An increase of stroke incidence occurs in women with the decline of estrogen levels following men... more An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio betwee...
Menopause leads to a decrease in estrogen production that increases central insulin resistance, c... more Menopause leads to a decrease in estrogen production that increases central insulin resistance, contributing to the development of neurodegenerative diseases. We have evaluated the influence of aging and estradiol or genistein treatments on some key stages of the insulin signaling pathway in the cerebral cortex. Young and aged female Wistar rats were ovariectomized and treated acutely with 17β-estradiol (1.4μg/kg body weight), two doses of genistein (10 or 40mg/kg body weight), or vehicle. The cortical expression of several key insulin signaling pathway components was analyzed by western blotting. Our results showed an age-related deterioration in the interactions between the regulatory subunit of phosphatidylinositol 3-kinase (p85α) and the activated form of insulin receptor substrate 1 (p-IRS1tyr612), as well as between p85α and the 46kDa isoform of the estrogen receptor α (ERα46). Moreover, aging also decreased the translocation of glucose transporter-4 (GLUT4) to the plasma membrane. 17β-Estradiol but not genistein reduced the negative impact of aging on central insulin sensitivity by favoring this GLUT4 translocation, and therefore could be neuroprotective against the associated neurodegenerative diseases. However, protein kinase B (Akt) activation by genistein suggests that other possible mechanisms are involved in the neuroprotective effects of this phytoestrogen during the aging process.
Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and c... more Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.
To evaluate antidiabetic and anti-inflammatory effects of resveratrol on the ovarian response to ... more To evaluate antidiabetic and anti-inflammatory effects of resveratrol on the ovarian response to controlled ovarian hyperstimulation (COH) in obesity-related infertility. Experimental. University laboratory. Sixteen female ob/ob mice and 16 female C57BL/6J mice undergoing COH. Wild-type placebo group; wild-type resveratrol group; ob/ob mice placebo group; ob/ob mice resveratrol group. Resveratrol 3.75 mg/kg daily for 20 days and undergoing COH protocol. Body and reproductive system weight, food intake, fasting blood glucose, plasma insulin and T levels, and Homeostatic Index of Insulin Resistance; interleukin-6 and tumor necrosis factor-α levels in adipose tissue by Western blot; assessment of quality and quantity of oocytes retrieved; and quantitative analysis of ovarian follicles. Plasma insulin and T levels decreased and Homeostatic Index of Insulin Resistance improved in ob/ob mice treated with resveratrol. Interleukin-6 and tumor necrosis factor-α levels were significantly reverted back to near normalcy after resveratrol treatment in obese mice. Administration of resveratrol resulted in a significantly higher number of oocytes collected in wild-type mice. The number of primary, growing, preovulatory, and atretic follicles was found to be decreased in the group of obese mice treated with resveratrol when compared with the obese control group. Resveratrol administration could exert benefits against loss of ovarian follicles, and these actions may be mediated, at least in part, via anti-inflammatory, insulin-sensitizing, and antihyperandrogenism effects. These observations further validate the therapeutic potential of resveratrol to preserve ovarian reserve in conditions associated with obesity. Our results suggest the possible clinical use of resveratrol to enhance the ovarian response to COH in normal-weight females.
Aging is associated with decreased insulin sensitivity and impaired cerebral glucose homeostasis.... more Aging is associated with decreased insulin sensitivity and impaired cerebral glucose homeostasis. These changes increase neural sensitivity to metabolic damage contributing to cognitive decline, being the decrease in plasma estrogen following menopause one of the main factors involved in aged females. Phytoestrogens as genistein are structurally similar to 17β-estradiol, bind to estrogen receptors, and can evoke both estrogenic and anti-estrogenic effects. Estrogens and phytoestrogens have neuroprotective potential, but the physiological mechanisms are not fully understood. Young and aged female Wistar rats were ovariectomized and treated acutely with 17β-estradiol (1.4μg/kg body weight), genistein (10 or 40 mg/kg body weight), or vehicle. Cortical expression of glucose transporter-3 (GLUT-3) and -4 (GLUT-4), cytochrome c oxidase (CO), estrogen receptor-α (ERα) and -β (ERβ) was measured by Western blotting. There was an age-related decline in GLUT-4, CO and ERβ levels. Both drugs, estradiol and genistein, were able to reverse GLUT-3 downregulation in the cortex following late ovariectomy. However, genistein was the only treatment able to restore completely GLUT-4 levels in aged rats. In contrast, estradiol was more potent than genistein at increasing CO, a marker of cerebral oxidative metabolism. As regards ER levels, estradiol increased the ERα67 quantity diminished by late ovariectomy, while genistein did the same with the other ERα isoform, ERα46, highlighting drug-specific differences in expression changes for both isoforms. On the other hand, no treatment-related differences were found regarding ERβ levels. Therefore, genistein like estradiol could be suitable treatments against cortical metabolic dysfunction caused by aging. These treatments may hold promise as neuroprotective strategies against diabetes and age-related neurodegenerative diseases.
Aging is characterized by decline in metabolic function and insulin resistance, and both seem to ... more Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.
American journal of physiology. Cell physiology, 2014
Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed in... more Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients...
Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance... more Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle. Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β(Ser9) and PKCδ(Thr505), two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment. An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.
An increase of stroke incidence occurs in women with the decline of estrogen levels following men... more An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio betwee...
Menopause leads to a decrease in estrogen production that increases central insulin resistance, c... more Menopause leads to a decrease in estrogen production that increases central insulin resistance, contributing to the development of neurodegenerative diseases. We have evaluated the influence of aging and estradiol or genistein treatments on some key stages of the insulin signaling pathway in the cerebral cortex. Young and aged female Wistar rats were ovariectomized and treated acutely with 17β-estradiol (1.4μg/kg body weight), two doses of genistein (10 or 40mg/kg body weight), or vehicle. The cortical expression of several key insulin signaling pathway components was analyzed by western blotting. Our results showed an age-related deterioration in the interactions between the regulatory subunit of phosphatidylinositol 3-kinase (p85α) and the activated form of insulin receptor substrate 1 (p-IRS1tyr612), as well as between p85α and the 46kDa isoform of the estrogen receptor α (ERα46). Moreover, aging also decreased the translocation of glucose transporter-4 (GLUT4) to the plasma membrane. 17β-Estradiol but not genistein reduced the negative impact of aging on central insulin sensitivity by favoring this GLUT4 translocation, and therefore could be neuroprotective against the associated neurodegenerative diseases. However, protein kinase B (Akt) activation by genistein suggests that other possible mechanisms are involved in the neuroprotective effects of this phytoestrogen during the aging process.
Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and c... more Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.
To evaluate antidiabetic and anti-inflammatory effects of resveratrol on the ovarian response to ... more To evaluate antidiabetic and anti-inflammatory effects of resveratrol on the ovarian response to controlled ovarian hyperstimulation (COH) in obesity-related infertility. Experimental. University laboratory. Sixteen female ob/ob mice and 16 female C57BL/6J mice undergoing COH. Wild-type placebo group; wild-type resveratrol group; ob/ob mice placebo group; ob/ob mice resveratrol group. Resveratrol 3.75 mg/kg daily for 20 days and undergoing COH protocol. Body and reproductive system weight, food intake, fasting blood glucose, plasma insulin and T levels, and Homeostatic Index of Insulin Resistance; interleukin-6 and tumor necrosis factor-α levels in adipose tissue by Western blot; assessment of quality and quantity of oocytes retrieved; and quantitative analysis of ovarian follicles. Plasma insulin and T levels decreased and Homeostatic Index of Insulin Resistance improved in ob/ob mice treated with resveratrol. Interleukin-6 and tumor necrosis factor-α levels were significantly reverted back to near normalcy after resveratrol treatment in obese mice. Administration of resveratrol resulted in a significantly higher number of oocytes collected in wild-type mice. The number of primary, growing, preovulatory, and atretic follicles was found to be decreased in the group of obese mice treated with resveratrol when compared with the obese control group. Resveratrol administration could exert benefits against loss of ovarian follicles, and these actions may be mediated, at least in part, via anti-inflammatory, insulin-sensitizing, and antihyperandrogenism effects. These observations further validate the therapeutic potential of resveratrol to preserve ovarian reserve in conditions associated with obesity. Our results suggest the possible clinical use of resveratrol to enhance the ovarian response to COH in normal-weight females.
Aging is associated with decreased insulin sensitivity and impaired cerebral glucose homeostasis.... more Aging is associated with decreased insulin sensitivity and impaired cerebral glucose homeostasis. These changes increase neural sensitivity to metabolic damage contributing to cognitive decline, being the decrease in plasma estrogen following menopause one of the main factors involved in aged females. Phytoestrogens as genistein are structurally similar to 17β-estradiol, bind to estrogen receptors, and can evoke both estrogenic and anti-estrogenic effects. Estrogens and phytoestrogens have neuroprotective potential, but the physiological mechanisms are not fully understood. Young and aged female Wistar rats were ovariectomized and treated acutely with 17β-estradiol (1.4μg/kg body weight), genistein (10 or 40 mg/kg body weight), or vehicle. Cortical expression of glucose transporter-3 (GLUT-3) and -4 (GLUT-4), cytochrome c oxidase (CO), estrogen receptor-α (ERα) and -β (ERβ) was measured by Western blotting. There was an age-related decline in GLUT-4, CO and ERβ levels. Both drugs, estradiol and genistein, were able to reverse GLUT-3 downregulation in the cortex following late ovariectomy. However, genistein was the only treatment able to restore completely GLUT-4 levels in aged rats. In contrast, estradiol was more potent than genistein at increasing CO, a marker of cerebral oxidative metabolism. As regards ER levels, estradiol increased the ERα67 quantity diminished by late ovariectomy, while genistein did the same with the other ERα isoform, ERα46, highlighting drug-specific differences in expression changes for both isoforms. On the other hand, no treatment-related differences were found regarding ERβ levels. Therefore, genistein like estradiol could be suitable treatments against cortical metabolic dysfunction caused by aging. These treatments may hold promise as neuroprotective strategies against diabetes and age-related neurodegenerative diseases.
Aging is characterized by decline in metabolic function and insulin resistance, and both seem to ... more Aging is characterized by decline in metabolic function and insulin resistance, and both seem to be in the basis of neurodegenerative diseases and cognitive dysfunction. Estrogens prevent age-related changes, and phytoestrogens influence learning and memory. Our hypothesis was that estradiol and genistein, using rapid-action mechanisms, are able to modify insulin sensitivity, process of learning, and spatial memory. Young and aged ovariectomized rats received acute treatment with estradiol or genistein. Aged animals were more insulin-resistant than young. In each age, estradiol and genistein-treated animals were less insulin-resistant than the others, except in the case of young animals treated with high doses of genistein. In aged rats, no differences between groups were found in spatial memory test, showing a poor performance in the water maze task. However, young females treated with estradiol or high doses of genistein performed well in spatial memory task like the control group. Only rats treated with high doses of genistein showed an optimal spatial memory similar to the control group. Conversely, acute treatment with high doses of phytoestrogens improved spatial memory consolidation only in young rats, supporting the critical period hypothesis for the beneficial effects of estrogens on memory. Therefore, genistein treatment seems to be suitable treatment in aged rats in order to prevent insulin resistance but not memory decline associated with aging. Acute genistein treatment is not effective to restore insulin resistance associated to the early loss of ovarian function, although it can be useful to improve memory deficits in this condition.
American journal of physiology. Cell physiology, 2014
Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed in... more Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients...
Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance... more Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle. Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β(Ser9) and PKCδ(Thr505), two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment. An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.
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Papers by Pablo Garrido