thapsigargin and Insulin-Resistance

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

Metabolic syndrome, which includes hypertension, hyperglycemia, obesity, insulin resistance, and dyslipidemia, has a negative impact on cognitive health. Endoplasmic reticulum (ER) stress is activated during metabolic syndrome, however it is not known which factor associated with metabolic syndrome contributes to this stress. ER stress has been reported to play a role in the development of insulin resistance in peripheral tissues. The role of ER stress in the development of insulin resistance in hippocampal neurons is not known. In the current study, we investigated ER stress in the hippocampus of 3 different mouse models of metabolic syndrome: the C57BL6 mouse on a high fat (HF) diet; apolipoprotein E, leptin, and apolipoprotein B-48 deficient (ApoE 3KO) mice; and the low density lipoprotein receptor, leptin, and apolipoprotein B-48 deficient (LDLR 3KO) mice. We demonstrate that ER stress is activated in the hippocampus of HF mice, and for the first time, in ApoE 3KO mice, but not LDLR 3KO mice. The HF and ApoE 3KO mice are hyperglycemic; however, the LDLR 3KO mice have normal glycemia. This suggests that hyperglycemia may play a role in the activation of ER stress in the hippocampus. Similarly, we also demonstrate that impaired insulin signaling is only present in the HF and ApoE 3KO mice, which suggests that ER stress may play a role in insulin resistance in the hippocampus. To confirm this we pharmacologically induced ER stress with thapsigargin in human hippocampal neurons. We demonstrate for the first time that thapsigargin leads to ER stress and impaired insulin signaling in human hippocampal neurons. Our results may provide a potential mechanism that links metabolic syndrome and cognitive health.

Topics: Animals; Apolipoprotein B-100; Apolipoproteins B; Apolipoproteins E; Diet, High-Fat; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Heat-Shock Proteins; Hippocampus; Humans; Hyperglycemia; Insulin Resistance; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Phenotype; Receptor, Insulin; Receptors, LDL; Signal Transduction; Thapsigargin

In this study, we quantitated kaempferol in water extract from Cudrania tricuspidata leaves (CTL) and investigated its effects on endoplasmic reticulum (ER) stress-induced inflammation and insulin resistance in HepG2 cells. The concentration of kaempferol in the CTL was 5.07 ± 0.08 mg/g. The HepG2 cells were treated with 300 µg/mL of CTL, 500 µg/mL of CTL, 1.5 µg/mL of kaempferol or 2.5 µg/mL of kaempferol, followed immediately by stimulation with 100 nM of thapsigargin for ER stress induction for 24 h. There was a marked increase in the activation of the ER stress and inflammation response in the thapsigargin-stimulated control group. The CTL treatment interrupted the ER stress response and ER stress-induced inflammation. Kaempferol partially inhibited the ER stress response and inflammation. There was a significant increase in serine phosphorylation of insulin receptor substrate (IRS)-1 and the expression of C/EBPα and gluconeogenic genes in the thapsigargin-stimulated control group compared to the normal control. Both CTL and kaempferol suppressed serine phosphorylation of IRS-1, and the treatments did not interrupt the C/EBPα/gluconeogenic gene pathway. These results suggest that kaempferol might be the active compound of CTL and that it might protect against ER stress-induced inflammation and hyperglycemia.

Topics: CCAAT-Enhancer-Binding Protein-alpha; Endoplasmic Reticulum Stress; Hep G2 Cells; Humans; Inflammation; Insulin Receptor Substrate Proteins; Insulin Resistance; Kaempferols; Moraceae; Phosphorylation; Plant Extracts; Plant Leaves; Serine; Signal Transduction; Thapsigargin; Water

β-aminoisobutyric acid (BAIBA) is a nature thymine catabolite, and contributes to exercise-induced protection from metabolic diseases. Here we show the therapeutical effects of BAIBA on hepatic endoplasmic reticulum (ER) stress and glucose/lipid metabolic disturbance in diabetes. Type 2 diabetes was induced by combined streptozotocin (STZ) and high-fat diet (HFD) in mice. Oral administration of BAIBA for 4 weeks reduced blood glucose and lipids levels, hepatic key enzymes of gluconeogenesis and lipogenesis expressions, attenuated hepatic insulin resistance and lipid accumulation, and improved insulin signaling in type 2 diabetic mice. BAIBA reduced hepatic ER stress and apoptosis in type 2 diabetic mice. Furthermore, BAIBA alleviated ER stress in human hepatocellular carcinoma (HepG2) cells with glucosamine-induced insulin resistance. Hepatic AMPK phosphorylation was reduced in STZ/HFD mice and glucosamine-treated HepG2 cells, which were restored by BAIBA treatment. The suppressive effects of BAIBA on glucosamine-induced ER stress were reversed by knockdown of AMPK with siRNA. In addition, BAIBA prevented thapsigargin- or tunicamycin-induced ER stress, and tunicamycin-induced apoptosis in HepG2 cells. These results indicate that BAIBA attenuates hepatic ER stress, apoptosis and glucose/lipid metabolic disturbance in mice with type 2 diabetes. AMPK signaling is involved to the role of BAIBA in attenuating ER stress.

Topics: Administration, Oral; Aminoisobutyric Acids; AMP-Activated Protein Kinases; Animals; Apoptosis; Blood Glucose; Blotting, Western; Carbohydrate Metabolism; Cholesterol; Diabetes Mellitus, Experimental; Diet, High-Fat; Endoplasmic Reticulum Stress; Glucosamine; Hep G2 Cells; Humans; Immunohistochemistry; Insulin Resistance; Lipid Metabolism; Liver; Mice; Phosphorylation; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction; Thapsigargin; Triglycerides; Tunicamycin

Bortezomib is an anti-cancer agent that induces ER stress by inhibiting proteasomal degradation. However, the effects of bortezomib appear to be dependent on its concentration and cellular context. Since ER stress is closely related to type 2 diabetes, the authors examined the effects of bortezomib on palmitic acid (PA)-induced ER stress in C2C12 murine myotubes. At low concentrations (<20nM), bortezomib protected myotubes from PA (750μM)-induced ER stress and inflammation. Either tunicamycin or thapsigargin-induced ER stress was also reduced by bortezomib. In addition, reduced glucose uptake and Akt phosphorylation induced by PA were prevented by co-treating bortezomib (10nM) both in the presence or absence of insulin. These protective effects of bortezomib were found to be associated with reduced JNK phosphorylation. Furthermore, bortezomib-induced AMPK phosphorylation, and the protective effects of bortezomib were diminished by AMPK knockdown, suggesting that AMPK activation underlies the effects of bortezomib. The in vivo administration of bortezomib at nontoxic levels (at 50 or 200μg/kg, i.p.) twice weekly for 5weeks to ob/ob mice improved insulin resistance, increased AMPK phosphorylation, reduced ER stress marker levels, and JNK inhibition in skeletal muscle. The study shows that bortezomib reduces ER stress, inflammation, and insulin resistance in vitro and in vivo, and suggests that bortezomib has novel applications for the treatment of metabolic disorders.

Topics: AMP-Activated Protein Kinases; Animals; Bortezomib; Cell Line; Cytoprotection; Endoplasmic Reticulum Stress; Enzyme Activation; Gene Knockdown Techniques; Inflammation; Insulin Resistance; Male; Mice, Obese; Models, Biological; Muscle Fibers, Skeletal; Palmitic Acid; Thapsigargin; Tunicamycin

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.

Topics: Abdominal Fat; Adipocytes; Animals; Carrier Proteins; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Fatty Acids; Homeostasis; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Lipase; Lipolysis; Male; MAP Kinase Signaling System; Obesity; p38 Mitogen-Activated Protein Kinases; Perilipin-1; Phosphoproteins; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Thapsigargin; Tunicamycin

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

Diet-induced obesity (DIO) is linked to peripheral insulin resistance-a major predicament in type 2 diabetes. This study aims to identify the molecular mechanism by which DIO-triggered endoplasmic reticulum (ER) stress promotes hepatic insulin resistance in mouse models.. C57BL/6 mice and primary hepatocytes were used to evaluate the role of LIPIN2 in ER stress-induced hepatic insulin resistance. Tunicamycin, thapsigargin, and lipopolysaccharide were used to invoke acute ER stress conditions. To promote chronic ER stress, mice were fed with a high-fat diet for 8-12 weeks. To verify the role of LIPIN2 in hepatic insulin signaling, adenoviruses expressing wild-type or mutant LIPIN2, and shRNA for LIPIN2 were used in animal studies. Plasma glucose, insulin levels as well as hepatic free fatty acids, diacylglycerol (DAG), and triacylglycerol were assessed. Additionally, glucose tolerance, insulin tolerance, and pyruvate tolerance tests were performed to evaluate the metabolic phenotype of these mice.. LIPIN2 expression was enhanced in mouse livers by acute ER stress-inducers or by high-fat feeding. Transcriptional activation of LIPIN2 by ER stress is mediated by activating transcription factor 4, as demonstrated by LIPIN2 promoter assays, Western blot analyses, and chromatin immunoprecipitation assays. Knockdown of hepatic LIPIN2 in DIO mice reduced fasting hyperglycemia and improved hepatic insulin signaling. Conversely, overexpression of LIPIN2 impaired hepatic insulin signaling in a phosphatidic acid phosphatase activity-dependent manner.. These results demonstrate that ER stress-induced LIPIN2 would contribute to the perturbation of hepatic insulin signaling via a DAG-protein kinase C ε-dependent manner in DIO mice.

Topics: Activating Transcription Factor 4; Animals; Blood Glucose; Blotting, Western; Cells, Cultured; Chromatin Immunoprecipitation; Dietary Fats; Endoplasmic Reticulum; Insulin Resistance; Lipopolysaccharides; Liver; Male; Mice; Mice, Inbred C57BL; Obesity; Phosphatidate Phosphatase; Polymerase Chain Reaction; Thapsigargin; Tunicamycin

Hepatic endoplasmic reticulum (ER) stress plays a key role in the development of obesity-induced insulin resistance. This study evaluated the effects of peptides from black soybean (BSP) on ER stress and insulin signaling in vitro and in vivo.. Using C2C12 myotubes or HepG2 cells, we evaluated the effects of BSP on the expression of proteins involved in insulin signaling and in the ER stress response in insulin-sensitive or insulin-resistant cells. BSP was given orally to db/db mice for 5weeks to investigate its antidiabetic effects in vivo and the underlying mechanisms.. BSP increased GLUT4 translocation and glucose transport in myotubes and stimulated Akt-mediated glycogen synthase kinase-3beta (GSK-3beta) and Foxo1 phosphorylation in HepG2 cells. BSP significantly restored the suppression of insulin-mediated Akt phosphorylation in insulin-resistant cells. BSP significantly inhibited the activation of ER stress-responsive proteins by thapsigargin. BSP also significantly reduced blood glucose and improved glucose tolerance in db/db mice. The serum lipid profile (triglyceride and high-density lipoprotein concentrations) improved concomitantly with the BSP-induced downregulation of hepatic fatty acid synthase expression in db/db mice. Consistent with the results observed in HepG2 cells, BSP downregulated the elevated hepatic ER stress response in diabetic mice concomitantly with an increased expression of phospho-Foxo1.. A peptide mixture, BSP, showed beneficial effects through multiple mechanisms involving the suppression of hepatic ER stress and restoration of insulin resistance, suggesting that it has potential as an antidiabetic agent.

Topics: Animals; Blood Glucose; Down-Regulation; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acid Synthases; Glucose Transporter Type 4; Glycine max; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Hypoglycemic Agents; Insulin Resistance; Lipoproteins, HDL; Liver; Male; Mice; Mice, Knockout; Muscle Fibers, Skeletal; Peptides; Phosphorylation; Plant Proteins; Proto-Oncogene Proteins c-akt; Stress, Physiological; Thapsigargin; Triglycerides

Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis. Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c. Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents. Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice. To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector. GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes. Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice. These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation. Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes. These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fatty Liver; Gene Expression; Glucose; Heat-Shock Proteins; Hepatocytes; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Lipid Metabolism; Liver; Male; Mice; Mice, Obese; Models, Biological; Molecular Chaperones; Nuclear Proteins; Obesity; Rats; Rats, Wistar; Rats, Zucker; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Thapsigargin; Transcription Factors

Chromium has gained popularity as a nutritional supplement for diabetic patients. This study evaluated the effect of chronic administration of a chromium complex of D-phenylalanine (Cr(D-phe)(3)) on glucose and insulin tolerance in obese mice. The study tested the hypothesis that Cr(D-phe)(3) suppresses endoplasmic reticulum (ER) stress and insulin resistance in these animals.. C57BL lean and ob/ob obese mice were randomly divided to orally receive vehicle or Cr(D-phe)(3) (3.8 mug of elemental chromium/kg/day) for 6 months. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Protein levels of phosphorylated pancreatic ER kinase (PERK), alpha subunit of translation initiation factor 2 (eIF2alpha) and inositol-requiring enzyme-1 (IRE-1), p-c-Jun, and insulin receptor substrate-1 (IRS-1) phosphoserine-307 were assessed by western blotting. In vitro ER stress was induced by treating cultured muscle cells with thapsigargin in the presence or absence of Cr(D-phe)(3).. ob/ob mice showed poor glucose and insulin tolerance compared to the lean controls, which was attenuated by Cr(D-phe)(3). Markers of insulin resistance (phospho-c-Jun and IRS-1 phosphoserine) and ER stress (p-PERK, p-IRE-1, p-eIF2alpha), which were elevated in ob/ob mice, were attenuated following Cr(D-phe)(3) treatment. Chromium treatment was also associated with a reduction in liver triglyceride levels and lipid accumulation. In cultured myotubes, Cr(D-phe)(3) attenuated ER stress induced by thapsigargin.. Oral Cr(D-phe)(3) treatment reduces glucose intolerance, insulin resistance, and hepatic ER stress in obese, insulin-resistant mice.

Topics: Animals; Blood Glucose; Chromium; Diabetes Mellitus, Type 2; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum; Glucose Intolerance; Insulin; Insulin Resistance; Leptin; Lipids; Liver; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Protein Serine-Threonine Kinases; Thapsigargin; Trace Elements

We clearly show that plasma membrane Ca2+ ATPase (PMCA) activity is lower in platelets from patients with non-insulin-dependent diabetes mellitus (NIDDM) than in those from healthy controls. The lower activity is likely due to reduced PMCA expression and increased tyrosine phosphorylation. These findings provide an explanation for the cellular ionic defects occurring in insulin resistant conditions.

Topics: Adult; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cation Transport Proteins; Diabetes Mellitus, Type 2; Female; Humans; Insulin Resistance; Ionomycin; Male; Phosphorylation; Phosphotyrosine; Plasma Membrane Calcium-Transporting ATPases; Platelet Activation; Protein Processing, Post-Translational; Thapsigargin

We have recently shown that insulin attenuates angiotensin II-induced intracellular Ca(2+) mobilization in human skin fibroblasts from normotensive subjects. This study was designed to investigate the effects of angiotensin II and the interactions between insulin and angiotensin II on intracellular Ca(2+) mobilization in skin fibroblasts from patients with essential hypertension. Fibroblasts were obtained from 9 normotensives and 18 hypertensives. Spectrofluorophotometric free Ca(2+) measurement was performed in monolayers of 24-hour serum-deprived cells. Resting intracellular Ca(2+) level and angiotensin II-stimulated intracellular Ca(2+) peak were higher in fibroblasts from hypertensives compared with those from normotensives. The effect of acute insulin exposure was evaluated in fibroblasts from hypertensives subdivided on the basis of insulin sensitivity. In insulin-sensitive hypertensives, insulin significantly blunted the effects of angiotensin II on intracellular Ca(2+) response, whereas in insulin-resistant patients, insulin did not modify intracellular Ca(2+) response to angiotensin II. Pertussis toxin, a G(ialpha)-inhibitor, reduced angiotensin II-stimulated Ca(2+) peak in insulin-sensitive but not in insulin-resistant hypertensives. In conclusion, the effects of angiotensin II on intracellular Ca(2+) mobilization are more pronounced in fibroblasts from hypertensives compared with those from normotensives, and the inhibitory effect of insulin is blunted in insulin-resistant hypertensives by a G(ialpha) pertussis toxin-sensitive abnormality.

Topics: Adult; Angiotensin II; Calcium; Cells, Cultured; Culture Media; Cytosol; Female; Fibroblasts; Humans; Hypertension; Insulin; Insulin Resistance; Male; Middle Aged; Pertussis Toxin; Skin; Thapsigargin; Virulence Factors, Bordetella