IV- New tools in research and their clinical interest

Gordon Weir (Boston, USA) described the methods used to investigate changes in gene expression in the cells that constitute the islets of Langerhans. Some of these methods enable the generation of cDNA, which may be used in gene cloning. The technique of laser capture microdissection (LCM) enables isolation of specific cells of interest from microscopic regions of tissue without altering or damaging the morphology or chemistry of the sample collected, nor the surrounding cells. LCM can also be performed on a variety of tissue samples including cytologic preparations, cell cultures, and aliquots of solid tissue. For these reasons, LCM can overcome the difficulty of isolating islet cells for gene expression analyses.

Leif Groop (Malmö, Sweden) reviewed the genetic factors that underlie beta-cell dysfunction. Single-gene (or Mendelian) diseases are generally more severe and easily detectable compared with complex polygenic diseases, which involve several interacting genetic and environmental factors. However, the sequencing and mapping of the human genome has enabled the introduction of whole genome-wide association studies. These have given some insights into the genetic factors that influence complex genetic diseases such as most variants of type 2 diabetes. Today, around 20 genetic variants have been identified that increase the risk of type 2 diabetes. Most of these variants are associated with problems in the capacity of insulin secretion to increase in response to increased body weight and insulin resistance. The current genetic data pertinent to type 2 diabetes are still very partial and future studies within the next few years will offer a more complete picture of the genetic complexity of the disease. In addition to increasing the understanding of genetic factors associated with future risk of the disease, this new data will also be used in selecting the most appropriate treatment according to the genetic variant of each patient. The genotyping of patients in order to select effective treatment is currently performed in certain cases of breast carcinoma.
In cases of type 2 diabetes, there is already evidence that patients of certain risk genotypes respond better to some antidiabetic medications than others. This new means of targeting treatment to specific patients is termed pharmacogenetics, and is one of the most promising future developments for the management of disease. The
genotype analysis may also be applied to identification of the risks of complications associated with type 2 diabetes.

Romano Regazzi (Lausanne, Switzerland) presented the potential therapeutic uses of microRNAs (miRNAs) in the treatment of diabetes. miRNAs are single stranded noncoding RNA molecules of 21 to 23 nucleotides in length and are processed from double-stranded hairpin DNA precursors in the nucleus. miRNAs have the potential to reduce gene expression through non-specific binding to messenger RNA, and it is estimated that approximately 30% of all protein coding genes are targets for miRNA. Specific expression profiles of serum miRNAs have been identified in cases of lung cancer, colorectal cancer, and diabetes, suggesting that these profiles represent blood biomarkers of various diseases.10 Interestingly, miR-375 was identified as a novel, evolutionarily conserved and islet-specific miRNA. Overexpression of miR-375 suppressed glucose-induced insulin secretion, while inhibition conversely enhanced insulin secretion. The mechanisms by which insulin secretion is modified by miR-375 are unrelated to changes in glucose metabolism or intracellular calcium signalling but rather correlated with a direct effect on insulin exocytosis.11 Transgenic mice lacking miR-375 (375KO) are hyperglycaemic and exhibit decreased beta-cell mass as a result of impaired proliferation and increased numbers of α cells.12 In agreement with this observation, expression of miR-375 is increased in pancreatic islets of obese mice, an animal model of increased beta-cell mass. Other miRNAs also affect insulin exocytosis; miR-9 is required for optimal insulin release in response to glucose and other secretagogues, while miR-124a modulates the expression of several components of the machinery governing insulin exocytosis. Culture of beta cells with elevated concentrations of FAs induces expression of miR-34a and miR-146a, and both of these miRNAs are increased in pancreatic islets isolated from diabetic obese mice. The discovery of miRNAs and their importance in regulating gene expression has opened the possibility of developing gene therapies that influence the course of disease through changing gene expression profiles in targeted tissues/cells. This would be achieved through targeted delivery of small oligonucleotide molecules that either block or mimick the function of miRNAs.
The different types of oligonucleotides that could be used for this strategy include siRNA-like oligonucleotides, pri-miRNA transcripts, antagomirs, and morpholinos.

Figure 4. Regulation of insulin secretion by microRNAs in the pancreatic beta cell (taken from Benoît R Gauthier, Department of Cellular Physiology and Metabolism, University of Geneva). The microRNAs miR-375, miR-124 and let-7b inhibit the translation of myotrophin (MTPN) messenger RNA. Myotrophin interacts with proteins associatied with the cytoskeleton in order to rearrange the actin coretex and enable fusion of the insulin secretory granules with the cell membrane. Furthermore, myotropin also acts as a transcription factor and regulates transcription of the nuclear factor kappa-light-chain (NF-κB) gene. At low concentrations, NF-κB increases insulin secretion, most likely through activation of genes involved in the transport and exocytosis of insulin secretory granules. RISC: RNA-induced silencing complex; ANK: ankyrin repeat motifs; GLP-1: glucagon like peptide-1.

Figure 4. Regulation of insulin secretion by microRNAs in the pancreatic beta cell (taken from Benoît R Gauthier, Department of Cellular Physiology and Metabolism, University of Geneva). The microRNAs miR-375, miR-124 and let-7b inhibit the translation of myotrophin (MTPN) messenger RNA. Myotrophin interacts with proteins associatied with the cytoskeleton in order to rearrange the actin coretex and enable fusion of the insulin secretory granules with the cell membrane. Furthermore, myotropin also acts as a transcription factor and regulates transcription of the nuclear factor kappa-light-chain (NF-κB) gene. At low concentrations, NF-κB increases insulin secretion, most likely through activation of genes involved in the transport and exocytosis of insulin secretory granules. RISC: RNA-induced silencing complex; ANK: ankyrin repeat motifs; GLP-1: glucagon like peptide-1.

Closing session: the future of treatment in type 2 diabetes

The limitations of current treatments for type 2 diabetes are related to the inevitable progressive decline of insulin secretion throughout the course of the disease and also to problems regarding the safety of antidiabetic agents. Kenneth Polansky (St Louis, USA) outlined 4 types of therapeutic strategies that promise to yield more effective treatments in the near future.

1. New incretins

The recently identified incretin hormone, xenin 25, is produced by K cells in the intestinal mucosa. Administration of xenin 25 to type 2 diabetes patients has the potential to increase glucose-dependent increase of circulating insulin if the treatment is combined with GIP. The effects of treatment with xenin 25 are currently being investigated in type 2 diabetes patients.

2. Inhibitors of beta-cell death

The development of inhibitors of beta-cell death as a therapeutic approach in diabetes is at present more concept than reality. A potential target for this therapeutic strategy is the pancreatic duodenal homebox 1 (Pdx1), a transcription factor that plays a central role in pancreatic beta-cell function and survival. Complete deficiency in Pdx1 is associated with severe beta-cell dysfunction and apoptosis in both rodents and humans. In agreement with these observations, mutations in the Pdx1 gene are the causal factor for type 4 MODY. Future therapies that improve Pdx1 deficiency could be a means of reducing beta-cell apoptosis in type 2 diabetes.

Other therapeutic targets for the inhibition of beta-cell death are the pathways involved in autophagy; a ubiquitous process in eukaryotic cells that involves the degradation of a cell’s own components through the lysosomal machinery (Figure 5). Autophagy is a tightly regulated process that plays a normal part in cell growth and development, and helps to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is also becoming increasingly clear that autophagy plays a broad role in disease pathogenesis. Transgenic mice studies, where genes in the autophagy pathway have been inactivated, have established that a basal level of autophagy is essential for beta-cell function and survival. However, with Pdx1deficiency, the rate of autophagy in the beta-cell is increased and possibly the cause of beta-cell death. In this scenario, inhibition of autophagy delays the onset of beta-cell death induced by reduced Pdx1. Further studies are needed to determine how autophagy regulates beta-cell survival and function.

Figure 5. Mechanisms of autophagy compared with phagocytosis

Figure 5. Mechanisms of autophagy compared with phagocytosis

3. Anti-inflammatory agents

The inflammatory nature of type 2 diabetes has been previously reviewed by Hotamisligil and coworkers,13 and it is now accepted that, as well as being a metabolic disorder, type 2 diabetes is an inflammatory disorder. Infiltration of inflammatory cells into the adipocyte modifies metabolism and increases the secretion of cytokines such as TNFα and IL-6, which are implicated in insulin resistance in the liver and muscle. Furthermore, secretion of cytokines that improve the sensitivity to insulin, such as adiponectin, are reduced. In established models of obesity and diabetes, interventions that block the cytokine-activated JNK/AP-1inflammatory pathway have been shown to improve systemic glucose homeostasis and insulin sensitivity, as well as reducing atherosclerosis. In addition, transgenic mice that are heterozygous for a null mutation in inhibitor κB kinase (IKK-beta), which inactivates the IKK–NF-κB inflammatory pathway, are partially protected from obesity-induced insulin resistance. In agreement with these observations, inhibition of IKK-beta using high doses of the nonsteroidal antiinflammatory drug, salicylate, improves insulin sensitivity, reduces glycemia, and may also improve inflammatory cardiovascular risk indexes in both animal models and humans.14 In addition, studies administering the nonsteroidal anti-inflammatory drug, salsalate, to type 2 diabetes patients have shown improvements of in vivo glucose and lipid homeostasis. Altogether, these findings support the targeting of inflammation as a therapeutic approach in type 2 diabetes.

4. Activation of glucokinase

Mutations in the glucokinase gene that reduce the functional efficiency of its enzyme product are the underlying cause of type 2 MODY. The defect in the glucokinase enzyme means that the sensitivity of beta-cells to rising levels of glucose is impaired, and insulin secretion only occurs above an abnormally high threshold. This produces a chronic, mild hyperglycemia which is usually asymptomatic. One hundred mutations in the glucokinase gene have been identified in around 42 different families. In mice models, targeted complete inactivation of the glucokinase gene in beta-cells causes a severe diabetes that results in death a few days after birth. Studies that aim to develop agents that activate the glucokinase gene in these animals could yield promising future therapies for the management of at least certain variations of type 2 diabetes.

FOCUS: Genetic variants

Genome-wide association studies have identified multiple loci at which common genetic variants modestly but reproducibly influence the risk of type 2 diabetes (T2D). Today, approximately 20 genetic variants that increase the risk of T2D have been described. Most appear to affect the capacity of beta-cells to cope with the increased insulin demand imposed by insulin resistance.
Among these variants is a single nucleotide polymorphism (SNP), rs10830963, on chromosome 11 in the MTNR1B gene, which encodes melatonin receptor 1B. The risk genotype of rs10830963 is associated with increased expression of melatonin receptor 1B in pancreatic islets and predicts future T2D. Melatonin therefore appears to be involved in the regulation of insulin secretion and blocking the melatonin ligand-receptor system could be a future therapeutic strategy in T2D.7
Rarer variants for increased susceptibility to T2D tend to have stronger clinical effects. These include copy-number variations, and epigenetic variants such as DNA methylation and histone acetylation. The transcriptional coactivator, PPARGC1A (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), is a master regulator of mitochondrial gene expression.
In T2D patients, reduced expression of PPARGC1A is associated with impaired oxidative phosphorylation in muscle and reduced insulin secretion in pancreatic islets. Reduced PPARGC1A expression is associated with the PPARGC1A Gly482Ser polymorphism and an increase in DNA methylation in islet cells from patients with T2D versus non-diabetic islets.8
Recently, a polymorphism in the SLC30A8 gene which encodes ZnT8, an islet-specific zinc transporter, has been associated with T2D.9 In patients with type 1 diabetes over two years after the onset of disease, progressive decline in circulating ZnT8 antibodies is highly correlated with declining levels of C-peptide.9 Further research of the polymorphisms and molecular function of ZnT8 is clearly vital to gain insights into the development of both forms of diabetes (Groop lecture).

I- Regulation and maintenance of beta-cell mass
II – Regulation and production of insulin
III- Factors underlying beta-cell dysfunction in type 2 diabetes
IV- New tools in research and their clinical interest
Conclusions
References