Carlos Larqué

Early endocrine and molecular changes in metabolic syndrome models

The twenty‐first century arrived in the middle of a global epidemic of metabolic syndrome (MS) and type 2 diabetes mellitus (DM2). It is generally accepted that an excess of nutrients linked to a low physical activity triggers the problem. However, the molecular features that interact to develop the MS are not clear. In an effort to understand and control them, they have been extensively studied, but this goal has not been achieved yet. Nonhuman animal models have been used to explore diet and genetic factors in which experimental conditions are controlled. For example, only one factor in the diet, such as fats or carbohydrates can be modified to better understand a single change that would be impossible in humans. Most of the studies have been done in rodents. However, it is difficult to directly compare them, because experiments are different in more than one variable; genetic strains, amount, and the type of fat used in the diet and sex. Thus, the only possible criteria of comparison are the relevance of the observed changes. We review different animal models and add some original observations on short‐term changes in metabolism and beta cells in our own model of adult Wistar rats that are not especially prone to get fat or develop DM2, treated with 20% sucrose in drinking water. One early change observed in pancreatic beta cells is the increase in GLUT2 expression that is located to the membrane of the cells. This change could partially explain the presence of insulin hypersecretion and hyperinsulinemia in these rats. Understanding early changes that lead to MS and in time to pancreatic islet exhaustion is an important biomedical problem that may contribute to learn how to prevent or even reverse MS, before developing DM2.

Modulation of ionic channels and insulin secretion by drugs and hormones in pancreatic beta cells

Pancreatic beta cells, unique cells that secrete insulin in response to an increase in glucose levels, play a significant role in glucose homeostasis. Glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells has been extensively explored. In this mechanism, glucose enters the cells and subsequently the metabolic cycle. During this process, the ATP/ADP ratio increases, leading to ATP-sensitive potassium (KATP) channel closure, which initiates depolarization that is also dependent on the activity of TRP nonselective ion channels. Depolarization leads to the opening of voltage-gated Na+ channels (Nav) and subsequently voltage-dependent Ca2+ channels (Cav). The increase in intracellular Ca2+ triggers the exocytosis of insulin-containing vesicles. Thus, electrical activity of pancreatic beta cells plays a central role in GSIS. Moreover, many growth factors, incretins, neurotransmitters, and hormones can modulate GSIS, and the channels that participate in GSIS are highly regulated. In this review, we focus on the principal ionic channels (KATP, Nav, and Cav channels) involved in GSIS and how classic and new proteins, hormones, and drugs regulate it. Moreover, we also discuss advances on how metabolic disorders such as metabolic syndrome and diabetes mellitus change channel activity leading to changes in insulin secretion.

Metabolic syndrome induces changes in KATP-channels and calcium currents in pancreatic β-cells

Metabolic syndrome (MS) can be defined as a group of signs that increases the risk of developing type 2 diabetes mellitus (DM2). These signs include obesity, hyperinsulinemia and insulin resistance. We are interested in the mechanisms that trigger hyperinsulinemia as a step to understand how β cells fail in DM2. Pancreatic β cells secrete insulin in response to glucose variations in the extracellular medium. When they are chronically over-stimulated, hyperinsulinemia is observed; but then, with time, they become incapable of maintaining normal glucose levels, giving rise to DM2. A chronic high sucrose diet for two months induces MS in adult male Wistar rats. In the present article, we analyzed the effect of the internal environment of rats with MS, on the activity of ATP-sensitive potassium channels (KATP) and calcium currents of pancreatic β cells. After 24 weeks of treatment with 20% sucrose in their drinking water, rats showed central obesity, hyperinsulinemia and insulin resistance, and their systolic blood pressure and triglycerides plasma levels increased. These signs indicate the onset of MS. KATP channels in isolated patches of β cells from MS rats, had an increased sensitivity to ATP with respect to controls. Moreover, the macroscopic calcium currents, show increased variability compared with cells from control individuals. These results demonstrate that regardless of genetic background, a high sucrose diet leads to the development of MS. The observed changes in ionic channels can partially explain the increase in insulin secretion in MS rats. However, some β cells showed smaller calcium currents. These cells may represent a β cell subpopulation as it becomes exhausted by the long-term high sucrose diet.

Metabolic syndrome and ionic channels in pancreatic beta cells.

Worldwide increase in the prevalence of metabolic syndrome and diabetes mellitus type 2 (DM2) during the past decades has converted them into a global epidemic disease. It is not well understood how these metabolic disorders initiate, but an increase in food consumption associated to low physical activity leads to increase in body weight and obesity. This in turn, elevates circulating lipids and cytokines release by adipose tissue, give the organism a chronic inflammation and potentiate insulin secretion, causing insulin resistance. Depending on genetics and probably other environmental factors, after a long period of hyperactivity, pancreatic beta cells become exhausted and DM2 overcomes. Pancreatic beta cells are the only source of insulin known in mammals. They are unique because of their ability to sense and transform fuels into a chemical signal, which affects mainly all the cells in the organism. Many other factors affect insulin secretion. We will focus on the alterations of glucose-induced insulin secretion coupling, particularly in ionic channels that have crucial importance in this process. Different channel types can be affected by metabolic syndrome. The most studied are K(ATP) and other potassium channels, calcium, sodium, and TRP channels. Much information comes from rodents that do not express exactly the same proportion and type of channels than humans. However, getting insight of how do they participate in insulin secretion and how to modulate them is important to completely understand beta-cell physiology and pathophysiological reactions to metabolic syndrome and diabetes, in order to stop the epidemic of these metabolic disorders.

Increased anxiety-like behavior is associated with the metabolic syndrome in non-stressed rats

Metabolic syndrome (MS) is a cluster of signs that increases the risk to develop diabetes mellitus type 2 and cardiovascular disease. In the last years, a growing interest to study the relationship between MS and psychiatric disorders, such as depression and anxiety, has emerged obtaining conflicting results. Diet-induced MS rat models have only examined the effects of high-fat or mixed cafeteria diets to a limited extent. We explored whether an anxiety-like behavior was associated with MS in non-stressed rats chronically submitted to a high-sucrose diet (20% sucrose in drinking water) using three different anxiety paradigms: the shock-probe/burying test (SPBT), the elevated plus-maze (EPM) and the open-field test (OFT). Behaviorally, the high-sucrose diet group showed an increase in burying behavior in the SPBT. Also, these animals displayed both avoidance to explore the central part of the arena and a significant increase in freezing behavior in the OFT and lack of effects in the EPM. Also, high-sucrose diet group showed signs of an MS-like condition: significant increases in body weight and body mass index, abdominal obesity, hypertension, hyperglycemia, hyperinsulinemia, and dyslipidemia. Plasma leptin and resistin levels were also increased. No changes in plasma corticosterone levels were found. These results indicate that rats under a 24-weeks high-sucrose diet develop an MS associated with an anxiety-like behavior. Although the mechanisms underlying this behavioral outcome remain to be investigated, the role of leptin is emphasized.

Transcriptome landmarks of the functional maturity of rat beta-cells, from lactation to adulthood.

Research on the postnatal development of pancreatic beta-cells has become an important subject in recent years. Understanding the mechanisms that govern beta-cell postnatal maturation could bring new opportunities to therapeutic approaches for diabetes. The weaning period consists of a critical postnatal window for structural and physiologic maturation of rat beta-cells. To investigate transcriptome changes involved in the maturation of beta-cells neighboring this period, we performed microarray analysis in fluorescence-activated cell-sorted (FACS) beta-cell-enriched populations. Our results showed a variety of gene sets including those involved in the integration of metabolism, modulation of electrical activity, and regulation of the cell cycle that play important roles in the maturation process. These observations were validated using reverse hemolytic plaque assay, electrophysiological recordings, and flow cytometry analysis. Moreover, we suggest some unexplored pathways such as sphingolipid metabolism, insulin-vesicle trafficking, regulation of transcription/transduction by miRNA-30, trafficking proteins, and cell cycle proteins that could play important roles in the process mentioned above for further investigation.

Rat Pancreatic Beta-Cell Culture

In this chapter, we describe the methods used to culture mainly rat pancreatic beta cells. We consider necessary to use this approach to get more information about physiological, biophysical, and molecular biology characteristics of primary beta cells. Most of the literature published has been developed in murine and human beta-cell lines. However, there are many differences between tumoral cell lines and native cells because, in contrast to cell lines, primary cells do not divide. Moreover, cell lines can be in various stages of the cell cycle and thus have a different sensitivity to glucose, compared to primary cells. Finally, for these reasons, cell lines can be heterogeneous, as the primary cells are. The main problem in using primary beta cells is that despite that they are a majority within a culture they appear mixed with other kinds of pancreatic islet cells. If one needs to identify single cells or has an only beta-cell composition, it is necessary to process the sample further. For example, one may obtain an enriched population of beta cells using fluorescence-activated cell sorting or identify single cells with the reverse hemolytic plaque assay. The other problem is that cells change with time in culture, becoming old and losing some characteristics, and so must be used preferentially during the first week. The development of human beta-cell cultures is of importance in medicine because we hope one day to be able to transplant viable beta cells to patients with diabetes mellitus type 1.

Changes in Insulin Secretion, KATP and Ba Currents in Rat Pancreatic Beta Cells from Rats with Metabolic Syndrome, Induced by a High Sucrose Diet

Metabolic syndrome (MS) increases the probability toh develop type 2 diabetes mellitus. We studied the effect of a high-sucrose diet on pancreatic beta cells physiology. Wistar adult male rats were fed with a 20 % sucrose solution in drinking water or control, which received plain water, for 24 weeks. After treatment, compared to controls, treated rats group increased body weight by 22%, due to peripancreatic and epididimal adipose tissue that increased by 135 and 154 %, respectively. Tey were also hyperglycemic, hyperinsulinemic and hypertriglyceridemic, without changes in plasmatic cholesterol. We concluded that this group developed metabolic syndrome (MS).We recorded the activity of ATP-sensitive potassium channels (KATP) in beta cells. Channel conductance was 50 ± 0.1 and 51 ± 7 pS for control and MS, respectively; however, the Kd for ATP was 10.1 ± 0.9 μM for MS cells, while the control was 19.3 ± 0.001 μM, indicating that KATP channels in MS rats were more sensitive to ATP.Beta cells from MS showed three behavior modes in whole cell-barium current (IBa). The peak current density in 50 % of the cells decreased by 40 %, while in 35 % of the cells, IBa increased by 90 %, compared to controls. In 15 % of the cells no IBa was recorded.These results indicate that a high sucrose diet induced MS and modified beta cell functions, leading to ATP hypersensitivity and altered IBa, and insulin secretion.Supported by Instituto de Ciencia y Tecnologia del Distrito Federal PICDS08-72.