exogenous metallothionein potentiates the insulin response at normal glucose concentrations in...

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Article Type: Short Communication Short Communication

Exogenous Metallothionein Potentiates the Insulin Response at Normal

Glucose Concentrations in INS-1E Beta Cells without disturbing Intracellular

ZnT8 Expression

S. B. Nygaarda, N.S. Lunda, A. Larsena, N. Pedersena, J. Rungbya and K. Smidta

aDepartment of Biomedicine - Pharmacology, Aarhus University, Aarhus, Denmark Running title: Exogenous metallothionein and insulin Author for correspondence:

Kamille Smidt

Department of Biomedicine, Aarhus University

Wilhelm Meyers Allé 4

8000 Aarhus

Denmark

E-mail: [email protected]

As a consequence of the global epidemic of obesity, the incidence of type 2 diabetes (T2D) is

increasing worldwide. T2D is characterized by hyperglycaemia, hyper-insulinaemia and a

reduced insulin response in muscular and fatty tissue. Over time, an increased insulin demand

leads to cellular fatigue and death of the insulin producing β-cells. In response, the T2D

patients become insulin-dependent and subjected to the boundaries of life-long insulin

This article is protected by copyright. All rights reserved.

treatment. Preservation of β-cell insulin secretion and a sufficient β-cell mass is thus a corner

stone in optimal TD2 treatment.

Physiologically, β-cell function and survival are closely related to zinc homeostasis. Zinc is

essential for the primary functions of β-cells; insulin biosynthesis, insulin storage and insulin

secretion and zinc are co-secreted with insulin in response to glucose stimulation.

Hypozincaemia accompanied by hyperzincuria are often present in diabetic subjects (1). On

the other hand, fluctuations in extracellular zinc levels can become toxic for β-cells if high

concentrations arise locally during stress or increased insulin secretion (2).

Regulation of cellular free zinc homeostasis is orchestrated by two categories of zinc-carrying

proteins; the zinc buffering metallothioneins (MTs) and the zinc transporters also known as

ZnTs (Zinc Transporters) and ZIPs, (Zrt-and Irt-like proteins). The ZnTs are responsible for

Zn2+ efflux from the cytoplasm to the extracellular matrix and into intra-cellular organelles

like insulin-containing vesicles whereas the ZIPs transport Zn2 + in the opposite direction (3).

MTs tightly regulate the intracellular level of free zinc and the levels of MTs are relatively

high in the pancreas (3, 4) suggesting that MTs are involved in the normal function of the

gland. New studies link dysregulation or dysfunction of zinc transporting proteins with

impaired insulin processing and impaired glucose metabolism (5, 6). Furthermore,

polymorphisms in genes encoding for isoforms of MTs have been related to the development

of type 2 diabetes and to the extent of diabetic complications (7). Transgenic mice, with β-

cell specific over-expression of MT-2 display significantly reduced β-cell death and the mice

have a better preservation of insulin production when exposed to β-cell toxic Streptozotozin

(8). Moreover, a specific neuroprotective role has been postulated for extracellular MT (9).

Here, we hypothesize that increasing MT levels by means of exogenous Zn7-MT-2A will be

beneficial for β-cell function by contributing to an optimal zinc supply and regulation, testing

this hypothesis in the glucose-sensitive, insulin-producing INS-1E β–cell culture.

Methods

Cell culture. INS-1E (rat), originally kindly provided by Prof. C.B. Wollheim, Switzerland,

was employed in this study. The INS-1E cells are cultured in a CO2 atmosphere in complete

RPMI 1640 supplemented with 11 mM glucose, 10% (v/v) heat inactivated foetal bovine

serum, 50 µM β-mercaptoethanoel, 2 mM L-Glutamine, 100 U/ml penicillin and 100 μg/ml

streptomycin as previously described (6).


Metallothionein exposure. INS-1E cells were plated in 6-well plates (NUNC) in standard 11

mM glucose cell medium for minimum 24 hr prior to experiments. To ensure stable

experimental conditions, 152 nM Zn7-Metallothionein-2A (Zn7-MT-2A) (Bestenbalt LLC,

Estonia) dissolved in 0.9% saline was supplied to the cell medium 6 hr before the

experimental start. The cells were then exposed to RPMI 1640 with either 6 or 21 mM

glucose and 152 nM Zn7-MT-2A for another 24 hr. In order to maintain the Zn7-MT-2A

concentration, an additional 152 nM of Zn7-MT-2A was supplied every 6th hour until cell

harvest. Control cells were not subjected to Zn7-MT-2A but exposed to similar changes of

media during the experimental period. Following the 24 hr of Zn7-MT-2A/glucose exposure,

INS-1E cells were either harvested for RT-PCR or used for measurements of insulin content

and insulin secretion. Samples and controls were performed in replicates of 5-6 for RT-PCR

and in 3-4 for insulin measurement.

β-cell insulin assay. Following the 6+24-hr of Zn7-MT-2A exposure, the INS1E- cells were

incubated for 2 hr in a Krebs-Ringer bicarbonate HEPES buffer (KRBH) at pH 7.4 containing

115 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.6 mM CaCl2, 1.2 mM KH2PO4, 20 mM

HEPES, 5mM NaHCO3, 0.1% (v/v) human serum albumin (HSA) (Sigma, Denmark) and

supplemented with 6.6 mM or 21 mM glucose. The incubation medium was collected for

analysis of insulin secretion whereas the cells were collected in Earle’s basal medium

(Invitrogen, Denmark) by scarping with a rubber policeman followed by centrifugation. The

pellet was split in to and re-suspended in a buffer comprising 0.75 % (v/v) glycine and 0.25%

(v/v) bovine serum albumin with pH 8.8 for intracellular insulin determination, or in 0.1% M

NaOH for protein determination. Total protein was determined using the BCA Protein Assay

Reagent Kit from Pierce, US (Bie & Berntsen A/S, Denmark). Insulin concentration was

determined using the ultra-sensitive Rat Insulin Elisa Kit from DRG Diagnostics (VWR,

Denmark).

RNA extraction and cDNA synthesis. RNA was extracted using RNeasy Mini Kit Qiagen

(VWR, Denmark) and treated with DNase (VWR, Denmark). Reverse transcription was

carried out using 500 ng total RNA, ImProm-IITM Reverse Transcription System (Promega,

Denmark) and oligo dT18 primers (TAC, Copenhagen).


Real-time PCR. Quantitative real-time PCR was performed in duplicate using IQ Sybr Green

Supermix (Bio-Rad, Denmark) in a MyiQ Two-Color Real-time PCR detection system (Bio-

Rad, Denmark). For all reactions, a melting curve was included. The results were analysed

with iQTM5 Optical System Software, Version 2.1. Starting quantities were calculated from a

standard curve. Values were normalized to two stable genes as previously described (10).

Statistical analysis. Data are presented as mean values with the standard error of the mean

(SEM). Unpaired T-test was used to determine statistical significance between two groups

with p< 0.05 as the level of statistical significance.

Results

Zn7-MT-2A increases β-cell insulin content and stimulates insulin secretion at normal

glucose levels

Zn7-MT-2A treatment induced the insulin secretion and elevated the intracellular insulin

content at 6.6 mM glucose (p=0.0001 and p=0.0325, respectively) whereas insulin secretion

at 21 mM glucose was unaffected by Zn7-MT-2A supplementation (fig. 1-A and B). As

expected, the level of insulin secretion was increased by 21 mM glucose compared to 6.6 mM

glucose in control cells while leaving the intracellular insulin content unaffected.

The transcriptional control of ZnT-3 and ZnT-8 remains sensitive to increasing glucose

concentration in the presence of exogenous MT-2A

Compared to normo-glucose levels (6.6 mM) exposure to a high glucose concentration (21

mM) is immediately reflected in the intracellular zinc homeostasis (fig. 1-C, D and E). At

high glucose concentrations, ZnT-3 was significantly up-regulated (p=0.0089), whereas ZnT-

5 (p=0.0075) and ZnT-8 (p=0.0061) were significantly down-regulated. Exposure to

exogenous Zn7-MT-2A did not seem to alter the zinc homeostatic response to increased

glucose.

Zn7-MT-2A exposure increases the glucose-induced down-regulation of MT-1A expression

There was no significant down-regulation of the MT-1A expression in INS-1E cells at high

glucose compared to normal glucose levels in the control cells, however, exposing INS-1E

cells to exogenous Zn7-MT-2A in a high glucose environment led to a significant down-

regulation of MT-1A transcription compared to a low glucose environment (p<0.0001) (fig.

1-F).


MT-3 gene expression is strongly up-regulated by high glucose independently of the presence

of exogenous MT-A2.

MT-3 gene expression was significantly up-regulated at 21 mM glucose in control cells (2.8-

fold, p=0.0010) as well as in Zn7-MT-2A exposed cells (3.0-fold, p<0.0001) compared to 6.6

mM glucose (fig. 1-G).

Discussion

The present study confirms that INS-1E is a physiologically functioning β-cell line displaying

a glucose-sensitive insulin response as well as glucose-induced alteration in intra-cellular

zinc homeostasis similar to previous report on the gene expression of ZnT-3, ZnT-5 and ZnT-

8 (5). Offering exogenous Zn7-MT-2A to this β-cell line, we observed direct effects on the

insulin production and insulin response; i.e. the presence of exogenous Zn7-MT-2A

dramatically induces insulin secretion and increases the intracellular insulin content at 6.6

mM glucose.

Increased insulin secretion is a cardinal feature of the sulfonylureas currently used in the

treatment of T2D. Pathway analysis has previously indicated that several genes involved in

increased activity of the secretory system such as SNARE 25 (11) could be involved in the

pharmacodynamics of sulfonylureas. The underlying mechanisms are, however, not known

but could include fluctuations in intra-cellular free zinc as such as known to be part of the

beta-cell response to glucose stimulation (12).

Much interest in the potential protective aspects of MTs towards T2D has focused on the

anti-oxidant properties of these proteins. Recent research has on the other hand indicated that

increased MT activity might not always be beneficial but in fact lead to an over-quenching of

free oxygen radicals which could lead to insulin resistance, e.g. through increased expression

of insulin signalling inhibitor PTP1B (13). So far, this phenomenon has only been described

in adipocytes, highlighting the substantial regional differences in the role of MTs in cellular

function.

Zn7-MT-2A can carry up to seven zinc ions potentially available for cellular demands and

could thus, supply extra zinc in the current experiments. In order to elucidate this effect, we

performed a pilot study showing no transcriptional effect of zinc in the concentration found

during the experimental conditions (data not shown). The main source of zinc during these

cell culturing experiments originates from the addition of 10% foetal calf serum which is 1/10

of the zinc concentration (14.0-18.0 µM) measured in plasma (14, 15), i.e. the zinc

concentration in our experiment is somewhat lower than the in vivo situation.


It is well known that the presence and function of the vesicular zinc transporters ZnT-3, and

ZnT-8 is of importance for optimal insulin production in β-cells and the expression of ZnT-3,

ZnT-5 and ZnT-8 respond to changes in glucose concentrations which we also describe in

this present study (6). Our group has recently established (16) that changing the ambient zinc

concentration affects the expression of the intracellular zinc transporters, ZnT-8 and ZnT-3,

yet in our present experiments, addition of Zn7-MT-2A did not affect these zinc-sensitive

intracellular zinc regulators. This indicates that exogenous MT does not act merely as a zinc

supplement. Supporting this hypothesis, we found no gene induction of MT-1A upon Zn7-

MT-2A addition. The MT-1A gene expression is known to be controlled by the metal

response element-binding transcriptional factor (MTF)-1 (17) and MT-1A expression is

readily induced by zinc supplementation (7).

It is possible that part of the effects of exogenous Zn7-MT-2A exposure is related to an

internalisation of the protein. Studies have shown that Megalin, a protein belonging to the

low density lipoprotein (LDL) receptor family, mediates endocytotic up-take of MT-2 and

MT-1 in neuron cultures (18). Additionally, recent studies suggest that sorLA/LR11, another

LDL-receptor subtype,- known to be expressed in the pancreas - could be responsible for the

uptake of MT-2A to collecting duct epithelial cells (194).

As MT and ZnT regulate the intra-cellular zinc homeostasis in concert with the ZIP family,

altered expression of these proteins regulating zinc influx to the cytoplasm might also

contribute to the response of the beta cells to exogenous Zn7-MT-2A. Up-regulation of key

ZIPs, i.e. ZIP6, 7 and 8 and the resultant increase in intracellular free zinc is part of the initial

response of beta cells towards high glucose (12). A higher baseline value of zinc e.g. caused

by high ambient zinc increasing the intracellular zinc level can ameliorate this response (12).

If Zn7-MT-2A is indeed internalized, the resulting increase in the zinc buffering capacities

of the cytoplasm might slightly reduce free cytoplasmatic zinc and hence potentiate the

glucose-induced ZIP up-regulation in this way increasing insulin secretion.

Although unaffected by extracellular MT supplements, we did on the other hand observe a

down-regulation of MT-1A at high glucose concentration compared to low glucose

concentration most likely caused by glucose-induced cAMP production known to have this

effect on MT expression (12). Together with the reduced intracellular insulin content at the

21 mM glucose concentration, these findings could reflect that the INS-1E cells are at their

maximum insulin secretion capacity when exposed to 21 mM glucose and might reflect

cellular stress under this condition. This concurs with previous findings that high glucose

levels increases β-cell stress and may induce apoptosis (6). Although not significantly


improving insulin secretion at high glucose concentrations, an increased MT presence might

still affect cellular survival due to the antioxidant scavenging properties of this protein.

Interestingly, high glucose levels are reflected by the concomitant increase in MT-3 gene

expression whereas MT-3 is unaffected by the presence of extra-cellular Zn7-MT-2A. This

result emphasizes that MT-3 and MT-1A react fundamentally different to stressors of β-cell

function and therefore might play different roles in protecting β-cell function.

In conclusion, this present study shows that exogenous, extra-cellular Zn7-MT-2A potentiates

insulin production and secretions, revealing a possible therapeutic potential of MT

administration for enhancing β-cell insulin processing capacity

Acknowledgements

The authors thank E. Cartsensen for her help with cell cultures and insulin measurements and

K. Skjødt for her help with the Q-PCR technique. INS-1E cells were kindly provided by

Professor Claes Wollheim and Pierre Maechler, Geneva, Switzerland.

Funding

This present study was funded by the A.P. Møller and Chastine Mc-Kinney Møller

Foundation and the Desirée and Niels Ydes Foundation.

Conflicts of interest

The authors declare that there are no conflicts of interest.

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13. Haynes V, Connor T, Tchernof A, Vidal H, Dubois S Metallothionein 2a gene expression is increased in subcutaneous adipose tissue of type 2 diabetic patients Mol Genet Metab 2013 (1) 90-4 10.1016/j.ymgme Epub 2012 Oct 18

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Fig. 1. Effects of exogenous MT-2A on the insulin response and on the gene expression

of specific metalloproteins INS-1E cells.

Intracellular insulin content (A), insulin secretion (B) and gene expression of ZnT-3 (C),

ZnT-5 (D), ZnT-8 (E), MT-1A (F) and MT-3 (G) after 6 hr of pre-conditioning with MT-2A

followed by 24 hr of stimulation of INS-1E cells with MT-2A at 6.6 mM or 21 mM glucose.

Data are mean and SEM (* p < 0.05, ** p < 0.01 or ***p < 0.001). N=3-6.


Insulin content

6.6 m

M

6.6 m

M + M

T-2A

21 m

M

21 m

M + M

T-2A

0

5

10

15

20

* **in

trac

ellu

lar

insu

lin,

μg/

mg

prot

ein

Insulin secretion

6.6

mM

6.6 m

M + M

T-2A

21 m

M

21 m

M + M

T-2A

0.0

0.5

1.0

1.5

2.0

***

P=0.0801

*

Insu

lin s

ecre

tion

,μ

g/m

g p

rote

in

ZnT-3 expression

6.6 m

M

6.6

mM

+ M

T-2A

21 m

M

21 m

M +

MT-2

A

0

1

2

3

4

5

6

7

8

*****

Gen

e ex

pre

ssio

n

ZnT-5 expression

6.6 m

M

6.6

mM

+ M

T-2A

21 m

M

21 m

M +

MT-2

A

0

1

2

3

**

Gen

e ex

pre

ssio

n

ZnT-8 expression

6.6 m

M

6.6 m

M + M

T-2A

21 m

M

21 m

M + M

T-2A

0.0

0.5

1.0

1.5

2.0 **

***

Gen

e ex

pre

ssio

n

MT-1A expression

6.6 m

M

6.6 m

M + M

T-2A

21 m

M

21 m

M + M

T-2A

0.0

0.2

0.4

0.6

0.8

***

Gen

e ex

pre

ssio

n

MT-3 expression

6.6

mM

6.6

mM

+ M

T-2A

21 m

M

21 m

M +

MT-2

A

0.0

2.5

5.0

7.5

10.0

*****

Gen

e ex

pre

ssio

n

E F

C D

A B

G

exogenous metallothionein potentiates the insulin response at normal glucose concentrations in...

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