Croatian guidelines for the pharmacotherapy of type 2 diabetes

INTRODUCTION: The Croatian Association for Diabetes and Metabolic Disorders of the Croatian Medical Association has issued in 2011 the first national guidelines for the nutrition, education, self-control, and pharmacotherapy of diabetes type 2. According to the increased number of available medicines and new evidence related to the effectiveness and safety of medicines already involved in the therapy there was a need for update of the existing guidelines for the pharmacotherapy of type 2 diabetes in the Republic of Croatia. PARTICIPANTS: as co-authors of the Guidelines there are listed all members of the Croatian Association for Diabetes and Metabolic Diseases, as well as other representatives of professional societies within the Croatian Medical Association, who have contributed with comments and suggestions to the development of the Guidelines. EVIDENCE: These guidelines are evidence-based, according to the GRADE system (eng. Grading of Recommendations, Assessment, Development and Evaluation), which describes the level of evidence and strength of recommendations. CONCLUSIONS: An individual patient approach based on physiological principles in blood glucose control is essential for diabetes' patients management. Glycemic targets and selection of the pharmacological agents should be tailored to the patient, taking into account the age, duration of disease, life expectancy, risk of hypoglyce- mia, comorbidities, developed vascular and other complications as well as other factors. Because of all this, is of national interest to have a practical, rational and applicable guidelines for the pharmacotherapy of type 2 diabetes.

Ca2+ signaling in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) from normal and catecholaminergic polymorphic ventricular tachycardia (CPVT)-afflicted subjects.

Derivation of cardiomyocytes from induced pluripotent stem cells (iPS-CMs) allowed us to probe the Ca(2+)-signaling parameters of human iPS-CMs from healthy- and catecholaminergic polymorphic ventricular tachycardia (CPVT1)-afflicted individuals carrying a novel point mutation p.F2483I in ryanodine receptors (RyR2). iPS-CMs were dissociated on day 30-40 of differentiation and patch-clamped within 3-6 days. Calcium currents (ICa) averaged ∼8pA/pF in control and mutant iPS-CMs. ICa-induced Ca(2+)-transients in control and mutant cells had bell-shaped voltage-dependence similar to that of ICa, consistent with Ca(2+)-induced Ca(2+)-release (CICR) mechanism. The ratio of ICa-activated to caffeine-triggered Ca(2+)-transients was ∼0.3 in both cell types. Caffeine-induced Ca(2+)-transients generated significantly smaller Na(+)-Ca(2+) exchanger current (INCX) in mutant cells, reflecting their smaller Ca(2+)-stores. The gain of CICR was voltage-dependent as in adult cardiomyocytes. Adrenergic agonists enhanced ICa, but differentially altered the CICR gain, diastolic Ca(2+), and Ca(2+)-sparks in mutant cells. The mutant cells, when Ca(2+)-overloaded, showed longer and wandering Ca(2+)-sparks that activated adjoining release sites, had larger CICR gain at -30mV yet smaller Ca(2+)-stores. We conclude that control and mutant iPS-CMs express the adult cardiomyocyte Ca(2+)-signaling phenotype. RyR2 F2483I mutant myocytes have aberrant unitary Ca(2+)-signaling, smaller Ca(2+)-stores, higher CICR gains, and sensitized adrenergic regulation, consistent with functionally altered Ca(2+)-release profile of CPVT syndrome.

Major histocompatibility complex class I-presented antigenic peptides are degraded in cytosolic extracts primarily by thimet oligopeptidase.

Nearly all peptides generated by proteasomes during protein degradation are digested rapidly to amino acids, but a few proteasomal products escape this fate and are presented to the immune system on cell surface major histocompatibility complex class I molecules. To test whether these antigenic peptides may be inherently resistant to cytosolic peptidases, six different antigenic peptides were incubated with HeLa cell extracts. All six were degraded rapidly by a process involving o-phenanthroline-sensitive metallopeptidases. One antigenic peptide, FAPGNYPAL, was rapidly destroyed in the extracts by a bestatin-sensitive exopeptidase, apparently by the puromycin-sensitive aminopeptidase. The disappearance of the other five was reduced 30-90% by a specific inhibitor of the cytosolic endopeptidase, thimet oligopeptidase (TOP) (EC ), whose physiological function(s) have been unclear and controversial. All these peptides were sensitive to pure recombinant TOP. Furthermore, upon fractionation of the extracts, the major peptidase peak that degraded the ovalbumin-derived epitope, SIINFEKL, co-purified with TOP. In the extracts, TOP also catalyzed rapid degradation of N-extended variants of SIINFEKL and of other antigenic peptides, which in vivo can serve as precursors of these major histocompatibility complex-presented epitopes. This enzyme (unlike cell proteins that promote production of antigenic peptides) is not regulated by interferon-gamma. TOP seems to be primarily responsible for the rapid breakdown of antigenic peptides in cytosolic extracts, and our related studies (A. X. Y. Mo, K. Lemerise, W. Zeng, Y. Shen, C. R. Abraham, A. L. Goldberg, and K. L. Rock, submitted for publication) indicate that TOP by destroying such peptides limits antigen presentation in vivo.

Identification of two distinct regions of allelic imbalance on chromosome 18Q in metastatic prostate cancer.

Like most cancers, prostate cancer (CaP) is believed to be the result of the accumulation of genetic alterations within cells. Previous studies have implicated numerous chromosomal regions with elevated rates of allelic imbalance (AI), using mostly primary CaPs with an unknown disease outcome. These regions of AI are proposed sites for tumor suppressor genes. One of the regions previously implicated as coding for at least one tumor suppressor gene is the long arm of chromosome 18 (18q). To confirm this observation, as well as to narrow the critical region for this putative tumor suppressor, we analyzed 32 metastatic CaP specimens for AI on chromosome 18q. Thirty-one of these 32 specimens (96.8%) exhibited AI at one or more loci on chromosome 18q. Our analysis using 17 polymorphic markers revealed statistically significant AI on chromosome 18q at 3 markers, D18S35, D18S64 and D18S461. Using these markers as a guide, we have been able to identify 2 distinct minimum regions of AI on 18q. The first region is between the genetic markers D18S1119 and D18S64. The second region lies more distal on the long arm of the chromosome and is between the genetic markers D18S848 and D18S58. To determine if 18q loss is a late event in the progression of CaP, we also examined prostatic intraepithelial neoplasia (PIN) and primary prostate tumors from 17 patients for AI with a subset of 18q markers. We found significantly higher AI in the metastatic samples. Our results are consistent with 18q losses occurring late in CaP progression.

Genetic pattern of prostate cancer progression.

Genetic alterations in primary prostate cancer (CaP) have been extensively studied, yet little is known about the genetic mechanisms underlying progression of primary CaP to metastatic prostate cancer. As a result, it is not possible to distinguish clinically indolent localized disease from potentially life-threatening tumors with high metastatic potential. To address this question, we collected tissue from 34 autopsy-derived metastases, samples rarely analyzed in previous studies. These were compared to a separate set of 17 prostatectomy specimens containing 22 foci of CaP associated with 49 examples of high-grade prostatic intraepithelial neoplasia (PIN), a histological precursor of CaP. We compared the loss of heterozygosity (LOH) profiles of high-grade PIN, primary CaP and metastases by analyzing 33 microsatellite markers previously found to have high frequencies of LOH in primary CaP. These markers were on chromosomes 5q, 6q, 7q, 8p, 9p, 10q, 11p, 13q, 16q, 17, 18q and 21q. In addition, markers on chromosomes 4p, 11q, 14q and 20q with no reported LOH in primary CaP were analyzed to determine the frequency of background LOH. In PIN lesions, the rate of LOH was significant only at D5S806 (20%) and D16S422 (29%). In addition, different PIN lesions within the same prostate gland were genetically diverse, indicating divergent evolution of synchronous neoplastic precursor lesions. LOH frequency was progressively higher in primary CaP and metastatic lesions. In primary CaP, significant losses occurred at the 8p, 10q, 11p, 16q, 17p, 18q and 21q loci (range 17-43%). Distinct patterns of LOH frequencies were observed in primary CaP compared with metastases. Although some loci (D16S422, D17S960, D21S156) showed similar frequencies of LOH in primary CaP and metastatic CaP, most other loci showed up to 7-fold metastasis-related increases. The metastatic samples revealed previously unrecognized prostate cancer LOH at D5S806, D6S262, D9S157, D13S133 and D13S227. These significant stage-specific differences in LOH frequency specify genetic loci that may play key roles in CaP progression and could represent clinically useful biomarkers for CaP aggressiveness.

Androgen regulation of prostate cancer cell FGF-1, FGF-2, and FGF-8: preferential down-regulation of FGF-2 transcripts.

Using quantitative RT-PCR, we found that T1 rat prostate cancer cell relative FGF-1 transcript content was about 180-fold greater than that of FGF-2. This difference in transcript content was not representative of T1 cell relative FGF-1 and FGF-2 protein content which showed, at most, only a 4- to 5-fold greater FGF-1 content. Testosterone caused time-dependent down-regulation of prostate cancer cell FGF-2 transcript content without influencing either FGF-1 or FGF-8 transcript content or T1 cell proliferation. Moreover, testosterone-mediated down-regulation of prostate cancer cell FGF-2 transcripts did not result in a statistically significant change in 21.5 or 17.0 kD FGF-2 isoform content. By contrast, an approximately 20% statistically significant decrement in 19.5 kD FGF-2 isoform content was demonstrable following 24 h testosterone treatment. However, following 72 h testosterone treatment, T1 cell 19.5 kD FGF-2 isoform content was not statistically significantly different from that of control. It is probable that the modest and variable decrement in 19.5 kD isoform content is not physiologically significant and is attributable to artifact resulting from difficulty quantifying this minor component of the FGF-2 isoforms. Transient transfection analysis showed that androgen caused concentration-dependent increases in MMTV-LTR regulated expression of chloramphenicol acetyl transferase activity. Consequently, the failure of androgen to affect either T1 cell FGF-1 and FGF-8 transcript content or T1 cell proliferation could not be attributed to defective androgen receptor function. Moreover, the absence of a close relationship between T1 cell FGF-2 transcript and FGF-2 protein content implies that FGF-2 transcript content is not the dominant determinant of prostate cancer cell FGF-2 protein content. Testosterone-mediated down-regulation of prostate-cancer-cell gene expression may have significance for clinical management of human disease that is treated by androgen ablation. The possibility that such ablation may enhance aggressiveness of "androgen-independent" cells by selective upregulation of gene expression merits further consideration.

Endogenous fibroblast growth factor-1 or fibroblast growth factor-2 modulate prostate cancer cell proliferation.

We constructed expression vectors containing either rat fibroblast growth factor (FGF)-1 for FGF-2 cDNA cloned in either the sense orientation or antisense orientation relative to the metallothionein promoter of plasmid pMTneo.1. Stable AXC/SSh rat prostate cancer cell transfectants expressing either chimeric FGF-1-sense, chimeric FGF-1-antisense, or chimeric FGF-2-antisense transcripts were obtained. Stable transfectants expressing chimeric FGF-2-sense transcripts were not obtained. Control, sense, and antisense transfectants expressed endogenous FGF-1 and endogenous FGF-2 transcripts, implying that transfection did not eliminate endogenous FGF transcripts. Control transfectants and sense transfectants contained FGF-1 isoforms having a mass of 16.4 or 17.3 kDa and FGF-2 isoforms having a mass of 17, 19.5, or 21.5 kDa. Significantly, adult AXC/SSh rat prostate contained only the 17.3 kDa FGF-1 isoform and the 17 kDa FGF-2 isoform, indicating that neoplastic transformation was associated with elaboration of novel, prostate epithelial cell-derived FGF-2 isoforms. FGF-1 antisense RNA expression eliminated transfectant FGF-1 isoforms without affecting FGF-2 isoform content. Similarly, FGF-2-antisense RNA expression eliminated the transfectant 21.5 kDa FGF-2 isoform, diminished the 19.5 kDa FGF-2 isoform content, and reduced the 17 kDa FGF-2 isoform content to barely detectable levels without affecting the FGF-1 isoform content. This established that FGF-antisense RNAs specifically inhibited translation of cognate, endogenous FGF transcripts. Doubling times of control transfectants and sense transfectants were indistinguishable and were not affected by including FGF-1 or FGF-2 in the culture medium. Doubling times of FGF-1-antisense or FGF-2-antisense transfectants were 1.3- to 1.4-fold greater than those of control transfectants or sense transfectants, and either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling times to values indistinguishable from those of control transfectants or sense transfectants. This established that with regard to prostate cancer cell proliferation: (a) endogenous FGF-1 cannot substitute for endogenous FGF-2 eliminated by FGF-2-antisense RNA expression; and (b) endogenous FGF-2 cannot substitute for endogenous FGF-1 eliminated by FGF-1-antisense RNA expression. In contrast, either exogenous FGF-1 or exogenous FGF-2 decreased antisense transfectant doubling time. The results of these studies establish that endogenous FGF-1 and endogenous FGF-2 modulate prostate cancer cell proliferation and imply that FGF-1 and FGF-2 of endogenous and exogenous origin conjointly control aspects of prostate cancer cell homeostasis. Our findings suggest complex interaction between components of prostate cancer cell regulatory processes and endogenously produced and exogenously accessible FGF-1 and FGF-2.

Detection of the substance immunologically cross-reactive with insulin in insulin RIA is an artifact caused by insulin tracer degradation: involvement of the insulin-degrading enzyme.

In previous literature, the existence of a new insulin-like substance found in tumor tissues, termed substance immunologically cross-reactive with insulin (SICRI), has been proposed. In these studies, insulin-specific radioimmunoassay (RIA) was the only detection method for SICRI. The mouse melanoma B16BL6 cell line was found to be a rich source of SICRI. In this paper, we show that SICRI is not expressed in B16BL6 cells. Previous RIA measurements were wrongly ascribed to SICRI. What was really measured was a positive artifact caused by insulin tracer degradation in RIA. Several lines of evidence indicate that protease responsible for insulin degradation in B16BL6 cells in insulin-degrading enzyme (IDE; EC 3.4.22.11). First, SICRI activity of B16BL6 cytosol measured by insulin RIA was inhibited by thiol protease inhibitor N-ethylmaleimide (NEM). Thiol active agents as well as metal chelators, both potent IDE blockers, inhibited also the insulin-degrading activity of the same sample. Second, cross-linking to 125I-labeled insulin of partially purified sample with highest insulin RIA activity specifically labeled only a single protein with molecular mass similar to IDE (110 kDa). Labeling was blocked by 'cold' insulin in excess. Third, kinetic studies of insulin degradation by RIA active chromatographic fractions revealed an apparent Kd of 90 nM which is very similar to the reported affinity of insulin for IDE (Kd = 100 nM). Additionally, in B16BL6 as well as in mouse myeloid leukemia cells, IDE gene is actively transcribed and this expression was found to be much stronger than in normal mouse tissues. In conclusion, our results strongly question the real existence of SICRI.(ABSTRACT TRUNCATED AT 250 WORDS)