The normal β-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with β-cells that are susceptible to failure. Here, we ...investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for β-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several β-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with β-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of β-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.
Aims/hypothesis
Mild islet inflammation has been suggested as a contributing factor to beta cell failure in type 2 diabetes. Macrophage levels are elevated in the islets of humans and mice with type ...2 diabetes, but their effects on beta cells are not understood. Our goal was to examine the gene expression changes in islet-associated macrophages in obesity models with opposing disposition to diabetes development and to assess their potential contribution to beta cell (mal)adaptation.
Methods
Islets were isolated from lean control mice, obese diabetes-prone
db/db
mice and obese diabetes-resistant
ob/ob
mice. Macrophages were sorted using flow cytometry. Islets were treated ex vivo with clodronate-containing liposomes to deplete macrophages. Gene expression was assessed by real-time RT-PCR.
Results
Macrophage levels were increased in islets from
db/db
mice but not in islets from
ob/ob
mice compared with lean control mice. Macrophages from
db/db
and
ob/ob
islets displayed distinct changes in gene expression compared with control islet macrophages, suggesting differential shifts in functional state. Macrophages from
db/db
islets displayed increased expression of interferon regulatory factor 5 (
Irf5
), IL-1 receptor antagonist (
Il1rn
) and mannose receptor C-type 1 (
Mrc1
), whereas macrophages from
ob/ob
islets showed elevated levels of transforming growth factor beta 1 (
Tgfb1
) and reduced IL-1β (
Il1b
). Clodronate-liposome treatment of islets depleted macrophages, as evidenced by reduced mRNA expression of
Cd11b
(also known as
Itgam
) and
F4/80
(also known as
Adgre1
) compared with PBS-liposome-treated islets. The depletion of macrophages in
db/db
islets increased the expression of genes related to beta cell identity. The mRNA levels of islet-associated transcription factors (
Mafa
and
Pdx1
), glucose transporter (
Glut2
also known as
Slc2a2
), ATP-sensitive K
+
channel (
Kcnj11
), incretin receptor (
Gipr
) and adaptive unfolded protein response (UPR) genes (
Xbp1
,
Hspa5
,
Pdia4
and
Fkbp11
) were increased in
db/db
islets after macrophage depletion, whereas the mRNA levels of the deleterious UPR effector,
Ddit3
, were reduced. In contrast, depletion of macrophages in islets of
ob/ob
mice did not affect beta cell identity gene expression.
Conclusions/interpretation
The findings of this study suggest that distinct alterations in islet macrophages of obese mice are critically important for the disruption of beta cell gene expression in diabetes.
Aims/hypothesis
Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) ...homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress.
Methods
Mouse islets and MIN6 cells were exposed to various oxygen (O
2
) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA;
Hspa5
was also overexpressed.
db/db
mice were used.
Results
Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic,
db/db
mice. In isolated mouse islets and MIN6 cells, O
2
deprivation (1–5% vs 20%; 4–24 h) markedly reduced the expression of adaptive UPR genes, including
Hspa5
,
Hsp90b1
,
Fkbp11
and spliced
Xbp1
. Coatomer protein complex genes (
Copa
,
Cope
,
Copg
also known as
Copg1
,
Copz1
and
Copz2
) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes (
Ddit3
,
Atf3
and
Trb3
also known as
Trib3
), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally,
Hspa5
overexpression significantly protected against hypoxia-induced cell death.
Conclusions/interpretation
Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress.
Aims/hypothesis
Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in ...islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress.
Methods
MIN6 cells were cultured in H
2
O
2
or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from
Id1
−/−
,
Id3
−/−
and diabetic
db/db
mice were used.
Results
ID1–4 expression was upregulated in vivo in the islets of diabetic
db/db
mice and stimulated in vitro by ribose and H
2
O
2
.
Id1/3
inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of
Tfam
, which encodes a mitochondrial transcription factor, and respiratory chain components.
Id1/3
inhibition also reduced the expression of small MAF transcription factors (
MafF
,
MafG
and
MafK
), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of
Id1/3
inhibition.
Conclusions/interpretation
Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant–mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2–small MAF pathway.
Aims/hypothesis
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.
Methods
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 Ca
2+
concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn
2+
concentration with Fluozin-3, and NAD(P)H by autofluorescence.
Results
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 Ca
2+
concentration and NAD(P)H levels, or intracellular Zn
2+
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.
Conclusions/interpretation
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.
The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study ...examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H
O
treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1β and IκBα) and NFκB phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes.
The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We ...have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 β-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.
Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 ...receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function.
Cytokine-Induced β-Cell Death Is Independent of Endoplasmic Reticulum Stress Signaling
Mia C. Åkerfeldt 1 ,
Jennifer Howes 1 ,
Jeng Yie Chan 1 ,
Veronica A. Stevens 1 ,
Nacer Boubenna 2 ,
Helen M. ...McGuire 2 ,
Cecile King 2 ,
Trevor J. Biden 1 and
D. Ross Laybutt 1
1 Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales,
Australia
2 Immunology and Inflammation Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South
Wales, Australia
Corresponding author: D. Ross Laybutt, r.laybutt{at}garvan.org.au
Abstract
OBJECTIVE— Cytokines contribute to β-cell destruction in type 1 diabetes. Endoplasmic reticulum (ER) stress–mediated apoptosis has been
proposed as a mechanism for β-cell death. We tested whether ER stress was necessary for cytokine-induced β-cell death and
also whether ER stress gene activation was present in β-cells of the NOD mouse model of type 1 diabetes.
RESEARCH DESIGN AND METHODS— INS-1 β-cells or rat islets were treated with the chemical chaperone phenyl butyric acid (PBA) and exposed or not to interleukin
(IL)-1β and γ-interferon (IFN-γ). Small interfering RNA (siRNA) was used to silence C/EBP homologous protein (CHOP) expression
in INS-1 β-cells. Additionally, the role of ER stress in lipid-induced cell death was assessed.
RESULTS— Cytokines and palmitate triggered ER stress in β-cells as evidenced by increased phosphorylation of PKR-like ER kinase (PERK),
eukaryotic initiation factor (EIF)2α, and Jun NH 2 -terminal kinase (JNK) and increased expression of activating transcription factor (ATF)4 and CHOP. PBA treatment attenuated
ER stress, but JNK phosphorylation was reduced only in response to palmitate, not in response to cytokines. PBA had no effect
on cytokine-induced cell death but was associated with protection against palmitate-induced cell death. Similarly, siRNA-mediated
reduction in CHOP expression protected against palmitate- but not against cytokine-induced cell death. In NOD islets, mRNA
levels of several ER stress genes were reduced (ATF4, BiP binding protein, GRP94 glucose regulated protein 94, p58, and
XBP-1 X-box binding protein 1 splicing) or unchanged (CHOP and Edem1 ER degradation enhancer, mannosidase α–like 1).
CONCLUSIONS— While both cytokines and palmitate can induce ER stress, our results suggest that, in contrast to lipoapoptosis, the PERK-ATF4-CHOP
ER stress–signaling pathway is not necessary for cytokine-induced β-cell death.
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 30 June 2008.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work
is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C Section 1734 solely to indicate this fact.
Accepted June 20, 2008.
Received December 20, 2007.
DIABETES
•Developmental gene PAX6 affects pancreatic and eye formation (aniridia).•Distal PAX6 enhancer region deletion segregates with diabetes and aniridia.•Clinically distinct from previous reports ...describing aniridia and glucose intolerance.•Aniridia patients with distal PAX6 deletions require early screening for diabetes.
Analyze cosegregation of aniridia and diabetes to identify genetic criteria for detection and early treatment of diabetes-susceptible aniridia patients.
We assessed a two-generation family: three individuals with aniridia, two previously diagnosed as type 2 diabetes. One individual with aniridia, with unknown diabetes status, was evaluated by oral glucose tolerance test. Genetic analysis of aniridia-associated genes was performed on all available family members. Candidate genes were functionally tested by gene silencing in MIN6 pancreatic β-cells.
A 25 year old male with aniridia had a diabetic oral glucose tolerance test despite a normal fasting blood glucose. A 484–630 kb deletion ∼120 kb distal to PAIRED BOX 6 (PAX6) showed dominant cosegregation with aniridia and diabetes in all affected family members. The deleted region contains regulatory elements for PAX6 expression and four additional coding regions. Knockdown of two of the deleted genes (Dnajc24 or Immp1l) with Pax6 impaired glucose-stimulated insulin secretion.
We demonstrate dominant cosegregation of diabetes and aniridia with a deletion distal to PAX6, which is clinically distinct from the mild glucose intolerance previously reported with PAX6 coding mutations. Asymptomatic aniridia individuals appear at risk of diabetes (and its complications) and could benefit from earlier diagnosis and treatment.
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