The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and (2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice.
Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (db/db mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn2+ concentration with Fluozin-3, and NAD(P)H by autofluorescence.
Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchanged. Ex vivo, islet Mt1 and Mt2 mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10–30 mmol/l vs 2–5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of MT1E, MT1X and MT2A were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice, Mt1-Mt2 double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-Mt1 mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both Mt1-Mt2 double-KO and Tg-Mt1 models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca2+ concentration and NAD(P)H levels, or intracellular Zn2+ concentration vs WT mice. In MIN6 cells, knockdown of MT1 but not MT2 potentiated GSIS, suggesting that Mt1 rather than Mt2 affects beta cell function.
These findings implicate Mt1 as a negative regulator of insulin secretion. The downregulation of Mt1 is associated with beta cell compensation in obesity, whereas increased Mt1 accompanies beta cell failure and type 2 diabetes.
Type 2 diabetes results from the complex interplay of genetic and environmental risk factors, among which obesity plays a predominant role. Interestingly, most obese individuals compensate for insulin resistance by increasing insulin secretion, thereby maintaining normoglycaemia at the price of hyperinsulinaemia. However, this compensation can be sidestepped by a phase of decompensation in which beta cells fail to uphold an adequate secretory response [1,2,3]. This leads to hyperglycaemia and subsequent glucotoxic alterations of beta cell mass and function [4, 5]. Identifying genes involved in beta cell compensation in obesity, and in beta cell failure in type 2 diabetes may provide new insights into beta cell pathophysiology and reveal novel therapeutic targets to preserve beta cell function in individuals with (pre) type 2 diabetes.
Metallothioneins (MTs) are a family of low molecular mass, cysteine-rich, metal-binding proteins, the (patho)physiological functions of which have not been fully characterised. Their predominant roles are heavy metal detoxification, metal ion (including zinc) homeostasis, and the regulation of cellular redox status and antioxidant defences. Among the four different murine genes encoding MTs, Mt1 and Mt2 are expressed ubiquitously, Mt3 is mainly expressed in neurons and Mt4 is expressed in squamous epithelium cells. In humans, in addition to MT2 (also known as MT2A), MT3 and MT4, there are eight MT1 isoforms for a total of 11 functional MT genes [6, 7]. Interestingly, polymorphisms in MT1A and MT2A have been associated with increased risk for type 2 diabetes and diabetic complications [8,9,10]. Moreover, MT1E, MT1M, MT1X and MT2A mRNA levels were significantly upregulated in islets obtained by laser-capture microdissection (LCM) of pancreatic sections from type 2 diabetes donors . However, the role of MTs in the pathophysiology of type 2 diabetes remains unclear. We, therefore, verified whether changes in Mt1 and/or Mt2 gene expression plays a role in the modulation of beta cell function.
Fura-2 leakage resistant (Fura-2 LR), diazoxide (Dz), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and ZnCl2 were from Sigma (St Louis, MI, USA). N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) was from Abcam (Cambridge, UK). Fluozin-3, control non-targeting small interfering RNA (siRNA), ON-TARGETplus SMARTpool siRNA and DharmaFECT3 (transfection reagent) were from Thermo Fisher Scientific (Lafayette, CO, USA).
Six- and 16-week-old male and female C57BL/KsJ db/db mice and age-matched lean control mice (C57BL/KsJ), 16-week-old male and female C57BL/6J ob/ob mice and age-matched lean control mice, and the 16-week-old wild-type (WT) male mice (C57BL/6JAusb) used in diet-induced obesity experiments were from the Garvan Institute breeding colonies (Australian BioResources, Moss Vale, NSW, Australia). Male Mt1-Mt2 double-knockout (KO) mice at 4–5 months of age (129S7/SvEvBrd-Mt1tm1BriMt2tm1Bri/J; herein referred to as KO mice) and their sex- and age-matched controls (129S1/SvImJ) originated from the Jackson Laboratory (Bar Harbor, MA, USA). Male transgenic mice overexpressing Mt1 under the control of its natural promoter at 3 and 9 months of age (B6.Cg-Tg(Mt1)174Bri/J; referred to as Tg-Mt1) and their sex- and age-matched controls (C57BL/6 J) were also from the Jackson Laboratory. All animals were bred in the local animal facility of the health sciences sector at UCLouvain. Mice were housed under a controlled temperature of 22°C and a 12 h light cycle, with ad libitum access to water and chow (8% energy from fat, 21% energy from protein and 71% energy from carbohydrate [wt/wt]; 10.9 kJ/g; Gordon’s Specialty Stock Feeds, Yanderra, NSW, Australia) or a high-fat diet (HFD; 23% energy from fat, 19.4% energy from protein and 57.6% energy from carbohydrate and fibres [wt/wt for all]; 20.1 kJ/g; catalogue no. SF03-020; Specialty Feeds, Glen Forest, WA, Australia). Mice were randomly assigned to experimental groups using an odd/even number method by unblinded experimenters. All experiments were approved by the Institutional Committee on Animal Experimentation of the Health Sciences Sector at UCLouvain (Project 2013/UCL/MD/016) and the Garvan Institute/St Vincent’s Hospital Animal Experimentation Ethics Committee.
Human islets were obtained from 24 non-diabetic and 12 diabetic individuals at the Tom Mandel Islet Transplant Program, Melbourne . Human islets were isolated from heart-beating, brain-dead donors and approved for use under the ethics reference HREC011/04 (St Vincent’s Hospital Human Research Ethics Committee). Characteristics of donors and islet preparations are listed in the electronic supplementary material (ESM) Table 1. To evaluate the effects of glucose on MT gene expression, islets were obtained from three non-diabetic donors through the JDRF award 31-2008-416 (European Consortium for Islet Transplantation [ECIT] Islet for Basic Research program) and approved for use under the ethics reference B403/2017/05JUL/355 (Comité d’éthique hospitalo-facultaire Saint-Luc, UCLouvain). Characteristics of these donors are indicated in ESM Table 2. Written informed consent was obtained from next of kin and all experiments were performed in accordance with relevant guidelines and regulations.
Glucose and insulin tolerance tests
Intraperitoneal (i.p.) glucose tolerance tests (i.p.GTTs) and fasting-refeeding tests were performed on overnight-fasted mice and i.p. insulin tolerance tests (i.p.ITTs) on 4 h-fasted mice. For fasting-refeeding tests, blood glucose levels were measured in fasted mice and 1 h after refeeding. For i.p.GTTs and i.p.ITTs, mice were injected i.p. with glucose (2 g/kg of body weight) or insulin (0.75 U/kg of body weight; Actrapid, Novo Nordisk, Bagsværd, Denmark) and blood glucose levels were measured at the indicated time points using a FreeStyle Precision Neo glucometer (Abbott, Wavre, Belgium).
Tissue collection and histological analysis
Mice were killed by cervical dislocation and trunk blood collected and centrifuged for measurement of plasma insulin levels by ultra-sensitive ELISA (Crystal Chem, Downers Grove, IL, USA). The pancreas, liver, white adipose tissue (WAT) fat pads (epidydimal, retroperitoneal and inguinal), interscapular brown adipose tissue (BAT), skeletal muscles of the right leg (tibialis anterior, extensor digitorum longus [EDL], gastrocnemius and soleus) and heart of each animal were dissected, weighed, and frozen or fixed in 4% (wt/vol.) paraformaldehyde for 24 h at 4°C and embedded in paraffin for further histological analysis. Liver and epidydimal fat pad sections were counter stained with haematoxylin and eosin (H&E) to assess morphology. Adipocyte size was analysed using Visiopharm software (Author module, Version 6.4.1; Hørsholm, Denmark). Insulin and glucagon immunostaining of pancreatic sections was performed as previously described [13, 14]. Beta cell mass was computed based on the relative cross-sectional beta cell area and pancreas weight. Three pancreatic sections at three different levels were analysed for each mouse. Pancreatic sections immunoprobed for insulin, glucagon and a nuclear stain (DAPI) were scanned using the panoramic 250 Flash III digital slide scanner (3DHISTECH, Budapest, Hungary) and analysed with Visiopharm.
Islet isolation and culture
Islets were isolated by collagenase digestion, separated by density gradient centrifugation and handpicked under a stereomicroscope. Islets were cultured in standard RPMI medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2 mmol/l glutamine, 5 g/l BSA, 100 U/ml penicillin and 100 μg/ml streptomycin.
Cell culture and treatment
MIN6 cells (passage 26–43; mycoplasma negative), originally provided by J. I. Miyazaki (Osaka University Medical School, Osaka, Japan) , were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 25 mmol/l glucose, 10 mmol/l HEPES, 10% FCS (vol./vol.), 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were transfected with 100 nmol/l control, Mt1 or Mt2 siRNA using DharmaFECT3 transfection reagent 24 h before experiments.
Glucose-stimulated insulin secretion (GSIS) tests
Isolated islets in batches of five, or MIN6 cells seeded in 24-well plates at 2 × 105 cell per well, were preincubated for 1 h in KRB buffer (120 mmol/l NaCl, 4.8 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l MgCl2, 24 mmol/l NaHCO3 and 1 g/l BSA) containing 3 mmol/l glucose and then incubated for 1 h in KRB buffer containing 3 mmol/l, 15 mmol/l or 30 mmol/l glucose. At the end of the incubation, the buffer was collected for measurement of insulin by RIA, and islets were collected and disrupted by sonication in 10 mmol/l Tris, 0.2 mol/l NaCl and 10 mmol/l EDTA for measurement of their DNA and insulin content. All secretion experiments were carried out in duplicate.
NAD(P)H autofluorescence (excitation/emission wavelength [λex/em], 360/470 nm) was measured every 5 s and expressed as the percentage of the fluorescence level measured after 15–20 min of treatment with 10 μmol/l FCCP in KRB buffer containing 30 mmol/l glucose. For measurements of intracellular Ca2+ concentrations, islets were loaded for 2 h with 2 μmol/l Fura-2 LR acetoxymethyl ester and the fluorescence ratio (λex/em, 340/510 to 380/510 nm) was measured every 5 s. For measurements of intracellular free Zn2+ levels ([Zn2+]i), islets were loaded for 2 h with 2 μmol/l Fluozin-3 acetoxymethyl ester and fluorescence (λex/em, 490/510 nm) was measured every 10 s. TPEN (a zinc chelator) was used as a negative control to lower [Zn2+]i and used at 50 μmol/l. Islets from control and KO/Tg-Mt1 mice were simultaneously perifused side by side with KRB buffer continuously gassed with 5% CO2 air mix to maintain pH 7.4 at a flow rate of 1 ml/min at 37°C on the stage of an inverted microscope.
Total RNA was extracted and reverse transcribed as previously described [16, 17]. Real-time RT-PCR was performed using the SYBR Green method and a 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) or a CFX96 optical cycler detection system (Bio-Rad, Hercules, CA, USA). Primer sequences are listed in ESM Table 3. The value obtained for a specific gene product was normalised to the control gene cyclophilin A or TBP and expressed as fold change of the value in the control condition. TaqMan assays were used to assess the mRNA levels of MT1E (Hs01938284_g1), MT1X (Hs00745167_sH), MT2A (Hs02379661_g1) and the control gene 18S rRNA (Hs03003631_g1) in human islets from control and type 2 diabetes donors (Applied Biosystems). AmpliTaq Gold reagents (Thermo Fisher Scientific) were used on a Light Cycler 480 Instrument II (Roche, Risch-Rotkreuz, Switzerland).
Islet MT1 and MT2 protein levels were quantified by ELISA (Frontier Institute, Ishikari, Japan). Absorbance values were normalised to total protein content measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
Results are means ± SEM for the indicated number of experiments. Statistical significance was assessed by unpaired two-tailed Student’s t test, one-way ANOVA with a Newman–Keuls post hoc test, or two-way ANOVA with a Bonferroni post hoc test.
Mt1 and Mt2 mRNA levels were differentially regulated between conditions of beta cell compensation and failure
Islet Mt1 and Mt2 mRNA levels were markedly downregulated in mice fed for 6 weeks with a HFD vs a chow diet. This was associated with increased body-weight gain and plasma insulin levels (compensation) (Fig. 1a,b and ESM Fig. 1). Similarly, islet Mt1 and Mt2 mRNA levels were significantly downregulated in 16-week-old ob/ob mice (Fig. 1c,d), a model of successful beta cell compensation [17, 18], vs control mice. In db/db mice, which progress from successful beta cell compensation to beta cell failure between 6 and 16 weeks of age , islet Mt1 and Mt2 mRNA levels were downregulated in 6-week-old mice (Fig. 1e,f) and upregulated in 16-week-old mice (Fig. 1g,h). These results reveal that, in vivo, beta cell compensation is associated with Mt1 and Mt2 downregulation, whereas beta cell failure is associated with Mt1 and Mt2 upregulation.
Glucose stimulation downregulated the expression of Mt1 and Mt2
WT mouse islets were cultured at various glucose concentrations, ranging from low, non-stimulating concentrations (2–5 mmol/l) to the optimal concentration for culture of rodent islets (10 mmol/l) to a very high glucose concentration (30 mmol/l) [16, 19, 20]. Mt1 and Mt2 mRNA and MT1/2 protein levels were markedly downregulated after culture at glucose at 10 mmol/l vs 2 and 5 mmol/l, with little or no further decrease at 30 mmol/l vs 10 mmol/l glucose (Fig. 2a–c). These effects were anti-paralleled by the stimulation of insulin secretion and the upregulation of the antioxidant genes Mt3, Srxn1 and Gpx2 (Fig. 2d–g). These findings show that, compared with other antioxidant genes, Mt1 and Mt2 have a specific gene expression pattern in response to glucose stimulation. They also show an ex vivo association between islet Mt1 and Mt2 downregulation and the stimulation of insulin secretion.
MT1X mRNA levels were upregulated in human islets from individuals with type 2 diabetes and were affected by glucose stimulation
MT1E, MT1X and MT2A were the most upregulated MT genes in islets obtained by LCM from pancreases of individuals with type 2 diabetes vs islets from non-diabetic donors . Among them, MT1X was the only isoform showing significantly upregulated mRNA levels in islets isolated from type 2 diabetes vs non-diabetic donors. Besides, MT1X displayed higher mRNA levels than MT1E and MT2A in islets isolated from non-diabetic donors (Fig. 3a–d). When islets from non-diabetic donors were cultured in the presence of a low, non-stimulating glucose concentration (2.2 mmol/l), the optimal glucose concentration for culture of human islets (5.5 mmol/l), a high glucose concentration (11.1 mmol/l) and a very high glucose concentration (22 mmol/l) [21, 22], MT1E, MT1X and MT2A mRNA levels were all downregulated between treatment with 2.2 mmol/l and 11.1 mmol/l glucose, while, with 22 mmol/l glucose, MT1E and MT1X mRNA levels returned to a similar level as with 5.5 mmol/l glucose (Fig. 3e–g). In parallel, treatment with glucose at 11.1 and 22 mmol/l significantly increased insulin secretion during culture vs treatment with 2.2 and 5.5 mmol/l glucose (Fig. 3h).
These results show that MT1X upregulation is associated with beta cell failure in human type 2 diabetes. They also reveal a specific gene expression pattern of human MT genes in response to glucose stimulation that partly resembles that of Mt1 and Mt2 in mouse islets.
Deletion of Mt1/Mt2 improved glucose tolerance
The potential role of Mt1 and/or Mt2 in the modulation of glucose homeostasis and beta cell function was investigated in a global Mt1-Mt2 double-KO mouse model . Compared with WT mice, KO mice displayed higher body weight and daily food intake (ESM Fig. 2a,b). They also showed increased liver weight (ESM Fig. 2c) without macroscopical or histological signs of steatosis (ESM Fig. 2d), and increased weight of different leg muscles, including tibialis anterior, EDL and gastrocnemius (ESM Fig. 2e–g). However, soleus muscle and heart weights were similar (ESM Fig. 2h,i). Interestingly, the weight of epidydimal, inguinal and retroperitoneal fat pads and the sum of the three fat pads (WAT), thereof, were reduced in KO mice (ESM Fig. 2j–m), whereas BAT weight was similar between KO and WT mice (ESM Fig. 2n). Histological sections of epidydimal fat pads also revealed reduced adipocyte surface in KO mice (ESM Fig. 2o,p).
Fed blood glucose levels were similar in KO and WT mice (Fig. 4a), overnight-fasted blood glucose levels were slightly higher in KO mice (Fig. 4b) and fed and fasted plasma insulin levels were not significantly different (Fig. 4c,d). Interestingly, glucose tolerance during i.p.GTT was markedly improved in KO mice, together with significantly increased plasma insulin levels (30 min following i.p.GTT) (Fig. 4e–g). Similarly, during an overnight fasting/1 h refeeding test, blood glucose levels after refeeding were lower in KO mice (Fig. 4h). On the other hand, insulin sensitivity during i.p.ITT was similar in KO and WT mice (Fig. 4i,j). Together, these findings suggest that Mt1-Mt2 deletion leads to improved glucose tolerance due to increased insulin secretion rather than changes in insulin action.
MT deletion potentiated GSIS
The mechanism underlying improved glucose tolerance in KO mice was further investigated in isolated islets. In WT islets, MT1/2 protein levels were expressed under control culture conditions and upregulated by treatment with ZnCl2 (a potent inducer of MT expression). In contrast, MT1/2 proteins were not detected in KO islets, even after treatment with ZnCl2, confirming the lack of Mt1-Mt2 expression (Fig. 5a). Interestingly, GSIS was potentiated in KO vs WT islets after acute stimulation with 15 mmol/l glucose, and to a stronger extent after stimulation with 30 mmol/l glucose (Fig. 5b), while islet insulin content was unchanged in KO vs WT mice (Fig. 5c). In agreement, MT1 but not MT2 knockdown in MIN6 cells potentiated GSIS (Fig. 5d–f), highlighting the role of Mt1, rather than Mt2, in the negative regulation of insulin secretion.
We also measured the effects of acute stepwise increases in glucose concentration on intracellular NAD(P)H and Ca2+ levels and found no differences between WT and KO islets (Fig. 5g,h). These findings indicate that the potentiation of GSIS in KO islets results from an effect downstream of the stimulation of glucose metabolism and Ca2+ influx.
MTs are known for their metal-binding properties and proposed to play a role in metal ion, including zinc, homeostasis. As zinc plays a key role in beta cell biology, we used Fluozin-3 to compare the dynamic changes in [Zn2+]i in response to glucose and zinc supplementation/chelation in islets from WT and KO mice (ESM Fig. 3a). In islets from both mouse types, [Zn2+]i was increased upon supplementation of 3 mmol/l glucose-KRB buffer with 10 μmol/l ZnCl2, slightly decreased upon subsequent stimulation with 30 mmol/l in the continued presence of 10 μmol/l ZnCl2, rapidly decreased upon ensuing zinc chelation using TPEN, and markedly increased upon final addition of 1 mmol/l ZnCl2 to the medium. These effects were almost identical in WT and KO islets. In agreement, the mRNA levels of the key beta cell zinc transporters Zip6 (also known as Slc39a6), Zip7 (Slc39a7), Znt1 (Slc30a1) and Znt8 (Slc30a8) were similar between KO and WT islets (ESM Fig. 3b–e). These results rule out a potential role of changes in [Zn2+]i levels in the potentiation of GSIS in KO islets.
Assessment of the pancreas morphology revealed no difference in islet architecture between islets from WT and KO mice (ESM Fig. 4a). There were no significant changes in pancreatic weight, beta and alpha cell masses, or the percentage of alpha/beta cells per islet area between KO and WT mice (ESM Fig. 4b–e). Moreover, islets from WT and KO mice displayed no difference in the mRNA levels of the beta cell-enriched genes preproinsulin, Pdx1, Glut2 (also known as Slc2a2), Pcx and Gpd2 (ESM Fig. 4f–j). Similarly, there were no changes in the mRNA levels of the endoplasmic reticulum (ER) stress-response genes Hspa5 and Ddit3 (ESM Fig. 4k,l). Importantly, the deletion of Mt1 and Mt2 was not compensated for by upregulation of other MT genes. Thus, Mt3 mRNA levels were unchanged in islets from KO vs WT mice (ESM Fig. 4m), while Mt4 mRNA levels were undetected after 40 cycles of PCR amplification in both islet types.
Altogether, these findings strongly support a role for Mt1 as a negative modulator of GSIS.
Mt1 overexpression attenuated GSIS
To further assess the implication of Mt1 in the negative regulation of GSIS, we examined islets isolated from global transgenic mice overexpressing mouse Mt1 under the control of its natural promoter (Tg-Mt1) [24, 25]. In comparison with WT mice, Tg-Mt1 mice exhibited similar body and liver weights (ESM Fig. 5a–c). They displayed no significant difference in the weight of epidydimal (p = 0.0519), inguinal and retroperitoneal fat pads or the sum of WAT, thereof (ESM Fig. 5d–g). There was also no difference in BAT weight (ESM Fig. 5h). Tg-Mt1 mice also displayed similar fed blood glucose levels (Fig. 6a) and plasma insulin levels (Fig. 6b) to WT mice, and a fasting-refeeding test revealed no significant differences between the two groups (Fig. 6c). Similarly, i.p.GTT tests were similar in 3-month-old and 9-month-old animals (Fig. 6d,e). In contrast, i.p.ITT tests revealed a notable difference between WT and Tg-Mt1 mice, with Tg-Mt1 mice displaying lower blood glucose levels during the test (Fig. 6f,g). This effect may stem from an impact of Mt1 overexpression on peripheral tissues.
In isolated islets, MT protein levels were markedly upregulated in Tg-Mt1 islets, confirming the overexpression of Mt1 (Fig. 7a). Interestingly, in contrast with its potentiation in KO vs WT islets, GSIS was significantly attenuated in Tg-Mt1 vs WT islets after acute stimulation with 30 mmol/l glucose (Fig. 7b), while islet insulin content was similar between the two islet types (Fig. 7c). This attenuation in GSIS occurred despite similar rises in intracellular NAD(P)H and Ca2+ levels in islets from WT and Tg-Mt1 mice in response to stepwise increases in glucose (Fig. 7d,e), indicating that the alteration of GSIS in Tg-Mt1 mice vs WT mice lies at a step downstream of the stimulation of glucose metabolism and Ca2+ influx.
Similarly, changes in [Zn2+]i in response to glucose and zinc supplementation/chelation were not different between islets from WT and Tg-Mt1 mice (ESM Fig. 6a). In agreement, the mRNA levels of zinc transporters Zip6, Zip7, Znt1 and Znt8 were unchanged in islets from Tg-Mt1 mice vs WT mice (ESM Fig. 6b–e).
Importantly, in comparison with islets from WT mice, Tg-Mt1 islets exhibited a similar expression level of the beta cell-enriched genes preproinsulin, Pdx1, Glut2 and Pcx (Fig. 8a–d). In addition, the mRNA levels of ER stress-response genes Hspa5 and Ddit3 were also not different between the two mouse models (Fig. 8e,f). On the other hand, the overexpression of Mt1 (Fig. 8g) resulted in reduced mRNA levels of Mt2 (p < 0.05) and Mt3 (p = 0.0704) in comparison with WT islets (Fig. 8h,i), while Mt4 mRNA levels were not detected after 40 cycles of PCR amplification in both types of islets.
Collectively, these findings further support the implication of Mt1 in the negative regulation of insulin secretion.
We have unveiled a novel role of Mt1 in beta cells as a negative regulator of insulin secretion. The key findings of the study are: (1) Mt1 and Mt2 islet gene expression in obese mice was downregulated with beta cell compensation and upregulated with beta cell failure; (2) MT1X islet mRNA levels were upregulated in human type 2 diabetes donors; (3) physiological and supraphysiological glucose stimulation downregulated mouse and human MT islet gene expression; (4) deletion of Mt1 and Mt2 improved glucose tolerance in vivo and potentiated GSIS in isolated islets; (5) knockdown of MT1, but not MT2, potentiated GSIS in MIN6 cells; and (6) Mt1 overexpression attenuated GSIS in isolated islets (Table 1). These cumulative findings strongly support the implication of Mt1 in the negative regulation of beta cell function.
Mt1 and Mt2 exhibit an atypical gene expression pattern in comparison with other antioxidant genes
MTs are known for their protective antioxidant properties [26,27,28]. Oxidative stress plays an important role in beta cell demise and islets of humans with diabetes and animal models display upregulated expression of many antioxidant genes and markers of oxidative damage . Interestingly, antioxidant genes like Hmox1, Gpx1, Gpx2, Sod1 and Nrf2 (also known as Nfe2l2) were also upregulated in the islets of compensating young db/db mice and mice fed an HFD [17, 29, 30], in sharp contrast with Mt1 and Mt2 downregulation (Fig. 1). Similarly, in isolated islets, treatment with glucose upregulated Mt3, Srxn1 and Gpx2, while it downregulated Mt1 and Mt2 (Fig. 2 and [31, 32]). Noteworthy, our previous studies in Wistar rats revealed that islet Mt1 expression was upregulated by fasting and downregulated upon refeeding (J-C Jonas, unpublished data). Furthermore, a recent study exploring beta cell heterogeneity by single-cell transcriptomics revealed that high activity of the insulin gene promoter was associated with low expression of Mt1 and Mt2 and vice versa . These observations demonstrate that Mt1 and Mt2 behave differently from other oxidative stress-response genes. Besides, Mt1-Mt2 deletion does not affect antioxidant/stress-response gene expression (ESM Fig. 4 and ESM Fig. 7), indicating no evident impact on islet redox status under physiological conditions. Thus, Mt1 and/or Mt2 may play a role in beta cell (patho)physiology that goes beyond their known antioxidant function.
Mt1 negatively regulates GSIS
Deletion of Mt1 and Mt2 improved glucose tolerance and potentiated GSIS in isolated islets from KO mice. These findings contrast with a previous study using islets from Mt1-Mt2 KO mice . Although differences in genetic backgrounds may contribute to this discrepancy, our study is more comprehensive than the previous investigation. Thus, besides our complementary in vivo and ex vivo results, the knockdown of MT1 in MIN6 cells reproduced the enhanced insulin secretory phenotype of the KO islets. Furthermore, in our study, islets overexpressing Mt1 exhibited the reciprocal phenotype.
Our results underscore the role of Mt1, rather than Mt2, in the modulation of insulin secretion. Although these genes exhibit similar expression patterns, Mt1 displays a higher expression level in comparison with Mt2 and Mt3 in primary mouse islets (ESM Fig. 8). In addition, mRNA sequence alignment analysis showed that Mt1 and Mt2 are only ~80% identical. Thus, the ~20% sequence difference may also underlie functional specificities. Such specificities may stem from different metal affinities and binding properties or from specific protein–protein interactions [6, 35].
How could Mt1 impact on insulin secretion?
We did not detect significant changes in classical metabolic (NAD(P)H) and ionic parameters (cytosolic free Ca2+ and Zn2+) in response to glucose stimulation between islets from WT mice and KO or Tg-Mt1mice. The expression of antioxidant and other stress-response genes was not different between WT and KO islets, thereby ruling out a potential impact on redox status (ESM Fig. 4 and ESM Fig. 7). Furthermore, Mt1-Mt2 deletion or Mt1 overexpression had no impact on cell death in islets cultured under control conditions (10 mmol/l glucose; ESM Fig. 9). Nevertheless, insulin secretion in response to high potassium was also potentiated in islets from KO vs WT mice (ESM Fig. 10). This result strongly suggests a potential impact of Mt1 on the beta cell exocytotic machinery. Interestingly, MT3 was previously shown to interact with Rab3A GTPase in neurons, thereby playing a role in presynaptic vesicle trafficking . One may, therefore, hypothesise that MT1 interacts with a yet-to-be-identified protein of the beta cell exocytotic machinery to modulate insulin secretion.
The mechanism(s) of induction of Mt1 in diabetes
Hyperglycaemia, per se, may not be the upstream factor involved in increased expression of Mt1 in diabetes. Indeed, although Mt1 mRNA expression transiently increases in rat islets cultured overnight with glucose at 30 mmol/l vs 10 mmol/l , prolonged exposure of mouse and rat islets to high glucose (30 mmol/l) had little or no impact (vs 10 mmol/l) on Mt1 and Mt2 mRNA and MT1/MT2 protein levels (Fig. 2 and ). In human islets, culture in the presence of the already supraphysiological glucose concentration of 11.1 mmol/l vs 2.2 and 5.5 mmol/l also downregulated the mRNA levels of MT genes. Upregulation beyond this concentration may result from differences in glucose sensitivity and metabolism between human and mouse islets and may involve the activation of glucotoxicity-related pathways . We have previously shown that rat islet Mt1 expression is markedly induced by exogenous H2O2, the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin, the cytokine IL-1β and hypoxia . However, the islet expression of genes induced by oxidative stress, ER stress and inflammatory stress are observed in the prediabetic stage in db/db mice , suggesting alternate mechanisms. On the other hand, evidence from several studies implicates a possible role of hypoxia: Mt1 mRNA expression is upregulated by hypoxia in mouse and human islets (ESM Fig. 11 and ), and a clear temporal in vivo association is observed between MT expression and an hypoxic gene expression signature in islets of db/db mice [39, 40].
Limitations of the study and perspectives
In this study, we used global KO animals. Since Mt1-Mt2 deletion may affect other metabolic tissues, one may argue that the observed secretory phenotype involves the effect of systemic factors. However, this is unlikely as we systematically precultured isolated islets for 1 week before GSIS tests. We also combined different models to demonstrate that Mt1 negatively regulates insulin secretion, i.e. by confirming the secretory phenotype after MT1 knockdown in MIN6 cells. Additionally, the attenuation of GSIS by Mt1 overexpression further supports our hypothesis. Although the Tg-Mt1 model is also global, it presents two important advantages: (1) the overexpression of the mouse Mt1 gene rather than human MT2A gene ; and (2) the control of Mt1 by its natural promoter rather than the insulin promoter, thereby avoiding the ER stress and oxidative stress observed in MT2A-transgenic mice . Indeed, our Tg-Mt1 islets displayed normal expression of ER stress-response genes (Fig. 8). Nevertheless, development of an Mt1-floxed mouse model is warranted for further exploration of these novel roles of Mt1 in beta cell biology.
Mt1 negatively regulates insulin secretion. Downregulation of islet Mt1 in obesity may, thus, contribute to beta cell compensation, and its upregulation in type 2 diabetes may contribute to beta cell failure. Inhibition of Mt1 may, therefore, represent an attractive therapeutic target to preserve and restore insulin secretion in type 2 diabetes.
All data points generated or analysed during the current study are shown in the figures of this published article (and its supplementary information files). Tabulated datasets are available from the corresponding author on reasonable request.
Brown adipose tissue
Extensor digitorum longus
- λex/em :
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
- Fura-2 LR:
Fura-2 leakage resistant
Glucose-stimulated insulin secretion
Small interfering RNA
- Tg-Mt1 :
Transgenic mice overexpressing Mt1 under the control of its natural promoter
White adipose tissue
- [Zn2+]i :
Intracellular free Zn2+ levels
Weyer C, Bogardus C, Mott DM, Pratley RE (1999) The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 104(6):787–794. https://doi.org/10.1172/JCI7231
Pratley RE, Weyer C (2002) Progression from IGT to type 2 diabetes mellitus: the central role of impaired early insulin secretion. Curr Diab Rep 2(3):242–248. https://doi.org/10.1007/s11892-002-0090-6
Kahn SE (2003) The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type 2 diabetes. Diabetologia 46(1):3–19. https://doi.org/10.1007/s00125-002-1009-0
Bensellam M, Laybutt DR, Jonas JC (2012) The molecular mechanisms of pancreatic beta-cell glucotoxicity: recent findings and future research directions. Mol Cell Endocrinol 364(1-2):1–27. https://doi.org/10.1016/j.mce.2012.08.003
Bensellam M, Jonas JC, Laybutt DR (2018) Mechanisms of beta-cell dedifferentiation in diabetes: recent findings and future research directions. J Endocrinol 236(2):R109–R143. https://doi.org/10.1530/JOE-17-0516
Zalewska M, Trefon J, Milnerowicz H (2014) The role of metallothionein interactions with other proteins. Proteomics 14(11):1343–1356. https://doi.org/10.1002/pmic.201300496
Kimura T, Kambe T (2016) The functions of metallothionein and ZIP and ZnT transporters: an overview and perspective. Int J Mol Sci 17(3):336. https://doi.org/10.3390/ijms17030336
Giacconi R, Bonfigli AR, Testa R et al (2008) +647 A/C and +1245 MT1A polymorphisms in the susceptibility of diabetes mellitus and cardiovascular complications. Mol Genet Metab 94(1):98–104. https://doi.org/10.1016/j.ymgme.2007.12.006
Yang L, Li H, Yu T et al (2008) Polymorphisms in metallothionein-1 and -2 genes associated with the risk of type 2 diabetes mellitus and its complications. Am J Physiol Endocrinol Metab 294(5):E987–E992. https://doi.org/10.1152/ajpendo.90234.2008
Raudenska M, Gumulec J, Podlaha O et al (2014) Metallothionein polymorphisms in pathological processes. Metallomics 6(1):55–68. https://doi.org/10.1039/C3MT00132F
Marselli L, Thorne J, Dahiya S et al (2010) Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes. PLoS One 5(7):e11499. https://doi.org/10.1371/journal.pone.0011499
O’Connell PJ, Holmes-Walker DJ, Goodman D et al (2013) Multicenter Australian trial of islet transplantation: improving accessibility and outcomes. Am J Transplant 13(7):1850–1858. https://doi.org/10.1111/ajt.12250
Bensellam M, Montgomery MK, Luzuriaga J, Chan JY, Laybutt DR (2015) Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells. Diabetologia 58(4):758–770. https://doi.org/10.1007/s00125-015-3503-1
Shi YC, Loh K, Bensellam M et al (2015) Pancreatic PYY is critical in the control of insulin secretion and glucose homeostasis in female mice. Endocrinology 156(9):3122–3136. https://doi.org/10.1210/en.2015-1168
Miyazaki J, Araki K, Yamato E et al (1990) Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127(1):126–132. https://doi.org/10.1210/endo-127-1-126
Bensellam M, Van Lommel L, Overbergh L, Schuit FC, Jonas JC (2009) Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations. Diabetologia 52(3):463–476. https://doi.org/10.1007/s00125-008-1245-z
Chan JY, Luzuriaga J, Bensellam M, Biden TJ, Laybutt DR (2013) Failure of the adaptive unfolded protein response in islets of obese mice is linked with abnormalities in beta-cell gene expression and progression to diabetes. Diabetes 62(5):1557–1568. https://doi.org/10.2337/db12-0701
Chan JY, Luzuriaga J, Maxwell EL, West PK, Bensellam M, Laybutt DR (2015) The balance between adaptive and apoptotic unfolded protein responses regulates beta-cell death under ER stress conditions through XBP1, CHOP and JNK. Mol Cell Endocrinol 413:189–201. https://doi.org/10.1016/j.mce.2015.06.025
Andersson A (1978) Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14(6):397–404. https://doi.org/10.1007/BF01228134
Ling Z, Pipeleers DG (1994) Preservation of glucose-responsive islet beta-cells during serum-free culture. Endocrinology 134(6):2614–2621. https://doi.org/10.1210/endo.134.6.7515006
Eizirik DL, Korbutt GS, Hellerstrom C (1992) Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function. J Clin Invest 90(4):1263–1268. https://doi.org/10.1172/JCI115989
Ling Z, Pipeleers DG (1996) Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation. J Clin Invest 98(12):2805–2812. https://doi.org/10.1172/JCI119108
Masters BA, Kelly EJ, Quaife CJ, Brinster RL, Palmiter RD (1994) Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc Natl Acad Sci U S A 91(2):584–588. https://doi.org/10.1073/pnas.91.2.584
Palmiter RD, Sandgren EP, Koeller DM, Brinster RL (1993) Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice. Mol Cell Biol 13(9):5266–5275. https://doi.org/10.1128/MCB.13.9.5266
Iszard MB, Liu J, Liu Y et al (1995) Characterization of metallothionein-I-transgenic mice. Toxicol Appl Pharmacol 133(2):305–312. https://doi.org/10.1006/taap.1995.1155
Chen H, Carlson EC, Pellet L, Moritz JT, Epstein PN (2001) Overexpression of metallothionein in pancreatic beta-cells reduces streptozotocin-induced DNA damage and diabetes. Diabetes 50(9):2040–2046. https://doi.org/10.2337/diabetes.50.9.2040
Li X, Chen H, Epstein PN (2004) Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J Biol Chem 279(1):765–771. https://doi.org/10.1074/jbc.M307907200
Li X, Chen H, Epstein PN (2006) Metallothionein and catalase sensitize to diabetes in nonobese diabetic mice: reactive oxygen species may have a protective role in pancreatic beta-cells. Diabetes 55(6):1592–1604. https://doi.org/10.2337/db05-1357
Roat R, Rao V, Doliba NM et al (2014) Alterations of pancreatic islet structure, metabolism and gene expression in diet-induced obese C57BL/6J mice. PLoS One 9(2):e86815. https://doi.org/10.1371/journal.pone.0086815
Hatanaka M, Anderson-Baucum E, Lakhter A et al (2017) Chronic high fat feeding restricts islet mRNA translation initiation independently of ER stress via DNA damage and p53 activation. Sci Rep 7(1):3758. https://doi.org/10.1038/s41598-017-03869-5
Bellomo EA, Meur G, Rutter GA (2011) Glucose regulates free cytosolic Zn(2)(+) concentration, Slc39 (ZiP), and metallothionein gene expression in primary pancreatic islet beta-cells. J Biol Chem 286(29):25778–25789. https://doi.org/10.1074/jbc.M111.246082
Duprez J, Roma LP, Close AF, Jonas JC (2012) Protective antioxidant and antiapoptotic effects of ZnCl2 in rat pancreatic islets cultured in low and high glucose concentrations. PLoS One 7(10):e46831. https://doi.org/10.1371/journal.pone.0046831
Modi HCH, Skovsø S, Hu X, et al (2018) Imaging Ins2 gene activity and single-cell RNA sequencing reveal heterogeneous beta cell states. In: Diabetologia (ed) 54th EASD Annual Meeting of the European Association for the Study of Diabetes. Springer Berlin Heidelberg, Berlin, Germany, p 17
Laychock SG, Duzen J, Simpkins CO (2000) Metallothionein induction in islets of Langerhans and insulinoma cells. Mol Cell Endocrinol 165(1-2):179–187. https://doi.org/10.1016/S0303-7207(00)00247-1
Artells E, Palacios O, Capdevila M, Atrian S (2013) Mammalian MT1 and MT2 metallothioneins differ in their metal binding abilities. Metallomics 5(10):1397–1410. https://doi.org/10.1039/c3mt00123g
Knipp M, Meloni G, Roschitzki B, Vasak M (2005) Zn7metallothionein-3 and the synaptic vesicle cycle: interaction of metallothionein-3 with the small GTPase Rab3A. Biochemistry 44(9):3159–3165. https://doi.org/10.1021/bi047636d
Jonas JC, Bensellam M, Duprez J, Elouil H, Guiot Y, Pascal SM (2009) Glucose regulation of islet stress responses and beta-cell failure in type 2 diabetes. Diabetes Obes Metab 11(Suppl 4):65–81. https://doi.org/10.1111/j.1463-1326.2009.01112.x
Gerber PA, Bellomo EA, Hodson DJ et al (2014) Hypoxia lowers SLC30A8/ZnT8 expression and free cytosolic Zn2+ in pancreatic beta cells. Diabetologia 57(8):1635–1644. https://doi.org/10.1007/s00125-014-3266-0
Bensellam M, Duvillie B, Rybachuk G et al (2012) Glucose-induced O(2) consumption activates hypoxia inducible factors 1 and 2 in rat insulin-secreting pancreatic beta-cells. PLoS One 7(1):e29807. https://doi.org/10.1371/journal.pone.0029807
Bensellam M, Maxwell EL, Chan JY et al (2016) Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells. Diabetologia 59(7):1492–1502. https://doi.org/10.1007/s00125-016-3947-y
Chen S, Han J, Liu Y (2015) Dual opposing roles of metallothionein overexpression in C57BL/6J mouse pancreatic beta-cells. PLoS One 10(9):e0137583. https://doi.org/10.1371/journal.pone.0137583
We thank F. Knockaert (UCLouvain, Belgium) for expert technical help and J.-C. Henquin (UCLouvain, Belgium) for valuable discussion and suggestions. We also thank J. Duprez (UCLouvain, Belgium) for pilot experiments carried out in Mt-KO mice. Some of the data were presented as an abstract at the 54th EASD meeting in Berlin, Germany, 1–5 October 2018.
MB was supported by a MOVE-in Louvain/EC Marie-Curie incoming postdoctoral fellowship and is currently supported by a fellowship from the ‘Fonds de Recherche Clinique’, Cliniques Universitaires Saint-Luc, Brussels, Belgium. This work was supported by a grant from the Société Francophone du Diabète, Paris, France (SFD/MSD 2016), the Action de Recherche Concertée 12/17–047 from the Communauté française de Belgique, and the ‘Fonds Spécial de Recherche 2016’ from UCLouvain to JCJ. JCJ and PG are Research Directors of the Fonds de la Recherche Scientifique-FNRS, Belgium. JYC is supported by an NHMRC Early Career Fellowship. DRL is supported by an Australian Research Council (ARC) Future Fellowship. St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.
The authors declare that there is no duality of interest associated with this manuscript.
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Bensellam, M., Shi, YC., Chan, J.Y. et al. Metallothionein 1 negatively regulates glucose-stimulated insulin secretion and is differentially expressed in conditions of beta cell compensation and failure in mice and humans. Diabetologia 62, 2273–2286 (2019). https://doi.org/10.1007/s00125-019-05008-3
- Beta cell compensation
- Beta cell failure
- Glucose-stimulated insulin secretion
- Type 2 diabetes