Pharmacogenetics of oral antidiabetic therapy.

Type 2 diabetes prevalence is still on the rise worldwide. Antidiabetic drugs are widely prescribed to patients with Type 2 diabetes. Most patients start with metformin which is mostly well tolerated. However, a high percentage of patients fail to achieve glycemic control. The effectiveness of metformin as well as most other antidiabetic drugs depends among other factors on interindividual genetic differences that are up to now ignored in the treatment of Type 2 diabetes. Interestingly, many genes influencing the effectiveness of antidiabetic drugs are Type 2 diabetes risk genes making matters worse. Here, we shed light on these interindividual genetic differences.

Common variation in the sodium/glucose cotransporter 2 gene SLC5A2 does neither affect fasting nor glucose-suppressed plasma glucagon concentrations.

AIM: Inhibition of sodium/glucose cotransporter 2 (SGLT2), the key transport protein in renal glucose reabsorption, promotes glucose excretion and represents a new concept in the therapy of type-2 diabetes. In addition, SGLT2 inhibition elevates circulating glucagon concentrations and enhances hepatic glucose production. Since SGLT2 is expressed in human pancreatic α-cells and regulates glucagon release, we tested whether common variants of the SGLT2 gene SLC5A2 associate with altered plasma glucagon concentrations in the fasting state and upon glucose challenge. METHODS: A study population of 375 healthy subjects at increased risk for type-2 diabetes, phenotyped by a 5-point oral glucose tolerance test (OGTT) and genotyped for recently described SLC5A2 tagging single nucleotide polymorphisms (SNPs), was selected for plasma glucagon measurements. RESULTS: After adjustment for gender, age, body mass index, and insulin sensitivity, the four tagging SNPs (rs9924771, rs3116150, rs3813008, rs9934336), tested separately or as genetic score, were neither significantly nor nominally associated with plasma glucagon concentrations at any time during the OGTT, with the inverse AUC of glucagon or the glucagon fold-change during the OGTT (p ≥ 0.2, all). Testing for genotype-related differences in the time course of the glucagon response using MANOVA did also not reveal any significant or nominal associations (p ≥ 0.5, all). CONCLUSION: We could not obtain statistically significant evidence for a role of common SLC5A2 variants in the regulation of glucagon release in the fasting state or upon glucose challenge. Moreover, the reported nominal effects of individual SLC5A2 variants on fasting and post-challenge glucose levels may probably not be mediated by altered glucagon release.

Granulocyte colony-stimulating factor (G-CSF): A saturated fatty acid-induced myokine with insulin-desensitizing properties in humans.

OBJECTIVE: Circulating long-chain free fatty acids (FFAs) are important metabolic signals that acutely enhance fatty acid oxidation, thermogenesis, energy expenditure, and insulin secretion. However, if chronically elevated, they provoke inflammation, insulin resistance, and ß-cell failure. Moreover, FFAs act via multiple signaling pathways as very potent regulators of gene expression. In human skeletal muscle cells differentiated in vitro (myotubes), we have shown in previous studies that the expression of CSF3, the gene encoding granulocyte colony-stimulating factor (G-CSF), is markedly induced upon FFA treatment and exercise. METHODS AND RESULTS: We now report that CSF3 is induced in human myotubes by saturated, but not unsaturated, FFAs via Toll-like receptor 4-dependent and -independent pathways including activation of Rel-A, AP-1, C/EBPα, Src, and stress kinases. Furthermore, we show that human adipocytes and myotubes treated with G-CSF become insulin-resistant. In line with this, a functional polymorphism in the CSF3 gene affects adipose tissue- and whole-body insulin sensitivity and glucose tolerance in human subjects with elevated plasma FFA concentrations. CONCLUSION: G-CSF emerges as a new player in FFA-induced insulin resistance and thus may be of interest as a target for prevention and treatment of type 2 diabetes.

Nor-1, a novel incretin-responsive regulator of insulin genes and insulin secretion.

B-cell failure at the onset of type 2 diabetes is caused by a decline in ß-cell function in the postprandial state and loss of pancreatic ß-cell mass. Recently, we showed an association between increased insulin secretion and a single nucleotide polymorphism (SNP), SNP rs12686676, in the NR4A3 gene locus encoding the nuclear receptor Nor-1. Nor-1 is expressed in ß-cells, however, not much is known about its function with regard to insulin gene expression and insulin secretion. Nor-1 is induced in a glucose-/incretin-dependent manner via the PKA pathway and directly induces insulin gene expression. Additionally, it stimulates insulin secretion possibly via regulation of potentially important genes in insulin exocytosis. Moreover, we show that the minor allele of NR4A3 SNP rs12686676 fully rescues incretin resistance provoked by a well-described polymorphism in TCF7L2. Thus, Nor-1 represents a promising new target for pharmacological intervention to fight diabetes.

The Akt substrate Girdin is a regulator of insulin signaling in myoblast cells.

Akt kinases are important mediators of the insulin signal, and some Akt substrates are directly involved in glucose homeostasis. Recently, Girdin has been described as an Akt substrate that is expressed ubiquitously in mammals. Cells overexpressing Girdin show an enhanced Akt activity. However, not much is known about Girdin's role in insulin signaling. We therefore analyzed the role of Girdin in primary human myotubes and found a correlation between Girdin expression and insulin sensitivity of the muscle biopsy donors, as measured by a hyperinsulinemic-euglycemic clamp. To understand this finding on a cellular level, we then investigated the function of Girdin in C2C12 mouse myoblasts. Girdin knock-down reduced Akt and insulin receptor substrate-1 phosphorylation. In contrast, stable overexpression of Girdin in C2C12 cells strikingly increased insulin sensitivity through a massive upregulation of the insulin receptor and enhanced tyrosine phosphorylation of insulin receptor substrate-1. Furthermore, Akt and c-Abl kinases were constitutively activated. To investigate medium-term insulin responses we measured glucose incorporation into glycogen. The Girdin overexpressing cells showed a high basal glycogen synthesis that peaked already at 1nM insulin. Taken together, we characterized Girdin as a new and major regulator of the insulin signal in myoblasts and skeletal muscle.

Common genetic variation in the SERPINF1 locus determines overall adiposity, obesity-related insulin resistance, and circulating leptin levels.

OBJECTIVE: Pigment epithelium-derived factor (PEDF) belongs to the serpin family of peptidase inhibitors (serpin F1) and is among the most abundant glycoproteins secreted by adipocytes. In vitro and mouse in vivo data revealed PEDF as a candidate mediator of obesity-induced insulin resistance. Therefore, we assessed whether common genetic variation within the SERPINF1 locus contributes to adipose tissue-related prediabetic phenotypes in humans. SUBJECTS/METHODS: A population of 1,974 White European individuals at increased risk for type 2 diabetes was characterized by an oral glucose tolerance test with glucose and insulin measurements (1,409 leptin measurements) and genotyped for five tagging SNPs covering 100% of common genetic variation (minor allele frequency ≥ 0.05) in the SERPINF1 locus. In addition, a subgroup of 486 subjects underwent a hyperinsulinaemic-euglycaemic clamp and a subgroup of 340 magnetic resonance imaging (MRI) and spectroscopy (MRS). RESULTS: After adjustment for gender and age and Bonferroni correction for the number of SNPs tested, SNP rs12603825 revealed significant association with MRI-derived total adipose tissue mass (p = 0.0094) and fasting leptin concentrations (p = 0.0035) as well as nominal associations with bioelectrical impedance-derived percentage of body fat (p = 0.0182) and clamp-derived insulin sensitivity (p = 0.0251). The association with insulin sensitivity was completely abolished by additional adjustment for body fat (p = 0.8). Moreover, the fat mass-increasing allele of SNP rs12603825 was significantly associated with elevated fasting PEDF concentrations (p = 0.0436), and the PEDF levels were robustly and positively associated with all body fat parameters measured and with fasting leptin concentrations (p<0.0001, all). CONCLUSION: In humans at increased risk for type 2 diabetes, a functional common genetic variant in the gene locus encoding PEDF contributes to overall body adiposity, obesity-related insulin resistance, and circulating leptin levels.

XPA-210: a new proliferation marker determines locally advanced prostate cancer and is a predictor of biochemical recurrence.

PURPOSE: XPA-210 is a proliferation marker derived from Thymidine kinase-1. It is of clinical significance in kidney, breast, and bladder cancer. There are no data available for XPA-210 in prostate cancer (PC). Herein, we aim to determine the clinical usefulness of XPA-210 in PC. MATERIALS AND METHODS: In a retrospective study, cancer and benign tissue samples of 103 patients (median age 65 years, median PSA 9.04 ng/ml, median Gleason score 6) who underwent prostatectomy were constructed to a tissue micro array and stained for XPA-210. Semi-quantitative results were correlated with pathological and clinical data by Wilcoxon-Kruskall-Wallis and linear regression analysis. Expression levels in PC were correlated between the time of biochemical recurrence and the time to development of metastasis by the Kaplan-Meier method. Multivariate analysis was done to correlate those with the resection status. RESULTS: Mean staining score was 0.51-0.14 for tumor and benign tissue (P < 0.0001). Tumor staining score was significantly associated with Gleason score <6/≥6 (P < 0.0001) and T2/T >2 (P = 0.0007). When dividing the tumor score by the mean value, higher expression of XPA-210 was associated with a shorter time to biochemical recurrence (P = 0.003) and time to development of metastasis (P = 0.0061). Tumor staining (P = 0.0371) was an independent prognostic factor for biochemical relapse regardless of resection status. CONCLUSIONS: XPA-210 is a new tissue-based prognostic marker for prostate cancer histopathology. It reliably differentiates tumor and normal prostatic tissue predicting biochemical relapse and onset of metastatic disease. XPA-210 might be clinically useful for individual decision-making in PC-treatment.

Insulin promotes glycogen storage and cell proliferation in primary human astrocytes.

INTRODUCTION: In the human brain, there are at least as many astrocytes as neurons. Astrocytes are known to modulate neuronal function in several ways. Thus, they may also contribute to cerebral insulin actions. Therefore, we examined whether primary human astrocytes are insulin-responsive and whether their metabolic functions are affected by the hormone. METHODS: Commercially available Normal Human Astrocytes were grown in the recommended medium. Major players in the insulin signaling pathway were detected by real-time RT-PCR and Western blotting. Phosphorylation events were detected by phospho-specific antibodies. Glucose uptake and glycogen synthesis were assessed using radio-labeled glucose. Glycogen content was assessed by histochemistry. Lactate levels were measured enzymatically. Cell proliferation was assessed by WST-1 assay. RESULTS: We detected expression of key proteins for insulin signaling, such as insulin receptor ß-subunit, insulin receptor substrat-1, Akt/protein kinase B and glycogen synthase kinase 3, in human astrocytes. Akt was phosphorylated and PI-3 kinase activity increased following insulin stimulation in a dose-dependent manner. Neither increased glucose uptake nor lactate secretion after insulin stimulation could be evidenced in this cell type. However, we found increased insulin-dependent glucose incorporation into glycogen. Furthermore, cell numbers increased dose-dependently upon insulin treatment. DISCUSSION: This study demonstrated that human astrocytes are insulin-responsive at the molecular level. We identified glycogen synthesis and cell proliferation as biological responses of insulin signaling in these brain cells. Hence, this cell type may contribute to the effects of insulin in the human brain.

In vitro responsiveness of human muscle cell peroxisome proliferator-activated receptor δ reflects donors' insulin sensitivity in vivo.

BACKGROUND: Peroxisome proliferator-activated receptor δ (PPARδ) activation enhances muscular fatty acid oxidation and oxidative phosphorylation, and muscle's oxidative capacity positively associates with whole-body insulin sensitivity. Therefore, we asked here whether human muscle cell PPARD expression is a determinant of donors' insulin sensitivity. MATERIALS AND METHODS: Skeletal muscle cells derived from 38 nondiabetic donors were differentiated in vitro to myotubes, and gene (mRNA) expression was quantified by real-time RT-PCR. Donors' insulin sensitivity was calculated from plasma insulin and glucose levels during oral glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp. RESULTS: Basal myotube PPARD expression was closely related to the expression of its target genes PDK4 and ANGPTL4 (P = 0·0312 and P = 0·0003, respectively). Basal PPARD, PDK4 and ANGPTL4 expression levels were not associated with donors' insulin sensitivity (P > 0·2, all). Treatment of myotubes with a selective high-affinity PPARδ agonist (GW501516) did not change mean PPARD, but enhanced mean PDK4 and ANGPTL4 expression 13- and 16-fold, respectively (P < 0·0001, both). The individual PDK4 and ANGPTL4 expression levels reached upon GW501516 treatment were associated with donors' insulin sensitivity neither (P > 0·2, both). However, GW501516-mediated fold increments in PDK4 and ANGPTL4 expression, reflecting PPARδ responsiveness, were positively associated with donors' insulin sensitivity derived from OGTT (P = 0·0182 and P = 0·0231, respectively) and hyperinsulinemic-euglycemic clamp (P = 0·0046 and P = 0·0258, respectively). CONCLUSIONS: Using a highly selective pharmacological tool, we show here that the individual responsiveness of human muscle cell PPARδ, rather than the absolute PPARD expression level, represents a major determinant of insulin sensitivity.

The myocyte expression of adiponectin receptors and PPARδ is highly coordinated and reflects lipid metabolism of the human donors.

Muscle lipid oxidation is stimulated by peroxisome proliferator-activated receptor (PPAR) δ or adiponectin receptor signalling. We studied human myocyte expression of the PPARδ and adiponectin receptor genes and their relationship to lipid parameters of the donors. The mRNA levels of the three adiponectin receptors, AdipoR1, AdipoR2, and T-cadherin, were highly interrelated (r ≥ 0.91). However, they were not associated with GPBAR1, an unrelated membrane receptor. In addition, the adiponectin receptors were positively associated with PPARδ expression (r ≥ 0.75). However, they were not associated with PPARα. Using stepwise multiple linear regression analysis, PPARδ was a significant determinant of T-cadherin (P = .0002). However, pharmacological PPARδ activation did not increase T-cadherin expression. The myocyte expression levels of AdipoR1 and T-cadherin were inversely associated with the donors' fasting plasma triglycerides (P < .03). In conclusion, myocyte expression of PPARδ and the adiponectin receptors are highly coordinated, and this might be of relevance for human lipid metabolism in vivo.