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Towards A Systems Biology Understanding of Metabolic Disorder

Cosmo

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Sodahead.com

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seqanswers.com

Illumina

Life Tech: Ion Torrent

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Transcriptomics – Expression Microarrays

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Proteins !

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Cell Signaling

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Genome

Proteome

Transcriptome

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A single cell ….

The Whole Body

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Insulin Resistance (Periphery)

Impaired Insulin Secretion

X

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What We Do

Before Exercise

After Exercise

Exercise

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Gene Expression Microarrays (Transcriptome)

Mass Spectrometry (Proteome)

Genome

Sequencing

X

Homogenization

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What can we learn from the genome?

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Genome Wide Association Study (GWAS)

Empire State Building

Of Type 2 Diabetes

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Blah

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Blah

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Transcriptomics – Expression Microarrays

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Metabolic Phenotypes

Subject
Sex	F	M	M	M	F	M	M	M	F	F	F	M	M	M
Age (yrs)	26	48	34	23	43	26	32	26	33	42	30	34	36	27
max HR pred	194	172	186	197	177	194	188	194	187	178	190	186	184	193
Height(in)	64.25	69.5	69.5	68.5	60.3	73	72	69.9	63.2	59.4	64.7	69.5	68.9	71.6
Weight (lbs)	129.5	205.5	201	177	133	201	189	177.6	142	147.8	152	205	231	195.5
Wt (kg)	58.86	93.41	91.36	80.45	60.45	91.36	85.91	80.73	64.55	67.04	69.1	92.99	104.9	88.8
BMI	22.1	29.9	29.3	26.5	25.7	26.5	25.6	23.5	25	30	25.5	29.8	34.2	26.8
M (mg/kg/min)	9.7	4.7	6.2	7.5	9	11.1	9.6	4.5	6.3	4.4	6.6	6.6	3.7	6.9
M (mg/min)	571	439	566.5	603.4	544.1	1014	824.7	363.3	406.6	295	456.1	613.7	388.1	612.7
M (mg/kg/ffm)	12.7	6.128	7.798	10.33	13.15	13.26	13.01	6.944	9.578	6.316	8.995	8.488	5.182	8.749
Body Fat % BAI	23.6	23.3	20.5	27.4	28.6	15	26.2	29.4	34.2	30.5	26.1	22.2	28.5	21
Fat mass (lbs)	30.6	47.9	41.2	48.5	36.5	29.7	49.5	47.9	48.6	45.2	39.5	45.5	65.8	41.1
Fat mass (kg)	13.91	21.77	18.73	22.05	16.59	13.5	22.5	21.77	22.09	20.5	17.9	20.6	29.8	18.6
Lean Body Mass (lbs)	98.9	157.6	159.8	128.5	91	168.3	139.5	115.1	93.4	102.9	111.8	159.5	165.2	154.4
Lean Body Mass (kg)	44.95	71.64	72.64	58.41	41.36	76.5	63.41	52.32	42.45	46.7	50.7	72.3	74.9	70.03
BMR	1365	2175	2205	1773	1256	2323	1925	1588	1289	1420	1540	2201	2280	2131
VO2 (l/m)	1.81	2.8	3.27	2.5	1.54	4.46	2.86	2.79	1.82	1.49	1.72	4.05	3.48	2.86
VO2max (ml/kg/min)	30.8	29.9	35.2	31.6	25.5	48.9	31.9	37.7	28.1	22.3	25.1	42	33.1	31.9
Heart Rate Max	191	174	185	197	183	199	173	186	190	172	179	189	183	173
Watt max (W)	165	200	200	175	125	260	200	175	125	100	125	230	175	200
Predicted70% max HR	133.7	121.8	129.5	137.9	128.1	139.3	121.1	130.2	133	120.4	125.3	132.3	128.1	121.1
Predicted90% max HR	171.9	156.6	166.5	177.3	164.7	179.1	155.7	167.4	171	154.8	161.1	170.1	164.7	155.7
Predicted70% max Watts	115.5	140	140	122.5	87.5	182	140	122.5	87.5	70	87.5	161	122.5	140
90% max watts	148.5	180	180	157.5	112.5	234	180	157.5	112.5	90	112.5	207	157.5	180
VO2 ml/kg/min	30.8	29.9	35.2	31.6	25.5	48.9	31.9	37.7	28.1	22.3	25.1	42	33.1	31.9
VO2 (ml kg FFM)	40.26	39.09	45.02	42.8	37.23	58.3	45.1	53.33	42.87	31.86	33.85	55.86	46.34	40.75
mean 70% max HR	136.5	123.4	144.9	158.6	134	151.7	131.9	136.3	144.4	127.4	138.5	136.3	129.9	129.7
mean 90% max HR	176.3	150.3	163.8	178.5	159.8	169.3	154.3	159.5	173.8	146	165.5	159.5	144.3	155.3
Chol	202	201	165	183	145	154	145	165	183	190	157	169	213	206
TG	56	227	114	78	55	44	94	119	98	93	152	57	204	258
HDL	84	46	52	64	56	47	42	40	60	51	40	48	33	41
LDL	109	117	94	106	80	100	87	105	107	124	92	112	146	113
VLDL	9	38	19	13	9	7	16	20	16	16	25	10	34	43
HA1C	5.1	5	5.3	5.1	5.7	5.5	5.8	5.5	5.7	5.4	4.9	5.5	5.8	5.7
Resting Heart Rate	69	66	58	76	64	67	58	66	76	61	62	64	66	71
SBP	113	143	122	130	108	131	94	112	113	104	106	141	126	144
DBP	69	94	74	83	64	69	69	72	78	61	62	86	66	81
FPG mg/dl	81.4	88.1	90	88.8	93.7	89.4	88.6	86.2	97.2	90.8	88.3	85.9	90.6	94.1
FPG mM	4.522	4.894	5	4.933	5.206	4.967	4.922	4.789	5.4	5.044	4.906	4.772	5.033	5.228
FPI (uU/ml)	2.9	10.67	6.35	5.15	6.7	4.52	3.74	2.79	3.66	7.75	16.67	8.41	13.53	1.89
FPI (pmol)	17.4	64.02	38.1	30.9	40.2	27.12	22.44	16.74	21.96	46.5	100	50.46	81.18	11.34
clamp ins uU	122	175	118	151	239	117	176	153	145	205	178	145	165	114
clamp pM	732	1050	708	906	1434	702	1056	918	870	1230	1068	870	990	684

Glucose

Uptake

Rate

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Distribution of Correlation Coefficients

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Correlations With Glucose Uptake

Correlation	ID	Description
0.86	GOPC	Homo sapiens golgi associated PDZ and coiled-coil motif containing (GOPC)
0.83	ZBED3	Homo sapiens zinc finger
0.83	MEA1	Homo sapiens male-enhanced antigen 1 (MEA1)
0.83	BC014384	Homo sapiens
0.83	GLUD1	Homo sapiens glutamate dehydrogenase 1 (GLUD1)
0.82	PDE4A	Homo sapiens phosphodiesterase 4A
0.81	EIF4ENIF1	Homo sapiens eukaryotic translation initiation factor 4E nuclear import factor 1 (EIF4ENIF1)
0.8	PPP2R3A	Homo sapiens protein phosphatase 2 (formerly 2A)
0.79	LOC401708	PREDICTED: Homo sapiens similar to Glutamine synthetase (Glutamate--ammonia ligase) (GS) (LOC401708)
0.79	PPP2CB	Homo sapiens protein phosphatase 2 (formerly 2A)
.
.
.
-0.78	ADRM1	Homo sapiens adhesion regulating molecule 1 (ADRM1)
-0.78	NFKB1	Homo sapiens nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) (NFKB1)
-0.78	UBE2E3	Homo sapiens ubiquitin-conjugating enzyme E2E 3 (UBC4/5 homolog
-0.78	PDXDC2	Homo sapiens mRNA; cDNA DKFZp686B0962 (from clone DKFZp686B0962). [BX647358]
-0.78	RPLP0	Homo sapiens ribosomal protein
-0.79	DYSF	Homo sapiens dysferlin
-0.8	EIF3S5	Homo sapiens eukaryotic translation initiation factor 3
-0.8	EEF1B2	Homo sapiens eukaryotic translation elongation factor 1 beta 2 (EEF1B2)
-0.8	RPS3A	Homo sapiens ribosomal protein S3A (RPS3A)
-0.8	RPLP0	Homo sapiens ribosomal protein
-0.81	A_24_P50489	Unknown
-0.83	FBXO32	Homo sapiens F-box protein 32 (FBXO32)
-0.85	LOC440350	Homo sapiens similar to nuclear pore complex interacting protein (LOC440350)
-0.85	NFKB1	Homo sapiens nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105) (NFKB1)
-0.87	FBXO9	Homo sapiens F-box protein 9 (FBXO9)

~40,000 OTHER Probes

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ZBED3

ZBED3 is an INTERNAL

Receptor for WNT Signaling!

Axin

ZBED3

Axin

ZBED3

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ZBED3

ZBED3 Overexpression ENHANCES

WNT Signaling

ZBED3 KnockDown BLUNTS

WNT Signaling

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ZBED3 Expression Correlates With Glucose Uptake !!!

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PP2A = Protein Phosphatase 2

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Top Correlate : GOPC

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GOPC

Frizzled

Golgi

8 Hours

20 Hours

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What About the Negative Correlates ?

WNT Signaling

Glucose Transportor trafficking

Muscle Atrophy / Wasting

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ADRM1 = RPN13 -> Activates Proteosome!

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Does this correlation make sense?

When starving, peripheral tissues should :

Stop stealing glucose from the brain

Send amino acids to the liver for glucose production

How can atrophy directly disable glucose uptake ?

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Can Atrogin switch off glucose uptake ?

GOPC

Frizzled

Golgi

Cell Membrane

Frizzled

S/T – X - V

Atrogin

BK Channels

S/T – X - V

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Hypothesis : �GOPC is on-switch / Atrogin is off-switch

GOPC

Frizzled

Golgi

Cell Membrane

Frizzled

S/T – X - V

Atrogin

S/T – X - V

X

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What about FBXO9 ?

FBXO9 turns on adipogenesis !

Kills B-catenin

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What about FBXO9 ?

FBXO9

?

Wnt signaling Anti - adipogenesis

FBXO9 Pro - adipogenesis !

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DYSF = Dysferlin : Muscular Dystrophy

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What About the Negative Correlates ?

WNT Signaling

Glucose Transportor trafficking

Muscle Atrophy / Wasting

Causes :

Disuse ?

Starvation / Fasting ?

Inflammation ?

Inhibition ?

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Correlations with Exercise Fold Change

Correlation	ID	Description
0.86	KCNMB2	potassium large conductance calcium-activated channel
0.86	ACP5	acid phosphatase 5
0.85	LIN28B	lin-28 homolog B (C. elegans) (LIN28B)
0.84	A_24_P941450	Unknown
0.84	HAO2	hydroxyacid oxidase 2 (long chain) (HAO2)
0.83	AL157439	mRNA; cDNA DKFZp434A225 (from clone DKFZp434A225). [AL157439]
0.81	PRKAG2	cDNA FLJ90194 fis
0.81	GPX4	glutathione peroxidase 4 (phospholipid hydroperoxidase) (GPX4)
0.80	AF349445	maxi-K channel HSLO mRNA
0.80	TJAP1	tight junction associated protein 1 (peripheral) (TJAP1)
0.79	TRIM43	tripartite motif-containing 43 (TRIM43)
0.79	AF116673	PRO1925 mRNA
0.79	DGKZ	diacylglycerol kinase
0.79	KIAA1772	KIAA1772 (KIAA1772)
0.79	SART3	squamous cell carcinoma antigen recognized by T cells 3 (SART3)
0.78	CBFA2T2	core-binding factor
0.78	A_23_P123234	Unknown
0.78	PFKFB3	6-phosphofructo-2-kinase/fructose-2
0.78	PFKFB3	6-phosphofructo-2-kinase/fructose-2

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Figure 1. A transgenic TRAP over expressing mouse subline displays hyperplastic obesity.

Lång P, van Harmelen V, Rydén M, Kaaman M, et al. (2008) Monomeric Tartrate Resistant Acid Phosphatase Induces Insulin Sensitive Obesity. PLoS ONE 3(3): e1713. doi:10.1371/journal.pone.0001713

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001713

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NFKB1 -> Atrophy pathway !

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Macrophage

avb3

OPN

M1 Polarization

Inflammatory

Cytokine Secretion

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Glucose Disposal

Rate !

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Figure 1. A transgenic TRAP over expressing mouse subline displays hyperplastic obesity.

OPN

ACP5/TRAP

TRAP dephosphorylates

Osteopontin

Prevents binding with avb-3 integrin receptors

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Macrophage

avb3

OPN

X

Inflammatory

Cytokines

X

OPN

ACP5/TRAP

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The Big Picture

FBXO9

FBXO32/Atrogin

(Maybe)

X

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What about correlation with other measurements ?

Subject
Sex	F	M	M	M	F	M	M	M	F	F	F	M	M	M
Age (yrs)	26	48	34	23	43	26	32	26	33	42	30	34	36	27
max HR pred	194	172	186	197	177	194	188	194	187	178	190	186	184	193
Height(in)	64.25	69.5	69.5	68.5	60.3	73	72	69.9	63.2	59.4	64.7	69.5	68.9	71.6
Weight (lbs)	129.5	205.5	201	177	133	201	189	177.6	142	147.8	152	205	231	195.5
Wt (kg)	58.86	93.41	91.36	80.45	60.45	91.36	85.91	80.73	64.55	67.04	69.1	92.99	104.9	88.8
BMI	22.1	29.9	29.3	26.5	25.7	26.5	25.6	23.5	25	30	25.5	29.8	34.2	26.8
M (mg/kg/min)	9.7	4.7	6.2	7.5	9	11.1	9.6	4.5	6.3	4.4	6.6	6.6	3.7	6.9
M (mg/min)	571	439	566.5	603.4	544.1	1014	824.7	363.3	406.6	295	456.1	613.7	388.1	612.7
M (mg/kg/ffm)	12.7	6.128	7.798	10.33	13.15	13.26	13.01	6.944	9.578	6.316	8.995	8.488	5.182	8.749
Body Fat % BAI	23.6	23.3	20.5	27.4	28.6	15	26.2	29.4	34.2	30.5	26.1	22.2	28.5	21
Fat mass (lbs)	30.6	47.9	41.2	48.5	36.5	29.7	49.5	47.9	48.6	45.2	39.5	45.5	65.8	41.1
Fat mass (kg)	13.91	21.77	18.73	22.05	16.59	13.5	22.5	21.77	22.09	20.5	17.9	20.6	29.8	18.6
Lean Body Mass (lbs)	98.9	157.6	159.8	128.5	91	168.3	139.5	115.1	93.4	102.9	111.8	159.5	165.2	154.4
Lean Body Mass (kg)	44.95	71.64	72.64	58.41	41.36	76.5	63.41	52.32	42.45	46.7	50.7	72.3	74.9	70.03
BMR	1365	2175	2205	1773	1256	2323	1925	1588	1289	1420	1540	2201	2280	2131
VO2 (l/m)	1.81	2.8	3.27	2.5	1.54	4.46	2.86	2.79	1.82	1.49	1.72	4.05	3.48	2.86
VO2max (ml/kg/min)	30.8	29.9	35.2	31.6	25.5	48.9	31.9	37.7	28.1	22.3	25.1	42	33.1	31.9
Heart Rate Max	191	174	185	197	183	199	173	186	190	172	179	189	183	173
Watt max (W)	165	200	200	175	125	260	200	175	125	100	125	230	175	200
Predicted70% max HR	133.7	121.8	129.5	137.9	128.1	139.3	121.1	130.2	133	120.4	125.3	132.3	128.1	121.1
Predicted90% max HR	171.9	156.6	166.5	177.3	164.7	179.1	155.7	167.4	171	154.8	161.1	170.1	164.7	155.7
Predicted70% max Watts	115.5	140	140	122.5	87.5	182	140	122.5	87.5	70	87.5	161	122.5	140
90% max watts	148.5	180	180	157.5	112.5	234	180	157.5	112.5	90	112.5	207	157.5	180
VO2 ml/kg/min	30.8	29.9	35.2	31.6	25.5	48.9	31.9	37.7	28.1	22.3	25.1	42	33.1	31.9
VO2 (ml kg FFM)	40.26	39.09	45.02	42.8	37.23	58.3	45.1	53.33	42.87	31.86	33.85	55.86	46.34	40.75
mean 70% max HR	136.5	123.4	144.9	158.6	134	151.7	131.9	136.3	144.4	127.4	138.5	136.3	129.9	129.7
mean 90% max HR	176.3	150.3	163.8	178.5	159.8	169.3	154.3	159.5	173.8	146	165.5	159.5	144.3	155.3
Chol	202	201	165	183	145	154	145	165	183	190	157	169	213	206
TG	56	227	114	78	55	44	94	119	98	93	152	57	204	258
HDL	84	46	52	64	56	47	42	40	60	51	40	48	33	41
LDL	109	117	94	106	80	100	87	105	107	124	92	112	146	113
VLDL	9	38	19	13	9	7	16	20	16	16	25	10	34	43
HA1C	5.1	5	5.3	5.1	5.7	5.5	5.8	5.5	5.7	5.4	4.9	5.5	5.8	5.7
Resting Heart Rate	69	66	58	76	64	67	58	66	76	61	62	64	66	71
SBP	113	143	122	130	108	131	94	112	113	104	106	141	126	144
DBP	69	94	74	83	64	69	69	72	78	61	62	86	66	81
FPG mg/dl	81.4	88.1	90	88.8	93.7	89.4	88.6	86.2	97.2	90.8	88.3	85.9	90.6	94.1
FPG mM	4.522	4.894	5	4.933	5.206	4.967	4.922	4.789	5.4	5.044	4.906	4.772	5.033	5.228
FPI (uU/ml)	2.9	10.67	6.35	5.15	6.7	4.52	3.74	2.79	3.66	7.75	16.67	8.41	13.53	1.89
FPI (pmol)	17.4	64.02	38.1	30.9	40.2	27.12	22.44	16.74	21.96	46.5	100	50.46	81.18	11.34
clamp ins uU	122	175	118	151	239	117	176	153	145	205	178	145	165	114
clamp pM	732	1050	708	906	1434	702	1056	918	870	1230	1068	870	990	684

Body Mass

Index

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That’s 2 GWAS

Connections !

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�Exercise:� Lowers LRP1B in thin people� Raises LRP1B in fat people ……

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Transcriptomics – Expression Microarrays

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Cell Signaling

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Proteomics – Mass Spectrometry

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Molecular Modeling Reveals Acetylation of Adenine Nucleotide Translocase (ANT)1 Regulates Affinity for ADP

Clinton Mielke, Banu Ozkan, Andrew Bordner, Jerez Te, Wayne T. Willis, Lawrence J. Mandarino

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Acetylation at K23 Correlates With Insulin Resistance

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m-state

c-state

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ANT1 is acetylated at lysines 10, 23, and 92 in vivo in human muscle.

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Making the Acetyl-Lysine

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Electrostatics

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ANT1 Gating Mechanism

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Simulation Movie

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Simulation with ADP

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Docking Experiments

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Yeast Mutagenesis Study

Site-directed mutagenesis of the yeast mitochondrial ADP/ATP translocator. Six arginines and one lysine are essential.

Nelson DR, Lawson JE, Klingenberg M, Douglas MG.

J Mol Biol. 1993 Apr 20;230(4):1159-70.

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Another Unfinished Story

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IRS1 Phosphorylation in Insulin Resistance

IRS1 : DGYMPMSPGVAPVPSGRKGSGDYMPMSPKSV

Y612 + S616

Y632 + S636

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COSMIC Database

ALL Mutations in COSMIC

7^th!

H1047R

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The last mutation

Mutation H1047R

Positive charge

Switches on PI3K → Cancer

Modification S612

Negative charge
SUPPOSEDLY switches off PI3K ….

Causes insulin resistance

Opposing actions !

Molecular Cell : 2011

IRS1 : DGYMPMSPGVAPVPSGRKGSGDYMPMSPKSV

Mutation 1047!

*

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Mutations (Genome) → “Source Code”

Modifications (Proteome) → “RAM”

Both can switch on/off proteins

How can we find all of these

molecular switches?

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AMASS (amass-db.org)

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~330 lbs

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If You’re Not Excited, You Should Be!