Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.

A mitochondrial protein called uncoupling protein (UCP1) plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy-consuming process. This pathway has been implicated in the regulation of body temperature, body composition and glucose metabolism. However, UCP1-containing brown adipose tissue is unlikely to be involved in weight regulation in adult large-size animals and humans living in a thermoneutral environment (one where an animal does not have to increase oxygen consumption or energy expenditure to lose or gain heat to maintain body temperature), as there is little brown adipose tissue present. We now report the discovery of a gene that codes for a novel uncoupling protein, designated UCP2, which has 59% amino-acid identity to UCP1, and describe properties consistent with a role in diabetes and obesity. In comparison with UCP1, UCP2 has a greater effect on mitochondrial membrane potential when expressed in yeast. Compared to UCP1, the gene is widely expressed in adult human tissues, including tissues rich in macrophages, and it is upregulated in white fat in response to fat feeding. Finally, UCP2 maps to regions of human chromosome 11 and mouse chromosome 7 that have been linked to hyperinsulinaemia and obesity. Our findings suggest that UCP2 has a unique role in energy balance, body weight regulation and thermoregulation and their responses to inflammatory stimuli.

[The study of therapeutic effect of synthetic bradykinin "gene" contained in retrovirus vector on spontaneously hypertensive rats].

Administration of DNA plasmid pPS-3-neo (brd) with synthetic bradykinin " gene " to 2-days old male spontaneously hypertensive rats (SHR) leads to 2 weeks delay in development of arterial hypertension. Lowering of SBP and positive results of PCR DNA of various organs observed in synthetic bradykinin " gene " transgenic SHR but not in control SHR confirm therapeutic effect of synthetic bradykinin " gene " . This data indicate one of possible ways of gene therapy of arterial hypertension as well as other pathological states by introduction of transgene directly into genome of the organism.

[Cardiomyocyte culture as a highly sensitive system for testing pharmacological activity of specific bradycardic agents].

Two antiarrhythmic agents were studied: verapamil and bradizole, a new bradycardic drug. It was found that both drugs exhibit a dose-dependent negative chronotropic effect on the culture of contractile cardiomyocytes of newborn rats. Bradizole showed more pronounced bradycardic properties than verapamil.

[Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes]. / Razlichnoe vliianie "rekombinantnogo" bradikinina na sokrashchenie kul'tiviruemykh kardiomiotsitov predserdii i zheludochkov.

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.

[Assessment of mitochondrial function in the surviving cytoplasts of cultured cells of pig embryonic kidney by using ethylrhodamine]. / Otsenka funktsional'nogo sostoianiia mitokhondrii v perzhivaiushchikh tsitoplastakh kul'tivirovannykh kletok pochki émbriona svin'i s pomoshch'iu étilrodamina.

A study was made of the functional state of chondriome in cytoplasts previously cultivated for a long time under vital staining with fluorescent dye ethylrhodamine (10 micrograms/ml) which is known to accumulate preferentially in mitochondria. The energization was estimated by the intensity of mitochondrial fluorescence. It was realized that 30 minutes after enucleation cytoplasts retained the same fluorescence as did the untreated cells, and that the mitochondrial distribution within the cell was very similar. Such a high intensity of fluorescence seen within one day after enucleation gives a strong evidence on the high degree of independence on the cell nucleus of organelles that provide the energy to metabolic processes. In the course of survival in the cultivation medium for 1-4 days the intensity of fluorescence is shown to fall, especially on the second day after enucleation. All these changes coincide with the changes in cytoplast shape: originally well spread bodies transform into squeezed, ball-shaped or strongly deformed ones. The adhesive ability is going down, and in result only single units of cytoplast can hardly be found on the cover slips by the 4th day after enucleation. These changes give evidence on the enhanced degeneration of cultured cytoplasts.

[Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability]. / Modifikatsiia metoda énukleatsii kletok s pomoshch'iu tsitokhalazina B i izuchenie zhiznesposobnosti tsitoplastov.

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.

[The creation of an SV40 virus-based vector as an experimental model for the study of gene expression]. / Sozdanie na osnove virusa SV40 vektora kak éksperimental'noi modeli dlia izucheniia ékspressii genov.

The purpose of the studies is to design the recombinant virus SV 40 where the C-end of the basic structural protein of virus P-1 was replaced by a synthetic sequence of the neuropeptide bradykinin. The recombinant virus SV (SV 40/Brd) was obtained. In this virus 60 n.p. with 3'-end of VP-1 gene was substituted for 36 n.p. synthetic gene of bradykinin without impairing the frame of translation. The biological activity of SV 40 (Brd) was tested on the cultured cells CV-1 permissive for this virus. An immunofluorescence method was used to detect T antigen and to examine the cytopathic action of this recombinant. The gene engineering design does not make the recombinant loose its biological properties typical of wild virus.

[Construction by using retrovirus vector of recombinant with synthetic bradykinin "gene" for investigations of human gene expression in mammalian cells]. / Konstruirovanie na osnove retrovirusnogo vektora rekombinanta s sinteticheskim "genom" bradikinina s tsel'iu izucheniia ékspressii genov cheloveka v kletkakh mlekopitaiushchikh.

The synthetic gene of bradykinin was built into the retrovirus vector pPS-3-neo under the guidance of LTR promotor, followed by pPS-3-neo (brd) vector transfection of strain 293 cells. The physiological activity of the expressed bradykinin was tested on cultured neonatal rat cardiomyocytes. The culture medium of strain 293 cells transferred by pPS-3-neo (brd) produces a positive chronotropic effect that is directly related to the time parameters of preparation of recombinant bradykinin, which are comparable with the curve of chronotropic effect of synthetic bradykinin at concentrations of 10(-17) to 10(-16) M. The control of bradykinin "gene" expression was due to the lack of chronotropic responses of cardiomyocytes to the kinin receptor blocker parmidine and the transfection of strain 293 cells with the retrovirus vector without bradykinin "gene".

[Morphologic and radioautographic study of reactivation of peritoneal exudate cell nuclei in heterokarya]. / Morfologicheskoe i radioavtograficheskoe issledovanie reaktivatsii iader kletok peritoneal'nogo ékssudata v geterokarionakh

The heterokaryons of undifferentiated mouse fibroblasts (L and 3T3-4E/TK-) and various cell elements of the rat peritoneal exudate were obtained under the treatment with inactivated Sendai virus. The reactivation of RNA and DNA synthesis in the nuclei of highly differentiated periotoneal exudate cells and the synthesis of thymidine kinase controlled by the nuclei of peritoneal exudate cells were shown to occur in the heterokaryons. During the process of reactivation, the ring-like nuclei of polymorphonuclear leucocytes acquired the form characteristic of the reactivated nuclei of mononuclears. The morphological changes of heparin-containing granules in the cytoplasm of the heterokaryons of mast cells and undifferentiated fibroblasts suggest the degeneration and breakdown of granules.

[Enucleation and reconstruction of murine L-cell fibroblasts]. / Enukleatsiia i rekonstruktsiia myshinykh fibroblastov linii L.

Two types of teflon chambers are suggested for the procedures of enucleation and reconstruction of cells cultivated in the monolayer. The first chamber simplifies the enucleation and permits performing it on the standard cover glasses with a small volume of medium with cytochalasin B. The second chamber permits purifying the karyoplast fraction from intact cells by their adhesion on the cover glasses. Then the karyoplasts are sedimented by centrifugation on cytoplasts located on the cover glasses. This procedure promotes sticking of fragments and their subsequent fusion in the presence of polyethylene glycol. In total the number of reconstructed cells increases many times.

[Changes in mitochondrial function in the cells and cytoplasts of a pig embryo kidney culture as affected by the action of actinomycin D]. / Izmenenie funktsional'nogo sostoianiia mitokhondrii v kletkakh i tsitoplastakh kul'tury SPEV pri deistvii aktinomitsina D.

Vital staining of PE kidney cells by fluorescent cationic dye, ethyl rhodamine, (accumulated inside mitochondria by the membrane potential and hence reflecting their functional state) shows rather close level of the fluorescence intensity both in cells and in their cytoplasts for 10 hours of culture survival in the standard medium. In cells and cytoplasts cultivated in the medium with 0.2 mg/ml of actinomycin D inhibiting RNA synthesis, fluorescence intensity of mitochondria sharply decreases after 10 hours as against the control patterns. It is concluded that mitochondria possess a significant degree of autonomy of the nucleus and it is supposed that a considerable part of mitochondrial RNA is under the nucleus control.

[The electron microscopic study of the cytoplast mitochondria of an ESK cell culture after the action of actinomycin D. Embryonic swine kidney]. / Elektronno-mikroskopicheskoe issledovanie mitokhondrii tsitoplastov kletok kul'tury SPEV posle deistviia aktinomitsina D.

The inhibitor of RNA synthesis actinomycin D substantially changed mitochondrial morphology and ultrastructure of cytoplasts in comparison with mitochondria of intact cells. These changes included an increase of matrix condensation, the number of cristae, and extension of intercristal and intracristal spaces. We suggest that mitochondria are characterized by significant independence from the nucleus that possibly controls a considerable part of mitochondrial RNA.

[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

[Delayed appearance of hypertension in spontaneously hypertensive rat (SHR) injected with CpG-rich DNA early in ontogenesis].

In this study we have investigated properties of blood serum extracellular DNA (cell-free DNA) from patients with essential arterial hypertension (AH). Cell-free DNA concentration was not changed in the control AH group compared to norma (healthy donors) but fragments of CpG-rich cell-free DNA marker content were increased at transcribed area of ribosomal repeat (TArDNA, CpG-DNA). To evaluate effect of CpG-DNA on AH development in 2-day SHR line and in control normotensive line (WKY), 700 ng of human TArDNA single subcutaneous injection were inoculated to obtain anti-CpG-DNA polyclonal antibodies. These antibodies could change CpG-DNA contents in total cell-free DNA. Blood pressure (BP) in 9-week SHR line rats immunized with CpG-DNA was equal to BP of WKY rats. Then BP of immunized SHR steadily increased with age and reached high value 8 weeks later compared to control SHR rats. Cell-free DNA analysis in 17-week SHR line rats showed significantly reduced concentrations of cell-free DNA and also showed decrease in small DNA fragments content, but increased content of CpG-DNA (rat TArDNA). These changes were accompanied with 3.5-fold blood endonuclease activity increase and decrease of free (unbound to cell-free DNA) anti-CpG-DNA antibodies quantity. Total anti-CpG-DNA antibodies quantity in immunized rats wasn't changed compared to control animals. Thus, observed effect of increase in stable BP elevation age in immunized SHR line rats doesn't relate to increase of anti-CpG-DNA antibody production. Possible reason of this effect is further discussed.

Effect of cell-free DNA of patients with cardiomyopathy and rDNA on the frequency of contraction of electrically paced neonatal rat ventricular myocytes in culture.

There is evidence that infarcted myocardium contributes to the increase of cell-free DNA levels (cfDNA). We studied the effect of different human DNA fragments on the rate of contraction of the electrically paced neonatal rat ventricular myocytes in culture (spontaneously hypertensive line SHR). AT-rich fragments of the human satellite 3 tandem repeat (1q12 region) at a concentration of 1 ng/mL increase the frequency of cardiomyocyte contractions by 2-2.5 times. GC-rich fragments of the rDNA at 0.5 ng/mL decrease the cardiomyocyte contraction frequency by 1.5-2 times. The serum cfDNA of patients with acute myocardial infarction decreases the frequency of the contraction of cardiomyocytes proportionally to the amount of the rDNA fragments it contains. The rDNA effect is similar to the E. coli DNA effect and is presumably being realized through TLR9. In the presence of the inhibitor of these receptors, rDNA operates the same way as the fragment of the satellite 3-increasing the frequency of the cardiomyocyte contractions. Accumulation of the GC-rich fragments of the cfDNA in the blood of the AMI patients might have an influence on the contractile function of myocardial cells.

Effect of hypoxia during early organogenesis on cardiac activity and noradrenergic regulation in the postnatal period.

Cardiac activity in rats during the postnatal period was studied in vitro and in vivo after exposure of rat pups to antenatal acute hypobaric hypoxia at the stage of organogenesis (day 9-10 of gestation). Cultured cardiomyocytes from rat pups exposed to antenatal hypoxia were characterized by increased rate of contractions and decreased reactivity to norepinephrine. Heart rate elevation, predominance of sympathetic influences on cardiac activity, and significant increase in norepinephrine concentration in the cerebral cortex were found in freely moving animals exposed to antenatal hypoxia. Our results indicate that hypoxia at the stage of organogenesis modulated cardiac activity during the postnatal period, which manifested at the level of effector structures in the heart and activity of regulatory systems.

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells.

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [3H]uridine and [3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3-4E cells with PE cells results in the incorporation of [3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.

Overexpression of muscle uncoupling protein 2 content in human obesity associates with reduced skeletal muscle lipid utilization.

Uncoupling proteins (UCP) may influence thermogenesis. Since skeletal muscle plays an important role in energy homeostasis and substrate oxidation, this study was undertaken to test the hypotheses that skeletal muscle UCP2 content is altered in obesity and could be linked to basal energy expenditure, insulin sensitivity, or substrate oxidation within skeletal muscle under postabsorptive (fasting) conditions. To examine these possibilities, limb basal energy expenditure and respiratory quotient (bRQ) were measured in 18 obese nondiabetic (Ob) and lean individuals (L). Total body fat (%) ranged from 11% to 46%. In addition, insulin-stimulated rates of glucose disposal (Rd) were measured under euglycemic hyperinsulinemic conditions. Biopsy of vastus lateralis muscle was used to measure cytochrome c oxidase (COX) enzyme activity and UCP2 content. Whereas low muscle COX activity was found in the Ob compared to L (6.9+/-1.6 vs. 9.6+/-1.2 U/g; P<0.001), skeletal muscle UCP2 content in Ob was significantly higher than in L (48+/-9 vs. 33+/-12 arbitrary units/g; P<0.05). Moreover, UCP2 content was positively correlated with percent of total body fat (r=0.57; P<0. 05) and bRQ (r=0.59; P<0.01), but not with visceral fat (r=0.17; P=0. 49), basal energy expenditure (r=0.07; P=0.79) or Rd (r=-0.23; P=0. 34). In summary, these results indicate that if development of obesity in humans is mediated by defective expression of UCP2 within skeletal muscle, then this effect is not observed in people with established obesity. The present study also suggests that skeletal muscle UCP2 content is not related to basal energy expenditure or insulin sensitivity in humans. However, the increased content of UCP2 within skeletal muscle in obesity appears to coincide with a reduced postabsorptive lipid utilization by muscle.