thapsigargin and Body-Weight

Increased endoplasmic reticulum (ER) stress has emerged as a vital contributor to dysregulated glucose homeostasis, and impaired function of sarco-endoplasmic reticulum Ca

Topics: Animals; Blood Glucose; Body Weight; Calcium-Transporting ATPases; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Flavonoids; Insulin Resistance; Lipogenesis; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Muscle Fibers, Skeletal; Muscle, Skeletal; Obesity; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Up-Regulation

Chronic endoplasmic reticulum (ER) stress was recently revealed to affect hypothalamic neuroendocrine pathways that regulate feeding and body weight. However, it remains unexplored whether brain ER stress could use a neural route to rapidly cause the peripheral disorders that underlie the development of type 2 diabetes (T2D) and the metabolic syndrome. Using a pharmacologic model that delivered ER stress inducer thapsigargin into the brain, this study demonstrated that a short-term brain ER stress over 3 d was sufficient to induce glucose intolerance, systemic and hepatic insulin resistance, and blood pressure (BP) increase. The collection of these changes was accompanied by elevated sympathetic tone and prevented by sympathetic suppression. Molecular studies revealed that acute induction of metabolic disorders via brain ER stress was abrogated by NF-κB inhibition in the hypothalamus. Therapeutic experiments further revealed that acute inhibition of brain ER stress with tauroursodeoxycholic acid (TUDCA) partially reversed obesity-associated metabolic and blood pressure disorders. In conclusion, ER stress in the brain represents a mediator of the sympathetic disorders that underlie the development of insulin resistance syndrome and T2D.

Topics: Animals; Blood Pressure; Blotting, Western; Body Weight; Diabetes Mellitus, Type 2; Eating; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Glucose Intolerance; Green Fluorescent Proteins; Hypothalamus; Immunoprecipitation; Insulin; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Neurosecretory Systems; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Stress, Physiological; Taurochenodeoxycholic Acid; Telemetry; Thapsigargin

Drosophila are tolerant of high levels of dietary salt and can provide a useful model for studies of the physiology of salt stress. The effects of NaCl- and KCl-rich diets on haemolymph ionoregulation and Malpighian tubule (MT) fluid secretion, Na(+) and K(+) secretion and transepithelial potential were examined in larval and adult Drosophila melanogaster. K(+) concentrations in the haemolymph of adults reared on the KCl-rich (0.4 mol l(-1)) diet did not differ from the values for insects reared on the control diet. In the haemolymph of larvae reared on the K-rich diet, K(+) concentrations increased from 23 to 75 mmol l(-1) after 6 h, then returned to the control value within 48 h. Na(+) concentrations in the haemolymph of adults or larvae reared for 1-7 days on the NaCl-rich (0.4 mol l(-1)) diet increased by ~50% relative to values for insects reared on the control diet. Rates of secretion of fluid, Na(+) and K(+) by MTs isolated from larvae reared on the Na-rich diet for >6 h and bathed in control saline containing 20 mmol l(-1) K(+) did not differ from the values for tubules of larvae reared on the control diet. Evidence of phenotypic plasticity was seen in the response of MTs isolated from larvae reared on the K-rich diet for >6 h and bathed in saline containing 60 mmol l(-1) K(+); secretion of fluid and K(+) increased by >50% relative to the values for tubules of larvae reared on the control diet. Secretion of fluid, Na(+) and K(+) increased when tubules were bathed in haemolymph collected from larvae reared on the Na- or K-rich diets. Secretion was further increased by addition of exogenous cAMP but not by addition of thapsigargin to the haemolymph. The results show that haemolymph ionoregulation in larvae reared on salt-rich diets involves both alterations in the basal secretion rates of Na(+) and/or K(+) as well as stimulatory effects of diuretic factors present in the haemolymph. The results suggest that such factors stimulate tubule fluid and ion secretion through increases in intracellular Ca(2+) in response to salt stress.

Topics: Animals; Body Fluids; Body Weight; Cyclic AMP; Diet; Drosophila melanogaster; Epithelial Cells; Hemolymph; Ion Transport; Larva; Malpighian Tubules; Phenotype; Potassium; Salts; Sodium; Stress, Physiological; Thapsigargin

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 micromol/L) or gadolinium (100 micromol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension.

Topics: Animals; Aorta; Blood Pressure; Body Weight; Calcium; Calcium Channels; Cytosol; Disease Models, Animal; Enzyme Inhibitors; Homeostasis; Hypertension; Male; Membrane Glycoproteins; Muscle Contraction; ORAI1 Protein; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Stromal Interaction Molecule 1; Thapsigargin

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.

Topics: Adenoviridae; Alkaline Phosphatase; Animals; Blotting, Western; Body Weight; Calcium; Calcium-Transporting ATPases; Calsequestrin; Dose-Response Relationship, Drug; Down-Regulation; Echocardiography; Electrophysiology; Enzyme Inhibitors; Fura-2; Heart Ventricles; Hypertrophy; Inhibitory Concentration 50; Kinetics; Lysophospholipase; Mice; Mice, Transgenic; Muscle Cells; Organ Size; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Reverse Transcriptase Polymerase Chain Reaction; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection; Transgenes; Up-Regulation

The leaner (tg(la)) mouse mutation occurs in the gene encoding the voltage-activated Ca(2+) channel alpha(1A) subunit, the pore-forming subunit of P/Q-type Ca(2+) channels. This mutation results in dramatic reductions in P-type Ca(2+) channel function in cerebellar Purkinje neurons of tg(la)/tg(la) mice that could affect intracellular Ca(2+) signaling. We combined whole cell patch-clamp electrophysiology with fura-2 microfluorimetry to examine aspects of Ca(2+) homeostasis in acutely dissociated tg(la)/tg(la) Purkinje cells. There was no difference between resting somatic Ca(2+) concentrations in tg(la)/tg(la) cells and in wild-type (+/+) cells. However, by quantifying the relationship between intracellular Ca(2+) elevations and depolarization-induced Ca(2+) influx, we detected marked alterations in rapid calcium buffering between the two genotypes. Calcium buffering values (ratio of bound/free ions) were significantly reduced in tg(la)/tg(la) (584 +/- 52) Purkinje cells relative to +/+ (1,221 +/- 80) cells. By blocking the endoplasmic reticulum (ER) Ca(2+)-ATPases with thapsigargin, we observed that the ER had a profound impact on rapid Ca(2+) buffering that was also differential between tg(la)/tg(la) and +/+ Purkinje cells. Diminished Ca(2+) uptake by the ER apparently contributes to the reduced buffering ability of mutant cells. This report constitutes one of the few instances in which the ER has been implicated in rapid Ca(2+) buffering. Concomitant with this reduced buffering, in situ hybridization with calbindin D28k and parvalbumin antisense oligonucleotides revealed significant reductions in mRNA levels for these Ca(2+)-binding proteins (CaBPs) in tg(la)/tg(la) Purkinje cells. All of these results suggest that alterations of Ca(2+) homeostasis in tg(la)/tg(la) mouse Purkinje cells may serve as a mechanism whereby reduced P-type Ca(2+) channel function contributes to the mutant phenotype.

Topics: Animals; Body Weight; Buffers; Calbindin 1; Calbindins; Calcium; Calcium-Transporting ATPases; Endoplasmic Reticulum; Enzyme Inhibitors; Female; Gene Expression; Heterozygote; Homeostasis; Homozygote; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Parvalbumins; Patch-Clamp Techniques; Phenotype; Purkinje Cells; RNA, Messenger; S100 Calcium Binding Protein G; Thapsigargin

1. The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared. 2. CPA (10 micronM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (-dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations. 3. In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations. 4. The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 micronM), mefenamic acid (100 micronM), lanthanum (La3+, 100 micronM), gadolinium (Gd3+, 100 micronM) and SK&F 96365 (100 micronM) but not by verapamil (10 micronM). 5. Thapsigargin (3 micronM), another SR Ca2+-ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction. 6. These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil-insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+](i) modulation due to CPA-sensitive SR Ca2+-ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.

Topics: Animals; Blood Pressure; Body Weight; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Esophagus; Hypertension; In Vitro Techniques; Indoles; Ion Channels; Male; Muscle Contraction; Muscle, Smooth; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin

1. The effect of experimental diabetes mellitus (DM; hyperglycaemic, non-ketototic; 2 months duration) in the rat on receptor-linked prostacyclin (PGI2) synthesis (measured as 6-oxo-PGF1 alpha by radioimmunoassay) was studied in the aorta and urinary bladder using adrenaline, angiotensin II (AII) and acetylcholine (ACh). Signal transduction systems were studied via stimulation of PGI2 synthesis with phorbol ester dibutyrate (PDBU; a protein kinase C activator [PKC]), Ca2+ ionophore A23187 (A23187) and thapsigargin (both elevate intracellular Ca2+, activating phospholipase A2 [PLA2]) and arachidonate (AA; substrate for PGI2 synthesis). 2. In response to adrenaline, AII and phorbol ester, aortic PGI2 release was markedly reduced (all > 75%) in diabetic rats compared to controls. EC50s of the dose-response curves for adrenaline, AII and PDBU were also markedly increased in aortae from DM rats compared to controls. Although there was decreased output of PGI2 in response to A23187 by aortae from diabetic rats compared to controls, there was no difference in the EC50s (mean +/- s.e. mean: diabetic, 2.7 +/- 0.2 x 10(-6) M; controls 2 +/- 0.18 x 10(-6) M). There were no differences in PGI2 release (or in the EC50s) in response to thapsigargin or AA between aortae from diabetic and control rats. 3. In the urinary bladder, there was a marked increase in PGI2 output in response to ACh and a marked decrease in EC50s for the ACh-PGI2 dose-response curves in diabetic rats (EC50 = 5.8 +/- 0.32 x 10(-7) M) compared to controls (EC50 = 2.2 +/- 0.15 x 10(-6) M). Although there was an increase in PGI2 output in the urinary bladders from diabetic rats in response to A23187, there were no differences in the EC50s (control, 1.8 +/- 0.2 x 10-6 M; diabetic, 1.1 +/- 0.15 X 10-6 M). In the urinary bladders, there were no differences in PGI2 output (or the EC50s) in response to PDBU, thapsigargin or AA between diabetic or control rats.4. These data indicate that: (i) reduced PGI2 synthesis coupled to adrenoceptors and AII receptors in the aortae of diabetic rats may be due to diminished PKC activity and not to changes in receptor density and/or affinity, Ca2+ stores, PLA2, cyclo-oxygenase or PGI2 synthase; (ii) the diametrically opposite effect of DM on ACh-stimulated PGI2 synthesis is not due to an increase in PKC activity, but possibly to an increase in muscarine receptor number and/or affinity; (iii) changes in receptor-linked PGI2 synthesis are not ubiquitous in experimental DM

Topics: Angiotensin II; Animals; Aorta, Thoracic; Arachidonic Acid; Body Weight; Calcimycin; Calcium-Transporting ATPases; Diabetes Mellitus, Experimental; Epoprostenol; In Vitro Techniques; Male; Muscle, Smooth; Muscle, Smooth, Vascular; Phorbol 12,13-Dibutyrate; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic; Receptors, Muscarinic; Terpenes; Thapsigargin; Urinary Bladder