Nadeeja Wijesekara

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Muscle-Specific Pten Deletion Protects against Insulin Resistance and Diabetes

ABSTRACT Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.

Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallisation and secretion

Aims/hypothesisZinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues.MethodsWe generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis.ResultsZnt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis.Conclusions/interpretationThis is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.

Inflammation and Oxidative Stress: The Molecular Connectivity between Insulin Resistance, Obesity, and Alzheimer’s Disease

Type 2 diabetes (T2DM), Alzheimers disease (AD), and insulin resistance are age-related conditions and increased prevalence is of public concern. Recent research has provided evidence that insulin resistance and impaired insulin signalling may be a contributory factor to the progression of diabetes, dementia, and other neurological disorders. Alzheimers disease (AD) is the most common subtype of dementia. Reduced release (for T2DM) and decreased action of insulin are central to the development and progression of both T2DM and AD. A literature search was conducted to identify molecular commonalities between obesity, diabetes, and AD. Insulin resistance affects many tissues and organs, either through impaired insulin signalling or through aberrant changes in both glucose and lipid (cholesterol and triacylglycerol) metabolism and concentrations in the blood. Although epidemiological and biological evidence has highlighted an increased incidence of cognitive decline and AD in patients with T2DM, the common molecular basis of cell and tissue dysfunction is rapidly gaining recognition. As a cause or consequence, the chronic inflammatory response and oxidative stress associated with T2DM, amyloid-β (Aβ) protein accumulation, and mitochondrial dysfunction link T2DM and AD.

Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 μg/ml) or full-length (5 μg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.

The Identification of Potential Factors Associated with the Development of Type 2 Diabetes A Quantitative Proteomics Approach

Type 2 diabetes (T2D) arises when pancreatic β-cells fail to compensate for systemic insulin resistance with appropriate insulin secretion. However, the link between insulin resistance and β-cell failure in T2D is not fully understood. To explore this association, we studied transgenic MKR mice that initially develop insulin resistance in skeletal muscle but by 8 weeks of age have T2D. In the present study, global islet protein and gene expression changes were characterized in diabetic MKR versus non-diabetic control mice at 10 weeks of age. Using a quantitative proteomics approach (isobaric tags for relative and absolute quantification (iTRAQ)), 159 proteins were differentially expressed in MKR compared with control islets. Marked up-regulation of protein biosynthesis and endoplasmic reticulum stress pathways and parallel down-regulation in insulin processing/secretion, energy utilization, and metabolism were observed. A fraction of the differentially expressed proteins identified (including GLUT2, DNAJC3, VAMP2, RAB3A, and PC1/3) were linked previously to insulin-secretory defects and T2D. However, many proteins for the first time were associated with islet dysfunction, including the unfolded protein response proteins (ERP72, ERP44, ERP29, PPIB, FKBP2, FKBP11, and DNAJB11), endoplasmic reticulum-associated degradation proteins (VCP and UFM1), and multiple proteins associated with mitochondrial energy metabolism (NDUFA9, UQCRH, COX2, COX4I1, COX5A, ATP6V1B2, ATP6V1H, ANT1, ANT2, ETFA, and ETFB). The mRNA expression level corresponding to these proteins was examined by microarray, and then a small subset was validated using quantitative real time PCR and Western blot analyses. Importantly ∼54% of differentially expressed proteins in MKR islets (including proteins involved in proinsulin processing, protein biosynthesis, and mitochondrial oxidation) showed changes in the proteome but not transcriptome, suggesting post-transcriptional regulation. These results underscore the importance of integrated mRNA and protein expression measurements and validate the use of the iTRAQ method combined with microarray to assess global protein and gene changes involved in the development of T2D.

Loss of Lkb1 in Adult β Cells Increases β Cell Mass and Enhances Glucose Tolerance in Mice

The Lkb1 tumor suppressor exerts its biological effects through phosphorylation and consequent activation of the AMP kinase (AMPK) family. Extensive genetic and biochemical evidence supports a role for Lkb1 in cell cycle arrest, establishment of cell polarity, and cellular energy metabolism. However, the role of Lkb1 and the AMPK family in beta cell function in vivo has not been established. We generated conditional knockout mice with a deletion of the Lkb1 gene in the beta cell compartment of pancreatic islets; these mice display improved glucose tolerance and protection against diet-induced hyperglycemia. Lkb1(-/-) beta cells are hypertrophic because of elevated mTOR activity; they also proliferate more and secrete more insulin in response to glucose. These data indicate that inhibiting Lkb1 activity in beta cells may facilitate beta cell expansion and glucose tolerance in vivo.

Zinc, a regulator of islet function and glucose homeostasis.

It is well known that zinc is required in pancreatic β‐cells in the process of insulin biosynthesis and the maturation of insulin secretory granules. In fact, the zinc level in pancreatic islets is amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. High concentrations of zinc can also be toxic because of enhanced oxidative damage. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genomewide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we explore the importance of both zinc and ZnT8 to islet biology and whole body glucose homeostasis.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic α-cells are exposed to Zn2+ released from insulin-containing secretory granules. Although maintenance of Zn2+ homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn2+-transporting pathways and the regulation of Zn2+ in α-cells. To examine the effect of Zn2+ on glucagon secretion and possible mechanisms controlling the intracellular Zn2+ level ([Zn2+]i), we employed a glucagon-producing cell line (α-TC6) and mouse islets where non-β-cells were identified using islets expressing green fluorescent protein exclusively in β-cells. In this study, we first confirmed that Zn2+ treatment resulted in the inhibition of glucagon secretion in α-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of KATP channels by Zn2+. We then determined that Zn2+ was transported into α-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca2+ channel blocker Gd3+ partially inhibited Zn2+ influx in α-TC cells, whereas the L-type voltage-gated Ca2+ channel inhibitor nitrendipine failed to block Zn2+ accumulation. To investigate Zn2+ transport further, we profiled α-cells for Zn2+ transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn2+ transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn2+]i, we examined the effects of oxidizing agents on Zn2+ mobilization within α-cells. 2,2′-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn2+]i in α-cells. Together these results demonstrate that Zn2+ inhibits glucagon secretion, and it is transported into α-cells in part through Ca2+ channels. Zn2+ transporters and the redox state also modulate [Zn2+]i.

miR-33a Modulates ABCA1 Expression, Cholesterol Accumulation, and Insulin Secretion in Pancreatic Islets

Changes in cellular cholesterol affect insulin secretion, and β-cell–specific deletion or loss-of-function mutations in the cholesterol efflux transporter ATP-binding cassette transporter A1 (ABCA1) result in impaired glucose tolerance and β-cell dysfunction. Upregulation of ABCA1 expression may therefore be beneficial for the maintenance of normal islet function in diabetes. Studies suggest that microRNA-33a (miR-33a) expression inversely correlates with ABCA1 expression in hepatocytes and macrophages. We examined whether miR-33a regulates ABCA1 expression in pancreatic islets, thereby affecting cholesterol accumulation and insulin secretion. Adenoviral miR-33a overexpression in human or mouse islets reduced ABCA1 expression, decreased glucose-stimulated insulin secretion, and increased cholesterol levels. The miR-33a–induced reduction in insulin secretion was rescued by cholesterol depletion by methyl-β-cyclodextrin or mevastatin. Inhibition of miR-33a expression in apolipoprotein E knockout islets and ABCA1 overexpression in β-cell–specific ABCA1 knockout islets rescued normal insulin secretion and reduced islet cholesterol. These findings confirm the critical role of β-cell ABCA1 in islet cholesterol homeostasis and β-cell function and highlight modulation of β-cell miR-33a expression as a means to influence insulin secretion.