Prevalence of diabetes in Poland: a combined analysis of national databases.

AIMS: To assess the number of people with diabetes in Poland using combined national sources and to evaluate the usefulness of data from an insurance system for epidemiological purposes. METHODS: The data were collected from four sources: 1) 2013 all-billing records of the national insurance system comprising people of all age groups undergoing procedures or receiving services in primary healthcare, specialist practices and hospitals and also those receiving drugs; 2) an epidemiological study, NATPOL, that involved the assessment of people with undiagnosed diabetes; 3) the RECEPTOmetr Sequence study on prescriptions; and 4) regional child diabetes registries. RESULTS: In 2013, 1.76 million people (0.98 million women and 0.79 million men) had medical consultations (coded E10-E14) and 2.13 million people (1.19 million women and 0.94 million men) purchased drugs or strip tests for diabetes. A total of 0.04 million people who used medical services did not buy drugs. In total, the number of people with diabetes in the insurance system was 2.16 million (1.21 million women and 0.95 million men), which corresponds to 6.1% (95% CI 6.11-6.14) of women and 5.1% (95% CI 5.12-5.14) of men. Including undiagnosed cases, the total number of people with diabetes in Poland was 2.68 million in 2013. CONCLUSION: The estimated prevalence of diabetes (diagnosed and undiagnosed cases) in Poland is 6.97%. Data from the national insurance system with full coverage of the population can be treated as a reliable source of information on diseases with well-defined diagnosis and treatment methods, combined with an assessment of the number of undiagnosed individuals.

Regional differences in diabetic patients' pharmacotherapy in Bulgaria.

BACKGROUND: The regional analyses play an important role in understanding a state of diabetes mellitus management and to support informed policy options. They need to be explored in more details in order to ensure an equal patients' access to health care services of the same value and quality. AIM: The aim of this study is to analyze regional differences in a cost of diabetes therapy for insulin users in Bulgaria. MATERIALS AND METHODS: It is a combined prospective and retrospective observational study with duration of 6 months. Diabetic patients on insulin therapy were recruited by 35 endocrinologists. Information about the health care resources used was collected within 3-prospective and 3 retrospective months in 2010 and 2011. The regional cost of illness analysis was performed by calculating the average cost attributable to each individual patient despite the fact that some might not use a particular health care resource. Subgroup analysis was performed for hospitalized patients. RESULTS: A detailed analysis revealed cost differences in the regions, especially with more vulnerable population like Burgas and Pleven regions. Another reason for the cost differences is the type of insulin or type of therapy. Our study confirms the fact that the hospitalizations are the major cost driver. Rising diabetes prevalence and associated costs, including hospitalizations and management of diabetes complications, are a growing concern. The last possible reason for regional differences is the patients' characteristics and therapy differences. We add evidence demonstrating that the patients on insulin and OAD consume more resources including hospitalizations and suffer from more complications of diabetes. CONCLUSIONS: Reasons for regional differences might have different origin as there are various population characteristics, type of therapy, socio economic status and others.

Insulin signalling mechanisms for triacylglycerol storage.

Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein ß isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research.

Antibacterial prescriptions for acute gastrointestinal infections: uncovering the iceberg.

A prospective survey was conducted in patients admitted to 11 randomly selected general practices and eight hospitals located in six provinces of Poland. For each patient meeting the international acute gastrointestinal infection (AGI) case definition criteria, information was collected on healthcare resources used. Antibacterial drug consumption was assessed using defined daily doses (DDD) and extrapolated to the national level using results from a parallel study of AGI incidence in the community. Additionally, a logistic multivariable model was fitted assessing determinants of antibacterial drug administration. Valid questionnaires were collected from 385 general practitioner (GP) consultations and 504 hospital admissions. Antibacterials for systemic use were prescribed during 60 (16%) GP consultations and 179 (36%) hospital admissions. The estimated societal AGI-related consumption of antibacterials amounted to 5·48 million DDD (95% uncertainty interval 1·56-14·12 million DDD). Antibacterial prescription was associated with work in large practices [adjusted odds ratio (aOR) 3·16] and hospital wards (aOR 2·87), compared to small general practices, referral for microbiological testing (aOR 2·88), presence of fever (aOR 2·50), presence of mucus or blood in stool (aOR 1·94), age >65 years vs. <5 years (aOR 1·88), and rural vs. urban residence (aOR 1·53). Despite the fact that antibacterials were prescribed to a minority of consulted AGI patients, their consumption in society was not negligible due to the high prevalence of AGI symptoms. Prescription of antibacterial drugs should be restricted to cases with specific indications, preferably following microbiological investigation of AGI aetiology. To achieve this, clear national recommendations should be widely disseminated to physicians, and included in medical training curricula.

RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity.

Analysis of insulin signalling by RNAi-based gene silencing.

Using siRNA-mediated gene silencing in cultured adipocytes, we have dissected the insulin-signalling pathway leading to translocation of GLUT4 glucose transporters to the plasma membrane. RNAi (RNA interference)-based depletion of components in the putative TC10 pathway (CAP, CrkII and c-Cbl plus Cbl-b) or the phospholipase Cgamma pathway failed to diminish insulin signalling to GLUT4. Within the phosphoinositide 3-kinase pathway, loss of the 5'-phosphatidylinositol 3,4,5-trisphosphate phosphatase SHIP2 was also without effect, whereas depletion of the 3'-phosphatase PTEN significantly enhanced insulin action. Downstream of phosphatidylinositol 3,4,5-trisphosphate and PDK1, silencing the genes encoding the protein kinases Akt1/PKBalpha, or CISK(SGK3) or protein kinases Clambda/zeta had little or no effect, but loss of Akt2/PKBbeta significantly attenuated GLUT4 regulation by insulin. These results show that Akt2/PKBbeta is the key downstream intermediate within the phosphoinositide 3-kinase pathway linked to insulin action on GLUT4 in cultured adipocytes, whereas PTEN is a potent negative regulator of this pathway.

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate.

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

PYK2 as a mediator of endothelin-1/G alpha 11 signaling to GLUT4 glucose transporters.

Endothelin-1 (ET-1) signaling through G alpha(q/11) stimulates translocation of intracellular GLUT4 glucose transporters to the plasma membrane of 3T3-L1 adipocytes by an unknown mechanism that requires protein tyrosine phosphorylation and ADP-ribosylation factor 6 (ARF6) but is independent of phosphatidylinositol 3 (PI3)-kinase. In contrast, insulin action on this process requires PI3-kinase but not ARF6. Here we report the identification of two proteins selectively tyrosine-phosphorylated in response to ET-1 but not insulin: the Ca(2+)-activated tyrosine kinase PYK2 and its physiological substrate, the adhesion scaffold protein paxillin. Endogenous paxillin as well as expressed Myc-tagged PYK2 or a Myc-tagged kinase-deficient PYK2 protein were acutely directed to F-actin-rich adhesion sites from the adipocyte cytoplasm in response to ET-1 but not insulin. CADTK-related non-kinase (CRNK) is a dominant negative form of PYK2 containing the C-terminal portion of the protein, which binds paxillin but lacks the PYK2 autophosphorylation site (Tyr(402)). CRNK expression in 3T3-L1 adipocytes inhibited ET-1-mediated F-actin polymerization and translocation of Myc-tagged GLUT4-enhanced green fluorescent protein (EGFP) to the plasma membrane without disrupting insulin action on these processes. These data reveal the tyrosine kinase PYK2 as a required signaling element in the regulation of GLUT4 recycling in 3T3-L1 adipocytes by ET-1, whereas insulin signaling is directed through a different pathway.

[Cost of depression: prospective analysis. Follow-up study in patients with recurrent depression]. / Ocena prospektywna przebiegu depresji i jej nawrotów. Skutki kliniczne, spoleczne i ekonomiczne--doniesienie tymczasowe.

This article presents a comparison of costs of the first, the second, the third or next episode of depression, based on preliminary results of an observation clinical trial.

Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1.

GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a (32)P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling.

Phox homology domains specifically bind phosphatidylinositol phosphates.

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane.

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways.

A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3-L1 adipocytes.

Insulin regulates glucose uptake in adipocytes and muscle by stimulating the movement of sequestered glucose transporter 4 (GLUT4) proteins from intracellular membranes to the cell surface. Here we report that optimal insulin-mediated GLUT4 translocation is dependent upon both microtubule and actin-based cytoskeletal structures in cultured adipocytes. Depolymerization of microtubules and F-actin in 3T3-L1 adipocytes causes the dispersion of perinuclear GLUT4-containing membranes and abolishes insulin action on GLUT4 movements to the plasma membrane. Furthermore, heterologous expression in 3T3-L1 adipocytes of the microtubule-binding protein hTau40, which impairs kinesin motors that move toward the plus ends of microtubules, markedly delayed the appearance of GLUT4 at the plasma membrane in response to insulin. The hTau40 protein had no detectable effect on microtubule structure or perinuclear GLUT4 localization under these conditions. These results are consistent with the hypothesis that both the actin and microtubule-based cytoskeleton, as well as a kinesin motor, direct the translocation of GLUT4 to the plasma membrane in response to insulin.

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase.

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.

Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains.

Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

Perinuclear localization and insulin responsiveness of GLUT4 requires cytoskeletal integrity in 3T3-L1 adipocytes.

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.