Carla Roberta de Oliveira Carvalho

Pleiotropic effects of fatty acids on pancreatic β‐cells

Hyperlipidemia is frequently associated with insulin resistance states as found in type 2 diabetes and obesity. Effects of free fatty acids (FFA) on pancreatic β‐cells have long been recognized. Acute exposure of the pancreatic β‐cell to FFA results in an increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. We recently showed that palmitate augments insulin release in the presence of non‐stimulatory concentrations of glucose. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose‐induced insulin release. These results imply that physiological plasma levels of FFA are important for β‐cell function. Although, it has been accepted that fatty acid oxidation is necessary for its stimulation of insulin secretion, the possible mechanisms by which fatty acids (FA) affect insulin secretion are discussed in this review. Long‐chain acyl‐CoA (LC‐CoA) controls several aspects of the β‐cell function including activation of certain types of protein kinase C (PKC), modulation of ion channels, protein acylation, ceramide‐ and/or nitric oxide (NO)‐mediated apoptosis, and binding to nuclear transcriptional factors. The present review also describes the possible effects of FA on insulin signaling. We showed for the first time that acute exposure of islets to palmitate upregulates the intracellular insulin‐signaling pathway in pancreatic islets. Another aspect considered in this review is the source of FA for pancreatic islets. In addition to be exported to the medium, lipids can be transferred from leukocytes (macrophages) to pancreatic islets in co‐culture. This process consists an additional source of FA that may plays a significant role to regulate insulin secretion.

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Objective We investigated the effects of high-fat diet-induced obesity on vascular proinflammatory factors and oxidative stress on endothelium-dependent relaxation of the aorta. Methods Female Swiss mice were submitted to a high-fat diet for 16 weeks. At the end of the experimental period, we evaluated blood pressure, relaxation in response to acetylcholine in aortic rings in the absence and the presence of the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), and the nuclear factor (NF)-κB inhibitor, sodium salicylate (5 mmol/l). Aortic protein expression of endothelial nitric oxide synthase, Cu/Zn-SOD, NF-κB, IκB-α, and proinflammatory cytokines were also evaluated. Results Obese mice presented higher systolic and diastolic blood pressure than control mice (P < 0.05). The relaxation of aortas to acetylcholine, but not to sodium nitroprusside, was significantly decreased in obese mice and was corrected by both SOD and sodium salicylate (P < 0.05). The protein expression of endothelial nitric oxide synthase and Cu/Zn-SOD was significantly decreased in aorta from obese mice (P < 0.05). Total p65 NF-κB subunit protein expression was not affected by obesity, but the protein expression of NF-κB inhibitor IκB-α was lower in aorta from obese mice (P < 0.05). There were no significant differences in the interleukin (IL)-1β and IL-6 protein expression between groups. In contrast, the expression of TNF-α was significantly increased in aortas from obese mice. Conclusion Our results suggest that the reduced antioxidant defense and the local NF-κB pathway play an important role in the impairment of endothelium-dependent relaxation in aorta from obese mice.

In vivo activation of insulin receptor tyrosine kinase by melatonin in the rat hypothalamus

Melatonin is the pineal hormone that acts via a pertussis toxin‐sensitive G‐protein to inhibit adenylate cyclase. However, the intracellular signalling effects of melatonin are not completely understood. Melatonin receptors are mainly present in the suprachiasmatic nucleus (SCN) and pars tuberalis of both humans and rats. The SCN directly controls, amongst other mechanisms, the circadian rhythm of plasma glucose concentration. In this study, using immunoprecipitation and immunoblotting, we show that melatonin induces rapid tyrosine phosphorylation and activation of the insulin receptor β‐subunit tyrosine kinase (IR) in the rat hypothalamic suprachiasmatic region. Upon IR activation, tyrosine phosphorylation of IRS‐1 was detected. In addition, melatonin induced IRS‐1/PI(3)‐kinase and IRS‐1/SHP‐2 associations and downstream AKT serine phosphorylation and MAPK (mitogen‐activated protein kinase) phosphorylation, respectively. These results not only indicate a new signal transduction pathway for melatonin, but also a potential cross‐talk between melatonin and insulin.

Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets

Abstract: The effect of melatonin (0.1 μM) on freshly isolated islets from adult rats was investigated. Melatonin caused a marked decrease of insulin secretion by islets in response to glucose. The mechanism involved was then examined. Melatonin did not interfere with glucose metabolism as indicated by the measurement of glucose oxidation. However, the content of the protein kinase A (PKA) catalytic α‐subunit was significantly decreased in islets exposed to melatonin for 1 hr in the presence of 8.3 mM glucose, whereas that of the protein kinase C (PKC) α‐subunit remained unchanged. Melatonin also inhibited forskolin‐induced insulin secretion, a well known activator of adenylate cyclase (AC) activity. This may explain the low content of insulin found in islets incubated in the presence of melatonin for 3 hr. In fact, 3′,5′ ‐cyclic adenosine monophosphate (cAMP), a product of AC activity, stimulates insulin synthesis. These findings led us to postulate that a down‐regulation of the PKA signaling pathway may be the mechanism involved in the melatonin inhibition of the process of glucose‐induced insulin secretion.

New Insights into Fatty Acid Modulation of Pancreatic β‐Cell Function

Insulin resistance states as found in type 2 diabetes and obesity are frequently associated with hyperlipidemia. Both stimulatory and detrimental effects of free fatty acids (FFA) on pancreatic β cells have long been recognized. Acute exposure of the pancreatic β cell to both high glucose concentrations and saturated FFA results in a substantial increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose‐induced insulin release but palmitate can augment insulin release in the presence of nonstimulatory concentrations of glucose. These results imply that changes in physiological plasma levels of FFA are important for regulation of β‐cell function. Although it is widely accepted that fatty acid (FA) metabolism (notably FA synthesis and/or formation of LC‐acyl‐CoA) is necessary for stimulation of insulin secretion, the key regulatory molecular mechanisms controlling the interplay between glucose and fatty acid metabolism and thus insulin secretion are not well understood but are now described in detail in this review. Indeed the correct control of switching between FA synthesis or oxidation may have critical implications for β‐cell function and integrity both in vivo and in vitro . LC‐acyl‐CoA (formed from either endogenously synthesized or exogenous FA) controls several aspects of β‐cell function including activation of certain types of PKC, modulation of ion channels, protein acylation, ceramide‐ and/or NO‐mediated apoptosis, and binding to and activating nuclear transcriptional factors. The present review also describes the possible effects of FAs on insulin signaling. We have previously reported that acute exposure of islets to palmitate up‐regulates some key components of the intracellular insulin signaling pathway in pancreatic islets. Another aspect considered in this review is the potential source of fatty acids for pancreatic islets in addition to supply in the blood. Lipids can be transferred from leukocytes (macrophages) to pancreatic islets in coculture. This latter process may provide an additional source of FAs that may play a significant role in the regulation of insulin secretion.

Cellular Mechanism by Which Estradiol Protects Female Ovariectomized Mice From High-Fat Diet-Induced Hepatic and Muscle Insulin Resistance

Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.

Time-Dependent Effects of Fatty Acids on Skeletal Muscle Metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin‐stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin‐stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA‐induced insulin resistance. J. Cell. Physiol. 210: 7–15, 2007.

Dehydroepiandrosterone protects against oxidative stress-induced endothelial dysfunction in ovariectomized rats

Non‐technical summary  It is well known that cardiovascular disease is more frequent in postmenopausal than in premenopausal women. Moreover, it has been shown that dehydroepiandrosterone (DHEA), a steroid hormone secreted by adrenal glands, reduces during ageing. Its reduced plasma level has been related to increased prevalence of obesity, insulin resistance and cardiovascular disease. We show that DHEA treatment in ovariectomized rats, an experimental model of menopause, reduces blood pressure and improves vascular function. Furthermore, DHEA reduced reactive oxygen species (ROS), correcting the reduced protein expression of Cu/Zn‐SOD, an antioxidant protein, and increased protein expression of NADPH oxidase, a pro‐oxidant protein. This work shows the potential effect of DHEA upon correction of endothelial dysfunction observed on oestrogen deprivation.

Melatonin improves insulin sensitivity independently of weight loss in old obese rats.

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8‐ and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1‐fold increase both after 8‐ and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin‐induced phosphorylation of the analyzed ISP proteins increased 1.3‐ and 2.3‐fold after 8‐ and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS‐1, 2) remained unchanged in all investigated tissues, except for the 2‐fold increase in the total amount of IRS‐1 in the periepididymal adipose tissue. Therefore, the known age‐related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle)

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs–Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 μM palmitate. Palmitate increased the insulin‐stimulated [14C]glycogen synthesis, decreased lactate production, and did not alter D‐[U‐14C]glucose decarboxylation and 2‐deoxy‐D‐[2,6‐3H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1‐14C]pyruvate decarboxylation and increased 14CO2 produced from [2‐14C]pyruvate. Palmitate reduced insulin‐stimulated phosphorylation of insulin receptor substrate‐1/2, Akt, and p44/42 mitogen‐activated protein kinases. Bromopalmitate, a non‐metabolizable analogue of palmitate, reduced [14C]glycogen synthesis. A strong correlation was found between [U‐14C]palmitate decarboxylation and [14C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6‐phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin‐stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin‐stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.