Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1

Jou

rnal

of

En

do

crin

olo

gy

ResearchE A FORSBERG and others Carnosine decreases IGFBP1

production in diabetes225 :3 159–167

Carnosine decreases IGFBP1production in db/db mice throughsuppression of HIF-1

Elisabete A Forsberg1, Ileana R Botusan1, Jing Wang1, Verena Peters3,

Ishrath Ansurudeen1, Kerstin Brismar1,2 and Sergiu Bogdan Catrina1,2

1The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden2Department of Endocrinology, Diabetes and Metabolism, Karolinska University Hospital, Stockholm, Sweden3Center for Pediatric and Adolescent Medicine, University of Heidelberg, Heidelberg, Germany

http://joe.endocrinology-journals.org � 2015 Society for EndocrinologyDOI: 10.1530/JOE-14-0571 Printed in Great Britain

Published by Bioscientifica Ltd.

Correspondence

should be addressed

to E A Forsberg

Email

[email protected]

Abstract

IGF binding protein 1 (IGFBP1) is a member of the binding proteins for the IGF with an

important role in glucose homeostasis. Circulating IGFBP1 is derived essentially from the liver

where it is mainly regulated negatively by insulin. Carnosine, a natural antioxidant, has been

shown to improve metabolic control in different animal models of diabetes but its

mechanisms of action are still not completely unraveled. We therefore investigate the effect

of carnosine treatment on the IGFBP1 regulation in db/db mice. Db/db mice and

heterozygous non-diabetic mice received for 4 weeks regular water or water supplemented

with carnosine. Igfbp1 mRNA expression in the liver was evaluated using qPCR and the

protein levels in plasma by western blot. Plasma IGF1 and insulin were analyzed using

immunoassays. HepG2 cells were used to study the in vitro effect of carnosine on IGFBP1.

The modulation of hypoxia inducible factor-1 alpha (HIF-1a) which is the central mediator

of hypoxia-induction of IGFBP1 was analyzed using: WB, reporter gene assay and qPCR.

Carnosine decreased the circulating IGFBP1 levels and the liver expression Igfbp1, through

a complex mechanism acting both directly by suppressing the HIF-1a-mediated IGFBP1

induction and indirectly through increasing circulating insulin level followed by a decrease

in the blood glucose levels and increased the plasma levels or IGF1. Reduction of IGFBP1

in diabetes through insulin-dependent and insulin-independent pathways is a novel

mechanism by which carnosine contributes to the improvement of the metabolic control

in diabetes.

Key Words

" carnosine

" IGFBP1

" liver

" HIF-1a

Journal of Endocrinology

(2015) 225, 159–167

Introduction

Insulin-like growth factor binding protein 1 (IGFBP1) is

one of the six proteins that binds and regulates the

bioavailability of IGF1. IGFBP1 binds IGF1 with high

affinity, and regulates IGF effects in a tissue-specific

manner either by enhancing or damping IGF activity.

IGFBP1 is expressed in the liver, kidney and decidua

(Rajaram et al. 1997). The liver is the major source for the

circulating IGFBP1 and its synthesis is centrally regulated

by insulin that represses IGFBP1 at the transcriptional

level (Brismar et al. 1994, Powell et al. 1995). Other factors,

including hypoxia, pro-inflammatory cytokines, cAMP,

glucocorticoids and oxidative stress stimulate the

synthesis of IGFBP1 (Mesotten et al. 2002). Hypoxia

inducible factor (HIF) is a transcription factor that binds

http://joe.endocrinology-journals.org

http://dx.doi.org/10.1530/JOE-14-0571

Jou

rnal

of

En

do

crin

olo

gy

Research E A FORSBERG and others Carnosine decreases IGFBP1production in diabetes

225 :3 160

to the hypoxia responsive elements (HRE) in the promoter

region of more than 100 genes including IGFBP1 and

mediates the adaptive response to hypoxia (Semenza

2011). HIF is a heterodimeric factor composed of two

subunits a and b, in which the a subunit is regulated by

oxygen. In the presence of oxygen, HIF is hydroxylated

by a specific Fe2C, oxoglutarate dependent prolyl

4-hydroxylases (PHD) allowing HIF-1a to bind to the von

Hippel-Lindau (VHL) tumor suppressor protein that acts as

an E3 ubiquitin ligase and targets HIF-1a for proteasome

degradation (Kaelin & Ratcliffe 2008).

Increased liver IGFBP1 synthesis is observed in states of

insulin resistance, such as diabetes type 2 (Munoz et al.

1996, Clauson et al. 1998). Compelling evidence suggests

that increased IGFBP1 is involved in the pathophysiology

of diabetes complications (Crossey et al. 2000, Heald et al.

2001, Schrijvers et al. 2004, Ezzat et al. 2008) either through

inhibition of IGF1 actions or by a direct effect of IGFBP1.

High levels of IGFBP1 contribute directly to impaired

metabolic control (Zachrisson et al. 2000) as illustrated in

transgenic mice where overexpression of Igfbp1 results in

fasting hyperglycemia (Murphy 2000) and long-term

IGFBP1 infusion leads to increased blood glucose levels in

rats (Lewitt et al. 1991). On the other side, in animal model

of diabetes high levels of IGFBP1 have been associated with

renal hypertrophy (Doublier et al. 2000, Van Buul-Offers

et al. 2000). Furthermore, a polymorphism in the IGFBP1

gene, which affects the activity of IGF1, was associated

with a decreased risk of developing diabetic nephropathy

(DN) (Stephens et al. 2005).

L-carnosine is a naturally occurring dipeptide, which is

endogenously synthesized from b-alanine and L-histidine

by an ATP-dependent carnosine synthase. Carnosine is

present in high concentrations in the skeletal muscle,

heart and nervous system and smaller quantities are

synthesized in other tissues such as kidney, liver, stomach

and lungs. Carnosine plays an important role in a number

of biological functions, through its antioxidant, anti-

inflammatory and anti-senescence properties (Lenz &

Martell 1964, Gallant et al. 2000, Guiotto et al. 2005).

Carnosine acts as a scavenger of reactive oxygen species

including peroxyl radicals and superoxide (Boldyrev et al.

2013). The importance of carnosine in diabetes and its

chronic complications was highlighted recently. A poly-

morphism in exon 2 of carnosinase (CN1), an enzyme that

degrades carnosine, was associated with susceptibility for

developing DN (Janssen et al. 2005). This polymorphism

associated with resistance against DN was demonstrated

in different populations (Freedman et al. 2007). Moreover,

exogenous carnosine improved the glucose levels in


different diabetic animal models (Yamano et al. 2001,

Sauerhofer et al. 2007). Additionally, experimental treat-

ment with carnosine in animal models has protective

effects on the development of chronic complications in

diabetes. Carnosine treatment decreases proteinuria and

renal damage in diabetic mice (Peters et al. 2012), inhibits

the production of fibronectin and TGF b in renal cells

(Janssen et al. 2005) and improves wound healing in

diabetes (Ansurudeen et al. 2012). It has been shown that

anserine (methylated carnosine) also has a positive effect

on blood glucose and plasma insulin concentration both

in rodents and humans (Kubomura et al. 2010a,b).

Furthermore, Peters et al. (2012) have shown that in

obese diabetic mice, renal CN1 activity is increased and

histidine dipeptide concentrations are reduced. Carnosine

supplementation mitigates DN, reduces renal vasculo-

pathy, and normalizes vascular permeability in diabetic

mice. In streptozotocin-induced, diabetic rats, carnosine

treatment prevents apoptosis of glomerular cells and

podocyte loss and vascular damage (Riedl et al. 2011).

Knowing IGFBP1 as a marker of hepatic insulin

resistance and as a potential pathogenic factor for chronic

complications of diabetes, we studied the influence of

exogenous carnosine on IGFBP1 production and circulat-

ing levels in db/db mice.

Materials and methods

Animals and experimental protocol

The db/db mouse was used as a model of type 2 diabetes

and its lean heterozygote, littermate as the control

(Charles River, Sulzfeld, Germany) (Stock 000662). Their

phenotype consists of obesity, insulin resistance and

diabetes, similar to type 2 diabetes in humans (Hummel

et al. 1966). Only male mice were used for this study and

housed in an animal facility that was maintained at 25 8C

with a 12 h light:12 h darkness cycle. Animals had free

access to water and standard rodent chow. The experi-

mental procedure was approved by the North Stockholm

Ethical Committee for Care and Use of Laboratory

Animals. Treatment was initiated at 6 weeks of age, before

the db/db mice developed hyperglycemia. Mice were

divided into four groups: i) diabetic mice with no

treatment; ii) diabetic mice that received 5 g/l of

L-carnosine (Sigma) in the drinking water for 4 weeks;

iii) control mice with no treatment; and iv) control mice

who received 5 g/l of L-carnosine in the drinking water for

4 weeks. Since L-carnosine was reported to be stable in the

water bottles over a period of minimum 5 days at room




Jou

rnal

of

En

do

crin

olo

gy


225 :3 161

temperature, we chose to replace the water every 5 days

(Sauerhofer et al. 2007). The water intake was estimated

by weighing the water bottles every 5 days. Unless stated

otherwise, each experimental group contained eight mice.

At the end of the experiment, body weights and blood

glucose levels were measured. Glucose levels were

determined in blood collected from the tail tip using

OneTouch Ultra Blood Glucose meter (LifeScan, Milpitas,

CA, USA). The liver was harvested and snap frozen. At the

end of the experiment, blood was collected in heparin

tubes (BD Vacutainer, Plymouth, UK) and snap frozen.

Plasma assays

Plasma samples for the determination of total IGF1

concentration were acid ethanol extracted prior to the

RIA, and to further eliminate major interactions with

IGFBPs, truncated IGF1 was used as ligand (Bang et al.

1991). The intra- and inter-assay coefficients of variations

(CV) were 4 and 8% respectively. The sensitivity of the RIA

was 3 mg/l and the intra- and inter-assay CV were 3 and

10% respectively.

Insulin was determined by using an Ultra-Sensitive

Mouse Insulin ELISA kit (Crystal Chem, Downers Grove,

IL, USA). Leptin was determined by using a Mouse Leptin

ELISA Kit (Crystal Chem).

Carnosine concentration

Carnosine concentrations were assayed in plasma and

in liver homogenates by fluorometic determination after

derivatization with carbazole-9-carbonyl chloride. Separ-

ation was performed by liquid chromatography according

to the method previously described (Peters et al. 2010).

RNA extraction and real-time RT-PCR

Liver tissues were harvested and quickly submerged in

RNA/later solution (Ambion, Austin, TX, USA). Total RNA

from liver or HepG2 cells was extracted by using RNeasy

Mini Kit (Qiagen). First-strand cDNA was synthesized

from 1 mg total RNA employing Superscript III reverse

transcriptase with UDG transacetylase (Invitrogen) accor-

ding to the manufacturer’ protocol. The cDNAs were

stored at K20 8C until use in quantitative real-time PCR.

The assay to semi-quantify specific mRNAs was carried

out using the SuperScript III Platinum Two-step Quan-

titative RT–PCR system according to manufacturer’s

instructions (Invitrogen). Real-time PCR was carried out

using gene specific primer pairs for Igfbp1 (forward,


5 0-ATCAGCCCATCCTGTGGAAC-3 0 and reverse 5 0-TGCA-

GCTAATCTCTCTAGCACTT-3 0), Vegf a (forward, 5 0-TTAT-

GCGGATCAAACCTCAC-3 0 and reverse 5 0-TCTTTCTTT-

GGTCTGCATTCAC-3 0), Pgk1 (forward, 5 0-AGTCGG-

TAGTCCTTATGAGCC-3 0 and reverse 5 0-TTCCCAGAAG-

CATCTTTTCCC-3 0), Bnip3 (forward, 5 0-ATTGGTCAAGTC-

GGCCAGAA-3 0 and reverse 5 0-AGTCGCTGTACGCTTTG-

GGT-3 0), and Pbdg (forward, 5 0-TCTCTGCTGCTACCT-

GCGT-3 0 and reverse 5 0-GTGGGAGCGGGTCATGTTC-3 0).

Real-time PCRs were carried out in the ABI Prism 7300

Sequence Detection System (Applied Biosystems). PCR

conditions were as follows: initial incubation for 2 min

at 50 8C and 2 min at 95 8C, and a two-step cycling PCR

protocol for 40 cycles at 94 8C for 15 s, at 60 8C for 30 s.

The melting curve analysis was done using the program

supplied by Applied Biosystems. The quality of the

quantitative PCR run was determined by standard curves

and melting curve analysis. Relative quantification was

carried out by using the 2KCT method.

Western blot analysis for IGFBP1 and HIF-1a

Liver tissues were homogenized in RIPA buffer

(150 mmol/l NaCl, 1% Igepal, 0.5% sodium deoxycholate,

0.1% SDS, 50 mmol/l Tris-HCl, pH 8.8, supplemented with

freshly made protease inhibitor cocktail) and centrifuged

at 4 8C with 20 000 g for 20 min. Proteins were quantified

(Bio-Rad) and after equally loading (50 mg) were electro-

phoresed in 7.5 or 12% SDS–PAGE gel, and transferred to

nitrocellulose membrane subsequently blocked with 5%

nonfat milk. The primary antibody against HIF-1a (1:500 –

Novus Biologicals, Littleton, Colorado, USA, NB 100-449)

or IGFBP1 (1:2000 – Abcam, Cambridge, UK, ab 4242) was

added and incubated overnight at 4 8C with gentle

shaking. Membranes were washed three times in PBS

containing 0.1% Tween 20. The secondary anti-rabbit

antibody conjugated to HRP was added at a concentration

of 1:3000 and incubated for 1 h at room temperature with

gentle shaking, after which membranes were washed three

times in PBS containing 0.1% Tween 20. Bound antibody

was detected by ECL western blotting detection system

(GE Healthcare, Piscataway, NJ, USA).

Protein for HepG2 cells were extracted and separated

by SDS–PAGE as described above, and transferred to PVDF

membranes (GE Healthcare). After blocking, membranes

were probed with related primary antibodies for HIF-1a

(Novus Biologicals) or human IGFBP1 (Abcam) for 2 h at

C22 8C. The membranes were then incubated with

fluorescent conjugated secondary antibody IRDye 800CW

goat (polyclonal) anti-rabbit IgG (HCL) (Li-Cor, Lincoln,




Jou

rnal

of

En

do

crin

olo

gy


225 :3 162

NE, USA). Results were developed with the Li-Cor Odyssey

system CLx (Li-Cor, Waltham, MA, USA). Band intensity of

western immunoblot was measured with Image Studio Lite

of the Li-Cor, Version 3.1.4 (Li-Cor). Protein concentration

of HepG2 extract was measured with a BCA protein kit

(Thermo Scientific, USA) to ensure equal loading.

Histological analysis

Frozen liver samples were placed in 4% paraformaldehyde

and embedded in paraffin. After deparaffinization and

dehydration, the sections were stained with hematoxylin

and eosin.

Cell culture

A human hepatocellular carcinoma cell line, HepG2,

obtained from the American Type Culture Collection

known to produce IGFBP1 (Hilding et al. 2003), was used

to investigate the effect of carnosine (Sigma–Aldrich) on

IGFBP1 secretion. HepG2 cells were grown at 37 8C in 95%

air-5% CO2 in 100-mm2 cell culture dishes and fed every

3–4 days with DMEM (Life Technologies) supplemented

with 10% fetal bovine serum, penicillin (100 U/ml) and

streptomycin (100 mg/ml). Before the experiments the

medium was replaced with serum-free DMEM containing

0.2% of albumin for 24 h. Hypoxia exposure was

performed by incubating the cells at 1% oxygen using

the Invivo 300 hypoxia chamber (Ruskinn, UK).

Carnosine dose used was 50 mmol/l carnosine, which

is the lowest dose that decreased IGFBP1 levels in

normoxia and this dose was further used for investigating

the effect on hypoxia-induced IGFBP1 (Supplementary

Figure 2, see section on supplementary data given at the

end of this article).
Table 1 Effects of carnosine supplementation for 4 weeks on
body weight, blood glucose, IGF1, insulin and leptin in control

and db/db mice. Values are meansGS.E.M. (nZ8)

Variable C CAR D DCCAR

Body weight (g) 26G0.6 26G0.5 38G2‡ 36G1Blood glucose

(mg/dl)149G10 165G11 407G36‡ 306G27§

IGF1 (ng/ml) 94G3 103G8 65G4† 90G5§

Insulin (ng/ml) 1.24G0.3 2G0.8 3.6G1.3 11.3G1.6§

Leptin (ng/ml) 4.6G0.6 6.6G1 61G7.4* 54G12Plasma carnosine

(nmol/l)163G15 397G107* 161G33 232G63§

*P!0.05 vs C, †P!0.01 vs C, ‡P!0.001 vs C, and §P!0.05 vs D. All data wereanalyzed by one-way ANOVA followed by Tukey’s multiple comparisonpost-test. P!0.05 was considered statistically significant. C, control; CAR,control treated with carnosine; D, db/db; DCCAR, db/db treated withcarnosine.

Luciferase experiments

HepG2 cells were transiently transfected with 300 ng HRE

– luciferase reporter gene plasmid (pT81/HRE-luc) using

Fugene reagent (Roche) according to the manufacturer’s

instructions. Renilla luciferase vector which provide

constitutive expression of Renilla luciferase was co-

transfected with HRE – luciferase plasmid and used as

internal control. The cells to be transfected were seeded

in six well plates and transfected at 75–80% confluency,

starved overnight and exposed then for 24 h to either

normoxia (21% O2) or hypoxia (1% O2) in the presence of

50 mmol carnosine solution or PBS, used as control. The

luminescence was measured in the cells extract using


Dual-Luciferase Reporter Assay Kit (Promega). Relative

light units were normalized to Renilla luciferase

expression.

Statistical analyses

Data are expressed as meanGS.E.M. Comparison among

groups was by ANOVA followed by Tukey’s multiple

comparison post-test. P!0.05 was considered statistically

significant.

Results

The data for water intake, baseline blood glucose and body

weight of the animals are shown in Supplementary

Table 1, see section on supplementary data given at the

end of this article. As shown in Table 1, the treatment with

carnosine for 4 weeks reduced the glucose levels in the

db/db mice by 25%. Carnosine increases by 38% the

plasma IGF1 levels in the db/db mice which have lower

levels than control mice. Treatment with carnosine

increased threefold plasma insulin concentration in the

db/db mice that had, as expected, higher levels than

controls (P!0.01). There was no effect of treatment on

leptin levels. Carnosine had no effect in the non-diabetic

control animals on any of the variables previously

mentioned.

Carnosine normalizes high levels of IGFBP1 in diabetes

The liver Igfbp1 mRNA levels were increased in the db/db

mice compared with non-diabetic control animals (Fig. 1).

Treatments of both control and db/db mice with carnosine


http://joe.endocrinology-journals.org/cgi/content/full/JOE-14-0571/DC1






**

†††

Liv

er I

gfbp

1gen

e ex

pres

sion

(arb

itrar

y un

it)

C CAR D D+CAR0.0

0.5

1.0

1.5

2.0

#

Figure 1

Liver samples from db/db and control mice treated and untreated with

carnosine were used to determine the effect of carnosine on the mRNA

expression of Igfbp1. Gene expression of Igfbp1 was significantly increased

in D compared to C. Treatment with carnosine decreased about 50% the

Igfbp1 expression in the liver after 4 weeks treatment. **P!0.01 vs C,#P!0.05 vs C, and †††P!0.001 vs D. C, non-diabetic control mice; CAR, non-

diabetic mice treated with carnosine; D, db/db; DCCAR, diabetic mice

treated with carnosine.

0.50

0.75

1.00

1.25

gf1

gene

exp

ress

ion

arbi

trar

y un

it)

***

†

Jou

rnal

of

En

do

crin

olo

gy


225 :3 163

caused a decrease in Igfbp1 mRNA expression (by 50%).

Carnosine increased the repressed levels of Igf1 mRNA

expression (Fig. 2) in concordance with the previously

mentioned effect on the circulating levels of IGF1 in

diabetic animals.

The effects of carnosine on IGF1 were restricted to the

diabetic mice since neither Igf1 expression or plasma

protein levels were modulated by carnosine treatment

in the non-diabetic mice.

The exogenous supplementation of carnosine

increased by almost twofold its accumulation in the liver

of the db/db mice that exhibited lower levels than the

non-diabetic mice (Fig. 3). The carnosine treatment had

no effect on the liver accumulation of carnosine in control

non-diabetic mice despite similar increase in plasma

carnosine levels in both the diabetic and the non-diabetic

animals. This was followed by a decrease in the liver

steatosis just in diabetic animals but not in non-diabetic

control mice (Supplementary Figure 1, see section on

supplementary data given at the end of this article).

C CAR D D+CAR0.00

0.25Liv

er I (

Figure 2

Liver samples from db/db and control mice treated and untreated with

carnosine were used to determine the effect of carnosine on the Igf1. Gene

expression for Igf1 was decreased in D compared to C. However, carnosine

treatment was able to increase Igf1 about 40%. ***P!0.001 vs C and†P!0.05 vs D.

Carnosine modulates IGFBP1 levels at multiple levels

The next step of our investigation was to study the

mechanisms by which carnosine modulated IGFBP1.

Having in mind the central role of insulin on IGFBP1

regulation, the noted increased in the circulating insulin

in the db/db mice after treatment with carnosine provides

an important explanatory mechanism for the repression of

IGFBP1 after treatment with carnosine.


However the impressive specific accumulation of

carnosine in the liver prompted us to continue our

investigation by studying additional mechanisms that

could be activated in the liver independent of insulin

levels. Having in mind the role of hypoxia in diabetes and

for IGFBP1 regulation we studied the effect of carnosine on

hypoxia-induced IGFBP1.

For this end, we have used HepG2 which are cells

extensively used for studies concerning both HIF and

IGFBP1 regulation. Exposure of HepG2 cells to hypoxia

was followed by an increase in Igfbp1 both at mRNA levels

(Fig. 4a) and protein levels (Fig. 4b and c). Carnosine

diminished this effect by normalizing the hypoxia-

induced levels of both Igfbp1 mRNA (Fig. 4a) and protein

(Fig. 4b and c).

Carnosine destabilizes HIF-1a and decreases its activity

Keeping in mind that the main adaptor of the cells to

hypoxia is HIF and that IGFBP1 is induced by HIF-1a

(Tazuke et al. 1998), we next investigated the effects of

carnosine on HIF stability and function. Carnosine

destabilized HIF-1a in hypoxia in the HepG2 cells as

shown in Fig. 5a. The functional inhibition of carnosine

treatment on HIF-1a activity was further proved using

a transient transfection with a HRE-reporter plasmid.

Carnosine decreased the hypoxia induced HRE-activity

(Fig. 5b) in concordance with the effects observed on

target genes (Fig. 6a, b and c).





Car

nosi

ne (

nmol

/mg)

C CAR D D+CAR0

5

10

15

20

***

†

Figure 3

Carnosine concentration in the liver was significantly reduced in diabetic

mice (D) compared to controls (C). Carnosine treatment did improve the

decreased levels of carnosine in the liver of diabetic animals but had no

effect on liver of control, non-diabetic animals. ***P!0.001 vs C and†P!0.05 vs D.

Nx Nx+CAR Hx Hx+CAR0.0

2.5

5.0

7.5

10.0

Igfb

p1 g

ene

expr

essi

on(a

rbitr

ary

unit)

***

†††

Nx CAR Hx Hx+CAR0

30

60

90

120

150

180

IGFB

P1 (

% o

f co

ntro

l)

***

†††

###

(b)

(a)

Nx

Nx + C

AR Hx

Hx + C

AR0

1

2

3

4

*

Fold

cha

nges

to N

x(I

GFB

P1)

IGFBP1β-actin

+ +––N H

(c)

Figure 4

Effect of hypoxia on Igfbp1 expression in HepG2 cells. HepG2 cells were

starved overnight then exposed to hypoxia for 24 h. Carnosine was added

just prior to placing the cells in hypoxia. (a) Total RNA was extracted, and

the Igfbp1 expression determined by qPCR. Hypoxia increase by eightfold

the mRNA expression of Igfbp1 and carnosine significantly blunted that

response. (b) The IGFBP1 secreted in the medium and (c) protein expression

in HepG2 cells were increased under hypoxia and carnosine treatment

significantly decreased IGFBP1. The values represent the meanGS.E.M.

of three independent experiments. *P!0.05 vs. Hx, ***P!0.001 vs. Nx,†††P!0.001 vs. Hx and ###P!0.001 vs. Nx.

Jou

rnal

of

En

do

crin

olo

gy


225 :3 164

Discussion

In this study, we report that carnosine, significantly

decreased the high levels of IGFBP1 seen in diabetes. We

therefore provide a new mechanism by which carnosine

improves the metabolic control in diabetes and protect

against the development of complications in diabetes.

Even though carnosine shown protective effect in several

animal models for complications of diabetes (Pfister et al.

2011, Riedl et al. 2011, Ansurudeen et al. 2012, Yapislar &

Aydogan 2012, Brown et al. 2014, Peters et al. 2014, Menini

et al. 2015) and strongly suggested to be relevant for

diabetes complications in humans (Ahluwalia et al. 2011,

Kurashige et al. 2013) the exact mechanism of action is

still unraveled. Here we suggest a potential mechanism by

showing that carnosine complexly modulates IGFBP1 by

different mechanisms, i.e. indirectly by increasing the

insulin levels and directly by interfering with the HIF

dependent IGFBP1 induction.

Increased IGFBP1 has been associated with risk of

development of cardiovascular diseases (Wallander et al.

2007), atherosclerosis (Wang et al. 2012) and kidney

disease (Lindgren et al. 1996). It is therefore tentative to

propose that at least part of the protective effect of

carnosine in diabetes is mediated through its inhibiting

effect on IGFBP1.

Carnosine has a combined effect by both decreasing

IGFBP1 and increasing circulating IGF1 levels with

potential beneficial effect on glucose metabolism since

it reverses the characteristic pathologic changes of both

IGFBP1 and IGF1 in diabetes (Clauson et al. 1998). The

mechanism by which carnosine increases the liver

expression of IGF1 is not clearly known. Insulin has a

known direct effect on IGF1 production (Brismar et al.


1994) but a direct effect of the dipeptide or of the IGFBP1

on IGF1 expression cannot be excluded.

Moreover, carnosine increases threefold plasma insu-

lin levels in db/db animals that contribute directly to the

improvement of glucose levels. The increase of insulin

levels secondary to carnosine treatment is in agreement

with previous observations in other models of diabetes

(Nagai et al. 2003). It definitely contributes to the

repression of the carnosine in IGFBP1 in vivo since insulin

is a major regulator of the production of IGFBP1 from

the liver.




*

Nx

Nx +CAR Hx

Hx + C

AR0

5

10

15

20

*

Fold

cha

nges

to N

x (H

IF1α

)

+ +– –

N H

HIF1α

β-actin

(a)

(b)

1200

1000

400

Rel

ativ

e lu

cife

rase

uni

ts (

% f

rom

Nx)

200

0Nx Nx+CAR Hx Hx+CAR

Figure 5

Carnosine decreases the functional activity of HIF-1a. (a) Carnosine

downregulates HIF-1a in hypoxia in the HepG2 cells, *P!0.05 vs Hx.

(b) HepG2 cells were transiently transfected with HRE-luciferase and CMV-

Renilla reporter vectors and relative luciferase activity was measured after

24 h incubation in the presence or absence of 50 mmol carnosine solution

and of hypoxia. Bars represent mean of three experimentsGS.E.M. of

relative luciferase activity (firefly luciferase activity/Renilla luciferase

activity) normalized to the relative luciferase activity in cells exposed only

to normoxia. *P!0.05 (significantly different from corresponding HepG2

cells non-exposed to carnosine). The values represent the meanGS.E.M. of

four independent experiments.

****

††

(a)

(b)

(c)

****

†

NX Nx+CAR Hx Hx+CAR0

5

10

15

20

Veg

f exp

ress

ion

(arb

itrar

y un

it)

****

††


5

10

15

20

Pgk

1 ge

ne e

xpre

ssio

n(a

rbitr

ary

unit)


5

10

15

20

Bni

p3 g

ene

expr

essi

on(a

rbitr

ary

unit)

Figure 6

qPCR analysis of endogenous HIF-1a target genes (a) Vegf, (b) Pgk1 and

(c) Bnip3 in HepG2 cells treated with carnosine and subjected to hypoxia.†P!0.05 vs. Hx, ††P!0.01 vs. Hx, ****P!0.001 vs. Nx. The values represent

the meanGS.E.M. of three independent experiments.

Jou

rnal

of

En

do

crin

olo

gy


225 :3 165

Furthermore, hypoxia was recently identified as an

additional pathogenic factor in diabetic complications

beside hyperglycemia (Catrina 2014). IGFBP1 is highly

stimulated by hypoxia (Tazuke et al. 1998) and is a direct

target of HIF, which is the main adaptor of the cells to

hypoxia. We demonstrate in this study that carnosine

reduces IGFBP1 in diabetes by a complex mechanism

involving also a direct decrease of HIF stability and

function.

This is in agreement with a recent study that found a

destabilizing effect of carnosine on HIF-1a in a cancer cell


line (HCT-116) (Iovine et al. 2014). Furthermore, in a

mouse model of retina ischemia it has been shown that

carnosine treatment decrease HIF-1a at the protein level

after 6 h ischemia (Ji et al. 2014).

In conclusion, we were able to show that carnosine

decreased the pathological levels of IGFBP1 in db/db mice.

This effect was both by modulating the hypoxia-driven

IGFBP1 increase and by increasing the insulin levels and

was followed by improvement of blood glucose levels.




Jou

rnal

of

En

do

crin

olo

gy


225 :3 166

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/

JOE-14-0571.

Declaration of interest

The authors declare that there is no conflict of interest that could be

perceived as prejudicing the impartiality of the research reported.

Funding

We thank the Family Erling Persson Foundation and DFG Collaborative

Research Center 1118 for the financial support.

Acknowledgements

Inga-Lena Wivall Helleryd and Elvi Sandberg are acknowledged for the

excellent technical assistance.

References

Ahluwalia TS, Lindholm E & Groop LC 2011 Common variants in CNDP1

and CNDP2, and risk of nephropathy in type 2 diabetes. Diabetologia 54

2295–2302. (doi:10.1007/s00125-011-2178-5)

Ansurudeen I, Sunkari VG, Grunler J, Peters V, Schmitt CP, Catrina SB,

Brismar K & Forsberg EA 2012 Carnosine enhances diabetic wound

healing in the db/db mouse model of type 2 diabetes. Amino Acids 43

127–134. (doi:10.1007/s00726-012-1269-z)

Bang P, Eriksson U, Sara V, Wivall IL & Hall K 1991 Comparison of acid

ethanol extraction and acid gel filtration prior to IGF-I and IGF-II

radioimmunoassays: improvement of determinations in acid ethanol

extracts by the use of truncated IGF-I as radioligand. Acta Endocrinolo-

gica 124 620–629.

Boldyrev AA, Aldini G & Derave W 2013 Physiology and pathophysiology

of carnosine. Physiological Reviews 93 1803–1845. (doi:10.1152/physrev.

00039.2012)

Brismar K, Fernqvist-Forbes E, Wahren J & Hall K 1994 Effect of insulin on

the hepatic production of insulin-like growth factor-binding protein-1

(IGFBP-1), IGFBP-3, and IGF-I in insulin-dependent diabetes. Journal of

Clinical Endocrinology and Metabolism 79 872–878. (doi:10.1210/jcem.

79.3.7521354)

Brown BE, Kim CH, Torpy FR, Bursill CA, McRobb LS, Heather AK, Davies MJ

& van Reyk DM 2014 Supplementation with carnosine decreases

plasma triglycerides and modulates atherosclerotic plaque composition

in diabetic apo E(K/K) mice. Atherosclerosis 232 403–409.

(doi:10.1016/j.atherosclerosis.2013.11.068)

Catrina SB 2014 Impaired hypoxia-inducible factor (HIF) regulation by

hyperglycemia. Journal of Molecular Medicine 92 1025–1034.

(doi:10.1007/s00109-014-1166-x)

Clauson PG, Brismar K, Hall K, Linnarsson R & Grill V 1998 Insulin-like

growth factor-I and insulin-like growth factor binding protein-1 in a

representative population of type 2 diabetic patients in Sweden.

Scandinavian Journal of Clinical and Laboratory Investigation 58 353–360.

(doi:10.1080/00365519850186544)

Crossey PA, Jones JS & Miell JP 2000 Dysregulation of the insulin/IGF

binding protein-1 axis in transgenic mice is associated with hyper-

insulinemia and glucose intolerance. Diabetes 49 457–465.

(doi:10.2337/diabetes.49.3.457)

DoublierS, SeurinD,FouquerayB,VerpontMC,CallardP,StrikerLJ,StrikerGE,

Binoux M & Baud L 2000 Glomerulosclerosis in mice transgenic for


human insulin-like growth factor-binding protein-1. Kidney Inter-

national 57 2299–2307. (doi:10.1046/j.1523-1755.2000.00090.x)

Ezzat VA, Duncan ER, Wheatcroft SB & Kearney MT 2008 The role of IGF-I

and its binding proteins in the development of type 2 diabetes and

cardiovascular disease. Diabetes, Obesity & Metabolism 10 198–211.

(doi:10.1111/j.1463-1326.2007.00709.x)

Freedman BI, Hicks PJ, Sale MM, Pierson ED, Langefeld CD, Rich SS, Xu J,

McDonough C, Janssen B, Yard BA et al. 2007 A leucine repeat in the

carnosinase gene CNDP1 is associated with diabetic end-stage renal

disease in European Americans. Nephrology, Dialysis, Transplantation 22

1131–1135. (doi:10.1093/ndt/gfl717)

Gallant S, Semyonova M & Yuneva M 2000 Carnosine as a potential anti-

senescence drug. Biochemistry 65 866–868.

Guiotto A, Calderan A, Ruzza P & Borin G 2005 Carnosine and carnosine-

related antioxidants: a review. Current Medicinal Chemistry 12

2293–2315. (doi:10.2174/0929867054864796)

Heald AH, Cruickshank JK, Riste LK, Cade JE, Anderson S, Greenhalgh A,

Sampayo J, Taylor W, Fraser W, White A et al. 2001 Close relation of

fasting insulin-like growth factor binding protein-1 (IGFBP-1) with

glucose tolerance and cardiovascular risk in two populations.

Diabetologia 44 333–339. (doi:10.1007/s001250051623)

Hilding A, Hall K, Skogsberg J, Ehrenborg E & Lewitt MS 2003 Troglitazone

stimulates IGF-binding protein-1 by a PPARg-independent mechanism.

Biochemical and Biophysical Research Communications 303 693–699.

(doi:10.1016/S0006-291X(03)00403-0)

Hummel KP, Dickie MM & Coleman DL 1966 Diabetes, a new mutation

in the mouse. Science 153 1127–1128. (doi:10.1126/science.153.

3740.1127)

Iovine B, Oliviero G, Garofalo M, Orefice M, Nocella F, Borbone N, Piccialli V,

Centore R, Mazzone M, Piccialli G et al. 2014 The anti-proliferative

effect of L-carnosine correlates with a decreased expression of hypoxia

inducible factor 1a in human colon cancer cells. PLoS ONE 9 e96755.

(doi:10.1371/journal.pone.0096755)

Janssen B, Hohenadel D, Brinkkoetter P, Peters V, Rind N, Fischer C,

Rychlik I, Cerna M, Romzova M, de Heer E et al. 2005 Carnosine as a

protective factor in diabetic nephropathy: association with a leucine

repeat of the carnosinase gene CNDP1. Diabetes 54 2320–2327.

(doi:10.2337/diabetes.54.8.2320)

Ji YS, Park JW, Heo H, Park JS & Park SW 2014 The neuroprotective effect of

carnosine (b-alanyl-L-histidine) on retinal ganglion cell following

ischemia-reperfusion injury. Current Eye Research 39 634–641.

(doi:10.3109/02713683.2013.855235)

Kaelin WG Jr & Ratcliffe PJ 2008 Oxygen sensing by metazoans: the central

role of the HIF hydroxylase pathway. Molecular Cell 30 393–402.

(doi:10.1016/j.molcel.2008.04.009)

Kubomura D, Matahira Y, Nagai K & Niijima A 2010a Effect of anserine

ingestion on hyperglycemia and the autonomic nerves in rats and

humans. Nutritional Neuroscience 13 183–188. (doi:10.1179/

147683010X12611460764363)

Kubomura D, Matahira Y, Nagai K & Niijima A 2010b Effect of anserine

ingestion on the hyperglycemia and autonomic nerves in rats and

humans. Nutritional Neuroscience 13 123–128. (doi:10.1179/

147683010X12611460764048)

Kurashige M, Imamura M, Araki S, Suzuki D, Babazono T, Uzu T, Umezono T,

Toyoda M, Kawai K, Imanishi M et al. 2013 The influence of a single

nucleotide polymorphism within CNDP1 on susceptibility to diabetic

nephropathy in Japanese women with type 2 diabetes. PLoS ONE 8

e54064. (doi:10.1371/journal.pone.0054064)

Lenz GR & Martell AE 1964 Metal complexes of carnosine. Biochemistry 3

750–753. (doi:10.1021/bi00894a002)

Lewitt MS, Denyer GS, Cooney GJ & Baxter RC 1991 Insulin-like growth

factor-binding protein-1 modulates blood glucose levels. Endocrinology

129 2254–2256. (doi:10.1210/endo-129-4-2254)

Lindgren BF, Odar-Cederlof I, Ericsson F & Brismar K 1996 Decreased

bioavailability of insulin-like growth factor-I, a cause of catabolism in

hemodialysis patients? Growth Regulation 6 137–143.




http://dx.doi.org/10.1007/s00125-011-2178-5

http://dx.doi.org/10.1007/s00726-012-1269-z

http://dx.doi.org/10.1152/physrev.00039.2012

http://dx.doi.org/10.1152/physrev.00039.2012

http://dx.doi.org/10.1210/jcem.79.3.7521354

http://dx.doi.org/10.1210/jcem.79.3.7521354

http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.068

http://dx.doi.org/10.1007/s00109-014-1166-x

http://dx.doi.org/10.1080/00365519850186544

http://dx.doi.org/10.2337/diabetes.49.3.457

http://dx.doi.org/10.1046/j.1523-1755.2000.00090.x

http://dx.doi.org/10.1111/j.1463-1326.2007.00709.x

http://dx.doi.org/10.1093/ndt/gfl717

http://dx.doi.org/10.2174/0929867054864796

http://dx.doi.org/10.1007/s001250051623

http://dx.doi.org/10.1016/S0006-291X(03)00403-0

http://dx.doi.org/10.1126/science.153.3740.1127

http://dx.doi.org/10.1126/science.153.3740.1127

http://dx.doi.org/10.1371/journal.pone.0096755


http://dx.doi.org/10.3109/02713683.2013.855235

http://dx.doi.org/10.1016/j.molcel.2008.04.009

http://dx.doi.org/10.1179/147683010X12611460764363

http://dx.doi.org/10.1179/147683010X12611460764363

http://dx.doi.org/10.1179/147683010X12611460764048

http://dx.doi.org/10.1179/147683010X12611460764048

http://dx.doi.org/10.1371/journal.pone.0054064

http://dx.doi.org/10.1021/bi00894a002

http://dx.doi.org/10.1210/endo-129-4-2254



Jou

rnal

of

En

do

crin

olo

gy


225 :3 167

Menini S, Iacobini C, Ricci C, Fantauzzi CB & Pugliese G 2015

Protection from diabetes-induced atherosclerosis and renal disease by

D-carnosine-octylester: effects of early vs late inhibition of advanced

glycation end-products in Apoe-null mice. Diabetologia 58 845–853.

(doi:10.1007/s00125-014-3467-6)

Mesotten D, Delhanty PJ, Vanderhoydonc F, Hardman KV, Weekers F,

Baxter RC & Van Den Berghe G 2002 Regulation of insulin-like growth

factor binding protein-1 during protracted critical illness. Journal of

Clinical Endocrinology and Metabolism 87 5516–5523. (doi:10.1210/jc.

2002-020664)

Munoz MT, Barrios V, Pozo J & Argente J 1996 Insulin-like growth factor I,

its binding proteins 1 and 3, and growth hormone-binding protein in

children and adolescents with insulin-dependent diabetes mellitus:

clinical implications. Pediatric Research 39 992–998. (doi:10.1203/

00006450-199606000-00011)

Murphy LJ 2000 Overexpression of insulin-like growth factor binding

protein-1 in transgenic mice. Pediatric Nephrology 14 567–571.

(doi:10.1007/s004670000347)

Nagai K, Niijima A, Yamano T, Otani H, Okumra N, Tsuruoka N, Nakai M &

Kiso Y 2003 Possible role of L-carnosine in the regulation of blood

glucose through controlling autonomic nerves. Experimental Biology

and Medicine 228 1138–1145.

Peters V, Kebbewar M, Jansen EW, Jakobs C, Riedl E, Koeppel H, Frey D,

Adelmann K, Klingbeil K, Mack M et al. 2010 Relevance of allosteric

conformations and homocarnosine concentration on carnosinase

activity. Amino Acids 38 1607–1615. (doi:10.1007/s00726-009-0367-z)

Peters V, Schmitt CP, Zschocke J, Gross ML, Brismar K & Forsberg E 2012

Carnosine treatment largely prevents alterations of renal carnosine

metabolism in diabetic mice. Amino Acids 42 2411–2416. (doi:10.1007/

s00726-011-1046-4)

Peters V, Riedl E, Braunagel M, Hoger S, Hauske S, Pfister F, Zschocke J,

Lanthaler B, Benck U, Hammes HP et al. 2014 Carnosine treatment in

combination with ACE inhibition in diabetic rats. Regulatory Peptides

194–195 36–40. (doi:10.1016/j.regpep.2014.09.005)

Pfister F, Riedl E, Wang Q, vom Hagen F, Deinzer M, Harmsen MC, Molema G,

Yard B, Feng Y & Hammes HP 2011 Oral carnosine supplementation

prevents vascular damage in experimental diabetic retinopathy.

Cellular Physiology and Biochemistry 28 125–136. (doi:10.1159/

000331721)

Powell DR, Allander SV, Scheimann AO, Wasserman RM, Durham SK &

Suwanichkul A 1995 Multiple proteins bind the insulin response

element in the human IGFBP-1 promoter. Progress in Growth Factor

Research 6 93–101. (doi:10.1016/0955-2235(95)00034-8)

Rajaram S, Baylink DJ & Mohan S 1997 Insulin-like growth factor-binding

proteins in serum and other biological fluids: regulation and functions.

Endocrine Reviews 18 801–831. (doi:10.1210/edrv.18.6.0321)

Riedl E, Pfister F, Braunagel M, Brinkkotter P, Sternik P, Deinzer M,

Bakker SJ, Henning RH, van den Born J, Kramer BK et al. 2011 Carnosine


prevents apoptosis of glomerular cells and podocyte loss in STZ diabetic

rats. Cellular Physiology and Biochemistry 28 279–288. (doi:10.1159/

000331740)

Sauerhofer S, Yuan G, Braun GS, Deinzer M, Neumaier M, Gretz N, Floege J,

Kriz W, van der Woude F & Moeller MJ 2007 L-carnosine, a substrate of

carnosinase-1, influences glucose metabolism. Diabetes 56 2425–2432.

(doi:10.2337/db07-0177)

Schrijvers BF, De Vriese AS & Flyvbjerg A 2004 From hyperglycemia to

diabetic kidney disease: the role of metabolic, hemodynamic, intra-

cellular factors and growth factors/cytokines. Endocrine Reviews 25

971–1010. (doi:10.1210/er.2003-0018)

Semenza GL 2011 Oxygen sensing, homeostasis, and disease. New England

Journal of Medicine 365 537–547. (doi:10.1056/NEJMra1011165)

Stephens RH, McElduff P, Heald AH, New JP, Worthington J, Ollier WE &

Gibson JM 2005 Polymorphisms in IGF-binding protein 1 are associated

with impaired renal function in type 2 diabetes. Diabetes 54 3547–3553.

(doi:10.2337/diabetes.54.12.3547)

Tazuke SI, Mazure NM, Sugawara J, Carland G, Faessen GH, Suen LF,

Irwin JC, Powell DR, Giaccia AJ & Giudice LC 1998 Hypoxia stimulates

insulin-like growth factor binding protein 1 (IGFBP-1) gene expression

in HepG2 cells: a possible model for IGFBP-1 expression in fetal

hypoxia. PNAS 95 10188–10193. (doi:10.1073/pnas.95.17.10188)

Van Buul-Offers SC, Van Kleffens M, Koster JG, Lindenbergh-Kortleve DJ,

Gresnigt MG, Drop SL, Hoogerbrugge CM, Bloemen RJ, Koedam JA &

Van Neck JW 2000 Human insulin-like growth factor (IGF) binding

protein-1 inhibits IGF-I-stimulated body growth but stimulates growth

of the kidney in snell dwarf mice. Endocrinology 141 1493–1499.

(doi:10.1210/endo.141.4.7418)

Wallander M, Norhammar A, Malmberg K, Ohrvik J, Ryden L & Brismar K

2007 IGF binding protein 1 predicts cardiovascular morbidity and

mortality in patients with acute myocardial infarction and type 2

diabetes. Diabetes Care 30 2343–2348. (doi:10.2337/dc07-0825)

Wang J, Razuvaev A, Folkersen L, Hedin E, Roy J, Brismar K & Hedin U 2012

The expression of IGFs and IGF binding proteins in human carotid

atherosclerosis, and the possible role of IGF binding protein-1 in the

regulation of smooth muscle cell proliferation. Atherosclerosis 220

102–109. (doi:10.1016/j.atherosclerosis.2011.10.032)

Yamano T, Niijima A, Iimori S, Tsuruoka N, Kiso Y & Nagai K 2001 Effect of

L-carnosine on the hyperglycemia caused by intracranial injection of

2-deoxy-D-glucose in rats. Neuroscience Letters 313 78–82. (doi:10.1016/

S0304-3940(01)02231-5)

Yapislar H & Aydogan S 2012 Effect of carnosine on erythrocyte

deformability in diabetic rats. Archives of Physiology and Biochemistry

118 265–272. (doi:10.3109/13813455.2012.714790)

Zachrisson I, Dahlquist G, Wallensteen M & Brismar K 2000 Insulin-like

growth factor binding protein-1 as glucose regulator in adolescent boys

with type 1 diabetes. Acta Paediatrica 89 1044–1049. (doi:10.1111/

j.1651-2227.2000.tb03348.x)

Received in final form 23 March 2015Accepted 30 March 2015Accepted Preprint published online 13 April 2015


http://dx.doi.org/10.1007/s00125-014-3467-6

http://dx.doi.org/10.1210/jc.2002-020664

http://dx.doi.org/10.1210/jc.2002-020664

http://dx.doi.org/10.1203/00006450-199606000-00011

http://dx.doi.org/10.1203/00006450-199606000-00011

http://dx.doi.org/10.1007/s004670000347

http://dx.doi.org/10.1007/s00726-009-0367-z

http://dx.doi.org/10.1007/s00726-011-1046-4

http://dx.doi.org/10.1007/s00726-011-1046-4

http://dx.doi.org/10.1016/j.regpep.2014.09.005

http://dx.doi.org/10.1159/000331721

http://dx.doi.org/10.1159/000331721

http://dx.doi.org/10.1016/0955-2235(95)00034-8

http://dx.doi.org/10.1210/edrv.18.6.0321

http://dx.doi.org/10.1159/000331740

http://dx.doi.org/10.1159/000331740

http://dx.doi.org/10.2337/db07-0177

http://dx.doi.org/10.1210/er.2003-0018

http://dx.doi.org/10.1056/NEJMra1011165


http://dx.doi.org/10.1073/pnas.95.17.10188

http://dx.doi.org/10.1210/endo.141.4.7418

http://dx.doi.org/10.2337/dc07-0825

http://dx.doi.org/10.1016/j.atherosclerosis.2011.10.032

http://dx.doi.org/10.1016/S0304-3940(01)02231-5

http://dx.doi.org/10.1016/S0304-3940(01)02231-5

http://dx.doi.org/10.3109/13813455.2012.714790

http://dx.doi.org/10.1111/j.1651-2227.2000.tb03348.x

http://dx.doi.org/10.1111/j.1651-2227.2000.tb03348.x



Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1

Documents

Transcript of Carnosine decreases IGFBP1 production in db/db mice through suppression of HIF-1