Aims/hypothesis
MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in ...insulin target tissues from three inbred rat strains that differ in diabetes susceptibility.
Methods
Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto–Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (
n
= 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions.
Results
We found 29 significantly differentiated microRNAs (
p
adjusted
< 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (
p
adjusted
= 0.0005) and miR-27a (
p
adjusted
= 0.006) were upregulated in adipose tissue; miR-195 (
p
adjusted
= 0.006) and miR-103 (
p
adjusted
= 0.04) were upregulated in liver; and miR-10b (
p
adjusted
= 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (
p
= 0.008), miR-27a (
p
= 0.02) and the previously reported miR-29a (
p
= 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes.
Conclusion
The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto–Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.
Aims/hypothesis
Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol
GCKR
) P446L variant, associated ...with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation.
Methods
Fluorescently tagged rat and human wild-type (WT)- or P446L-
GCKR
and
GCK
were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency.
Results
P446L-GKRP had a decreased degree of nuclear localisation, ability to sequester GCK and direct interaction with GCK as measured by FRET compared with WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high compared with low glucose, but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localisation or GCK sequestration.
Conclusions/interpretation
Our study suggests the common human P446L-GKRP variant protein results in elevated hepatic glucose uptake and disposal by increasing active cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.
Heterozygous activating mutations in KCNJ11, which encodes the Kir6.2 subunit of the pancreatic ATP-sensitive potassium (K(ATP)) channel, cause both permanent and transient neonatal diabetes. A ...minority of patients also have neurological features. The identification of a KCNJ11 mutation has important therapeutic implications, as many patients can replace insulin injections with sulfonylurea tablets. We aimed to determine the age of presentation of patients with KCNJ11 mutations and to examine if there was a relationship between genotype and phenotype.
KCNJ11 was sequenced in 239 unrelated patients from 21 countries, who were diagnosed with permanent diabetes before 2 years of age.
Thirty-one of the 120 patients (26%) diagnosed in the first 26 weeks of life had a KCNJ11 mutation; no mutations were found in the 119 cases (0%) diagnosed after this age. Fourteen different heterozygous mutations were identified, with the majority resulting from de novo mutations. These include seven novel mutations: H46Y, R50Q, G53D C166Y, K170T, L164P and Y330S. All 11 probands with the most common mutation, R201H, had isolated diabetes. In contrast, developmental delay in addition to diabetes was seen in four of five probands with the V59M mutation and two of four with the R201C mutation. Five patients with developmental delay, epilepsy and neonatal diabetes (DEND) syndrome had unique mutations not associated with other phenotypes.
KCNJ11 mutations are a common cause of permanent diabetes diagnosed in the first 6 months and all patients diagnosed in this age group should be tested. There is a strong genotype-phenotype relationship with the mutation being an important determinant of associated neurological features.
Aims/hypothesis
An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to ...prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype–phenotype relationship and compare different hsCRP assays.
Methods
High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (
n
= 457), glucokinase (GCK)-MODY (
n
= 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (
n
= 54) and type 2 diabetes (
n
= 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic.
Results
In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (
z
score −21.8,
p
< 5 × 10
−105
). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l,
p
< 5 × 10
−5
). High-sensitivity CRP values between assays were strongly correlated (
r
2
≥ 0.91,
p
≤ 1 × 10
−5
). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy.
Conclusions/interpretation
In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes.
Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment. We recently identified a locus on chromosome 10p13-p12.1 ...involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family. Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1α, as disease-causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development, and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a−/− mice.
Inwardly rectifying potassium channels (Kir channels) control cell membrane K+fluxes and electrical signaling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the ...pore-forming subunit of the ATP-sensitive ( KATP) channel, cause permanent neonatal diabetes mellitus (PNDM). For some mutations, PNDM is accompanied by marked developmental delay, muscle weakness, and epilepsy (severe disease). To determine the molecular basis of these different phenotypes, we expressed wild-type or mutant (R201C, Q52R, or V59G) Kir6.2/sulfonylurea receptor 1 channels in Xenopus oocytes. All mutations increased resting whole-cell KATPcurrents by reducing channel inhibition by ATP, but, in the simulated heterozygous state, mutations causing PNDM alone (R201C) produced smaller KATPcurrents and less change in ATP sensitivity than mutations associated with severe disease (Q52R and V59G). This finding suggests that increased KATPcurrents hyperpolarize pancreatic beta cells and impair insulin secretion, whereas larger KATPcurrents are required to influence extrapancreatic cell function. We found that mutations causing PNDM alone impair ATP sensitivity directly (at the binding site), whereas those associated with severe disease act indirectly by biasing the channel conformation toward the open state. The effect of the mutation on ATP sensitivity in the heterozygous state reflects the different contributions of a single subunit in the Kir6.2 tetramer to ATP inhibition and to the energy of the open state. Our results also show that mutations in the slide helix of Kir6.2 (V59G) influence the channel kinetics, providing evidence that this domain is involved in Kir channel gating, and suggest that the efficacy of sulfonylurea therapy in PNDM may vary with genotype.
Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-βH1 and EndoC-βH2 are increasingly used by the research community. However, ...little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of Ca
, membrane depolarisation and increased action potential firing. Similar to human primary beta cells, K
channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the K
channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca
channels with some small contribution of TTX-sensitive Na
channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca
-activated K
channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and thus represent a useful experimental model.
Permanent neonatal diabetes (PND), requiring insulin within the first months of life, is unexplained at the molecular level in most cases. It has very recently been shown that heterozygous activating ...mutations in the KCNJ11 gene, encoding the Kir6.2 subunit of the pancreatic ATP-sensitive K(+) channel involved in the regulation of insulin secretion, cause PND. In the present study, we screened the KCNJ11 gene for mutations in French patients with PND. Patients were recruited through the French network for the study of neonatal diabetes. Seventeen at-term babies with a median age at diagnosis of diabetes of 64 days (range 1-260) were included. We identified in nine patients seven heterozygous nonsynonymous mutations: three of them (V59M, R201C, and R201H) were already described, and the four novel mutations resulted in an amino acid change of Kir6.2 at positions F35L, G53N, E322K, and Y330C. More patients with a Kir6.2 mutation (six of nine) were reported to have a smaller birth weight than those without mutation (two of eight). Although Kir6.2 mutation carriers do not represent a phenotypically specific form of PND, an impaired function of Kir6.2 is associated with in utero insulin secretory insufficiency and growth retardation. In conclusion, we confirmed that Kir6.2 mutations are a common cause (53%) of PND in Caucasians.
Finding novel causes for monogenic forms of diabetes is important as, alongside the clinical implications of such a discovery, it can identify critical proteins and pathways required for normal beta ...cell function in humans. It is increasingly apparent that there are significant differences between rodent and human islets. One example that has generated interest is the relative importance of the glucose transporter GLUT2 in rodent and human beta cells. The central role of GLUT2 in rodent beta cells is well established, but a number of studies have suggested that other glucose transporters, namely GLUT1 and GLUT3, may play an important role in facilitating glucose transport into human beta cells. In this issue of
Diabetologia
Sansbury et al (DOI:
10.1007/s00125-012-2595-0
) report homozygous loss of function mutations in
SLC2A2
, which encodes GLUT2, as a rare cause of neonatal diabetes. Evidence for a beta cell defect in these subjects comes from very low birthweights, lack of endogenous insulin secretion and a requirement for insulin therapy. Neonatal diabetes is not a consistent feature of
SLC2A2
mutations. It is only found in a small percentage of cases (~4%) and the diabetes largely resolves before 18 months of age. This discovery is significant as it suggests that GLUT2 plays an important role in human beta cells, but the interplay and relative roles of other transporters differ from those in rodents. This finding should encourage efforts to delineate the precise role of GLUT2 in the human beta cell at different developmental time points and is a further reminder of critical differences between human and rodent islets.
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