[Obesity and diabetes - are they always together?]

Obesity and type 2 diabetes mellitus (DM 2) are two interrelated metabolic diseases widespread throughout the developed world. However, up to 30% of individuals with a long history of obesity do not have a carbohydrate metabolism disorder. This article presents the results of a multi-year study of adipose tissue biology in obese individuals with DM 2 compared with individuals with the same history of obesity without DM 2. Comparative analysis of hormonal, cellular, and genetic factors in two groups of patients showed that DM 2 occurs in individuals with abnormal proliferation and adipogenic differentiation of mesenchymal stem cells (MSCs) of adipose tissue. It leads to adipocyte hypertrophy and inflammatory infiltration of adipose tissue macrophages, resulting in increased insulin resistance and diabetogenic effects. These disorders are due to abnormal expression of genes responsible for the proliferation and adipogenic differentiation of MSCs. The study of the possible reversibility of abnormal changes in adipose tissue MSCs in obese patients after significant weight loss and DM 2 remission appears to be a promising research direction. The ability to control adipose tissue progenitor cells may represent a new target for treating and preventing metabolic disorders in obesity.

[Obesity without diabetes: the role of hormonal regulation].

AIM: Obese patients without diabetes present an interesting phenotype to explore protective mechanisms against type 2 diabetes (T2D) development. In our study we looked for specific hormonal features of obese patients without T2D. MATERIALS AND METHODS: We included 6 groups of patients with different metabolic profiles (n=212): controls with BMI25 kg/m2, HbA1c6%, age 30 years; patients with 25BMI30 kg/m2and HbA1c6%; patients with 25BMI30 kg/m2and HbA1c6%; patients with BMI30 kg/m2and HbA1c6% (+ Obesity - T2D) obese patients without T2D or prediabetes; patients with BMI30 kg/m2and newly-diagnosed T2D/prediabetes, HbA1c6%; patients with known history of T2D on glucose-lowering drugs with BMI30 kg/m2. Insulin, GLP-1, GIP were measured during glucose-tolerance test at 0, 30 and 120 minutes; insulin resistance (IR) was assessed by HOMA-IR. RESULTS: Waist circumference was bigger in patients with obesity despite their metabolic profile comparing to patients without obesity (p0.001). Waist-to-hip ratio was similar in patients with different metabolic status. According to IR + Obesity - T2D group had intermediate position: IR was higher in that group comparing to people without obesity, but was less that in patients with obesity and HbA1c6% (p0.001). + Obesity - T2D group had the most potent baseline insulin secretion, assessed by НОМА-%band the highest postprandial secretion, measured by insulinogenic index among all patient groups with obesity (p0.001). There was no significant difference in GLP-1 secretion; GIP secretion was higher in patients with BMI30 kg/m2comparing to people with BMI30 kg/m2(p0.01).

[BRCA1 and Estrogen Receptor α Expression Regulation in Breast Cancer Cells].

BRCA1 (breast cancer 1) protein is involved in the genome stability maintenance participating in homologous recombination-dependent DNA repair. Disruption of BRCA1 functioning is associated with breast and ovarian cancer. Despite the important role of BRCA1 in DNA repair in all cell types, the development of BRCA1-associated cancer takes place mainly in estrogen-dependent tissues such as breast and ovarian ones. Using breast cancer cell line MCF-7 it was demonstrated in in vitro experiments that the estrogen 17ß-estradiol (E2), phytoestrogens (genistein and apigenin) and antiestrogens (tamoxifen and fulvestrant) inhibited estrogen receptor (ERα) expression while only genistein influenced BRCA1 increasing its expression. In hypoxia, that is an important factor of solid tumors progression, the decrease of BRCA1 and ERα expression was demonstrated in MCF-7 cells. Therefore, hypoxia influences both BRCA1-dependent DNA repair and hormonal regulation of breast cancer cell growth. Taken together, obtained results demonstrate a relationship between BRCA1 and steroid hormones signal transduction pathways in breast cancer cells and point out to the importance of complex BRCA1 and ERa expression regulation mechanisms studies including epigenetic gene expression regulation.

[Glycemia normalization in patients with obesity and type 2 diabetes mellitus: bariatric surgery vs pharmacological therapy].

AIMS: To compare glucose - lowering and weight reduction capacity of bypass operations (gastric bypass (GB), biliopancreatic diversion (BPD) vs GLP-1 agonist liraglutide 3.0 mg (models of maximum incretin effect) for 6 months. MATERIALS AND METHODS: 46 patients with type 2 diabetes and long history (≥10 years) of obesity were divided into 2 groups: surgery - group (n=23) and liraglutide - group (n=23), where liraglutide 3.0 mg in dose - escalation manner was added to baseline glucose - lowering therapy. Anthropometric parameters, HbA1c and insulin resistance (IR) by hyperinsulinemic euglycemic clamp (M-value) were measured before and 16 weeks after the intervention. With the stabilization of glycemia (≤6.5 mmol/l at fasting state, ≤8 mmol/l postprandial) the initial glucose - lowering therapy was canceled. RESULTS AND DISCUSSION: Both surgery and liraglutide 3.0 mg provided target HbA1c in 16 weeks. Bypass operations led to elimination of glucose - lowering therapy in 82.6% patients due to a more significant weight reduction and decrease in IR. In liraglutide - group previous glucose - lowering therapy was cancelled in 78.3% patients, mainly receiving baseline mono - and two - component therapy. The most significant difference between interventions was achieved in BMI (-8.9 kg in surgery group vs -3.8 kg in liraglutide group, p.

[Streptozocin-induced Alzheimer's disease as an independent risk factor for the development of hyperglycemia in Wistar rats].

BACKGROUND: In recent years the theme of the relationship of Alzheimers disease (AD) and metabolic disorders has been widely discussed. Nevertheless, it remains unclear whether AD is a direct cause of carbohydrate metabolism disorders or it is the presence of classical risk factors for type 2 diabetes mellitus (DM 2), primarily obesity, that significantly increases the risk of AD. AIM: To evaluate the separate contribution of two factors to the development of disorders of carbohydrate metabolism: (1) weight gain due to a high-calorie diet and (2) experimental-induced AD. METHODS: Male Wistar rats were injected with streptozocin (STZ) in the lateral ventricles of the brain to induce AD or saline (sham operated animals - SO) during stereotactic operations. After 2 weeks, the animals were divided into four groups: 1) the SO group, which was assigned to the normal calorie (NCD) diet (SO NCD); 2) the SO group, which was assigned to the high-calorie diet (SO HCD); 3) the group to which the norm-calorie diet was prescribed after the administration of STZ into the lateral ventricles of the brain (STZ NCD); 4) the group to which the HCD was assigned after the administration of STZ (STZ HCD). The animals were on a diet for 3 months. Intraperitoneal glucose tolerance tests were carried out before the diet and after 3 months. At the end of the study, a morphological assessment of brain tissue, pancreas, and liver was performed. RESULTS: 3 months after surgical interventions and the appointment of diets, the glycemic curves significantly differed in the 4 studied groups: normoglycemia persisted only in the SO + NCD group, while HCD and the STZ administration were accompanied by the development of hyperglycemia (p = 0.0001). The STZ + NСD group, which represented the isolated effect of AD, was also characterized by impaired carbohydrate metabolism. A morphological study showed that HCD leads to a more pronounced ectopic accumulation of fat in the liver and pancreas tissue than NCD. The administration of STZ, regardless of the diet, led to changes typical for the AD model an increase in the size of the ventricles of the brain, degeneration of white matter, and the accumulation of -amyloid in the hypothalamus. CONCLUSIONS: The STZ-induced brain damage typical for AD led to impaired carbohydrate metabolism regardless of diet and was an independent risk factor for hyperglycemia.

[Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification].

Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.

[Mass spectrometry analysis of blood plasma lipidome as method of disease diagnostics, evuation of effectiveness and optimization of drug therapy].

A new method for the analysis of blood lipid based on direct mass spectrometry of lipophilic low molecular weight fraction of blood plasma has been considered. Such technique allows quantification of hundreds of various types of lipids and this changes existing concepts on diagnostics of lipid disorders and related diseases. The versatility and quickness of the method significantly simplify its wide use. This method is applicable for diagnostics of atherosclerosis, diabetes, cancer and other diseases. Detalization of plasma lipid composition at the molecular level by means of mass spectrometry allows to assess the effectiveness of therapy and to optimize the drug treatment of cardiovascular diseases by phospholipid preparations.

[Secretion of incretin hormones in people having risk factors for type 2 diabetes mellitus].

AIM: To study the secretion of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP),and glucagon- like peptide 2 (GLP-2) in response to a carbohydrate load in people with risk factors for type 2 diabetes mellitus (DM2) in relation to the type of carbohydrate metabolic disturbances and age. SUBJECTS AND METHODS: One hundred and twenty-seven patients having DM2 risk factors who had not previously received glucose- lowering therapy underwent an oral glucose tolerance test (OGTT). The plasma levels of glucose, insulin, glucagon, GLP-1, GIP, and GLP-2 were determined at 0, 30, and 120 minutes of the test. RESULTS: According to the findings, the patients were divided into 3 groups: 1) normal glucose tolerance; 2) prediabetic states (impaired glucose tolerance and/or impaired fasting glycemia); 3) new-onset DM2. OGTT showed that the secretion of GLP-1 was lower and that of GIP and GLP-2 was higher in patients with DM2. GLP-1 secretion decreased with patient age. CONCLUSION: During OGTT, there is a statistically significantly difference in the secretion of incretin hormones in persons with varying degrees of carbohydrate metabolic disturbances: the peak GLP-1 secretion is the highest in healthy individuals and lowest in the patients with DM2; on the contrary, the peak GLP2 and GIP secretions are the highest in the patients with DM2. This may suggest that GLP-1 and the two other hormones (GLP-2 and GIP) show opposite effect in the regulatory mechanisms of carbohydrate metabolism. GLP-1 secretion is decreased with age, which may be one of the reasons for the higher prevalence of DM2 among the elderly.

The physiological significance of beta -actin mRNA localization in determining cell polarity and directional motility.

beta-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3' untranslated region, the "zip code." Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3' untranslated region leads to delocalization of beta-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after beta-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of beta-actin protein. These results indicate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Correlation of beta-actin messenger RNA localization with metastatic potential in rat adenocarcinoma cell lines.

The actin cytoskeleton is involved in the motility of tumor cells. It has been shown in several cell types that beta-actin mRNA is localized in the protrusions of cells in which actin is actively polymerized, and the ability to localize mRNA is correlated with the efficiency of motility. In this context, we studied the distribution of beta-actin mRNA in two different tumor cell lines and correlated it with their metastatic potential. The two cell lines used were the highly metastatic MTLn3 cells and nonmetastatic MTC cells. Nonmetastatic MTC cells have two different pools of beta-actin mRNA (perinuclear and at the leading edge), whereas highly metastatic MTLn3 cells have only a perinuclear distribution of beta-actin mRNA. These differences in mRNA localization are correlated with profound differences in the polarity and plasticity of cell motility of these cells in culture and the histopathology of primary breast tumors derived from these cells. In particular, MTLn3 cells are unpolarized by all cell shape and motility criteria in culture and in their histopathological organization in primary tumors. By comparison, MTC cells are polarized in all identical measurements. These results suggest that the increased plasticity of cell locomotion and the invasiveness of MTLn3 cells result from the failure of metastatic cells to localize beta-actin mRNA properly, causing them to be less polarized and therefore more flexible in their direction of motility. Thus, differences in the polarized organization of cells in the primary tumor that are correlated with beta-actin mRNA localization may have prognostic value in predicting metastatic potential.

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2, ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2- carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.

Co-localization of components of the protein-synthesizing machinery with the cytoskeleton in G0-arrested cells.

The distribution of eukaryotic elongation factor 2 (eEF-2) in G0-arrested fixed human skin diploid fibroblasts was studied by indirect immunofluorescent microscopy. It was found earlier that the main part of eEF-2 in cycling cells was located near the nucleus in the endoplasm (Gavrilova et al., 1987). It has been demonstrated here that the transition from proliferation to the G0 phase of the cell cycle leads to the distribution of eEF-2 mainly along the intermediate filaments and/or microtubules. Both in cycling and in G0-arrested fibroblasts a portion of eEF-2 is also co-localized with actin microfilament bundles. The reversion of the cells from the G0 phase to proliferation is accompanied by rearrangement of the actin cytoskeleton and reversal to the original pattern of eEF-2 distribution. It is likely that the different types of cytoskeleton in eukaryotic cells can be involved in organization of protein-synthesizing machinery.

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts.

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.

Phosphorylation of elongation factor 2 by EF-2 kinase affects rate of translation.

A new Ca2+/calmodulin-dependent protein kinase has been recently discovered in mammalian cells. The major substrate of this kinase, a protein of relative molecular mass (Mr) approximately equal to 100,000 (100K), has been identified as elongation factor 2 (EF-2), which participates in protein synthesis. The in vivo activity of the EF-2 kinase depends upon growth factors and other agents affecting the level of Ca2+ and cAMP. Its effect on EF-2 activity, however, remained obscure. This work shows that the phosphorylation of EF-2 by the EF-2 kinase results in a drastic inhibition of polyphenylalanine synthesis in poly(U)-directed translation. Phosphorylated EF-2 is completely inactive in translation and, moreover, inhibits the activity of non-phosphorylated EF-2. Dephosphorylation of EF-2 by phosphatase restores its activity. Hence, the phosphorylation of EF-2 directly affects the elongation stage of translation and thus represents a novel mechanism of translational control.

Phosphorylation of the elongation factor 2: the fifth Ca2+/calmodulin-dependent system of protein phosphorylation.

Elongation factor 2 (EF-2) has been recently shown to be extensively phosphorylated in a Ca2+/calmodulin-dependent manner in extracts of mammalian cells (A. G. Ryazanov (1987) FEBS Lett. 214, 331-334). In the present study, we partially purified the protein kinase which phosphorylates EF-2 from rabbit reticulocytes. The molecular weight of the enzyme determined by gel filtration was about 140,000. Unlike the substrate, the EF-2 kinase had no affinity for RNA and therefore could be separated from EF-2 by chromatography on RNA-Sepharose. After chromatography on hydroxyapatite, the kinase activity became calmodulin-dependent. Two-dimensional separation of the phosphorylated EF-2 according to O'Farrell's technique revealed that there were two phosphorylation sites within the EF-2 molecule; in both cases, the phosphorylated amino acid was threonine. The EF-2 kinase differed from the four known types of Ca2+/calmodulin-dependent protein kinases. Thus, the system of EF-2 phosphorylation represents the novel (fifth) Ca2+/calmodulin-dependent system of protein phosphorylation. This system is supposed to be responsible for the regulation of the elongation rate of protein biosynthesis in eukaryotic cells.

cAMP-dependent activation of protein synthesis correlates with dephosphorylation of elongation factor 2.

The addition of 5 mM cAMP to a cell-free translation system from rabbit reticulocytes increases the rate of protein synthesis 3 5-fold. Lower concentrations of cAMP (0.005, 0.05 and 0.5 mM) have no effect on translation in this system. cAMP at all the concentrations tested stimulates the phosphorylation of the same pattern of polypeptides, while 5 mM cAMP additionally stimulates dephosphorylation of the 95 kDa polypeptide identified as elongation factor 2 (EF-2). Testing of the preparations of EF-2 with a different content of the phosphorylated form in poly(U)-directed poly(Phe) synthesis reveals that the EF-2 activity correlates with the fraction of non-phosphorylated EF-2. Thus cAMP-dependent activation of protein synthesis seems to be due to dephosphorylation of EF-2.