Esther Velázquez

Insulin in the Brain: Its Pathophysiological Implications for States Related with Central Insulin Resistance, Type 2 Diabetes and Alzheimer’s Disease

Although the brain has been considered an insulin-insensitive organ, recent reports on the location of insulin and its receptors in the brain have introduced new ways of considering this hormone responsible for several functions. The origin of insulin in the brain has been explained from peripheral or central sources, or both. Regardless of whether insulin is of peripheral origin or produced in the brain, this hormone may act through its own receptors present in the brain. The molecular events through which insulin functions in the brain are the same as those operating in the periphery. However, certain insulin actions are different in the central nervous system, such as hormone-induced glucose uptake due to a low insulin-sensitive GLUT-4 activity, and because of the predominant presence of GLUT-1 and GLUT-3. In addition, insulin in the brain contributes to the control of nutrient homeostasis, reproduction, cognition, and memory, as well as to neurotrophic, neuromodulatory, and neuroprotective effects. Alterations of these functional activities may contribute to the manifestation of several clinical entities, such as central insulin resistance, type 2 diabetes mellitus (T2DM), and Alzheimer’s disease (AD). A close association between T2DM and AD has been reported, to the extent that AD is twice more frequent in diabetic patients, and some authors have proposed the name “type 3 diabetes” for this association. There are links between AD and T2DM through mitochondrial alterations and oxidative stress, altered energy and glucose metabolism, cholesterol modifications, dysfunctional protein O-GlcNAcylation, formation of amyloid plaques, altered Aβ metabolism, and tau hyperphosphorylation. Advances in the knowledge of preclinical AD and T2DM may be a major stimulus for the development of treatment for preventing the pathogenic events of these disorders, mainly those focused on reducing brain insulin resistance, which is seems to be a common ground for both pathological entities.

Evidence That Circadian Variations of Circulating Melatonin Levels in Fetal and Suckling Rats Are Dependent on Maternal Melatonin Transfer

Although the circadian variation of melatonin content in the pineal gland appears during the 3rd week of extrauterine life, recent studies suggest that the fetus perceives the day length through maternal melatonin transfer. Accordingly, we determined serum melatonin concentrations in pregnant and lactating rats and in their offsprings during the day (D) and at night (N). As compared with nonpregnant adult female rats (D: 6.0 +/- 0.5 and N: 112.0 +/- 5.0 pg/ml), significant increases of serum melatonin concentrations, both during the day and at night, were observed in 21-day pregnant (D: 21.0 +/- 2.6 and N: 222.0 +/- 7.2 pg/ml) and in 10-day (D: 20.0 +/- 2.6 and N: 145.0 +/- 12.3 pg/ml) and 20-day (D: 19.0 +/- 1.2 and N: 140.0 +/- 8.0 pg/ml) lactating animals, while a profound decrease was found in 5-day (D: 13.0 +/- 1.0 and N: 50.0 +/- 5.6 pg/ml) lactating rats. In addition, melatonin levels were significantly higher during the night than during the day in all the experimental groups. High levels and circadian variation of serum melatonin were detected also in 21-day-old fetuses (D: 13.0 +/- 0.8 and N: 108.0 +/- 4.8 pg/ml) and in 5- and 10-day-old suckling (in 5-day-old, D: 13.0 +/- 0.6 and N: 71.0 +/- 3.1 pg/ml; in 10-day-old, D: 17.0 +/- 1.4 and N: 63.0 +/- 3.8 pg/ml) rats.(ABSTRACT TRUNCATED AT 250 WORDS)

25-hydroxycholesterol has a dual effect on the proliferation of cultured rat astrocytes

We examined the effects of 25-OH-cholesterol on the growth of cultured rat astrocytes in the presence of lipoprotein-deficient serum (LPDS). 25-OH-cholesterol at 0.5-8 microM induced an increase in DNA synthesis as measured by [3H]thymidine incorporation into DNA, staining the cells with crystal violet, or counting the number of cells in different phases of the cell cycle by flow cytometry; however, at higher doses, an inhibition of cell proliferation was produced. Similar dose-dependent effects were found in media containing albumin (alone or with added EGF, PDGF, IGF-I or insulin), fetal bovine serum (FBS), or cholesterol-enriched LPDS. Mevalonate, and partially 25-OH-cholesterol, reversed the decrease in cell viability caused by mevinolin (lovastatin). However, mevalonate did not have any effect on 25-OH-cholesterol-stimulated proliferation. Finally, in media with albumin alone or in the presence of fetal bovine serum, growth factors, insulin or forskolin, 25-OH-cholesterol did not affect the expression of either c-fos mRNA or c-fos protein, as measured by real-time quantitative PCR or by Western blot, respectively. These results suggest that 25-OH-cholesterol has a dual effect on the proliferation of cultured rat astrocytes through an AP-1-independent mechanism. This could be of interest for gaining a better knowledge of the pathophysiological processes occurring in these cells.

Synergistic Effect of Glucagon-Like Peptide 2 (GLP-2) and of Key Growth Factors on the Proliferation of Cultured Rat Astrocytes. Evidence for Reciprocal Upregulation of the mRNAs for GLP-2 and IGF-I Receptors

The aim of this work was to determine whether the stimulating effect of glucagon-like peptide (GLP)-2 on astrocyte proliferation could be reinforced by proliferating substances, including growth factors such as EGF, platelet-derived growth factor, insulin-like growth factor type I (IGF-I) or a hormone such as insulin. Both DNA synthesis and astrocyte density, as well as the expression of c-Fos, Ki-67, proliferating cell nuclear antigen and glial fibrillary acidic proteins, were found to be higher in the presence of GLP-2 than in its absence. In an attempt to get a better understanding of this process, intracellular cyclic adenosine monophosphate (cAMP) production, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and the expression of GLP-2R and IGF-I receptor (IGF-IR) mRNAs were studied in response to growth factors. Our results indicate that, in the presence of different growth factors, GLP-2 does not increase cAMP production but raises ERK 1/2 phosphorylation. In addition, GLP-2R mRNA expression was increased by IGF-I, whilst mRNA expression of IGF-IR was higher in cells incubated with GLP-2 than in control cells. These results suggest for the first time that GLP-2 and several growth factors show synergistic effects on the proliferation of rat astrocytes, a process in which an enhanced expression of GLP-2R and IGF-IR may be involved, providing additional insights into the physiological role of this novel neuropeptide, specially during astroglial regeneration.

Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol‐specific phospholipase C and glycosyl phosphatidylinositol‐specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 nM insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration‐dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C‐mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin‐like growth factor (IGF)‐I and/or IGF‐II receptors, as demonstrated by affinity cross‐linking studies. Also, the effects of 5 nM IGF‐I or 5 nM IGF‐II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet‐derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl‐glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF‐I, and especially with IGF‐II, for 10 min. Despite the remarkable homology among insulin, IGF‐I, and IGF‐II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.

Insulin induces a similar reduction in the concentrations of its own receptor and of an insulin‐sensitive glycosyl‐phosphatidylinositol in isolated rat hepatocytes

We have used isolated rat hepatocytes to study whether the insulin‐induced reduction of its own receptors may modify the transduction of hormone signals by changes in the content of a glycosyl‐phosphatidylinositol. Both subsequent insulin binding and glycosyl‐phosphatidylinositol concentrations markedly decreased as a function of time and insulin concentration during preincubation of hepatocytes with insulin. The modifications observed in insulin binding were due to changes in receptor concentration. These results show that insulin regulates both the number of its own receptors and glycosyl‐phosphatidylinositol concentrations in target cells, which may be of interest in many pathophysiological situations.

Insulin Does Not Induce the Hydrolysis of a Glycosyl Phosphatidylinositol in Rat Fetal Hepatocytes

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.

Isolation of a glycosylphosphatidylinositol (GPI) from rat brain

Brain lipids were labelled with [1-14C]-isethionyl acetimidate and purified by sequential thin layer chromatography. Four labelled peaks were obtained, the first ones migrating with the same Rf as glycosyl-phosphatidylinositol (GPI). Further proof of the isolation of GPI was obtained by the observations that 44.8% of the radioactivity associated with the lipid in peak I was converted to the water phase by the effect of a PI-specific phospholipase C, and that the soluble material so obtained produced a dose-dependent inhibition of cAMP-dependent protein kinase activity. These findings indicate a biological equivalence between GPI and its polar head group from rat brain and those described in other cell types, and are consistent with the proposed role of these molecules in cellular signalling.

Free cholesterol transfer from human lower-density lipoproteins (d < 1.063) to lipoprotein-deficient serum and high-density lipoproteins

Abstract The in vitro transfer of free cholesterol (FC) between human serum lipoproteins in the absence of lecithin: cholesterol acyltransferase (LCAT) activity has been examined. The results show that the amount of FC that the high-density lipoprotein (HDL) and lipoprotein-deficient serum (LDS) fractions were able to capture from low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) was proportional to the amount of FC present in d