE PROMOTOR.
BUT IF WE BLOCK ERK1 AND 2 UNDER
THESE CONDITIONS, SO THIS IS A
EXACERBATED CHRONIC GLUCOSE
SITUATION, EXACERBATED WITH THE
CYTOKINE, THEN WE NO LONGER LOSE
THE INTERACTION BY BLOCKING ERK1
AND 2 ACTIVITY.
AND THIS IS MORE OF THE SAME.
THIS IS IN HUMAN ISLETS.
AGAIN, PREINCUBATED IN A RESTING
GLUCOSE CONCENTRATION WE SEE
PRETTY NORMAL BEHAVIOR WITH
STIMULATION OF HISTONE
ACETYLATION AS WE ADD GLUCOSE,
BUT IF WE INCUBATE THESE ISLETS
IN HIGH GLUCOSE FOR 3 DAYS, WE
GET THE SAME KIND OF REVERSAL I
WAS SHOWING YOU BEFORE.
INSTEAD OF INCREASED ACETYLATION
AS WE INCREASED GLUCOSE
CONCENTRATION, WE SEE DECREASED
ACETYLATION AND SO BASICALLY,
WHAT WE ARE SEEING HERE IS THAT
THE CONDITION OF THE BETA CELL,
NOT THE ACTIVATION OF THE KINASE
PATHWAY, IS A MAJOR DETERMINANT
WHAT HAVE IS ON THE PROMOTOR AND
HOW IT CAN BE REGULATED BY THE
SIGNALING PATHWAY.
AND THIS MIGHT BE THE MOST
OBVIOUS SET OF EXPERIMENTS
SHOWING THAT.
SO HERE WE LOOKED IN MORE DETAIL
AT WHAT HAPPENS WHEN CELLS ARE
TREATED WITH EITHER RESTING OR
STIMULATORY GLUCOSE UNDER
CHRONIC CONDITIONS ALONG WITH
IL1 BETA.
AND SO ERK1 AND 2 IS ACTIVATED
BY GLUCOSE BUT IT'S ALSO
ACTIVATED BY THE CYTOKINE.
PERHAPS A LITTLE BIT DIFFERENTLY
UNDER EACH CONDITION BUT
BASICALLY, ALL OF THESE
CONDITIONS WILL CAUSE ERK
ACTIVATION.
THE STRESS ASSOCIATED MAP
KINASES P38 AND JUNK, THESE ARE
ACTIVATED BY THE CYTOKINE.
SO YOU CAN SEE HERE ADDITION OF
THE CYTOKINE INDEPENDENT OF THE
GLUCOSE CONCENTRATION, ACTIVATES
THESE PROTEIN KINASES ALTHOUGH
ERK IS ACTIVATED UNDER ALL OF
THESE CONDITIONS.
SO, NOW WHAT HAPPENS?
WHEN WE ACTIVATE ALL OF THESE
PATHWAYS?
THESE ARE TIME COURSES
CHROMATIN, IMMUNOPRECIPITATION
AND WHAT YOU CAN SEE HERE IS
THAT THE ERK1 TWO BIND RIGHT
AWAY AND STILL ASSOCIATED EVEN
UNDER THIS CONDITION WHICH I'LL
SHOW YOU IN A MOMENT.
HAS TURNED OFF THIS PROMOTOR
COMPLETELY.
P38 LIKEWISE JNK LIKEWISE AND
RISK LIKEWISE AND ALL THE
SIGNALING MOLECULES BIND AWAY
WHEN THEY ARE STIMULATED IN
ASSAY AND THEY STAY THERE.
WHAT ABOUT FACTORS THAT ARE
IMPORTANT FOR INSULIN GENE
TRANSCRIPTION?
YOU CAN SEE MAFA BINDS BUT IN
THE PRESENCE OF THE CYTOKINE
WITH HIGH GLUCOSE, IT'S GONE
AFTER AN HOUR.
NEUROD DOES STAY ON.
PDX ONE ALSO DROPS OFF.
P300 AND POLYMERASE ARE ALSO
GONE.
SO ONE OF THE THINGS THAT
HAPPENS, IT TAKES ABOUT A DAY OF
HIGH GLUCOSE TO INDUCE THE BP
BETA BUT IF WE ADD IL1 BETA, WE
SEE INCREASE IN THIS MOLECULE BY
A COUPLE OF HOURS.
HERE IS THE 4 HOUR TIME POINT.
SO IT'S A MUCH FASTER INDUCTION
OF THIS INHIBITORY TRANSCRIPTION
FACTOR.
IF WE DID NUCLEAR RUNON AT 4 AND
24 HOURS JUST TO SEE WHAT WAS
ACTUALLY HAPPENING TO
TRANSCRIPTION IN THE INSULIN
GENE PROMOTOR AND AT 4 HOURS IN
THE PRESENCE OF THE CYTOKINE BUT
AT MODERATE AMOUNT OF GLUCOSE,
YOU CAN SEE WE STILL HAVE
TRANSCRIPTION INITIATION ONGOING
SO WE COULD SEE NUCLEAR RUN ON.
BUT IF WE LOOKED AT 16 MILL I
MOLAR GLUCOSE AT 4 HOUR TIME
POINT WHICH CORRESPONDS TO THIS
LAST COLUMN, YOU CAN SEE A
SUPPRESSION OF TRANSCRIPTION.
THERE WASN'T ANY ONGOING
INITIATION SO THERE WAS NO
NUCLEAR RUN ON.
BUT WE COULD MEASURE.
SO, I GUESS THE MAIN POINT HERE
IS THAT THE KINASE MAY BE BOUND
WITH THE PROMOTOR AND THEY ARE
REGULATED THE SAME WAY
REGARDLESS OF THE CONDITIONS IN
THE BETA CELL.
SO IF YOU LOWER GLUCOSE ERK1 AND
2 ACTIVITY WILL GO DOWN.
IF YOU INCREASE GLUCOSE, THE
ACTIVITY WILL GO UP.
BUT IF THE CELLS ARE TREATED
UNDER CIRCUMSTANCES THAT PROMOTE
THEIR RESISTENCE, THEIR GLUCOSE
INNOCENCE ACTIVITY, THEN WHAT
ERK DOES IN THE SIGNALING
STANDPOINT WILL NOT BE THE SAME
BECAUSE THE PROTEIN COMPLIMENT
IN THESE CELLS IS DIFFERENT.
OKAY.
SO, A POINT OF GREAT INTEREST TO
ME BUT NOT NECESSARILY
IMMEDIATELY RELEVANT TO BETA
CELL FUNCTION IS WHAT IS THE
MOLECULAR BASIS FOR ERK ERK1 AND
2 CHROMATIN ASSOCIATION?
WE ARE WORKING HARD TO,000
BETTER.
FIRST ERK1 AND 2 BINDS ALL KINDS
OF DNA ASSOCIATED FACTORS.
THEY BIND A LOT OF HLH PROTEINS,
COMPLEX FACTORS, P300 AND MANY
OTHER BINDING PROTEINS.
THEY ALSO BIND OTHERS.
FSK, ANOTHER HISTONE KINASE SO
IT WAS REPORTED AT 3 YEARS AGO
NOW THAT UNPHOSPHORYLATED ERK2
CAN BIND TO DNA.
WE FIND THAT PHOSPHORYLATED ERK2
IS THE BETTER DNA BINDER BUT WE
ALSO SEE DIRECT BINDING TO A
NUMBER OF DIFFERENT REGIONS OF
DNA.
SO WE ARE EXPLORING WHAT THAT
MIGHT MEAN.
AND ONE IDEA IS THAT IT MAY
OCCUR THROUGH BASIC RESIDUES IN
THE MAP KINASE INSERT SUGGESTED
BY THIS PAPER BUT ALSO BEFORE
THAT BY A PAPER FROM MARIA
SCHUMACHER WHO DETERMINED THE
CRYSTAL STRUCTURE OF A BACTERIAL
KINASE HAS A EUKARYOTIC KINASE
FOLD ASSOCIATED WITH BACTERIAL
DNA.
AND WHAT SHE FOUND IS THIS IS
THE PART OF THE KINASE, THE
STRUCTURE OF THE KINASE LOOKS
JUST LIKE THIS.
THIS IS THE PART OF THE KINASE
THAT WAS TOUCHING THE DNA
DIRECTLY.
AND THIS IS THE MAP KINASE
INSERT WHICH IS UNIQUE TO THIS
PARTICULAR BRANCH OF KINASE.
SO FAR WE HAVE DONE A LOT OF
SEQUENCING AND WE HAVE
IDENTIFIED A NUMBER OF
INTERACTIONS IN BETA CELLS.
THERE ARE A NUMBER OF
INTERACTIONS THAT OVERLAP WITH
P300 BINDING SITES.
THIS MAY ACCOUNT FOR SOME
INTERACTIONS WE CAN DETECT.
SO OVERALL, ERK1 AND 2 AND
TRANSCRIPTION.
MAP K ACTIVITIES REFLECT IN
COMING SIGNALS.
THEY ARE ON IF THEY HAVE BEEN
STIMULATED TO BE ON AND THEY ARE
NOT IF THEY ARE NOT.
THEY ARE PRESENT IN
TRANSCRIPTION COMPLEXES.
RESPONSES DEPENDS ON WHAT IS
THERE.
CHANGING GLUCOSE CONCENTRATION
AND BETA CELLS ALTERS THE
PROFILE OF THE TRANSCRIPTION
FACTORS.
SO ERKON AND TWO REGULATE
CONTACTS HISTONE MODIFICATIONS
AND OTHER KINDS OF
TRANSCRIPTIONAL RESPONSES AND
REDUCTION IN BETA CELLS.
AND BRIEFLY FOR THE LAST COUPLE
OF MINUTES, I'D LIKE TO TELL BUT
STUDIES THAT HAVE BEEN CARRIED
OUT BY A POSTDOCTORAL FELLOW IN
THE LAB.
AND THE RESULTS OF THESE
EXPERIMENTS WERE QUITE SHOCKING
TO ME, VERY GRATIFYING AND I
HOPE WE CAN TURN THEM INTO
SOMETHING MORE SIGNIFICANT EVEN
THEN.
THEY MIGHT APPEAR HERE.
SO THIS IS A COLLABORATION WITH
SOMEBODY IN CARDIOLOGY AND
PROFESSOR J SNIDER.
THE CHEMICAL STRENE ABOUT 10
YEARS AGO ALMOST FOR MOLECULES
THAT ENHANCE CARDIAC
DIFFERENTIATIONS AND REPORTER
ASSAY AND HE FOUND A GROUP OF
COMPOUNDS THAT STARTED LOOKING
AT HOW SOME OF THEM WORK.
HE NOTICED THE COMPOUNDS COULD
INCREASE EXPRESSION OF NEUROD
CELLS.
SO WE THOUGHT IT WOULD BE A GOOD
IDEA TO LOOK SINCE NEUROD REALLY
IS BETA TWO.
SO WE TRIED THESE COMPOUNDS ON
ISLETS AND WE GET AS MANY
ISLETS, HUMAN ISLETS AS WE CAN
HANDLE AND WHEN WE CAN'T USE
THEM ALL ACUTELY FOR
EXPERIMENTS, WE PUT THEM IN THE
INCUBATOR AND WHEN HE GOT THIS
COMPOUND TO WORK ON, WE ACTUALLY
HAD ISLETS THAT HAD BEEN IN THE
INCUBATOR FOR A YEAR.
HUMAN ISLETS SITTING THERE IN
THE INCH BEIRUT.
A THIS POINT THEY DON'T MAKE A
LOT OF
INSULIN -- INCUBATOR -- THEY
MIGHT SECRETE JUST A LITTLE.
HE STARTED LOOKING AT THEM AND
HE FOUND -- THIS HAPPENS TO BE A
6-MONTH-OLD SET OF ISLETS.
HE FOUND THAT WITHIN TWO DAYS
INSULIN mRNA WENT UP BY MORE
THAN 60 FOLD AND THIS EXPERIMENT
BY TREATING WITH THIS COMPOUND
AND THAT WAS PRETTY SHOCKING.
INTERESTINGLY, GLUCOGON WENT
DOWN SEVERAL FOLD.
HE DID OR LOOKED AT INSULIN
SECRETION AND YOU CAN SEE HERE
THE COMPOUND INCREASED THE
AMOUNT OF PROTEIN SECRETION
QUITE SUBSTANTIALLY.
AND SO, A QUESTION IS REALLY
THIS IS AN INCREASE BUT IS IT
SIGNIFICANT?
HOW DOES TO COMPARE TO WHAT A
FRESH ISLET WOULD SECRETE?
SO HE COMPARED ISLETS THAT HAD
BEEN IN INCUBATORS THIS TIME FOR
THREE MONTHS WITH ONES THAT WE
HAD FRESH FROM THE TRANSPLANT
FACILITY AT UAB.
AND HE EITHER TREATED WITH THE
DRUG IN THE LIGHT BARS OR NOT.
AND THEN HE COMPARED INSULIN
CONTENT.
PROTEIN NOT mRNA.
AND YOU CAN SEE HERE THIS IS THE
AMOUNT IN THE FRESH ISLETS AND
AFTER TREATING THE OLD ISLETS
WITH THE DRUGS FOR TWO DAYS.
YOU CAN SEE THAT IT WAS IN THE
RIGHT ORDER OF MAGNITUDE FOR
INSULIN PRODUCTION FROM FRESH
ISLETS AND THE COMPOUND
INCREASED INSULIN CONTENT OF THE
FRESH ISLETS AFTER ONLY TWO
DAYS.
SO, THIS WAS A REALLY AMAZING
RESULT TO ME.
SO HE ANALYZED THE ACTIONS OF
THIS COMPOUND IN A NUMBER OF
WAYS AND HE LOOKED AT A LOT OF
CHANGES IN GENE EXPRESSION AND
WE START OUT WITH THE THREE
GENES THAT ARE ESSENTIAL FOR
INSULIN GENE TRANSCRIPTION,
NEUROD/BETA 2.
PDX ONE AND MAFA.
THEY ARE ALL IN CREASED AS A
FUNCTION OF TIME AFTER EXPOSURE
TO THE COMPOUND.
THAT WAS REALLY GOOD.
BUT MORE SURPRISING STILL WAS
AFTER AGAIN A TIME COURSE OF
TREATMENT WITH THE COMPOUND HE
LOOKED AT GLUKOKINAISE AND
GLUCOSE SENSING APPARATUS
BASICALLY BETA CELLS AND THEY
ARE INCREASED A LOT.
REALLY HUGE INCREASE IN
GLUKOKINAISE EXPRESSION.
THEN HE STOOD BACK AND LOOKED AT
THE GENES INVOLVED AND THE BETA
CELL DIFFERENTIATION PATHWAY AND
THEY ARE LISTED HERE AND MORE OR
LESS IN SEQUENCE OF THEIR
EXPRESSION DURING BETA CELL
DIFFERENTIATION AND THEY LOOKED
AT EVERYTHING ON HERE AND SOME
OF THEM ARE SHOWN HERE.
A LOT OF THEM SHOW UP IN
MULTIPLE STATES OF COURSE.
AND YOU CAN SEE HERE, HUGE
INCREASES NKX6.1 AND 6.2.
HUGE INCREASES IN NEUROJEN PATH
4.
THESE TWO FACTORS ARE NOT HIGHLY
EXPRESSED IN MATURE ISLETS AS
SOME OF THE OTHER OTHERS AND
THEY WENT UP VERY TREMENDOUSLY
RIGHT AT THE BEGINNING AND THEN
BEGAN TO DROP SLIGHTLY FOLLOWING
FURTHER TIME OF TREATMENT WITH A
COMPOUND.
SO, EVERYTHING HE LOOKED AT
BASICALLY INCREASED WITH TIME OF
TREATMENT WITH THIS DRUG.
AND SO HE DID LOOK A LITTLE BIT
AT HISTONE ACETYLATION SOME
SIGNALING IN THESE CELLS
FOLLOWING DRUG TREATMENT.
AND THIS IS A TIME COURSE.
AND I WANT TO POINT OUT THAT
THIS TIME COURSE LOOKS QUITE
DIFFERENT FROM WHAT I SHOWED YOU
WITH GLUCOSE OR PROTEIN COUPLED
RECEPTORS AND BETA CELLS WHERE
WE SAW ACUTE RESPONSES WITHIN
THE FIRST FEW MINUTES.
YOU CAN SEE A LITTLE BIT OF
ACTIVATION OF ERK2 HERE, 15
MINUTES.
BUT TREMENDOUS ACTIVATION AT
ABOUT 4 HOURS THAT IS SOMEWHAT
PERSISTENT ALTHOUGH DROPPED
SOMEWHAT.
THIS LOOKS LIKE NGF-TYPE OF
RESPONSE ALTHOUGH IT'S NOT VERY
PRONOUNCED EARLY.
SO IT'S QUITE DELAYED.
DOESN'T LOOK LIKE ANY OBVIOUS
SIGNALING TO ME.
YOU CAN SEE A TIME DEPENDENT
INCREASE IN THE ACETYLATION OF
H3 AND H4 AND HERE NOT SUCH A
BIG CHANGE IN H3K27
TRIMETHYLATION.
AND SO THIS IS MEN 6 MOUSE BETA
CELLS AND HERE WE HAVE HUMAN
ISLETS AND YOU CAN SEE TO THE
EXTENT THAT HE LOOKED, THESE
CHANGES ARE QUITE SIMILAR.
INTERESTINGLY, WITH WHEN I DEAL
WITH THIS COMPOUND IT WOULD
EFFECT HISTONE TRANSFERASE
ACTIVITY BUT AN ACTUAL FACT
THERE WAS NO EFFECT ON -- SORRY.
ONE IDEA THAT EVERYBODY EXPECTED
WAS THAT IT WOULD EFFECT HDAC
ACTIVITY.
IT DIDN'T.
BUT IT DID INCREASE HAT
ACTIVITY, TREATMENT WAY DRUG
COMPARED TO TSA.
AND YOU CAN SEE BIG INCREASE IN
HAT ACTIVITY AND NUCLEAR
EXTRACTS FROM THE CELL.
AND WHEN HE RECONSTITUTED P300
AND LOOKED AT A EFFECTS OF ERK
ON ACTIVITY AS WELL AS, YOU CAN
SEE DRUG INCREASED ACTIVITY
RELATIVE TO UNTREATED AND THAT
ERK1 AND 2 DID SOMEWHAT SUPPRESS
ACTIVITY COMPARED TO THE DRUG
TREATED WITHOUT ERK INHIBITION.
SO, WE HAVE COMPOUNDS THAT SEEM
TO REALLY ENHANCE BETA CELL
FUNCTION.
THE IMPROVED ISLET FUNCTION IN
CULTURE AND TRANSPLANT EF SEAS
AND DIABETIC MICE.
WE HAVEN'T DONE ENOUGH ANIMALS
YET, TREATED THEM TO HAVE
STATISTICAL SIGNIFICANTS BUT IT
SEEMS VERY LIKELY BASED ON OUR
CURRENT DATA THAT THE DRUG CAN
BE USED TO TREAT ANIMALS AND
INCREASE THEIR GLUCOSE
TOLLERRENCE.
WHAT WE DON'T KNOW THAT THE
POINT IS WHAT ARE THE MECHANISMS
OF ACTION.
WE ARE LOOKING AT THIS VERY
INTENSELY AND WE'D ALSO LIKE TO
KNOW HOW LONG THE EFFECTS ARE
STABLE.
IF WE TAKE THE DRUG AWAY, DO THE
EFFECTS STOP?
ONCE WE REMOVE THE DRUGS, HOW
LONG WILL BETA CELL FUNCTION
REMAIN IMPROVED?
AND FINALLY WE ARE LOOKING AT
SIMILAR COMPOUNDS THAT SEEM TO
WORK AT LOWER CONCENTRATIONS.
WE WERE LOOKING AT 10 AND 20
MICROMOLAR COMPOUNDS.
WE WOULD LIKE TO BE LOOKING AT
NANOMOLAR CONCENTRATIONS SO
SIMILAR COMPOUNDS TO SEE IF THEY
IMPROVE TYPE II DIABETES OR
PERHAPS REJUVENATE ISLETS FOR
TRANSPLANTATION OR STABILIZE
THEM FOR TRANSPLANTATION.
SO, JUST ALL OF THIS CAME FROM
THE INTEREST IN ERK SIGNALING
AND BETA CELLS AND WE ARE HOPING
IT WILL GO FURTHER THAN THAT.
SO PEOPLE WHO DID THE WORK, HERE
THEY AND ARE A LOT OF PEOPLE IN
THE LAB HAVE CONTRIBUTED IN
PARTICULAR, KATHY WHO IS
ACTUALLY MAINTAINED ISLETS IN
CULTURE FOR US FOR ABOUT 10
YEARS.
THANK YOU VERY MUCH.
[ APPLAUSE ]
>> THANK YOU VERY MUCH.
WE HAVE TIME FOR QUESTIONS.
PLEASE USE THE MICROPHONES IN
THE AISLES SO INDIVIDUALS
WATCHING BY VIDEO CAN ALSO HEAR.
THIS IS VERY INTERESTING STORY.
IT CERTAINLY RAISES THE QUESTION
OF WHAT IS THE EARLIEST RESPONSE
THAT HAPPENS WHEN YOU HAVE THE
COMPOUNDS?
YOU SHOWED US A VARIETY OF
TRANSCRIPTS THAT SEEM TO BE
GOING UP FAIRLY DRAMATICALLY.
HAVE YOU BASICALLY DONE A
COMPLETE GLOBAL ANALYSIS?
TO SEE WHAT HAPPENS IN THE VERY
EARLIEST MOMENTS AFTER EXPOSE
AND YOU ARE SEE WHETHER YOU CAN
IDENTIFY WHAT MIGHT BE DRIVING
THIS IN TERMS OF THE PRIMARY
OFFENSE.
>> NO.
WE HAVEN'T DONE THAT.
WE STARTED -- WE HAVE DONE SOME
INITIAL MICROAWAY EXPERIMENTS SO
WE HAVEN'T DONE ANYTHING GLOBAL
OR QUANTITATIVE.
WHAT WE TRIED TO DO IS IDENTIFY
DIRECT TARGETS BY AFFINITY
CHROMATOGRAPHY AND WE HAVE SOME
CANDIDATES BUT I'M SKEPTICAL SO
FAR BECAUSE I'D LIKE COMPOUNDS
THAT BIND A LOT BETTER THAN MORE
EFFICACIOUS BEFORE WE -- MORE
PELT ENT BEFORE WE REALLY GET
SERIOUS ABOUT -- POTENT -- WHAT
POTENTIAL TARGETS MIGHT BE.
BUT I THINK THAT IS REALLY OUR
GOAL AT THIS POINT.
INITIALLY, THEY ARE TO SHOW
EFFICACY IN ANIMALS AND TRY TO
IDENTIFY DIRECT BINDERS.
>> THANK YOU FOR COMPLETE
COVERAGE OF THE KINASES
INVOLVED.
SO WE UNDERSTAND ABOUT THE
SIGNALING PATHWAYS.
WHY ARE THERE SO MANY DRUGS FOR
TYPE II DIABETES?
BESIDES METFORMIN?
I'M INTERESTED.
>> WHY ARE THERE SO FEW DRUGS
FOR TYPE TYPE II DIABETES?
>> AND CARDIOVASCULAR
COMPLICATIONS.
>> AND COMPLICATIONS.
I'LL TELL YOU ONE REASON WHY I
THINK THERE ARE SO FEW DRUG IS
BECAUSE WE DON'T REALLY
UNDERSTAND MUCH ABOUT BETA
CELLS.
I HAVE PRECONCEIVED NOTION BUSY
HOW THESE CELLS WOULD BE
REGULATED AND THEY ARE
INCREDIBLY FASCINATING BUT THEY
DIDN'T EXACTLY MATCH MY
PRECONCEIVED NOTIONS IN A NUMBER
OF WAYS.
SO, THEY DON'T HAVE PROPERTIES
THAT EXACTLY MATCH ANYTHING ELSE
THAT HAS BEEN WELL
CHARACTERIZED.
AND THERE AREN'T EXACTLY A LOT
OF THEM.
OTHER THAN LOOKING AT THESE
CULTURED CELL MODELS IN RODENTS,
IT'S QUITE DIFFICULT TO LOOK AT
BETA CELL REGULATIONS,
PARTICULARLY BECAUSE THE OTHER
CELLS IN THE ISLETS DON'T WORK
THE SAME WAY.
SO I THINK IT'S, WE JUST PLAIN
OLD DON'T KNOW ENOUGH.
THE MORE WE KNOW THE BETTER OFF
WE WILL BE.
BUT UNDERSTANDING MORE ABOUT
RECEPTORS ON BETA CELLS IS VERY
IMPORTANT AND LEARNING HOW TO
MANIPULATE THOSE RECEPTORS SO WE
DON'T DESENSITIZE THE FILM.
ONE OF THE THINGS THAT IS QUITE
STRIKING ABOUT BETA CELLS IS YOU
TREAT A BETA CELL FOR 10 MINUTES
WITH SOMETHING AND THE EFFECT
CAN BE VERY GOOD TO TREAT FOR AN
HOUR AND IT WILL START TO BE
BAD.
NOT GOOD.
SO, THEY GET DESENSITIZED OR
REWIRED OR WHATEVER OR HOW FAR
YOU LIKE TO THINK OF IT, VERY
QUICKLY.
WHICH MEANS PROLONGED THERAPIES
MAY BE DIFFICULT TO ACHIEVE.
IT'S NOT A DRUG COMPANY CONCEPT
TO LOOK FOR SHORT ASSAY
AGONISTS.
>> AND REGARDING THE KINASE, AND
SPECIFICITY.
DO YOU HAVE ANY EFFECT?
[ INDISCERNIBLE ]
>> I CONTINUED UNDERSTAND THAT.
>> ON THE
KINASE -- INHIBITORS --
>> OKAY.
ABOUT KINASE INHIBITORS.
I THINK ONE OF THE MAJOR THINGS
I HAVE LEARNED IS THAT KINASE
INHIBITORS PROBABLY AREN'T A
GOOD IDEA HERE BECAUSE THE
SIGNALING IS VERY RESPONSIVE TO
WHAT IS OUTSIDE BUT BETA CELLS
CHANGE DEPENDING UPON WHAT IS
OUTSIDE.
SO YOU CAN STILL GROW BETA CELLS
IN 25 MILL I MOLAR GLEE COWS,
TAKE IT AWAY FOR 15
MINUTES -- GLUCOSE AND ADD
GLUCOSE BACK AND ERK WILL BE
TURNED ON BUT THE THE BETA CELL
WILL HAVE NEGATIVE REGULATORY
FACTORS AND WON'T HAVE ANY OF
THE THINGS -- SOME OF THE THINGS
THEY NEED IN ORDER TO CONTROL
WHAT THEY DO PROPERLY.
SO UNDER THOSE CONDITIONS, ONLY
BAD STUFF WILL HAPPEN.
INHIBITING THE KINASE MAY OR MAY
NOT MAKE A DIFFERENCE.
SO I THINK WHAT I LEARNED IS
THAT THE GLUCOSE REGULATION OF
WHAT IS IN THIS CELL AND THE
KINASE REGULATION ARE DISTINCT.
ERK1 AND 2 REGULATE INSULIN GENE
TRANSCRIPTION BUT THEY DON'T
CONTROL THE COMPLIMENT OF
FACTORS EXPRESSED IN THE BETA
CELL THAT CAN REGULATE ANY OF
THESE TRANSCRIPTIONAL EVENTS.
>> THAT WAS RELATED TO MY
QUESTION.
SO YOU SAW THAT WITH LONG TERM
GLUCOSE TREATMENT, YOU SEE THE
CHANGE IN TRANSCRIPTION FACTOR
PROFILE.
SO IF YOU TREAT WITH YOUR SMALL
MOLECULE ISX, DO YOU SEE AN
INHIBITION OF THAT CHANGE THAT
YOU SEE WITH LONG TERM GLUCOSE?
BECAUSE IT SEEMED TO BE THE
OPPOSITE EFFECT WITH YOUR
MOLECULE.
>> SO I WILL SAY THAT WE DIDN'T
LOOK AT CEBT BETA, AT LEAST I
CAN'T RECALL THE DATA IF WE DID.
WE PROBABLY DID BUT I DON'T
REMEMBER IT.
BUT ALL OF THE FACTORS THAT ARE
IMPORTANT TO STIMULATE OR
STIMULATORY TRANSCRIPTION, THEY
ARE ALL INITUDES.
AND WHAT HAPPENS?
THESE CELLS -- INDUCED -- THE
CELLS AND ISLETS ARE MAINTAINED
IN HIGH GLUCOSE.
SO ADDING THE DRUGS STILL HAS
POSITIVE EFFECTS EVEN THOUGH THE
CELLS ARE MAINTAINED IN HIGH
NEWICOSE.
>> OKAY.
ONE FOLLOW-UP.
IS THE SMALL MOLECULE THE
EFFECTS YOU SEE WITH THAT, ARE
THEY SENSITIVE TO CALLS NEURIN
INHIBITION?
>> LET US THANK OUR SPEAK ARE
FOR AN ELEGANT PRESENTATION.
[ APPLAUSE ]