Brain GLP-1/IGF-1 Signaling and Autophagy Mediate Exendin-4 Protection Against Apoptosis in Type 2 Diabetic Rats.

Type 2 diabetes (T2D) is a modern socioeconomic burden, mostly due to its long-term complications affecting nearly all tissues. One of them is the brain, whose dysfunctional intracellular quality control mechanisms (namely autophagy) may upregulate apoptosis, leading to cognitive dysfunction and Alzheimer disease (AD). Since impaired brain insulin signaling may constitute the crosslink between T2D and AD, its restoration may be potentially therapeutic herein. Accordingly, the insulinotropic anti-T2D drugs from glucagon-like peptide-1 (GLP-1) mimetics, namely, exendin-4 (Ex-4), could be a promising therapy. In line with this, we hypothesized that peripherally administered Ex-4 rescues brain intracellular signaling pathways, promoting autophagy and ultimately protecting against chronic T2D-induced apoptosis. Thus, we aimed to explore the effects of chronic, continuous, subcutaneous (s.c.) exposure to Ex-4 in brain cortical GLP-1/insulin/insulin-like growth factor-1 (IGF-1) signaling, and in autophagic and cell death mechanisms in middle-aged (8 months old), male T2D Goto-Kakizaki (GK) rats. We used brain cortical homogenates obtained from middle-aged (8 months old) male Wistar (control) and T2D GK rats. Ex-4 was continuously administered for 28 days, via s.c. implanted micro-osmotic pumps (5 µg/kg/day; infusion rate 2.5 µL/h). Peripheral characterization of the animal models was given by the standard biochemical analyses of blood or plasma, the intraperitoneal glucose tolerance test, and the heart rate. GLP-1, insulin, and IGF-1, their downstream signaling and autophagic markers were evaluated by specific ELISA kits and Western blotting. Caspase-like activities and other apoptotic markers were given by colorimetric methods and Western blotting. Chronic Ex-4 treatment attenuated peripheral features of T2D in GK rats, including hyperglycemia and insulin resistance. Furthermore, s.c. Ex-4 enhanced their brain cortical GLP-1 and IGF-1 levels, and subsequent signaling pathways. Specifically, Ex-4 stimulated protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/Akt signaling, increasing cGMP and AMPK levels, and decreasing GSK3ß and JNK activation in T2D rat brains. Moreover, Ex-4 regulated several markers for autophagy in GK rat brains (as mTOR, PI3K class III, LC3 II, Atg7, p62, LAMP-1, and Parkin), ultimately protecting against apoptosis (by decreasing several caspase-like activities and mitochondrial cytochrome c, and increasing Bcl2 levels upon T2D). Altogether, this study demonstrates that peripheral Ex-4 administration may constitute a promising therapy against the chronic complications of T2D affecting the brain.

Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes.

Diabetes mellitus is a degenerative disease that has deleterious effects on male reproductive function, possibly through an increase in oxidative stress. This study was conducted in order to clarify the mechanisms by which oxidative stress influences animal models for both type 1 (streptozotocin-treated rats, STZ) and type 2 (Goto-Kakizaki (GK) rats) diabetes. We determined the extent of lipid peroxidation, protein oxidation, lactate levels, adenine nucleotides, adenylate energy charge and the activity of glutathione peroxidase, glutathione reductase and lactate dehydrogenase, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with sperm count and motility. Sperm concentration and motility were decreased in STZ-treated rats. ATP levels were lower in rats treated with STZ for 3 months, in contrast to GK and rats treated with STZ for 1 month, suggesting an adaptative response. STZ-treated rats showed increased lipid peroxidation after 1 week and 3 months of treatment. Glutathione reductase (G-red) activity was found to be higher in GK rats. Glutathione peroxidase activity was lower in GK and rats treated with STZ for 1 month, which is in accordance with the proposal of functional recovery in these animals. We conclude that hyperglycemia has an adverse effect in sperm concentration and motility via changes in energy production and free radical management. Furthermore, both animal models, particularly GK rats and rats treated with STZ for 1 month, present some metabolic adaptations, increasing the efficiency of mitochondrial ATP production, in order to circumvent the deleterious effects promoted by the disease.

Gut-brain connection: The neuroprotective effects of the anti-diabetic drug liraglutide.

Long-acting glucagon-like peptide-1 (GLP-1) analogues marketed for type 2 diabetes (T2D) treatment have been showing positive and protective effects in several different tissues, including pancreas, heart or even brain. This gut secreted hormone plays a potent insulinotropic activity and an important role in maintaining glucose homeostasis. Furthermore, growing evidences suggest the occurrence of several commonalities between T2D and neurodegenerative diseases, insulin resistance being pointed as a main cause for cognitive decline and increased risk to develop dementia. In this regard, it has also been suggested that stimulation of brain insulin signaling may have a protective role against cognitive deficits. As GLP-1 receptors (GLP-1R) are expressed throughout the central nervous system and GLP-1 may cross the blood-brain-barrier, an emerging hypothesis suggests that they may be promising therapeutic targets against brain dysfunctional insulin signaling-related pathologies. Importantly, GLP-1 actions depend not only on the direct effect mediated by its receptor activation, but also on the gut-brain axis involving an exchange of signals between both tissues via the vagal nerve, thereby regulating numerous physiological functions (e.g., energy homeostasis, glucose-dependent insulin secretion, as well as appetite and weight control). Amongst the incretin/GLP-1 mimetics class of anti-T2D drugs with an increasingly described neuroprotective potential, the already marketed liraglutide emerged as a GLP-1R agonist highly resistant to dipeptidyl peptidase-4 degradation (thereby having an increased half-life) and whose systemic GLP-1R activity is comparable to that of native GLP-1. Importantly, several preclinical studies showed anti-apoptotic, anti-inflammatory, anti-oxidant and neuroprotective effects of liraglutide against T2D, stroke and Alzheimer disease (AD), whereas several clinical trials, demonstrated some surprising benefits of liraglutide on weight loss, microglia inhibition, behavior and cognition, and in AD biomarkers. Herein, we discuss the GLP-1 action through the gut-brain axis, the hormone's regulation of some autonomic functions and liraglutide's neuroprotective potential.

Insulin affects synaptosomal GABA and glutamate transport under oxidative stress conditions.

In this study, we investigated the in vitro effect of exogenously administered insulin on the susceptibility to oxidative stress and on the accumulation of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in a synaptosomal fraction isolated from male Wistar rat brain cortex. Insulin (1 microM) did not affect synaptosomal lipid peroxidation induced by the oxidant pair ascorbate/Fe(2+), although under these conditions an increase in thiobarbituric acid reactive substances (TBARS) levels was observed. Under control conditions, the presence of insulin did not change the uptake of [3H]GABA or [3H]glutamate. In contrast, under oxidizing conditions, we observed a 1.8- and a 2.2-fold decrease in [3H]GABA and [3H]glutamate accumulation, respectively, and insulin reverted the lower levels of both [3H]GABA and [3H]glutamate accumulation (to 86.74+/-6.26 and 67.01+/-6.65% of control, respectively). Insulin also increased the extrasynaptosomal levels of GABA and glutamate, determined both in control and oxidizing conditions. From this study, we can conclude that insulin is a modulator of amino acid neurotransmitter transport, either directly, as seems to occur under normal conditions, or via the decrease in ATP levels and the subsequent reversion of the amino acid transporters, as seems to occur under oxidative stress conditions. The modulation of both GABA and glutamate transport might be implicated in the neuroprotective role of insulin.

Carvedilol protects ischemic cardiac mitochondria by preventing oxidative stress.

Ischemia negatively affects mitochondrial function by inducing the mitochondrial permeability transition (MPT). The MPT is triggered by oxidative stress, which occurs in mitochondria during ischemia as a result of diminished antioxidant defenses and increased reactive oxygen species production. It causes mitochondrial dysfunction and can ultimately lead to cell death. Therefore, drugs able to minimize mitochondrial damage induced by ischemia may prove to be clinically effective. We analyzed the effect of carvedilol, a beta-blocker with antioxidant properties, on mitochondrial dysfunction. Carvedilol decreased levels of TBARS (thiobarbituric acid reactive substances), an indicator of oxidative stress, which is consistent with its antioxidant properties. Regarding cell death by apoptosis, although ischemia did increase caspase-8-like activity, there were no changes in caspase-3-like activity, which is activated downstream of caspase-8; this may indicate that the apoptotic cascade is not activated by 60 minutes of ischemia. We conclude that carvedilol protects ischemic mitochondria by preventing oxidative mitochondrial damage, and, by so doing, it may also inhibit the formation of the MPT pore.

Vascular, oxidative, and synaptosomal abnormalities during aging and the progression of type 2 diabetes.

Alterations in brain structure and function are a well-known long-term complication of type 2 diabetes (T2D). Although the mechanism(s) by which T2D lead(s) to cognitive dysfunction and neuronal cells degeneration continue(s) to be a matter of debate, vascular alterations emerged as major players in this scenario. This study was aimed to evaluate the antioxidant defenses and oxidative markers present in brain vessels and synaptosomes from 3- and 12-month-old Goto- Kakizaki (GK) rats, a spontaneous non-obese model of T2D, and Wistar control rats. A significant increase in manganese superoxide dismutase (MnSOD) activity and vitamin E levels and a significant decrease in aconitase and glutathione reductase (GR) activities, glutathione (GSH)/glutathione disulfide (GSSG) ratio, and GSH and malondialdehyde (MDA) levels were observed in brain vessels and synaptosomes from GK rats, and these effects were not significantly affected by aging. However, an age-dependent increase in hydrogen peroxide (H2O2) levels in both diabetic synaptosomes and vessels was observed. No significant alterations were observed in the activity of glutathione peroxidase (GPx) and GR in both brain vessels and synaptosomes from diabetic animals. In control rats, an age-dependent increase in the activity of GPx, GR, and MnSOD and vitamin E and MDA levels and an age-dependent decrease in GSH levels were observed in brain vessels. In contrast, a significant age-dependent increase in GSH levels and a decrease in vitamin E levels were observed in synaptosomes from control animals. Altogether, our results show that T2D and aging differently affect brain vessels and synaptosomes. However, both conditions increase the vulnerability of brain structures to degenerative events.

"MitoTea": Geranium robertianum L. decoctions decrease blood glucose levels and improve liver mitochondrial oxidative phosphorylation in diabetic Goto-Kakizaki rats.

Several chemical compounds found in plant products have proven to possess beneficial properties, being currently pointed out due to their pharmacological potential in type 2 diabetes mellitus complications. In this context, we studied the effect of Geranium robertianum L. (herb Robert) leaf decoctions in Goto-Kakizaki (GK) rats, a model of type 2 diabetes. Our results showed that oral administration of G. robertianum leaf decoctions over a period of four weeks lowered the plasma glucose levels in diabetic rats. Furthermore, the treatment with G. robertianum extracts improved liver mitochondrial respiratory parameters (state 3, state 4 and FCCP-stimulated respiration) and increased oxidative phosphorylation efficiency.

Diabetes and mitochondrial oxidative stress: a study using heart mitochondria from the diabetic Goto-Kakizaki rat.

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe2+ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O2 consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O2 consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determining alpha-tocopherol, GSH and coenzyme Q content. Although no difference of alpha-tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q9, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.