>> GOOD MORNING.

I'M VERY PLEASED FOR THIS

INTERGROUP MEETING AND ALSO I'M

HAPPY TO INTRODUCE DR. ALEXANDRA

MCPHERRON.

DR. MCPHERRON IS A -- 2005 AND

SHE HAS A BACHELOR'S DEGREE FROM

UC DAVIS -- JOHNS HOPKINS SCHOOL

OF MEDICINE.

SHE CONTINUED HER POST DOC

 TRAINING AT HOPKINS

UNTIL SHE JOINED NIH IN 2005.

AND SHE HAS A VERY INTERESTING

PUBLICATION INCLUDING -- MANY

OTHER DISEASE JOURNALS.

AND SHE HAS BEEN MANY LECTURES

AND SERVED AS A REVIEWER PEER

REVIEW JOURNALS AND ALSO --

SHE'S ALSO ON THE BOARD FOR

THE -- JOURNAL AND SHE WROTE A

NUMBER OF -- AND SHE INCLUDE --

SCIENTIST AWARD IN 2010 AND

AWARD FOR SCIENTISTS AND

ENGINEERS IN 2006 AND EARLY --

AND SHE HOLDS A NUMBER OF TILES

TOO.

TODAY SHE'LL BE PRESENTING HER

TEAM'S WORK ME METABOLOMICS

WORKS -- THIS IS A TGF PROTEIN

DISCOVERED BY HER TEAM AND SHE

HAS EXTENSIVE -- THANK YOU.

>> THANK YOU VERY MUCH.

THANK YOU FOR INVITING ME.

GOOD MORNING EVERYONE.

TODAY I'M GOING TO TALK ABOUT

TGF-BETA FAMILY MEMBER GROWTH

FACTOR CALLED MYOSTATIN AND I'LL

BRIEFLY TALK ABOUT MYOSTATIN

SIGNALING BECAUSE SOME OF THIS

WILL COME UP LATER.

MYOSTATIN IS FIRST SYNTHESIZED

BY OTHER TGF-BETAS AS A

PROPEPTIDE -- SORRY, TO SEPARATE

THE PROPEPTIDE FROM THE MATURE

SULFIDE LINK DIMER.

IT ACTUALLY BINDS TO THE

RECEPTOR.

ONE OF THE RECEPTORS IS ACTIVE

IN TYPE TWO B AND ALSO BINDS TO

TYPE TWO A RECEPTOR BUT HAS A

STRONG AFFINITY FOR THE TYPE 2B

RECEPTOR.

ONCE THIS BINDING OCCURS,

SIGNALING DOES NOT OCCUR UNTIL

ITSING COMPLEX ASSOCIATES WITH A

TYPE ONE RECEPTOR, IN THIS CASE

KINASE 4 OR 5.

AND THE HERET HETERO MAKER COMPLEX

THAT SIGNALS.

MYOSTALTEN IS FOUNMYOSTATIN IS FOUND IN

CIRCULATION HIGHER IN MOUSE THAN

IN HUMANS.

IT'S NOT ACTIVE, IT'S ACTUALLY

IN AN INACTIVE COMPLEX MOST OF

WHICH IS THE PROPEPTIDE BOUND TO

9 TERMINATOTHE TERMINAL MATURE REGION.

IT BREAKS IN HALF AND THIS

ALLOWS MYOSTATIN TO BIND TO THE

RECEPTOR.

SO IN THEORY, MYOSTATIN COULD

ACT ON ANY TARGET SITE THAT HAS

A PROTEASE THAT COULD CLEAR THE

PROPEPTIDE.

WE KNOW FROM MUTATING THE

PROPEPTIDE DONE AT JOHNS HOPKINS

SO THAT THAT SITE DOES NOT

EXIST, THAT YOU THEN GET THE

SAME PHENOTYPE AS MYOSTATIN.

IT'S ALSO FOUND COMPLEX TO FULL

STATIN LIKE 3 AND GAS ONE TO

OTHER PROTEINS THAT CAN BE BOUND

TO MYOSTATIN IN SERUM TO INHIBIT

BINDING TO THE RECEPTOR.

SO MYOSTATEMENT I MYOSTATIN -- MOUSE EMBRY
O,

AND THE PURPLE THAT YOU SEE IS

FROM THE HYBRIDIZATION WITH THE

MYOSTATIN PROBE AND IT

CORRESPONDS TO THE MYOTOME IN

THE EMBRYO WHICH WILL GO ON TO

FORM THE MUSCLES OF THE BODY.

ON THE RIGHT IS THE BLOT OF

ADULT TISSUES AND YOU CAN SEE A

VERY STRONG SIGNAL IN SKELETAL

MUSCLE IN DELTA 12.

YOU MIGHT BE ABLE TO SEE THE

SAME SIGNAL IN WHITE FAT.

SO THE FUNCTION OF MYOSTATIN, WE

KNOCKED IT OUT IN MICE AND ON

THE UPPER LEFT, THOSE TWO PANELS

SHOW YOU THE WALL TYPE AND

MYOSTATIN KNOCKOUT MOUSE.

WE WERE EXPECTING PERHAPS

KNOCKING IT OUT WOULD INHIBIT,

WOULD CAUSE THE DECREASE OR

DEFECT IN SKELETAL MUSCLE

DIFFERENTIATION.

INSTEAD WE FOUND THE OPPOSITE

AND SKULL TEL SKELETAL MUSCLES ARE TWICE

THE SIZE -- IT TURNS OUT THERE

ARE NATURALLY OCCURRING

MUTATIONS IN OTHER SPECIES --

HAVE FOUND NO MUTATIONS IN THESE

CATTLE.

THEY'RE EXTREMELY MUSCULAR --

MUTATIONS IN SPLICE THAT CAUSES

SPLICING DEFECTS.

HERE AT NIH IN THE NDRI FOUND

MUTATIONS IN SOME WHIPPET DOGS.

THEY LOOK LIKE GRAY HOUND

THEY'RE VERY FAST BUT THEY CARRY

THE MUTATION IN THE MYOSTAT

ISSUE TEAMS.

THE ONE I'M SHOWING YOU IS HIGH

MUSCULAR BUT THE HIGHER STATIN

IS IN BETTER RACERS.

IN THE LOWER LEFT IS THE ONLY

PERSON WHO DATE WHO HAS BEEN

IDENTIFIED WITH MYOSTATIN

MUTATION.

AND THIS IS ONE BORN IN GERMANY

THAT IS VERY MUSCULAR AT BIRTH.

HE'S HOMOZYGOUS.

HIS MOTHER WAS HETEROZYGOUS AND

WAS A PROFESSIONAL ATHLETE.

THOSE ARE ALL DEVELOPMENTAL

DELETIONS.

OBVIOUSLY A LOT OF PEOPLE WOULD

BE INTERESTED IN INCREASING

MUSCLE MASS IN PATIENTS WHO HAVE

MUSCLE WASTING DISEASES, WHETHER

ATROPHY OR MUSCULAR DISTROPHY.

SO WHY THE FARM SUIT KULZ

DEVELOP IN NEUTRALIZING

MONOCLONALS TO SEE IF YOU COULD

INCREASE MUSCLE MASS IN AN ADULT

BY INHIBITING MYOSTATIN.

AND ON THE LEFT IN THE BLACK BAR

SHOWS YOU THE ANTIBODY TREATMENT

AND THEY DO GET AN INCREASE IN

MUSCLE MASS.

ALTHOUGH IT'S NOT AS HIGH AS

WHEN YOU HAVE A KNOCKOUT.

SO THAT WAS EVIDENCE THAT IT

COULD POTENTIALLY BE A THERAPY

EVEN THOUGH IT WASN'T DURING

DEVELOPMENT.

ON THE RIGHT IS A STUDY DONE BY

GRADUATE STUDENTS -- AND SHE

IMPLANTED CHO CELLS THAT WERE

SECRETING MYOSTATIN INTO NEW

MICE.

AND YOU CAN SEE ON THE LOWER

ANIMAL HAS A TUMOR THAT'S

DEVELOPED WHICH ARE CHO CELLS

INJECTED INTO THAT ARE NOT

UTILITYINSECRETING MYOSTATIN.

IN THE TOP MOUSE YOU CAN SEE

IT'S WASTING.

THESE MICE HAVE SEVERE WEISSING

SYNDROME THAT DEVELOPS IN TWO

WEEKS.

VERY HIGH MYOSTATIN LEVELS.

THESE LEVELS ARE PROBABLY WAY

HIGHER THAN YOU WOULD EVER

NORMALLY GET BUT IT AND SHOW

THAT MYO STATIN IS AGAIN ON

ADULT MUSCLE MASS.

LET'S NO REAL EVIDENCE THAT

MYOSTATIN IS POSITIVELY INVOLVED

IN CANCER TAX YEAH.

I MENTIONED PHARMACEUTICAL

COMPANIES ARE INTERESTED IN

DEVELOPING INHIBITORS TO

MYOSTATIN FOR DISEASES THAT

MYOSTATIN ITSELF PROBABLY HAS

NOTHING TO DO WITH.

BUT THE RATIONALE IS THAT FOR

SAY MUSCULAR DISTROPHY AS A

CHILD AGES STRENGTH DECLINES AND

THEY BECOME DISABLED, WHEELCHAIR

BOUND, FOR INSTANCE, AND THEN

EVENTUALLY THIS LEADS TO DEATH

BECAUSE OF BREATHING PROBLEMS.

BUT IF YOU CAN INCREASE MUSCLE

MASS OF MYOSTATIN INHIBITION AT

SOME POINT, PERHAPS YOU CAN

DELAY DISABILITY OR EVEN DEATH.

SO CURRENTLY ELI LILLY FROM

CLINICAL TRIALS GOING FROM

PATIENTS GETTING HIP

REPLACEMENTS AND THEY'RE LOCALLY

INJECTING MYOSTATIN TRYING TO

INCREASE MUSCLE MASS IN THE

OPERATED HIP.

WE USE BASICALLY TWO APPROACHES

IN ADDITION TO THE KNOCKOUT.

ONE IS TO MAKE TRANSGENIC MICE

THAT ARE EXPRESSING AN INHIBITOR

OF MYOSTATIN AND THE INHIBITOR

WE USE SHOWN ON THE RIGHT IS THE

EXTRACELLULAR PORTION OF THE

RECEPTOR THAT STILL HAS A

TRANSMEMBRANE DOMAIN BUT IS

MISSING THE KINASE DOMAIN.

SO THIS BASICALLY ACTS AS A

STAIN TO STOP UP THE MYOSTATIN

BUT IT BLOCKS SIGNALING.

WE EXPECT THESE VENDOR TISSUES

SPECIFIC PROMOTER.

ANOTHER METHOD THAT WE HAVE IS

BY OVER EXPRESSING THE

EXTRACELLULAR DEMAND OF THE

RECEPTOR FUSED TO FC FOR

STABILITY AND THIS WAS A COME

LABS HAVE MADE THIS AND WE GOT

OURS FROM A GIFT FROM -- SO WE

GROW IT IN CELL CULTURE, COLLECT

THE CONDITION MEAD U MEDIUM AND PURIFY

THE PROTEIN THERE.

WE CAN INJECT THE INTO MICE AND

GET AN INCREASE IN MUSCLE MASS.

SO IN THAT CASE, WE'RE BLOCKING

THE CIRCULATING MYO STATIN WHERE

IT'S A TRANS GENIC JUST AT A

SPECIFIC TISSUE BUT MUSCLE IS

STILL PRODUCING MYO STATIN AND

STILL GETTING INTO CIRCULATION.

SO I WANT TO DISCUSS A LITTLE

BIT ABOUT WHAT MUSCLE LOOKS

LIKE.

SO WHEN I SAY AN INCREASE IN

MUSCLE MASS WHAT EXACTLY AM I

TALKING ABOUT.

ON THE LEFT SHOWS YOU A

CROSS-SECTION OF A SKELETAL

MUSCLE, AND YOU CAN SEE THE

PURPLES THERE ARE THE NUCLEI.

BUT THE MUSCLE FIBER IS A SINGLE

CELL BUT IT'S WORN BY FUSION OF

MYOBLASTS SO IT'S THE

INTERSTITIAL CELLS.

IN THE DIGITORUM LONGEST THE

MUSCLE WE LOOKED THERE ARE 260

OF THESE MYONUCLEI IN ONE FIBER.

THE OTHER CELL TYPE FOUND IN

MUSCLE, THEIR STEM CELLS THAT

ARE CALLED SATELLITE CELL IN THE

ADULT, AND THERE ARE FEWER OF

THESE FOUND UNDERNEATH THE BASAL

LAMINA OUTSIDE THE PLASMA

MEMBRANE OF THE FIBER.

AND YOU CAN SEE ON THE RIGHT IS

A PICTURE MADE BY CHEN WANG, THE

BLUE ARE STAINED MYONUCLEI AND

THE GREEN ARROW POINTS TO A

SATELLITE CELL EXPRESSING A

MARKER PACK SEVEN.

SO WHEN WE LOOK AT THE MYOSTATIN

KNOCKOUT MOUSE, ONE THING WE SEE

IN THE HISTOGRAM OF THE DIAMETER

OF MUSCLE FIBERS IN CLASS

SECTION IN PINK SHIFTED TO THE

RIGHT, SHIFTED TO LARGER FIBERS.

HOWEVER IT'S NOT REALLY DRAMATIC

AND THE TIBIALIS IN THE KNOCKOUT

IS TWICE THE WEIGHT OF THE WILD

TYPE SO THAT DOESN'T SEEM TO BE

ENOUGH TO ACCOUNT FOR THAT

WEIGHT.

SO IN COUNT FIBERS THOUGH, WE

ALSO SEE AN INCREASE ON A PINK

COMPARED TO WILD TYPE IN BLACK.

AND THE SOLEUS WHICH IS THE

MUSCLE, THE FAT MUSCLE, THEY ARE

BOTH REALLY SMALL MUSCLE AND YOU

DON'T SEE AS BIG AN INCREASE IN

THE NUMBER OF FIBERS AS WE DO

WITH THE TA TIBIALIS WHICH IS A 67%

INCREASE.

MUSCLE FIBER COUNTS FOR A LOT OF

THE INCREASE IN THE KNOCKOUT

MOUSE.

THEY HAVE BOTH HYPERTROPHY OF

THE FIBERS AND WHEN HYPERPLASIA.

IF WE LOOK SPECIFICALLY AT FIBER

TYPES, IDENTIFIED BY THE TYPE OF

MYO SIN, EACH FIBER IS

EXPRESSING, FIBER TENDS TO

EXPRESS JUST ONE, WE FIND THAT

THERE'S A DECREASE IN SLOWER,

MORE OX DATE OF FIBERS WHICH IS

ONE AND 2A.

IN THE SOLEUS TYPE ONE.

BUT AN INCREASE IN THE TYPE TWO

OR FASTER CONTRACTING FIBERS,

AND THEY TEND TO BE MORE PSYCHO

LYTIC.

THE SOLEUS MAY HAVE A DIFFERENT

COMPOSITION BUT IT'S THE SAME

BUT LESS IN AN THE TYPE 2A WHICH

IS BOAST PSYC MOSTLY PSYCHOTIC.

THERE'S A SHIFT TO THE RIGHT FOR

PSYCHO LYTIC AND MUSCLE

CONTRACTION IN THE FIBERS.

ONE EXPLANATION WE THINK FOR

THIS IS THAT THERE ARE MORE

PROGENITOR CELLS DURING

DEVELOPMENT.

THIS IS A STUDY DONE BY

CATHERINE SAVAGE, FORMER POST

DOC IN MY LAB.

WHEN SHE LOOKED AT PACK 7 WHICH

IS A MARKER HOW FAR PROGENITOR

CELLS IN THE DEVELOPING EMBRYO

AND IN TH THE ADULT.

IF YOU LOOK AT E17 AND-A-HALF ON

THE FAR LEFT YOU SEE A HUGE

INCREASE IN THE NUMBER OF THESE

PROGENITOR CELLS IN THE MUTANT.

HOWEVER, 17 AND-A-HALF, IS THIS

BEFORE BIRTH ON THE RIGHT,

THERE'S NO DIFFERENCE IN THE

NUMBER OF FIBERS IN THE EDO.

HOWEVER, AS DEVELOPMENT

CONTINUES, THERE'S A BIG

DECREASE IN THE NUMBER OF

PROGENITORS IN BOTH WILD TYPE

AND IN THE MUTANT BUT ESPECIALLY

IN THE MUTANT.

AND THEN THE FIBER NUMBER

SUBSEQUENTLY GOES UP.

THEY ALSO NOTICED POST NASAL

BASE 7 TO OUT EIGHT WEEKS.

NOW THE SAME NUMBER OF FIBERS IN

THE WILD TYPE AND IN THE MUTANT.

SO THE TIME IS SUSTAINED.

WHAT WE KNOW FOR WILD IS A ROUND

BIRTH, YOU HAVE THE SAME NUMBER

OF FIBERS AS YOU DO AS AN ADULT.

WHEN YOU'RE ABLING AGING GROWING YOU

GET AN INCREASE IN FIBER SIZE

RATHER THAN THE FIBER NUMBER.

AS IT HAPPENS, THIS DISPLAYS

AROUND 17 AND-A-HALF, THESE

CELLS ARE KNOWN TO TEND TO

BECOME FAST WITH AN EARLIER WAVE

A FEW DAYS EARLIER THE

PROGENITOR ARE FIBERS THAT TEND

TO BECOME SLOW.

THIS MAY BE THE EXPLANATION IN

BOTH THE INCREASED FIBER NUMBER

AND THE SHIFT TOWARD MORE GLYCO

LYTIC FIBERS.

WE SEE SOMETHING VERY DIFFERENT

WHEN WE TREAT WITH A FIBER

RECEPTOR IN ADULTS.

INSTEAD WE SEE ONLY HYPERTROPHY.

QUITE A FEW LABS HAVE LOOKED AT

THIS AND NOBODY'S SEEING AN

INCREASE IN FIBER NUMBER.

THERE WAS A DEBATE OF WHETHER

THE SATELLITE CELLS THAT I

SHOWED YOU EARLIER, WHETHER THEY

NEEDED TO BE ACTIVATED OR NOT TO

SEE THIS HYPERTROPHY, AND THERE

WAS SEVERAL CONFLICTING STUDIES

CHEN WANG A POST DOC IN MY LAB

DID A VERY SENSITIVE STUDY

INJECTING BRDU DAILY AND

INJECTING THE FIBER RECEPTOR

INTO ADULT MICE.

AND SHE ISOLATES THE EDL, WEIGH

IT AND THEN ISOLATE INDIVIDUAL

FIBERS FROM THE EDL AND EXAMINE

THE BRDU FOR INCORPORATATION

INTO THE FIBER AND THAT WOULD BE

THE MARKER OF THE SATELLITE CELL

PROLIFERATION AND THEN FUSION TO

THE FIBER.

AND LOTS OF HYPERTROPHIC STIMULI

BY DOING EXERCISE FOR INSTANCE

IN CHEC CHICKENS OR MICE OR RATS

WOULD SHOW AN INCREASE IN BRDU

NUMBER.

THE QUESTION IS WHETHER THE

MYOSTATIN WOULD CAUSE THAT.

WHAT CHEN FOUND IN DOING

EXPERIMENTS JUST SHOWING YOU A

TIME COURSE, YOU SEE HYPERTROPHY

SIGNIFICANT HYPERTROPHY AT FOUR

DAYS OR SIX DAYS AFTER THIS

INJECTION WHICH IS SHOWN IN THE

BAR GRAPH ON THE LEFT.

IF YOU LOOK AT THE NUMBER OF

BRDU NUCLEI, BRUD POSITIVE

NUCLEI IN THE FIBER WHICH IS THE

LINE ON THE LEFT, YOU DON'T

REALLY SEE A SIGNIFICANT

INCREASE UNTIL SIX DAYS.

IF YOU NOTICE THE GRAPH TO THE

RIGHT OF THE GRAPH ON THE LEFT,

THE LABEL'S TO THE RIGHT ON THAT

GRAPH, THERE'S ONLY ABOUT TWO TO

TWO AND-A-HALF BRDU POSITIVE

NUCLEI IN THE WHOLE FIBER AND

THERE'S SOMETHING LIKE 260

NUCLEI IN THAT FIBER.

SO THAT DOESN'T SEEM TO BE

ANYWHERE NEAR ENOUGH TO ACCOUNT

FOR THE INCREASE IN MUSCLE MASS.

AND CHEN ALSO IN THE FIBER AREA

IN EACH OF THOSE ANIMALS IN THE

CONTRA LATERAL MUSCLE AND YOU

SEE A SHIFT TO THE RIGHT IN THE

RECEPTOR TWO DAYS BEFORE YOU SEE

THIS BRDU INCORPORATION.

WHAT'S HAPPENING IS THE FIBERS

ARE GETTING BIGGER WITHOUT ANY

INCORPORATION OF NEW NUCLEI.

AND SEVERAL LABS HAVE LOOKED AT

THE INTERACTION OF MYO STATIN

SIGNALING WITH ITF SIGNALING

BECAUSE ITF IS KNOWN TO TURN ON

PROTEIN SYNTHESIS AND CAUSE

HYPERTROPHY IN MUSCLE.

THIS DIAGRAM FROM DAVID GLASS

AND SEVERAL OTHERS HAVE FOUND

SIMILAR RESULTS SHOW THAT

MYOSTATIN SIGNALING WHICH TURNS

ON THE MEDIATORS OF THE

SIGNALING 2 AND 3 SOMEHOW

INHIBIT ACTIVATION OF AKT WHICH

IS DOWN STREAM OF IDF1 SNALG.

WHEN YOU REMOVE MYOSTATIN YOU

INCREASE ACTIVITY OF AKT

INSTANTLY TO THE PROTEIN

SYNTHESIS.

AND THIS PATHWAY WILL COME UP

AGAIN WHEN WE TALK ABOUT INSULIN

SIGNALING BECAUSE THAT USES A

SIMILAR PATHWAY.

I WANT TO MENTION ALSO HERE AT

THE TOP IN RED IT SAYS MY I

DON'T STATIN GS11.

GS11 IS 90% IDENTICAL TO

MYOSTATIN AND LESS HOMOLOGOUS

BUT THEY CAN ALL BIND TO THESE

RECEPTORS.

SO WHEN WE USE THE RECEPTOR OR

WE USE A DOMINANT NEGATIVE

RECEPTOR TRANSGENIC, WE ARE

BLOCKING OTHER FACTORS BESIDES

MYOSTATIN.

ACTIVE N IN PARTICULAR ALSO

SEEMS TO INCREASE MUSCLE MASS

WHEN IT'S INHIBITED.

WHAT I WANT TO TALK TODAY ABOUT

IS FIRST THE REGULATION OF BODY

COMPOSITION AND THEN WE'LL GET

INTO A DIABETIC MODEL, MICE THAT

HAVE NO WHITE FAT.

AND FINALLY THE -- ANALYSIS.

THE FIRST PART BRIEFLY, ONE

THING I NOTICED WHEN I WAS

KILLING A BUNCH OF OLDER MICE IS

THAT THEY HAVE LESS ADIPOSE

TISSUE.

ON THE LEFT THE BODY COMPOSITION

ANALYSIS AND IN PINK IT SHOWS

THE MY OH STATIN KNOCKOUT HAS A

LOT MORE LEAN MASS THAN YOU

WOULD EXPECT.

THEY ALSO HAVE LESS FAT MASS.

THIS ISN'T THE PERCENT, IT'S THE

ACTUAL GRAMS OF FAT MEASURED.

WHEN WE PUT THEM ON A HIGH DIET

THEY GAINED LESS WEIGHT SHOWN IN

PINK COMPARED TO THE BLACK BARS

AS WELL.

AND ON THE RIGHT ARE NORMAL

ANIMALS THAT'S SHOWING YOU THE

SMALLER FAT CELLS IN THE

EPIDERMAL FAT PAD.

AND IT'S BEEN REPLICATED BY

QUITE A FEW LABS THAT WE SENT

THE MICE TO.

HERE AT NIDEK -- WHO RUNS THE

METABOLIC CORE AND -- IN HER LAB

DID THE HYPER IN SHINMIC LAB FOR

US.

THIS IS MEASURING THE

SENSITIVITY.

THEY ARE INFUSING INSULIN SO

EVERY ANIMAL GETS THE SAME

AMOUNT OF INSULIN AND

CIRCULATION.

AND THEN THEY INFUSE GLUCOSE AND

THE MORE GLUCOSE THEY INFUSE,

THE MORE SENSITIVE THE ANNAL IS

TO -- ANIMAL IS TO INSULIN.

IF THE ANIMAL IS COMPLETELY

INSENSITIVE TO INSULIN YOU DON'T

HAVE TO USE ANY GLUCOSE AT ALL.

TRYING TO KEEP THE BLOOD GLUCOSE

AT A CONSPHEN CONTENT LEVEL.

THIS INCREASE WITH THE INFUSION

AND THE MY OH STATIN GLUCOSE

SHOWING THEY ARE MUCH MORE

INSULIN SENSITIVE THAN THE WILD

TYPE ANIMALS.

AND THEY ALSO INJECT LABEL

GLUCOSE AND FOUND AN INCREASE IN

WHOLE BODY GLUCOSE UP TAKE SHOWN

IN THE UPPER RIGHT AND A LOWER

LEFT AN INCREASE IN MUSCLE

GLUCOSE UP TAKE IN FAT.

SO IT SUGGESTS THERE ARE MORE

INSULIN SENSITIVE TAKING UP MORE

GLUCOSE AND ALSO PUTTING ON LESS

FAT.

SO I TOLD YOU IN THE BEGINNING

MYOSTATIN'S EXPRESSED IN MUSCLE

AND ALSO EXPRESSED IN FAT.

SO SOME OF THIS PHENOTYPE DUE TO

FAT.

I DON'T THINK WE CAN SAY FROM

LOOKING AT THE KNOCKOUT MOUSE

BECAUSE THERE WERE SEVERAL OTHER

LINES OF MICE FOUND IN THE

LITERATURE IS THAT ON MUSCULAR

SUCH AS TRANSGENIC OVER

EXPRESSING THE ONYX GENE AND

OVER EXPRESSING IGF ONE

SPECIFICALLY IN SKELETAL MUSCLE

AND ALSO OVER EXPRESSING THE

ACTIVE AKT TO KEEP THE IGF1

PATHWAY ACTIVATED WITH THE

SKELETAL MUSCLE SPECIFIC

PROMOTER.

AND THOSE LAST TWO IN PARTICULAR

ARE IMPORTANT.

SO ALL OF THESE HAVE LESS

MUSCLE.

SO THEY HAVE LESS FAT.

SO IT SUGGESTS IF YOU INCREASE

MUSCLE MASS, PERHAPS THE MUSCLE

IS TAKING THE ENERGY AND THERE'S

NUT ENOUGH STORAGE FAT.

THAT COULD BE A PHENOTYPE IN THE

KNOCKOUT OR IT COULD BE THAT

MYOSTATIN HAS A ROLE IN

ONCOGENESIS.

USING THE TISSUE SPECIFIC

TRANSGENICS TO TRY TO ANALYZE

THIS, AND SOMETIME BEFORE HE HAD

MADE A DOMINANT NEGATIVE

RECEPTOR AND YOU CAN SEE ON THE

RIGHT, THEY'VE SEEN A COPY OF

THE KNOCKOUT AND EXTREMELY

MUSCULAR.

AND WE MADE IN MY LAB THE DNA2

PROMOTER TO EXPRESS THE SAME

DOMINANT NEGATIVE RECEPTOR

CONSTRUCTING FAT IN WHITE FAT

AND BROWN FAT.

SO THEN WE COMPARED THESE TWO

AND LOOKING AT THE BODY

COMPOSITION, IF WE HAVE

EXPRESSED A DOMINANT NEGATIVE

RECEPTOR IN FAT, YOU GET

ABSOLUTELY NO EFFECT AT ALL.

AND IF WE LOOK AT THE DOMINANT

NEGATIVE RECEPTOR IN FAT IT'S

VERY MUSCULAR WE GET THE SAME

PHENOTYPE AS THE KNOCKOUT IN

LOWER FAT.

WE THINK ABOUT THE LOWER FAT

PHENOTYPE IS NOT BY DIRECT

EFFECTS OF MYOSTATIN ON FAT.

ALTHOUGH IF YOU DO LOOK IN

VITRO, YOU CAN SEE MYOSTATIN

INHIBITING DIFFERENTIATION.

SO IT MAY BE MORE SUBTLE

EFFECTS.

WE TRIED PUTTING THEM ON HIGH

FAT DIET FOR INSTANCE AND

NOTHING, YOU DIDN'T SEE ANYTHING

WITH THE FAT SPECIFIC DOMINANT

NEGATIVE EFFECTOR BUT THE MUSCLE

SPECIFIC EFFECTIVE DOMINANT

NEGATIVE RECEPTOR IS THE COPY

KNOCKOUT PHENOTYPE.

SO THAT'S OBVIOUSLY NOT A WAY

THAT WE COULD HELP TREAT

OBESITY.

THE QUESTION IS IF WE INCREASE

MUSCLE MASS IN MICE THAT ARE

ALREADY OBESE, COULD WE GET THEM

TO LOSE FAT.

SOME STUDIES HAVE BEEN DONE ON

EARLY.

I DON'T KNOW HOW MUCH OF THAT IS

PREVENTING AND OUCH OF THAT

COULD ACTUALLY BE MAKING A MOVE.

ON THE RIGHT, THE INDUCIBLE

SKELETAL MUSCLE SPECIFIC ACTIVE

AKT IN KEN WOUL WALSH'S LAB HAS A

DRAMATIC PHENOTYPE.

YOU GET HYPERTROPHY AND GLUCOSE

UP TAKE IN THE MUSCLE.

YOU SEE INCREASED OXIDATION AND

YOU SEE A DECREASE IN FAT MASS.

THEY SAW THIS MAINLY IN MICE

THAT WERE ALREADY OBESE FROM A

HIGH FAT DIET.

SO INCREASING MUSCLE HYPERTROPHY

CAUSE A BIG LOSS IN FAT MASS.

HOWEVER, ACTIVE AKT MAYBE

THERE'S SOME OTHER SIGNALING

GOING ON IN THE MUSCLE MYOKIND

ACTING ON THE STAT.

PLUS THAT'S NOT A

PHARMACOLOGICAL WAY TO DO THIS.

SO WE TRY TO USE MICE PUT ON A

HIGH FAT DIET FOR 24 WEEKS BUT

OF 12 WEEKS WE INJECTED THE

SOLUBLE REJERK.

WE FOUND THE INCREASE IN THE

MASS THAT'S WHAT WE EXPECTED

SHOWN IN RED.

BUT THE BODY WEIGHT KEPT GOING

UP PARTLY DUE TO THE MUSCLE

MASS, AND THEN THE FAT WEIGHT

KEPT GOING UP.

NOT ONLY DID THEY NOT LOSE IT,

THEY KEPT RIGHT ON GAINING FAT.

AND WE THEN TRIED GLUCOSE

TOLERANCE, AND BY JENNIFER -- IN

MY LAB, WE DIDN'T SEE ANY

DIFFERENCE IN GLUCOSE TOLERANCE.

SO THAT WAS SOMEWHAT

DISAPPOINTING BUT THE SAME

RESULTS ALSO FOUND THAT FIBER

BY -- USING AN ANTI-MYOSTATIN

ANTIBODY, THEY ALSO DID NOT IN

OBO MICE -- AND THE SAME THING,

THEY DID NOT SEE A LOSS OF FAT

MASS.

THEY DID SEE SOME IMPROVEMENTS

IN THE GLUCOSE METABOLISM,

HOWEVER.

SO I DON'T KNOW WHETHER WE HAD A

MILDER DIET THIS WAS 60% FAT OR

BUT IT DIDN'T SEEM TO WORK AFTER

THE FACT.

SO WE THOUGHT WE SHOULD BE

LOOKING AT MAYBE A DIABETIC

MODEL INSTEAD OF AN OBESE MODEL.

SO WE PUT MICE ON HIGH FAT DIET

AND THEY REALLY DIDN'T BECOME

VERY DIABETIC.

SO ONE MODEL THAT WAS AVAILABLE

IN NIDDK WAS A LIPO DISTROPHY

MODEL AND LIPO DISTROPHY IS A

DISEASE WHERE WHITE FAT DOES NOT

DEVELOP.

SO YOU ARE MISSING THE STORAGE

SITES FOR LIPIDS.

SO INSTEAD, IT GETS INTO THE

CIRCULATION AND VERY HIGH

TRIGLYCERIDE LEVELS AND YOU ALSO

HAVE HIGH FAT IN THE LIVER AND

MUSCLES SO THEY GET HEPATIC

PSYCHOSIS AND THIS CAUSES

INSULIN RESISTANCE WHICH LEADS

TO HYPERGLYCEMIA, HYPER IN

INSULINEMIA.

ANOTHER PHENOTYPE OF BOTH THE

PATIENTS AND THE MOUSE MODELS IS

THAT THEY OVEREAT WHICH IS

CALLED HYPERPLASIA.

SO THE MODEL IS CALLED FATLESS

MICE AND IT'S OVER EXPRESSING A

DOMINANT NEGATIVE IN A TRANSGENE

THAT INTERFERES WITH SOME OF THE

TRANSCRIPTION FACTORS IMPORTANT

FOR LIPID SITE DIFFERENTIATION

AND THIS IS USING A -- SO HERE'S

WHAT THE MOUSE LOOKS LIKE ON THE

LEFT.

COMPARED WILD TYPE AND YOU'RE

MISSING ALL OF THAT WHITE FAT.

YOU CAN'T SEE THE SUBCUTANEOUS

WHITE FAT.

AND ON THE RIGHT SHOWS HOW FAST

THE LIVER CAN GET, IT CHANGES

COLOR AND IT HAS THESE WHITE FAT

DROPLETS.

SO THE FIRST THING THAT THE POST

DOC WHO WAS DOING THIS PROJECT

DID IS HE LOOKED AT THE

EXPRESSION OF MYO STATIN IN THE

MUSCLE.

AND MICE DO NOT HAVE AN INCREASE

IN MUSCLE MASS BUT THEY HAVE AN

INCREASE IN MYO STATIN GENE

EXPRESSION WHICH IS QUITE HIGH.

THERE ARE SEVERAL PAPERS IN THE

LITERATURE THAT HAVE CHANGES IN

MYO STATIN EXPRESSION IN MUSCLE

BUT I'M STRICTLY CORRELATED TO

THE MUSCLE MASS.

FOR INSTANCE, IN MICE, OBOB HAVE

AN INCREASE IN MYOSTATIN THEY

HAVE A DECREASE IN MUSCLE MASS

AND THEY MIGHT EXPECT THAT.

HOWEVER, IF YOU PUT MICE ON A

HIGH FAT DIET YOU ALSO SEE AN

INCREASE IN MYOSTATIN BUT YOU

DON'T SEE A CHANGE IN THE MASS.

IN HUMANS, THERE HAVE BEEN A FEW

STUDIES LOOKING AT PATIENTS THAT

GET SURGERY, STOMACH BYPASS FOR

OBESITY AND ONE OF THOSE

PHENOTYPES BESIDES LOSING FAT IS

THEY ALSO LOSE SOME MUSCLE BUT

NOT A WHOLE LOT.

THEY HAVE A DECREASE IN

MYOSTATIN.

WE THINK A DECREASE IN MYOSTATIN

WOULD INCREASE MUSCLE MASS.

THE OTHER THING THAT HAPPENS

AFTER SURGERY IS THEY HAVE AN

INCREASE IN INSULIN SENSITIVITY

IT CAN OFTEN GO OFF INSULIN.

AND ANOTHER STUDY BY THE

UNIVERSITY OF CALGARY FOUND THAT

THERE'S AN INCREASE IN MYO

STATIN INCREASE FROM MYOTUBES

MADE FROM OBESE PATIENTS LOOKING

AT MUSCLE MASS.

AND FINALLY THE MICRO -- STUDY

SHOWING AN INCREASE IN MYOSTATIN

INCREASE IN MUSCLE RELATIVE TO

TYPE TWO DIE BOAT BOTHICS.

THEY HAVE NOT YET DEVELOPED DIE --

DIDIABETES.

THERE'S NO SIZE OF THE MUSCLE IN

OVERALL LEAN MASS BUT IT DOES

SEEM TO BE PODDIVELY CORRELATED

BUT YET THERE'S NO CONVINCING

EVIDENCE THAT MY OH STATIN CAN

ACTUALLY CAUSE INSULIN

RESISTANCE.

I WANT TO BRING THIS UP BECAUSE

IT'S POSSIBLE THAT THERE'S

SOMETHING DIRECT WITH MYOSTATIN

SIGNALING THAT COULD BE

INFLUENCING INSULIN SIGNALING AT

THE LEVEL OF AKT SOMETHING OTHER

THAN JUST INCREASING MUSCLE

MASS.

OWE WHAT DO THEY LOOK LIKE?

HERE WE HAVE THE CROSSES USING A

MUSCLE SPECIFIC DOMINANT

NEGATIVE TRANSGENE.

WE STILL GOT AN INCREASE IN

MUSCLE MASS, YOU MIGHT BE ABLE

TO SEE THE PECTORAL MUSCLES IN

THE MOUSE ON THE FAR RIGHT WITH

A DOUBLE TRANSGENIC AND THERE'S

NO WHITE FAT IN THOSE MICE

VERSUS WHITE FAT IN THE WILD

TYPE AND A LITTLE BIT LESS IN

THE MUSCLE DOMINANT NEGATIVE.

SO WHAT HE DID IS FOLLOWED THE

BLOOD GLUCOSE OVER TIME STARTING

AT WEANING AND THIS IS FIRST

WILD TYPE AND THEN VERY SIMILAR

BLOOD GLUCOSE TO THE MUSCLE

DOMINANT NEGATIVE SHOWN IN

ORANGE.

AND EXTREMELY DIABETIC AFTER

FIVE WEEKS, VERY HIGH BLOOD

GLUCOSE LEVELS.

AND THEN WE GET PRETTY NORMAL

BLOOD GLUCOSE.

IT'S MUCH MORE DRAMATIC THAN I

WOULD HAVE THOUGHT.

ESPECIALLY CONSIDERING WE HAVE

AN INCREASE IN GLYCOLYTIC

MUSCLE.

YOU THINK PERHAPS YOU WANT THE

MUSCLE THAT BURNS A LOT OF FAT.

SO THIS SEEMED TO COMPLETELY

PREVENT THE DEVELOPMENT OF

HYPOGLYCEMIA IN THESE FATLESS

MICE.

AND HE LOOKED MORE CLOSELY AND

THE HYPER INSULINEMIA SHOWN IN

BLUE WAS NORMALIZED IN THE

DOUBLE TRANSGENIC SHOWN IN

PURPLE AND DID INFLUENCE

TOLERANCE TEST WHERE YOU MEASURE

GLUCOSE OVER TIME.

AND IF YOU'RE SENSITIVE TO

GLUCOSE, IT SHOULD DROP.

AND GET THE BIGGER DROP, A

NORMAL DROP IN THE DOUBLE

TRANSGENIC, VERY SIMILAR TO THE

WILD TYPE MICE.

AND THE OTHER PHENOTYPE WE SEE

IS AN IMPROVEMENT IN SIGNALING

AND LEFT A VERY BASIC SCHEMATIC

OF THE INSULIN RECEPTOR

SIGNALING ALSO ACTIVATES AKT

WHICH CAN TURN ON PROTEIN

SYNTHESIS AS WELL AS GLUCOSE

METABOLISM IN GENES.

SO ON THE RIGHT, THERE'S AN

EXPERIMENT WHERE TEACHING

INJECTED ANIMALS WITH A HIGH

DOSE OF THIS I INSULIN AND TEN MINUTES

LATER THEY HAD A MUSCLE REMOVED.

WE EXPECT TO SEE AN INCREASE IN

PHOSPHOLATE AKT IN THE MUSCLE

AND ESPECIALLY STRONG IN THE

MUSCLE SPECIFIC DOMINANT

NEGATIVE MICE.

IN THE MICE THAT'S SO INSULIN

RESISTENT IN MUSCLE BASICALLY

NOTHING HAPPENED.

ON THE FAR RIGHT THE DOUBLE

TRANSGENIC HAVE RESTORED

RESPONSIVENESS TO INSULIN.

AND THE OTHER STRONG PHENOTYPE

GLUCOSE HANDLING IN THESE MICE

IS HIGH LIPID LEVELS.

AND DOUBLE MUTANTS INCREASE

MUSCLE MASS HAD NORMAL FREE

FATTY ACIDS, NORMAL TRY GLIS

RIDS AND ON THE RIGHT MUCH MORE

MUSCLE TRIGLYCERIDES.

IT SEEMS TO IMPROVE BOTH THE

GLUCOSE AND LIPID DEFECTS THAT

PREVENT DEVELOPMENT OF BOTH THE

GLUCOSE AND LIPID DEFECT IN

THESE MICE.

IF WE LOOK SPECIFICALLY AT THE

LIVER YOU SEE IN THE CENTER IS

THE A ZIP WHITE OR GLAK DROPLETS

CONTAINED IN THE LIVER.

ON THE WHITE THE LIVER LOOKS

NORMAL.

TRIGLYCERIDES ARE QUANTIFIED ON

THE LOWER LEFT AS NORMAL IN THE

DOUBLE MUTANT.

THE WHITE MUSCLES, THE LIVER HAS

RESTORED INSULIN SIGNALING THE

INCREASE IN RESPONSE TO INSULIN

IN THE DOUBLE TRANSGENIC MUSCLE

COMPARED TO THE A ZIP MUSCLE.

SO THIS COULD BE DO TO AN

INCREASE IN LIPID OXIDATION,

HOWEVER WHEN WE DID SOME LIPID

OXIDATION ASSAYS IN LIVE ANIMALS

ON THE LEFT OR SPECIFICALLY IN

THE SOLE YUTIO SOLEUS MUSCLE SHE DID NOT

SEE AN INCREASE IN OXIDATION.

HOWEVER SHE DID SEE A DIFFERENCE

IN TRIGLYCERIDE SYNTHESIS.

AND THIS IS DONE BY BLOCKING --

AND THEN LOOKING AT THE

DEVELOPMENT OF THE INCREASE IN

PLASMA TRIAL GLYCERIDES OVER

TIME   AND THE A ZIP MICE HAVE A

MUCH BIGGER INCREASE THAN THE

DOUBLE TRANSGENIC.

SO IT SEEMED TO BE TRY LOS

ANGELES RIDE SYNTHESIS --

TRIGLYCERIDE SYNTHESIS CHANGED

IN THESE ANIMALS.

I DON'T WANT TO GO INTO TOO MUCH

DETAIL.

WE DON'T REALLY UNDERSTAND IT

BUT WE ARE SURPRISED WE ALSO

NORMALIZED THE HYPER PHAGIA.

THERE ARE SOME EXPERIMENTS

SHOWING IT IS NOT JUST TURNING

ON -- BUT SOMEHOW WE DON'T

UNDERSTAND THEY DID NOT DEVELOP

THE INCREASE OF FOOD INTAKE

WHETHER IT'S THE ABSOLUTE FOOD

INTAKE OR NORMALIZED TO THE BODY

WEIGHT.

SO IT LARGELY PREVENTED A LOT OF

THE PHENOTYPES IN THE LIPO

DISTROPHIC MICE.

SO WE DID, WE WERE CONCERNED

THEY DID SOMETHING WEIRD ABOUT

THE TRANSGENICS IN THE BRAIN OR

SOMETHING LIKE THAT.

SO WE CROSSED THEM TO THE

KNOCKOUT AND WE GET THE SAME

PHENOTYPE.

WE ALSO CROSSED THEM TO OUR FAT

DOMINANT NEGATIVE MICE

EXPRESSING THE DOMINANT NEGATIVE

RECEPTOR IN BROWN FAT AND STILL

PRESENT IN THESE ANIMALS.

AND THAT DID NOT HELP THE A ZIP

PHENOTYPE AT ALL.

WE'RE PRETTY SURE IT HAD

SOMETHING TO DO WITH THE

INCREASE MUSCLE MASS.

POSSIBLY IN ADDITION TO BLOCKING

MYOSTATIN SIGNALING IN MUSCLE.

SO I PUT THE HIGH FAT DIET WHAT

WE REALLY WANT TO KNOW IS CAN WE

DRAW THE BLOOD GLUCOSE AFTER THE

MICE BECOME DIABETIC.

WE INJECTED THE RECEPTOR IN THE

A ZIP MICE AFTER FIVE OR SIX

WEEKS OF AGE AND HAD BLOOD

GLUCOSE AROUND 300.

WHAT WE FOUND IT DID DROP A

LITTLE, DEFINITELY PREVENTED THE

INCREASE AND DID SEEM TO LEVEL

OFF.

THE GLUCOSE TOLERANCE LOOKED A

LITTLE BIT BETTER.

HOWEVER, SURPRISE THE INSULIN

WAS HIGHER THOUGH.

IT'S NOT SIGNIFICANT BECAUSE

IT'S SO VARIABLE.

AND THE TRIGLYCERIDES ALSO WERE

NOT IMPROVED IN SERUM OR LIVER

ALTOUGH THEY DID GO DOWN

TYPICALLY IN MUSCLE.

LOOKING AT THE FOOD INFAKE AND

BODY WEIGHT, I ALREADY SHOWED

THE UPPER LEFT GLUCOSE ONE THING

WE NOTICED IS THIS SEPARATES

FROM THE PDF CONTROLS BETWEEN

TWO AND THREE WEEKS OF AGE WHEN

THEY WERE GETTING HEAVIER AND WE

KNOW THIS IS ABOUT THE TIME WE

GET THE MAXIMUM MUSCLE

HYPERTROPHY.

AND IF YOU LOOK AT THE FOOD

INTAKE, IT'S HARD TO SEE WHAT'S

GOING ON IN THE UPPER RGHT WITH

FOOD IN TAKE.

BUT YOU NORMALIZE THE BODY

WEIGHT IT SEEMS LIKE AT THE

POINT WHERE THEY GET THE MAXIMUM

MUSCLE HYPERTROPHY, THEY HAVE A

DROP IN FOOD INTAKE AND THEN

THAT LEVELS OFF.

SO IT SUGGESTS SOME CONNECTION

PERHAPS THE SAME MECHANISM

BETWEEN THE GLUED GLUCOSE

AMELIORATION OF THE

HYPERGLYCEMIA AND THE IMPROVED

FOOD INTAKE.

SO HOWEVER WE DIDN'T IMPROVE

EVERY PHENOTYPE IN THIS CASE.

TO MY KNOWLEDGE THAT'S THE FIRST

DEMONSTRATION THAT MICE

INHIBITION CAN LOWER BLOOD

GLUCOSE IN A DIABETIC ANIMAL.

ONE QUESTION THAT NICOLE IN MY

LAB HAD WAS WHETHER WE COULD

DECREASE THE BLOOD GLUCOSE AND

GET A DECREASE IN FOOD INTAKE.

SHE INJECTED -- IMPLANTED --

CREATING FLOOR SIN -- PHLORIDZIN

EIGHTY AND NOT THE WILD TYPE

BECAUSE OF THE GLUCOSE TO BEGIN

WITH.

YET SHOWN BELOW THAT DIDN'T DO

ANYTHING TO THE FOOD INTAKE.

THE REVERSE EXPERIMENT HAS BEEN

DONE BY A GROUP USING THE SAME

MICE IN JAPAN DOING PARAFEEDING

SO THEY ARE PURPOSELY FED WILD

TYPE FOOD BUT THAT DID NOT GET

RID OF THE INSULIN RESISTANCE OR

HYPERGLYCEMIA IN THE MICE.

WE DON'T KNOW WHAT THE MECHANISM

IS, IT'S JUST CURING ONE AND THE

OTHER FOLLOWS.

SO IT MIGHT BE INTERESTED --

FROM THE AMERICAN COLLEGE OF

SPORTS MEDICINE AND THE AMERICAN

DIABETES ASSOCIATION HAS

SUGGESTED DIABETICS DO BOTH

AEROBIC AND RESISTANCE TRAINING

BECAUSE THERE HAVE BEEN SEVERAL

CLINICAL TRIALS SAYING THAT

RESISTANCE TRAINING HELPS IT AS

WELL.

WE'VE KNOWN FOR SOME TIME THAT

AEROBIC EXERCISE WITH IMPROVE

INSULIN ACTION.

SO THE MYOSTATIN INHIBITOR

PERHAPS COULD BE EXERCISING THE

MODEL AND SOME PEOPLE CAN'T

EXERCISE BUT MANY OF US REALLY

DON'T LIKE TO.

OKAY.

TO SUMMARIZE JUST THIS PART, WE

SHOWED THAT MYOSTATIN INHIBITION

PRIOR TO THE METABOLIC CHALLENGE

OR DISEASE DID THE DIABETES AND

HIGH FAT DIET CAN OIL PROVE THE

PHENOTYPE BUT IT DOES NOT WORK

AFTER ANIMALS ARE OBESE TO LOWER

OBESITY.

IT DID HELP IN THE LIPO

DISTROPHIC DIABETIC MODEL.

THIS LED US TO SOME QUESTIONS

SPECIFICALLY WHAT BIOCHEMICAL

PATHWAYS ARE CHANGED IN THE

MUSCLE DURING AND AFTER

HYPERTROPHY AND THEN HOW DOES

THAT SECONDARILY CAUSE CHANGES

IN OTHER TISSUES PARTICULARLY

METABOLICALLY IMPORTANT TISSUES.

SO THE FILM PART I'M GOING TO

TALK ABOUT SOME ANALYSIS WE DID

WITH -- WE INJECTED THE RECEPTOR

INTO ADULT MICE AND WE TOOK THEM

AT THREE DAYS AFTER THE FIRST

INJECTION, AND AT THIS POINT,

THEY ARE STARTING TO HYPERTROPHY

THE MUSCLE.

WE'VE GOT ABOUT A 10% INCREASE

IN MUSCLE MASS.

WE TOOK THEM AGAIN AT 21 DAYS

AND THIS IS AFTER THE MUSCLE

MASS IS STABILIZED, EVEN THOUGH

YOU KEEP INJECTING YOU DON'T GET

ANY MORE MUSCLE.

THAT WAS ROUGHLY 50%

HYPERTROPHY.

SO WE HAD THESE FOUR GROUPS AND

SENT SERUM TISSUES TO -- THEY

DID A NON-TARGETED APPROACH TO

GET BIO MOLECULE.

SO THIS SHOWS YOU MUSCLE WEIGHT

AND THE RIGHT BARS ARE THE NANO

FAT PAD.

YOU CAN'T REALLY TELL FOR THREE

DAYS BECAUSE I WANTED TO PUT

THEM ON THE SAME SCALE BUT

THERE'S A SMALL INCREASE IN

MUSCLE MASS, 13 IN EACH GROUP.

AND THEN ON THE RIGHT, A BIGGER

INCREASE IN MOST OF THE MUSCLES.

AND THERE STILL WAS NO CHANGE IN

THE FAT MASS.

SO THIS IS JUST A GENERAL

SUMMARY BEFORE I GO INTO

SPECIFIC RESULTS, AND I'M

SHOWING THE FIRST ROW IS, THE

TOTAL NUMBER OF METABOLIZE THEY

FOUND IT CHANGED SIGNIFICANTLY

IN THE SECOND THE NUMBER THAT

WENT UP AND THE THIRD THE NUMBER

THAT WENT DOWN.

SO IF WE LOOK AT MUSCLE FIRST AS

YOU'D EXPECT A LOT OF CHANGES,

THERE ARE ACTUALLY MORE ON DAY

THREE THAN ON DAY 21.

AND YOU GET A LOT MORE THINGS

GOING UP THAN GOING DOWN.

IN SERUM, A LOT OF THINGS

CHANGED AS WELL, ALTHOUGH ABOUT

TWICE AS MANY ARE GOING DOWN AS

GOING UP.

AND THERE ARE A LOT OF CHANGES

IN SERUM MORE THAN ANYWHERE

EFLTION AT 2 --THINGS HAVE STABILIZED.

NOT ANYWHERE NEAR DAY THREE WHEN

MUSCLES BECOME HYPERTROPHY BUT

DAY 21, WE'RE GOING DOWN THAN

WE'RE GOING UP.

IN FACT AT DAY THREE, VERY

LITTLE HAPPENS.

14 METABOLITES WERE CHANGED IN

DAY 3.

BUT DAY 21 THERE WERE A LOT MORE

IN 69.

ALL OF THEM WENT UP AND NOTHING

WENT DOWN IN DAY 21.

SO I'LL START WITH THE MUSCLE

AND THE SERUM.

I'LL SHOW ALL OF THESE AMINO

ACID PATHWAYS SO YOU CAN

UNDERSTAND.

I'LL JUST LIST THEM OPERATED AT

NON-E SO MUCH FIRST EVENTUAL --

NON-ESSENTIAL VERSUS ESSENTIAL

AMINO ACIDS.

THERE'S AN INCREASE IN THE

MUSCLE AND A LOT OF OF THE

ESSENTIAL AMINO ACIDS.

THE NON-ESSENTIAL IS MORE MIXED

A FEW GOING UP AND A FEW GOING

DOWN.

AND AT DAY 21, YOU HAVE EVEN

MORE OF THE ESSENTIAL AMINO

ACIDS THAN BOTH GOING DOWN.

BUT STILL THERE'S NOT THAT MUCH

OF A CHANGE OF THE NON-ESSENTIAL

AMINO ACIDS.

ONE THING THAT WAS CHANGED IN

RELATION TO AMINO ACIDS WERE THE

POLY AMINES AND THESE ARE -- AND

THE WAY I'M SHOWING THIS, WORDS

THAT ARE IN GRAY ARE THINGS THAT

WERE NOT DETECTED IN THIS

PARTICULAR TISSUE BY METABOLITES

BUT THEY ARE IN THEIR LIBRARY.

ACTUALLY I'M NOT SURE ALL OF

THEM WERE IN THE LIABLE BUT IN

THIS PARTICULAR TISSUE THEY WERE

NOT DETECTED.

AND IF THEY'RE STILL IN BLACK,

THERE'S NO CHANGE.

SO THE RED IS MUSCLE DAY THREE

INCREASE AND -- WENT UP THREE

AND-A-HALF WHICH IS CONSIDERABLE

AND SPERM DEAN WENT UP 1.8.

THE POLY MEANS TEND TO GO UP

WHEN THE TISSUE IS GROWING

WHETHER IT'S DIVIDING OR IT'S

JUST INCREASING IN SIZE.

AND TYPICALLY A MUSCLE THEY'VE

BEEN SEEN TO INCREASE WHEN

MUSCLE IS BECOMING HYPERTROPHIED

SAY BY AN GEN STIMULATION OR BY

EXERCISE.

IT'S ALSO BEEN SEEN TO INCREASE

IN REGENERATING MUSCLE AND

CONVERSELY IF YOU'RE UNDER GOING

ATROPHY THEY GO DOWN.

BUT WHAT THE ACTUAL - ROLE IS IT'S

NOT AT ALL CLEAR.

SO THEN AT DAY 21, THE PRECURSOR

IS ACTUALLY GOING DOWN.

IT IS STILL ELEVATED COMPARED TO

WILD TYPE BUT IT'S LESS THAN IT

WAS AT DAY THREE WHEN MUSCLE IS

UNDER GOING HYPERTROPHY AND

SPERM DEAN IS EVEN HIGHER HAS

NOT IT WAS IN DAY THREE.

THIS MAKES SENSES GIVEN WE'RE

GETTING A BIG INCREASE IN

SKELETAL MUSCLE MASS.

SO I WANT TO GO THROUGH SOME

PATHWAYS HERE AND THE FIRST

THING GLYCOLYSIS BECAUSE WE

WOULD EXPECT SOMETHING TO HAPPEN

GIVEN OUR OTHER RESULTS WITH THE

DIABETIC MICE AND IMPROVED

INSULIN HANDLING OF THE KNOCKOUT

MICE.

SO ALSO LINK IT UP TO THE TCA

CYCLE.

SO STARTING WITH MUSCLE ON DAY

THREE SOME OF THESE IN GRAY WERE

NOT DETECTABLE.

AND THOSE IN RED HERE ARE BIO

MOLECULES THAT INCREASED.

AS YOU NOTICED NEAR THE TOP

GLUCOSIX PHOSPHATE INCREASED 5.1

TIMES THREE DAYS AFTER THE

INJECTIONS OF THE CYTORECEPTOR

COMPARED TO PDF INJECTION.

AND THE PHOSPHATE FOUR

AND-A-HALF TIMES.

SO WE WEREN'T EXPECTING ANYTHING

QUITE THAT DRAMATIC.

WE'RE DOWN THREE PHOSPHOGLIS

RATE INCREASE BY 20%.

BUT THERE WAS NO INCREASE IN --

WHICH YOU MIGHT THINK THERE'S AN

INCREASE IN THIS PATHWAY.

ALSO WANT TO NOTICE IS THE

INCREASE IN GLUCOSE IN THE

MUSCLE ITSELF, 1.6 SOMETIMES JOY TIMES

WILD TYPE.

AND YOU LOOKED AT GLYCOGEN AND

GLUCOSE SIX PHOSPHATE CAN BE

CONVERTED TO GLUCOSE ONE

PHOSPHATE REVERSIBLE REACTION

AND YOU ALSO SEE A BIG INCREASE

THERE AT 3.7 INCREASE AT DAY

THREE.

AND YOU CAN'T DETECT GLYCOGEN IN

THE PLATFORM BUT THEY CAN INJECT

SMALL CHAINS OF MALTOSE WHICH WE

INTERPRET AS GLYCOGEN BREAK DOWN

WHEN IT'S NEARLY COMPLETELY

BROKEN DOWN.

AND EXPANDING THE GLYCOGEN MATT

WAY, WE -- PATHWAY, WE CAN SEE

DEXTROSE WHICH IS FOUR GLUCOSE

MODULES TOGETHER INCREASE 4.3

AND THEN BELOW THAT, THREE

TOGETHER IS 3.3 AND MALTOSE

WHICH IS JUST TWO DISACCHARIDE

INCREASED 3 BYT.3.

THIS IS A RELEASE OF THE GLUCOSE

ONE PHOSPHATE.

SO GLUCOSE SIX FAUST IF A IT TO

GLUCOSE ONE PHOSPHATE TO

GLYCOGEN, THAT IS ELEVATED QUITE

A BIT, JUST AT THE MUSCLE ON DAY

THREE.

THE BIG INCREASE IN FRUCTOSE

WHICH CAN BE CONVERTED TO

FRUCTOSE SIX PHOSPHATE AND A BIG

INCREASE IN IN AN HE'S PHOSPHATE

CAN BE CONVERTED TO FRUCTOSE.

THE OTHER GLUCOSE SIX PHOSPHATE

IS PEN TOE PHOSPHATE PATHWAY AND

THIS CAN PRODUCE A LOT OF --

THAT CAN BE USED FOR BY OH SENT

THITTIC PATHWAYS WITH FATTY

ACIDS CHOLESTEROL.

AND THERE'S AN INCREASE IN --

LIE BOAST WHICH IS NEEDED FOR

NUCLEOTIDE SYNTHESIS.

SO SOME OF THIS GLUCOSE

PHOSPHATE IS IN THAT DIRECTION.

HERE I'VE CIRCLED A ALL OF THE

METABOLITES THAT ARE AT LEAST

THREE FOLD INCREASE.

I REALLY WAS NOT EXPECTING TO

GET RESULTS THIS DRAMATIC BUT IT

DOES SEEM LIKE IT'S KIND OF

CLOTTING UP THERE GLUCOSE SIX

PHOSPHATE FRUCTOSE SIX

PHOSPHATE.

SO THEN IF WE LOOK AT DAY 21,

AND THESE ARE, THESE NUMBERS ARE

WRITTEN BELOW IN ITALICS.

SO AS YOU SEE MOST OF THESE

NUMBERS OF THOSE REALLY HIGH

VALUES ARE A LITTLE BIT LOWER

BUT THEY'RE STILL PRETTY HIGH.

AND THE ONLY WUBLES THAT ARE ONES THAT ARE

HIGHER THAN DAY THREE ARE THE

MOLECULES FURTHER DOWN IN

GLYCOLYSIS PATHWAY, PHOSPHOGLIS

RATES, TWO PHOSPHOGLYPHS RATES

SIGNIFICANTLY INCREASED AND --

SIGNIFICANTLY INCREASED.

NOW YOU SEE A LITTLE BIT OF

INCREASE IN LARK TASTE 10%.

THERE'S A LITTLE BIT MORE SIGH

CALL JUST.

YOU CAN'T EVEN SEE MORE OF THE

METABOLITES IN THE -- FOR SOME

REASON.

WE NOTICED AT THE VERY TOP, THE

SERUM GLUCOSE WHICH IS THE BLUE

BAR.

AND THAT WAS NOT SIGNIFICANTLY

DIFFERENT AT DAY THREE.

IT WAS ACTUALLY ELEVATED 1.3 AT

DAY 21.

AND THAT ONE WE'RE NOT

EXPECTING.

SO GLYCOGEN CAN BREAK DOWN AND

MUSCLE DOES NOT INCREASE GLUCOSE

IN CIRCULATION, IT CAN'T BE

RELEASED SO THAT'S NOT THE

SOURCE OF THAT.

THAT SEEMS TO BE THE OPPOSITE OF

WHAT WE WERE FINDING WITH THE

LIPO DISTROPHIC MICE WE WERE

DECREASING GLUCOSE.

SO ANOTHER THING THAT WE FOUND

MORE AMINO ACID PATHWAY, IN

GLUCOSE SIX PHOSPHATE IS

CONVERTED TO -- LACK TONE

INCREASE IN NADPH.

THAT ALSO, THE REVERSE REACTION

HELPS CATALYZE GLUCOSE OXIDIZED

FORM TO THE REDUCED FORM.

SO THERE'S AN INCREASE IN THE

OVERALL ROOTS VERSUS OXIDIZED

GLOOGLOOT THIGH OWN AND OXIDATIVE

TRESS AND SIMILARLY AT DAY 2 1.

AFTER THE INCREASE IN GLUCOSE IN

SERUM, IT ALSO FOUND A CHANGE IN

A MONOSACCHARIDE CALLED ONE

FIVE -- THAT'S ACTUALLY FOUND IN

FOOD AND IT DOESN'T GET BROKEN

DOWN BY THE BODY.

IT'S JUST SOMETHING THAT IT'S

TAKEN UP INTO THE BLOOD STREAM

WHEN YOUR FOOD IS DIGESTED.

AND THEN IT CAN BE TAKEN UP INTO

TISSUES, GET BACK INTO THE

BLOOD.

THE KIDNEY WILL REABSORB IT THE

SODIUM GLUCOSE TRANSPORTERS AND

SOME OF IT WILL BE EXSECRETED

AND SOME WILL GO BACK INTO THE

BLOOD.

IF YOU'RE HYPER GLYCEMIC,

GLUCOSE WILL COMPETE WITH ONE

FIVE AG FOR THOSE TRANSPORTERS

IN THE KIDNEY.

SO THEN YOU ACTUALLY GET LESS

FILTERED BACK INTO THE BLOOD AND

MORE WILL BE EXCRETED.

SO A DECREASE IN ONE FIVE AG

TENDS TO HAPPEN WHEN YOU HAVE

HYPERGLYCEMIA ABOVE 180 IN

HUMANS ANYWAY.

AND SO IT TAKES A WHILE TO BRING

THAT BACK DOWN SO IT'S A

MEASUREMENT OF HOW MUCH

VARIATION YOU MIGHT HAVE IN

GLUCOSE.

SAY YOU CAN HAVE NORMAL BLOOD

GLUCOSE HAPPENING IN THE DOCTOR

BUT IF YOU HAVE LOWER

[INDISCERNIBLE] MAY HAVE HAD

SOME TIRE GLYCEMIC EPISODES.

SO AS I MENTIONED THE GLUCOSE

ITSELF WAS HIGHER.

HOWEVER IN SERUM, THE ANTI---

WAS ALSO HIGHER WHICH SUGGESTS

THE GLUCOSE IS HIGHER, IT'S NOT

FLUCTUATING ABOVE 180, IT'S MORE

STABLE.

SO IF WE LOOK AT LIPIDS, NOT

MUCH OF A CHANGE IN THE MUSCLE

JUST SOME LONG CHAIN MOLECULES

WENT UP.

MIGHT NOT HAVE EXPECTED THAT.

AND IN SERUM HOWEVER, SOME LIPO

LIPIDS WERE CHANGE BEEN MIGHT BE

SUGGESTING SOME TISSUES WERE

REMODELING.

AT DAY 21, REALLY NOTHING

HAPPENED IN THE MUSTAL AS FAR AS

THE LIPIDS.

BUT IN SERUM, ALL OF THE

ESSENTIAL AMINO ACIDS WENT DOWN.

AND 11 OUT OF 19 OF THE LONG

CHAIN FATTY ACIDS WENT DOWN AND

WITH MORE THAN HALF.

AND THE SKI TOKES HYDROXY

BUTYRATE WENT DOWN SIGNIFICANTLY

AT DAY 21 AND SUGGESTS LESS BETA

OXIDATION IN THE ANIMAL.

AND AGAIN THERE ARE SOME LIPO

LIPIDS CHANGES.

SO TO SUMMARIZE THE SERUM IN

MUSCLE, YOU MIGHT EXPECT THERE

ARE SOME CHANGES IN AMINO ACIDS

WHICH MAKES SENSE IN TERMS OF

THE HYPERTROPHIC GROWTH IN

MUSCLE.

AND SOME CARBOHYDRATES WERE

CHANGED MORE THAN DRAMATICALLY

THAN BE THOUGHT AND DIFFERENTLY

THAN WE THOUGHT IN INCREASING

GLUCOSE BUT NOT -- INCREASE IN

GLYCO LYTIC MAYBE THE BREAK DOWN

IN THE MUSCLE ITSELF.

AND WITH THE LIPIDS, REDUCED

FATTY ACIDS IN SERUM AFTER THE

MUSCLE HAD HYPERTROPHY HAD

STABILIZED IN LOWER KETONES

SUGGESTING LESS LIPID OXIDATION.

SO ANOTHER POSSIBILITY IS BEING

ABSORBED TAKEN UP BY OTHER

TISSUE.

SO NEXT WE'LL LOOK AT LIVER, AND

IN THIS CASE, VERY LITTLE CHANGE

AT DAY THREE OR AT DAY 21 IN

TERMS OF THE AMINO ACIDS.

THE CARBOHYDRATES AT DAY THREE

NOT MUCH CHANGED EITHER.

INCREASE IN LACTATE, THERE WAS

SOME MORE GLYCOLYSIS SO WE CAN'T

SEE IT IN OTHER MOLECULES AND AN

INCREASE IN CITRATE.

IN DAY 21, IT'S KIND OF HARD TO

FIGURE OUT WHAT WENT OUT HERE ON

THE UPPER LEFT, IT DID LOOK LIKE

MORE UDP GLUE COAST AT DAY 21

WHICH MIGHT SUGGEST MORE

GLYCOGEN SYNTHESIS.

THE DECREASE OF SEVERAL OUT OF

OF THE OTHER SUGARS DECREASE OF

LACTATES WAS THE ONLY MOLECULE

THAT WENT IN DIFFERENT

DIRECTIONS IN THE TWO DAYS.

AND THE TPA CYCLE SEEMS TO BE

BLOCKED.

THERE'S LESS FLUX THROUGH THERE.

LOOK AT SOME OF THESE SAME

MOLECULES BUT IN DETERMINES OF

GLUE OGLUCOGENESIS IN DAY THREE, IT

CAN BE A PRECURSOR FOR

GLUCOGENESIS.

-- WENT DOWN LATER.

THERE'S INCREASE IN -- PHOSPHATE

ONLY AT DAY 21 BUT

SIMULTANEOUSLY A DECREASE IN

GLUCOSE.

SO SOME REGULATION AT THE POINT

OF FRUCTOSE PHOSPHATASE,

SOMETHING LIKE THAT.

IN GLOOT THIGH OWN HOWEVER THE

REDUCED FORM WENT DOWN AND THIS

MIGHT SUGGEST -- IF WE LOOK AT

THE LIPIDS WHICH IS PROBABLY

MOST INTERESTED IN THE LIVER,

SOME INCREASES AT DIE THREE.

ONE OF THE ESSENTIAL OF THE LONG

CHAIN FATTY ACIDS.

IN ONE OF THE KETONES WENT DOWN

TO .82.

AT 21 WE THOUGHT THE MA TAB

LITTLE GO DOWN AND MOST OF THE

ESSENTIAL LONG CHAIN FATTY ACIDS

WENT DOWN.

THIS MIGHT MAKE SENSE IN SOME OF

OUR PHENOTYPES IN GENERAL IN THE

MUTANTS WHERE WE SEE LESS TRY

GLIS RAIDS FOR SURE --

TRIGLYCERIDES IN THE LIVER

ALTHOUGH WE DIDN'T SEE IT IN THE

MICE EXTREME MODELS AND BOTH KEY

TONES WERE DOWN WITH LESS

OXIDATION IN THE LIVER AT DAY

21.

AND THE FAT.

SO NOTHING HAPPENS AT DAY THREE.

NO CHANGES IN AMINO ACIDS.

BUT AT DAY 21, MOST OF THEM WERE

INCREASED.

YOU DID NOT SEE THAT COMING AND

I'M REALLY NOT SURE WHY THEY

WOULD NECESSARILY BE INCREASED.

THAT THEY'RE BLOCKING SYNTHESIS

OF PROTEINS PERHAPS.

IF WE LOOK AT THE POLY AMEANS

AND THE YOU'RE AWE CYCLE THAT

DAY 21 IS THE TIME WE ACTUALLY

SAW A BIG CHANGE FOR ANY OF

THESE MOLECULES.

WE SEE AN INCREASE IN SMER SPERM

DEAN -- THERE'S AN INCREASE IN

PROLIEN AND THE SPAR TASTE.

THE BIGGEST AND MOST DRAMATIC

CHANGE HERE IS IN OWNER THEME

INCREASED 5.1.

THE UREA CYCLE GETS RID OF

NITROGEN AND SOME PROTEINS ARE

BROKEN DOWN.

SO I'M NOT SURE WHY THE OWNER

THEME WOULD BE UP 5.1.

SOMETHING TO DO WITH THE

INCREASE IN AMINO ACIDS AND KIND

OF STRESS.

OKAY.

IF WE LOOK AT THE GLYCO LYTIC

PATHWAY NOTHING CHANGED AT DAY 3

BUT AT DAY 21 THE GLUCOSE IS

ALSO UP IN FAT AS WAS MAN HOST

AND GLUCOSE SIX PHOSPHATE --

PHOSPHATE, SORRY.

AND THEN THE LIPIDS IN FACT

ALMOST NOTHING HAPPENED AT DAY

THREE AGAIN AND THEN AT DAY 21,

IN ADDITION TO INCREASE IN AMINO

ACIDS INCREASE IN THE FATTY

ACIDS, PARTICULARLY THE MEDIUM

CHAIN FATTY ACIDS AND AN

INCREASE IN LIP TOE LIPIDS.

MAYBE FAT IS THE TISSUE THAT'S

ACTUALLY HAVING SOME KIND OF

MEMBRANE MODELING OCCUR.

SO IF YOU TRY TO PUT IT

TOGETHER, WHAT WE SAW IN

CIRCULATION, OVERALL DECREASE IN

AMINO ACIDS WHICH MAKES SENSE IF

THEY GOT SHIFTED OVER INTO

GROWING CELLS IN THE MUSCLE.

AN INCREASE IN GLUCOSE AT DAY 21

BUT AN INCREASE IN -- WHICH

INDICATES MORE STABLE GLUCOSE

LEVELS.

AND A DECREASE IN KETONES

ESSENTIAL AND LONG CHAIN FATTY

ACIDS WHICH IS A DECREASE

OXIDATION IMPROVING THE FATTY

ACID PROFILES SHOULD BE HELPFUL

FOR THE INFLUENCE SENSITIVITY.

COULD THIS MEAN REDUCED

OXIDATION, REDUCE HIGH PAUL

SUSOR SOME EFFECT ON LIQUID

ABSORPTION IN MUSCLE DECREASE IN

SOME OF THE AMINO ACIDS, THE

AMINO ACID POOL IS STABLE.

AND A BIG INCREASE IN MANY OF

THE CARBOHYDRATES.

SO THE QUESTION IS GLYCOGEN

BROKEN DOWN IN MUSCLE.

THE LIVER IS A DECREASE IN

GLUCOSE UNLIKE EVERY OTHER

TISSUE OR SERUM AND DECREASE IN

LACTASE SO PROBABLY LESS

GLYCOGEN, SORRY, LESS GLYCOLYSIS

IN THE LIVER MIGHT BE SEEING AN

INCREASE IN GLYCOGEN SYNTHESIS

AND IN THAT TISSUE WE ALSO SEE

AN INCREASE IN -- STABLE GLUCOSE

LEVELS.

AND DECREASE IN KETONES

ESSENTIAL AND LAWMPING FATTY

ACIDS JUST LIKE IN CIRCULATION.

SO THEY ALSO WILL BE HAVING,

COULD THAT TISSUE BE HAVING BETA

OXIDATION.

YOU MIGHT EXPENSE AN APRIL

CORROSION ACTUALLY.

FINALLY ADIPOSE ON THE LOWER

RIGHT BASED ON INCREASE AMINO

ACIDS -- UREA CYCLE AN INCREASE

IN A FEW CARBOHYDRATES HAD AN

INCREASE IN FATAL AND FATTY

ACIDS -- SO THE ADIPOSE IN SOME

KIND OF CATABOLIC STAGE,

MEMBRANE REMODELING.

IF WE COULD KEEP THIS EXPERIMENT

GOING FOR MONTHS, WOULD WE SEE

LESS FAT.

LESS FAT SIGNS THAN IN WILD TYPE

ANIMALS.

SO THE FINAL QUESTIONS THAT

WE'RE TRYING TO FOLLOW UP, WHAT

ARE THE GLYCOGEN CONCENTRATIONS

IN MUSCLE AND LIVER.

SO MUSTAL MUSCLES THAT INCREASE FLUX OR

DECREASE IN GLYCOGEN.

AND TRIGLYCERIDE CONCENTRATIONS

IN THE LIVER.

SO THE QUESTION IS PHOSPHOLIGHT

ACTIVITY IN FAT INCREASEDDED IN

DAY 21 WITH MORE FATTY ACIDS YET

NOT INCREASED IN SERUM SO WHY

THAT.

THIS MYOSTATIN REGULATES SOME OF

THIS METABOLIC ENZYMES DIRECTLY

WHERE WE SEE A BIG DIFFERENCE IN

METABOLITES.

AND FINALLY CAN WE TAKE SOME OF

THESE CHANGES AND GO BACK TO OUR

DIABETIC MICE THAT ARE TREATED

WITH MYOSTATIN INHIBITORS AND

GET SOME INSIGHTS THERE AS TO

WHAT'S GOING ON IN THE ANIMAL

FAT IN DECREASED GLUCOSE.

SO I THINK I MENTIONED MOST OF

THE PEOPLE AS I WAS GOING ALONG,

I WANT TO THANK PEOPLE IN THE

LAB, JEN LANG, NICOLE --

JENNIFER -- WHO DID A LOT OF THE

LIPO DISTROPHIC WORK AND WE GET

A LOT OF WORK THROUGH THIS --

METABOLIC CORE, THE NIDDK AND

SOME REAGENTS AND MY FORMER

MENTOR -- I WANT TO THANK PEOPLE

AT MA TAB LAWN WHO HELPED US A

LOT IN ORDER TO DO THE LAST

PART.

THANK YOU.

I'LL TAKE ANY QUESTIONS.

[APPLAUSE]

>> [INDISCERNIBLE]

>> THE ONLY NATURAL ONES KNOWN

ARE THE FULL STATIN 3 --

PROTEINS THAT THE BODY PRODUCES

TO NORMALLY REGULATE IT.

AS FAR AS LIKE A SUPPLEMENT

SOMETHING LIKE THAT, THERE WAS

SOMETHING ON THE MARKET BRIEFLY,

SOME EXTRACT AND SOME SUPPLEMENT

CALLED MYO STAT OR SOMETHING

LIKE THAT CLAIMING IT WAS MYO

STATIN BUT IT WASN'T FOR REAL --

SO FAR NOTHING IS KNOWN.

>> [INDISCERNIBLE]

>> MYOSTATIN AND THE -- THEY'VE

BEEN SHOWN TO COMPETE FOR

BINDING.

THAT IS A REALLY COMPLICATED

QUESTION AND I DON'T KNOW

ANYBODY WOULD GET INCREASED EMP

SIGNALING IN SOME OF THESE

TISSUES.

POSSIBLY.

I DON'T KNOW THAT IT WOULD

NECESSARILY TAKE IT ON --

ALREADY THERE.

MAYBE WHAT WE'RE SEEING IN THE

MUTANTS FOR INSTANCE IS THAT

WHEN NEW ENERGY IS TAKEN IN,

IT'S GOING INTO MAINTAINING THAT

LARGER MUSCLE MASS AS THERE'S

LESS TO, THE MUSCLE TAKES YOU

MORE GLUCOSE AS THERE'S LESS

GLUCOSE TAKEN OVER BY FAT SO

THERE'S LESS ENERGY STORED AS

FAT.

>> [INDISCERNIBLE]

>> RIGHT.

SO IF YOU INCREASE MUSCLE MASS

AFTER THE FACT, AT LEAST BY THIS

METHOD YOU DON'T SEE AS BIG A

DIFFERENCE, YOU DON'T SEE MUCH

OF A DIFFERENCE AT ALL IN THE

OVERALL FAT MASS PADS YOU DO IF

YOU'VE GOT IT IN A TRANSGENIC

MOUSE.

ACTUALLY IN THE MUTANTS IF WE

LOOK REALLY EARLY WHEN WILD TYPE

HAS VERY LITTLE FAT, THERE'S NUT

MUCH OF A DIFFERENCE AT ALL.

SO WHAT REALLY HAPPENS IS THE

WILD TYPE IS GAINING FAT AS THEY

AGE WHEREAS THE MUTANTS ARE NOT

GAINING FAT AS THEY AGE.

>> [INDISCERNIBLE]

>> THE MUTANTS SOMEHOW REALLY

BALANCE THEIR ENERGY EXPENDITURE

AND THEIR ENERGY INTAKE

PERFECTLY SO THEY'RE NOT GAINING

WEIGHT.

WILD TYPES HAVE A SLIGHTLY

POSITIVE ENERGY BALANCE WHICH

OVER TIME MAKES THEM FARTHE FATTER AND

FATTER.

>> [INDISCERNIBLE]

SOMEONE ELS>> THERE'S A SIMILAR IDEA AND

DID MICRO RAY ON THE MUSCLE AT

TWO DAYS AND TWO WEEKS INSTEAD

OF THREE DAYS AND THREE WEEKS

LIKE WE DID.

SO IT WILL BE GOOD TO GET AT

LEAST THOSE TWO TOGETHER AND SEE

WHAT'S GOING ON.

NOBODY TO MY KNOWLEDGE HAS DONE

LIVER AND FAT.

>> [INDISCERNIBLE]

>> WE DO HAVE SOME MICRO RAY

FROM THE DOMINANT NEGATIVE

TRANSGENICS.

SO WE HAVE LOOKED AT SOME OF

THEM AND FOR INSTANCE OWNER

THEME DECAR BOX LACE IN MUSCLE

WENT UP LIKE THREE FOLD AND THAT

CONVERTS OWNER THEME --

ORNITHINE ALSO INCREASES FAT.

>> [INDISCERNIBLE]

>> NOBODY LOOKED AT ANY

PREVENTION TYPE.

>> [INDISCERNIBLE]

>> DID THEY PROVE WHAT?

>> [INDISCERNIBLE]

>> THEY DEFINITELY REPRODUCED.

I WOULD SAY THAT THE KNOCKOUT

FEMALES DON'T REPRODUCE ALL THAT

WELL.

THE MALES ARE BETTER.

THE DOMINANT NEGATIVE

TRANSGENICS STILL REPRODUCE

REASONABLY WELL.

ONE THING THAT WE CAN'T REALLY

EXPLAIN IS THE KNOCKOUT

HETEROZYGOUS WE DON'T GEL A FULL

25% WE GET 18% HOMOZYGOUS.

IT SEEMS TO BE RIGHT AROUND

BIRTH.

THERE'S A GENETIC BACKGROUND

WHEN WE CROSS IT INTO THE DBA

BACKGROUND WE CAN'T GET ANY

KNOCKOUT PAST TWO DAYS.

THEY DEFINITELY CAN MOVE SO THE

MUSCLE'S FUNCTIONAL SO MAYBE

THERE IS SOME METABOLIC PROBLEM

THAT'S MORE EXTREME THAN THE DBA

IN SOME OF THE OTHER GENETIC

BACKGROUNDS.

THERE ARE A FEW BUT NOT ALL OF

THEM.

>> [INDISCERNIBLE]

>> THAT WAS LIVER.

>> [INDISCERNIBLE]

>> DECREASED OXIDATION.

AT BOTH 21 AND A LITTLE BIT AT

DAY 3.

>> [INDISCERNIBLE]

>> I GUESS THEY DIDN'T HAVE ANY

QUESTIONS ON-LINE.

THANK YOU VERY MUCH FOR YOUR