bmi (kg/m qualifying hba1c (%) dpp4 activity (mu/ml)10.1038... · supplementary table 3....

Supplementary Table 1: Baseline participant characteristics according to randomized treatment and baseline metformin

Sitagliptin and no metformin (N=146)

Sitagliptin and metformin

(N=149)

Placebo and no metformin

(N=149)

Placebo and metformin

(N=148)

Age at Randomization (yrs) 68 (60, 72) 67 (61, 73) 68 (62, 73) 70 (62, 74) Female sex 39 (26.7%) 43 (28.9%) 42 (28.2%) 53 (35.8%) BMI (kg/m2)

≤30 72 (49.3%) 74 (49.7%) 74 (49.7%) 73 (49.3%) >30 74 (50.7%) 75 (50.3%) 75 (50.3%) 75 (50.7%)

Qualifying HbA1c (%) 7.3 (7.0, 7.7) 7.2 (6.9, 7.7) 7.3 (6.8, 7.7) 7.4 (6.9, 7.7) Duration of type 2 diabetes (yrs) 12 (7, 21) 13 (6, 20) 13 (8, 22) 12 (6, 20) DPP4 activity (mU/mL) 3.7 (3.2, 4.3) 3.4 (3.0, 3.9) 3.8 (3.3, 4.3) 3.6 (3.1, 4.1) sDPP4 protein (ng/mL) 569 (479, 700) 525 (445, 631) 582 (514, 697) 556 (450, 693) CRP (µg/mL) 2.5 (1.1, 5.5) 1.8 (0.8, 5.0) 2.9 (1.1, 7.1) 2.4 (0.9, 4.0) IL-6 (pg/mL) 1.2 (0.8, 1.9) 0.9 (0.6, 1.3) 1.3 (0.8, 2.0) 1.0 (0.8, 1.5) TNFa (pg/mL) 2.7 (2.2, 3.3) 2.7 (2.1, 3.2) 2.9 (2.1, 3.5) 2.7 (2.2, 3.3) MCP-1 (pg/mL) 171 (135, 208) 166 (144, 197) 182 (149, 213) 161 (137, 189) Data are summarized as number (percentage) for categorical variables and median (25th, 75th percentile) for continuous variables. BMI, body mass index.

Supplementary Table 2: Association of baseline metformin and baseline biomarkers adjusted for clinical covariates

Baseline log base-2 transformed biomarker N

Least squares means for no

baseline metformin

Least squares means for baseline

metformin use

Difference in least squares

means F p

sDPP4 592 9.20 (9.15, 9.25) 9.11 (9.06, 9.16) -0.09 (-0.16, -0.02) 7.27 0.007

DPP4 activity 592 1.88 (1.84, 1.93) 1.80 (1.75, 1.84) -0.09 (-0.15, -0.02) 6.73 0.010

CRP 592 1.51 (1.32, 1.71) 1.12 (0.92, 1.32) -0.39 (-0.67, -0.11) 7.59 0.006

IL-6 592 0.35 (0.23, 0.47) 0.05 (-0.07, 0.17) -0.30 (-0.48, -0.12) 11.28 <0.001

TNFa 592 1.49 (1.43, 1.55) 1.37 (1.31, 1.43) -0.12 (-0.20, -0.03) 7.02 0.008

MCP-1 592 7.46 (7.41, 7.52) 7.37 (7.32, 7.43) -0.09 (-0.17, -0.01) 5.31 0.022

P values are two-sided and derived from multiple linear regression models. No adjustments were made for multiple comparisons.

Supplementary Table 3. Correlation of sDPP4 protein with other markers at 12 months

Month 12 biomarker N

Spearman correlation

p-value

DPP-4 activity (mU/mL) 592 0.38 <0.001

CRP (µg/mL) 592 -0.06 0.158

IL-6 (pg/mL) 592 -0.11 0.005

TNFa (pg/mL) 592 -0.05 0.261

MCP-1 (pg/mL) 592 -0.03 0.430

P values are two-sided and derived from Spearman correlation. No adjustments were made for multiple comparisons.

Supplementary Table 4: qPCR primers for Taqman gene epression

Gene Description Assay ID

Adgre1 Adhesion G protein-coupled receptor E1 Mm00802529_m1

Alpi Alkaline phosphatase, intestinal Mm01285814_g1

Cav 1 Caveolin 1 Mm00483057_m1

Ccl2 C-C motif chemokine ligand 2 Mm00441242_m1

CD36 Cluster of differentiation 36 Mm00432403_m1

Col1a1 Collagen, type 1, alpha 1 Mm00801666_g1

Cyp7a1 Cytochrome P450 Family 7 Subfamily A Member 1 Mm00484152_m1

Egfr Epidermal growth factor receptor Mm00433023_m1

Fgfr4 Fibroblast growth factor receptor 4 Mm01341852_m1

Gip Glucose-dependent insulinotropic polypeptide Mm00433601_m1

Havcr1 Hepatitis A Virus Cellular Receptor 1 Mm00506686_m1

Icam1 Intercellular Adhesion Molecule 1 Mm00516023_m1

Ifng Interferon gamma Mm01168134_m1

Il10 Interleukin 10 Mm99999062_m1

Il12b Interleukin 12b Mm99999067_m1


Il1b Interleukin 1b Mm01336189_m1




Lcn2 Lipocalin 2 Mm01324470_m1

Lipc Lipase C, hepatic type Mm01171487_m1

Ocln Occludin Mm00500912_m1

Ppia Peptidylprolyl isomerase A Mm02342430_g1

Reg3a Regenerating family member 3 alpha Mm00441127_m1

Reg3b Regenerating family member 3 beta Mm00440616_g1

Reg3g Regenerating family member 3 gamma Mm00441127_m1

Rpl32 Ribosomal protein L32 Mm02528467_g1

Tbp Tata box binding protein Mm00446973_m1

Tff2 Trefoil factor 2 Mm00447491_m1

Tff3 Trefoil factor 3 Mm00495590_m1

Tgfbb1 Transforming growth factor beta 1 Mm01178820_m1

Tjp1 (ZO-1) Tight junction protein 1 Mm00493699_m1

Tlr4 Toll-like receptor 4 Mm00445273_m1

Tnfa Tumour necrosis factor alpha Mm00443258_m1

Tslp Thymic stromal lymphopoietin Mm00498739_m1

All primers are from Thermo-Fisher Scientific.

8 wk-old C57BL/6 male mice:

0 8 18 20 weeks10% Fat

10% Fat

60% Fat

60% Fat

10% Fat

10% Fat

HFFC

HFFC

10% Fat

10% Fat + DPP4i

HFFC

HFFC + DPP4i

HFFC Diet = 40% kcal fat (mostly Primex), 20% kcal fructose, and 2% cholesterol

DPP4i = DPP4 inhibitor (15.5 mg of MK-0626 per 4057 kcal diet)

3210% Fat

HFFC

HFFC + DPP4i

Sac (1) Sac (2)

10% Fat + DPP4i

0 1 2 3 4 5 6 720

25

30

35

40

45

10% Fat Diet60% Fat Diet

Bo

dy

Wei

gt

(g)

8 9 10 11 12 13 14 15 16

Control DietHFFC Diet

ControlControl + DPP4i

HFFC Diet

HFFC + DPP4i

Control HFFC25

30

35

40

45

50

55Body Weight (g)

14 wks of DPP4i

- DPP4i

+ DPP4i

A

B

D

18 19 20

- DPP4i + DPP4i + DPP4i

CD HFFC

0

10

20

30

40

50

60

Act

ive

GIP

(p

M) ***

***

CD HFFC

0

1

2

3

4

5

6

Act

ive

GL

P-1

(p

M)

**** ****

Week Week Week

Supplementary Figure 1

C

14 wks of DPP4i

CD HFFC4

5

6

7

8

9

10

Blood Glucose

mM

##

- DPP4i + DPP4i

n=8

n=8

n=8

Supplementary Figure 1. Long‐term inhibition of DPP4 increases plasma levels of active GIP and GLP‐1but does not modify HFFC diet‐induced weight gain in WT mice. (A) Schematic depicting the diet regimenand time of sacrifice (Sac) in the WT mouse studies. (B) Five hour fasted plasma levels of active gastric inhibitory polypeptide (GIP) and glucagon‐like peptide ‐1 (GLP‐1) in WT mice that were maintained on controldiet (CD) or high fat, fructose and cholesterol (HFFC) diet supplemented with (+) or without (‐) the DPP4inhibitor (DPP4i) MK‐0626 for 14 wks. (C) Non‐fasting blood glucose levels at the time of sacrifice in WT micethat were maintained on CD or HFFC diet supplemented with or without DPP4i. (D) Weekly body weightsand body weights at the time of sacrifice in WT mice that were maintained on CD or HFFC diet supplementedwith or without DPP4i. Data shown are mean SEM. n=9 or 10 mice per group, except where n=8 asindicated on the graph above the data set.***P<0.001,****P<0.0001 vs. DPP4i‐treated. ##P<0.01 for CD vs.HFFC. Data in (B‐D) were analyzed by two‐way ANOVA. Source data are provided as a Source data file.

0

1

2

3

4

DP

P4

acti

vity

(m

U/m

g) ****

CD HFFC0

1

2

3

4

DP

P4

ac

tiv

ity

(m

U/m

g) ****

****

CD HFFC0

1

2

3

DP

P4

ac

tiv

ity

(m

U/m

g)

****

****

####

CD HFFC0

1

2

DP

P4

ac

tiv

ity

(m

U/m

g) **** ****

##

0

50

100

150

200

250

DP

P4

pro

tein

(ng

/mg

)

**

CD HFFC0

50

100

150

DP

P4

pro

tein

(ng

/mg

)

**##

CD HFFC0

50

100

150

DP

P4

pro

tein

(ng

/mg

) *******

###

CD HFFC0

50

100

150

DP

P4

pro

tein

(ng

/mg

)####

*

CD HFFC0

5

10

15

20

DP

P4

pro

tein

(ng

/mg

)

CD HFFC0

2

4

6

DP

P4

ac

tiv

ity

(m

U/m

g)

**** ****

CD HFFC0.0

0.1

0.2

0.3

0.4

0.5

DP

P4

ac

tiv

ity

(m

U/m

g) ***

**

CD HFFC0.0

0.2

0.4

0.6

0.8

1.0

DP

P4

ac

tiv

ity

(m

U/m

g)

*******

CD HFFC0

50

100

150

200

250

DP

P4

pro

tein

(ng

/mg

)

********

CD HFFC0

5

10

15

20

25

30

DP

P4

pro

tein

(ng

/mg

)

##

Lung Jejunum

Liver Ventricle

Spleen

Kidney Pancreas

A B

C D

E F

G

#

#

- DPP4i

+ DPP4i

- DPP4i

+ DPP4i

HFFC HFFC

Supplementary Figure 2. DPP4 activity and protein levels in tissues after 2 or 4 wks of DPP4i. Levels ofDPP4 activity (left panels) and protein (right panels) in (A) lung, (B) jejunum, (C) liver, (D) whole ventricle, (E) kidney, (F) pancreas and (G) spleen from mice that were maintained on control diet (CD) or a high fat, fructose and cholesterol (HFFC) diet for 10 weeks and then the same diets supplemented with (+) orwithout (‐) the DPP4 inhibitor (DPP4i) MK‐0626 for 4 (A) or 2 (B‐G) wks. Data shown are mean SEM. ForA, n=7 or 8 mice per group. For B‐G, n=9 or 10 mice per group, except where n=8 as indicated on the graphabove the data set. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. DPP4i‐treated. #P<0.05, ##P<0.01,###P<0.001, ####P<0.0001 for CD vs. HFFC. Data were analyzed by two‐sided unpaired Student’s t‐test (A)or two way ANOVA (B‐G). Source data are provided as a Source data file.

n=8n=8

n=8

n=8

n=8

n=8

PBSSita LPS

LPS + S

ita

0.0

0.2

0.4

0.6

0.8

Bone Marrow DPP4 Activity

mU

nit

s/m

g

*

**

PBSSita LPS

LPS + S

ita

0

20

40

60

80

Bone Marrow DPP4 Protein

ng

/mg

*** *

A B

PBSSita LPS

LPS + S

ita

0

1

2

3

4

5

Plasma DPP4 Activitym

Un

its/

mL

**

**

****

PBSSita LPS

LPS + S

ita

0

100

200

300

400

500

Plasma DPP4 Protein

ng

/ml

** *

Supplementary Figure 3. Sitagliptin reduces DPP4 activity and increases DPP4 protein levels in plasma and bone marrow of LPS‐treated mice. Levels of DPP4 activity (top panel) and protein (bottom panel)in (A) plasma and (B) bone marrow from WT mice that were fed a HFFC diet for 4 days and treated with LPS (two injections of 35 g each, one on the evening prior to sacrifice and the second one hour before sacrifice) or PBS vehicle. n=5 mice per group. Data shown are mean SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the indicated comparator. Data were analyzed by two‐way ANOVA. Sourcedata are provided as a Source data file.

CD HFFC0.000

0.005

0.010

0.015

Re

lati

ve

mR

NA

le

ve

ls

CD HFFC0.000

0.002

0.004

0.006

0.008

Rel

ativ

e m

RN

A le

vels

***

CD HFFC0.0000

0.0005

0.0010

0.0015

Re

lati

ve

mR

NA

le

ve

ls

Kidney - 14 weeks + DPP4i


CD HFFC0.0000

0.0005

0.0010

0.0015

Rel

ativ

e m

RN

A le

vels

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

CD HFFC0.0000

0.0005

0.0010

Rel

ativ

e m

RN

A le

vels

##

CD HFFC0.00

0.01

0.02

0.03R

elat

ive

mR

NA

leve

ls

#

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.002

0.004

0.006

0.008

Rel

ativ

e m

RN

A le

vels

** ****

Liver - 14 weeks + DPP4i

- DPP4i + DPP4i

CD HFFC0.0

0.2

0.4

0.6

0.8

Rel

ativ

e m

RN

A le

vels

##

#

Il1b

CD HFFC0.000

0.002

0.004

0.006

0.008

Rel

ativ

e m

RN

A le

vels

Il6

CD HFFC0.00

0.01

0.02

0.03

0.04

0.05

Rel

ativ

e m

RN

A le

vels

########

Tnfa

CD HFFC0.00

0.01

0.02

0.03

Rel

ativ

e m

RN

A le

vels

#

Ifng

CD HFFC

0.00

0.02

0.04

0.06

Rel

ativ

e m

RN

A le

vels

####

###

Il10

CD HFFC0

1

2

3

Rel

ativ

e m

RN

A le

vels

Cxcl1

CD HFFC0.000

0.001

0.002

0.003

Rel

ativ

e m

RN

A le

vels

Il2

CD HFFC

0.00

0.05

0.10

0.15

0.20

Rel

ativ

e m

RN

A le

vels

####

####

Il12b

n=8

n=5

n=8

Supplementary Figure 4. Gene expression in mouse liver and kidney after 14 wks of DPP4i.mRNA levels of genes central to inflammation were examined in liver (upper panels) and kidney (lower panels) samples from WT mice that were maintained on control diet (CD) or a high fat, fructose and cholesterol (HFFC) diet for 10 weeks and then the same diets supplemented with (+) or without (‐) theDPP4 inhibitor (DPP4i) MK‐0626 for 14 wks. Data were normalized to Tbp (liver) or Rpl32 (kidney) expressionand are mean SEM. n=9 or 10 mice per group, except where the specific n is indicated on the graph abovethe data set. *P<0.05, **P<0.01, ****P<0.0001 vs. DPP4i‐treated. #P<0.05, ##P<0.01, ###P<0.001,####P<0.0001 for CD vs. HFFC. Data were analyzed by two‐way ANOVA. Source data are provided as a Source data file.


CD HFFC0.00

0.01

0.02

0.03

0.04

0.05

Re

lati

ve

mR

NA

le

ve

ls *

CD HFFC0.000

0.003

0.006

0.009

0.012

Re

lati

ve

mR

NA

le

ve

ls

CD HFFC0.000

0.005

0.010

0.015

0.020

Re

lati

ve

mR

NA

le

ve

ls

*

CD HFFC0.00

0.01

0.02

0.03

0.04

Re

lati

ve

mR

NA

le

ve

ls

Jejunum - 14 weeks + DPP4i

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0000

0.0005

0.0010

0.0015

0.0020

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

Spleen - 14 weeks + DPP4i

- DPP4i + DPP4i

CD HFFC0.00

0.05

0.10

0.15

0.20

Rel

ativ

e m

RN

A le

vels

CD HFFC0.00

0.05

0.10

0.15

0.20

0.25

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

Rel

ativ

e m

RN

A le

vels **

#

CD HFFC0.000

0.005

0.010

0.015

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.002

0.004

0.006

0.008

0.010

Rel

ativ

e m

RN

A le

vels

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

CD HFFC0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.001

0.002

0.003

0.004

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0

0.1

0.2

0.3

Rel

ativ

e m

RN

A le

vels

n=8

n=7

n=8

n=8

n=8

n=6

n=7n=6

n=7 n=7

n=7

n=6

n=4

n=6

Supplementary Figure 5. Gene expression in mouse spleen and jejunum after 14 wks of DPP4i. mRNA levels of genes central to inflammation were examined in spleen (upper panels) and jejunum(lower panels) samples from WT mice that were maintained on control diet (CD) or a high fat, fructose andcholesterol (HFFC) diet for 10 weeks and then the same diets supplemented with (+) or without (‐) the DPP4 inhibitor (DPP4i) MK‐0626 for 14 wks. Data were normalized to Rpl32 expression and are mean SEM.n=9 or 10 mice per group, except where the specific n is indicated on the graph above the data set. *P<0.05, **P<0.01 vs. DPP4i‐treated. #P<0.05 for CD vs. HFFC. Data were analyzed by two‐way ANOVA. Source data are provided as a Source data file.

CD HFFC0.000

0.005

0.010

0.015

0.020

Re

lati

ve

mR

NA

le

ve

ls

*

CD HFFC0.0000

0.0001

0.0002

0.0003

Re

lati

ve

mR

NA

le

ve

ls

*

CD HFFC0.0000

0.0006

0.0012

0.0018

0.0024

Rel

ativ

e m

RN

A le

vels

*

**

#

####

Liver - 2 weeks + DPP4i

- DPP4i + DPP4i

CD HFFC0.0

0.2

0.4

0.6

0.8

1.0

Re

lati

ve

mR

NA

le

ve

ls

*##

CD HFFC0.00

0.01

0.02

0.03

0.04

Re

lati

ve

mR

NA

le

ve

ls

#

CD HFFC0.000

0.005

0.010

0.015

0.020

Re

lati

ve

mR

NA

le

ve

ls

**

Kidney - 2 weeks + DPP4i

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

CD HFFC0.000

0.002

0.004

0.006

0.008

0.010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.00

0.01

0.02

0.03

0.04

0.05

Rel

ativ

e m

RN

A le

vels

##

##

CD HFFC0

1

2

3

Rel

ativ

e m

RN

A le

vels

*

##

CD HFFC0.000

0.001

0.002

0.003

0.004

Rel

ativ

e m

RN

A le

vels

CD HFFC0.00

0.05

0.10

0.15

Rel

ativ

e m

RN

A le

vels

##

##

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.002

0.004

0.006

0.008

Rel

ativ

e m

RN

A le

vels

** *

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.001

0.002

0.003

0.004

0.005

Rel

ativ

e m

RN

A le

vels


n=8n=8 n=8

n=8

n=6

n=7n=6

n=6

n=8

n=8

n=8

n=8

Supplementary Figure 6. Gene expression in mouse liver and kidney after 2 wks of DPP4i.mRNA levels corresponding to genes important for inflammation were examined in liver (upper panels and kidney (lower panels) samples from WT mice that were maintained on control diet (CD) or a high fat, fructose and cholesterol (HFFC) diet for 10 weeks and then the same diets supplemented with (+) or without (‐) the DPP4 inhibitor (DPP4i) MK‐0626 for 2 wks. Data were normalized to Tbp (liver) or Rpl32(kidney) expression and are mean SEM. n=9 or 10 mice per group, except where the specific n is indicatedon the graph above the data set. *P<0.05, **P<0.01 vs. DPP4i‐treated. #P<0.05, ##P<0.01, ####P<0.0001 forCD vs. HFFC. Data were analyzed by two‐way ANOVA. Source data are provided as a Source data file.

- DPP4i + DPP4i

Il1b

Il6

Tnfa

Ifng Il10 Cxcl1

Il2 Il12b

Il1bTnfa

Ifng Il10 Cxcl1

Il2 Il12b

Spleen - 2 weeks + DPP4i

Jejunum - 2 weeks + DPP4i

CD HFFC0.000

0.005

0.010

0.015

0.020

Rel

ativ

e m

RN

A le

vels #

CD HFFC0.00

0.01

0.02

0.03

0.04

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.001

0.002

0.003

0.004

0.005

Rel

ativ

e m

RN

A le

vels

*

CD HFFC0.000

0.001

0.002

0.003

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0

0.2

0.4

0.6

0.8

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0

0.2

0.4

0.6

0.8

1.0

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.001

0.002

0.003

0.004

Rel

ativ

e m

RN

A le

vels

CD HFFC0.0

0.1

0.2

0.3

Rel

ativ

e m

RN

A le

vels #

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.001

0.002

0.003

0.004

0.005

Rel

ativ

e m

RN

A le

vels

#

CD HFFC0.0000

0.0005

0.0010

0.0015

0.0020

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.006

0.012

0.018

Rel

ativ

e m

RN

A le

vels

#

CD HFFC0.00

0.01

0.02

0.03

0.04

Rel

ativ

e m

RN

A le

vels #

CD HFFC0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

Rel

ativ

e m

RN

A le

vels

CD HFFC0.000

0.002

0.004

0.006

Rel

ativ

e m

RN

A le

vels

**

#

Supplementary Figure 7. Gene expression in mouse spleen and jejunum after 2 wks of DPP4i. mRNA levelsof genes central to inflammation were examined in spleen (upper panels) and jejunum (lower panels)samples from WT mice that were maintained on control diet (CD) or a high fat, fructose and cholesterol (HFFC) diet for 10 weeks and then the same diets supplemented with (+) or without (‐) the DPP4 inhibitor (DPP4i) MK‐0626 for 2 wks. Data were normalized to Rpl32 expression and are mean SEM. n=9 or 10 miceper group, except where the specific n is indicated on the graph above the data set. *P<0.05, **P<0.01 vs.DPP4i‐treated. #P<0.05 for CD vs. HFFC. Data were analyzed by two‐way ANOVA. Source data are providedas a Source data file.

n=8n=8

n=7 n=8

n=7

n=8 n=8

n=4

n=5

n=7

n=7

0 9 11weeks10% Fat

HFFC

HFFC

10% Fat

HFFC

HFFC+DPP4i

8 wk-old Glp1r+/+ or Glp1r-/- male mice:

Sac

-1 0 1 2 3 4 5 6 7 8 9 10 11

0

3

6

9

12

15

Time (wks)

Bo

dy

We

igh

t (g

)n

orm

ali

zed

to

ba

se

lin

e

A B Glp1r+/+ CD

Glp1r+/+ HFFC

Glp1r+/+ HFFC + DPP4i

Glp1r-/- CD

Glp1r-/- HFFC

Glp1r-/- HFFC + DPP4i

Glp1r+/+ Glp1r-/-0.0

0.2

0.4

0.6

DP

P4

ac

tiv

ity

(m

U/m

g)

Glp1r+/+ Glp1r-/-0

10

20

30

40

50

60

70

DP

P4

pro

tein

(n

g/m

g)

CD HFFC HFFC + DPP4i

****

****

****

** ****

************

C

D Bone Marrow

Plasma

Glp1r+/+ Glp1r-/-0

1

2

3

4

5

6

7

8

DP

P4

acti

vity

(m

U/m

L)

Glp1r+/+ Glp1r-/-0

100

200

300

400

500

600sD

PP

4 p

rote

in (

ng

/mL

)****

****

********

********

***

**** ********

DPP4i = DPP4 inhibitor (15.5 mg ofMK-0626 per 4057 kcal diet)

Supplementary Figure 8. (A) Schematic depicting the diet regimen and time of sacrifice (Sac) in the Glp1r+/+

and Glp1r‐/‐ mouse studies. (B) Weekly body weights of Glp1r+/+ and Glp1r‐/‐mice maintained on a control diet (CD) or a high fat, fructose and cholesterol (HFFC) diet for 11 wks, or HFFC diet for 9 wks and thenswitched to HFFC diet supplemented with (+) the DPP4 inhibitor (DPP4i) MK‐0626 (MK) for 2 wks. (C‐D)Levels of DPP4 activity (left panels) and protein (right panels) in (C) plasma and (D) bone marrow fromGlp1r+/+ and Glp1r‐/‐ mice maintained on CD or HFFC diet for 11 wks +/‐ DPP4i for the final 2 wks. Data shown are mean SEM. For C and D, n=6 or 7, except where the specific n is indicated on the graph above the data set. *P<0.05, **P<0.01, ****P<0.0001 vs. the indicated comparator. Data were analyzed by two‐way ANOVA. Source data are provided as a Source data file.

(n=6)

(n=8) (n=7)

(n=6)

(n=7) (n=7)

n=5

n=8

WT Glp1r -/-0

10

20

30

40

50

Rel

ativ

e m

RN

A le

vels

WT Glp1r -/-0

20

40

60

WT Glp1r -/-0

40

80

120

160

WT Glp1r -/-0.0

0.6

1.2

1.8

WT Glp1r-/-0.00

0.05

0.10

0.15

0.20

0.25

WT Glp1r-/-0.0

0.2

0.4

0.6

0.8

1.0

Jejunum:

* ***

************

****

*

** *

*

**** *****

***

**

**

**

**

Ileum:

WT Glp1r -/-0.00

0.01

0.02

0.03

0.04

0.05

Rel

ativ

e m

RN

A le

vels

WT Glp1r -/-0.000

0.003

0.006

0.009

0.012

0.015

WT Glp1r-/-0.00

0.02

0.04

0.06

0.08

WT Glp1r -/-0

300

600

900

1200

1500

WT Glp1r -/-0.0

0.2

0.4

0.6

0.8

WT Glp1r-/-0.0000

0.0005

0.0010

0.0015

0.0020

** ***** *

** *

*

**

*

*

***

WT Glp1r-/-0

2

4

6

8

10

Rel

ativ

e m

RN

A le

vels

WT Glp1r-/-0

1

2

3

WT Glp1r-/-0.00

0.05

0.10

0.15

WT Glp1r-/-0.0000

0.0005

0.0010

0.0015

WT Glp1r -/-0.00

0.05

0.10

0.15

WT Glp1r -/-0.00

0.05

0.10

0.15

Colon:

Control Diet HFFC Diet HFFC + DPP4i

*** *** *

*****

***

Reg3a

Reg3a

CD36 Alpi Egfr Ocln Il18

Il10 Il25 Reg3g Tlr4 Gip Il18

Reg3gReg3b Il12b Tff2 Tslp

Supplementary Figure 9. Gene expression in Glp1r+/+ and Glp1r‐/‐ mouse intestine.mRNA levels of genesimportant for inflammation or epithelial protection and repair were assessed in jejunum, ileum and colon samples from Glp1r+/+ (WT) and Glp1r‐/‐ mice maintained on a control diet (10% fat) or a high fat, fructose and cholesterol (HFFC) diet for 11 wks, or HFFC diet for 9 wks and then switched to HFFC diet supplementedwith (+) the DPP4 inhibitor (DPP4i) MK‐0626 for 2 wks. Data were normalized to Ppia or Tbp and are mean SEM. n=6 or 7 mice per group, except where the specific n is indicated on the graph above the data set.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. the indicated comparator. Data were analyzed by two‐way ANOVA. Source data are provided as a Source data file.

n=8

n=8n=8

n=7

n=5

n=8

n=8

n=8

n=8

n=8n=8

n=5

n=8

n=5

n=5

n=5

n=5

n=5n=5n=5

n=5

Baseline 12 months

0.0

0.2

0.4

0.6

0.8

2

4

6

8

DPP4 Activitym

Un

its/

ml

****

Basel

ine

12 m

onths

0

200

400

600

ng

/ml

Basel

ine

12 m

onths

0

100

200

300

400

500

600

ng

/ml

Basel

ine

12 m

onths

-100

0

100

200

300

% C

han

ge

Fro

m B

asel

ine

sDPP4 Protein (Thermo Fisher):

sDPP4Protein (RayBiotech):

Basel

ine

12 m

onths

0

500

1000

1500

2000

2500

ng

/ml

Basel

ine

12 m

onths

0

500

1000

1500

2000

2500

ng

/ml

Basel

ine

12 m

onths

-40

-20

0

20

40

% C

han

ge

Fro

m B

asel

ine

B

A

C

Supplementary Figure 10. Plasma levels of sDPP4 vary widely among TECOS trial patients. (A) DPP4 activity and (B, C) sDPP4 protein levels at baseline and month 12 in plasma samples from asubset of patients from the TECOS trial (n=40 patients) that received sitagliptin. Plasma sDPP4 levels were measured using assay kits from two independent sources (B, C). In B and C, data are shown as individualvalues in a scatter plot (left panels), summary data with a line connecting the baseline data to the 12 monthdata (center panels), or percent change from baseline (right panels). Data are mean SEM. ****P<0.0001vs. the indicated comparator. Data were analyzed by two‐sided unpaired Student’s t‐test.

bmi (kg/m qualifying hba1c (%) dpp4 activity (mu/ml)10.1038... · supplementary table 3....

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