Final Report Summary - THE DIABETIC BRAIN (The diabetic brain)
Diabetes mellitus is a widespread disease that has become one of the main health problems worldwide. There were an estimated 171 million people affected in 2000 in the world, and that number is expected to increase to 366 million by 2030 (Wild et al., 2004). Of the different types of diabetes, type 2 is by far the most common, accounting for more than 90 % of the cases worldwide (Zimmet et al., 2001). It is not known how the body becomes resistant to insulin; importantly, however, insulin resistance is often seen as part of other metabolic imbalances, such as obesity, dyslipidaemia, and hypertension (Alberti et al., 2006). In fact, most diabetics are also obese, which suggests that an imbalance between energy intake, expenditure, and storage can lead to weight gain and eventually to the development of insulin resistance (Levin, 2006; Schwartz and Porte, 2005). Thus, to gain insight into the origins of diabetes it is essential to understand how the body senses its metabolic status and how it integrates that information to effectively achieve energy homeostasis.
Objective 1.2.1
Glucose-sensing mechanisms in glucose-excited neurones of the lateral hypothalamus
In the hypothalamus, specific groups of neurones react to physiological changes in extracellular glucose, but the mechanisms used to sense glucose are different across different neuronal phenotypes. Here, we investigated the mechanisms allowing cells of the lateral hypothalamus that express melanin-concentrating hormone (MCH) to detect variations in glucose. To identify MCH-expressing cells, a transgenic mouse that expresses green fluorescent protein in MCH cells was used as well as in vitro experiments using electrophysiological and pharmacological tools.
A class of drugs (thiazolidinediones, (TZD)) that are widely used in the treatment of type 2 diabetes for their insulin-sensitising properties induce weight gain as a side effect. This has been shown to result from neuronal actions of TZDs, in particular its effects on the Paraventricular nucleus (PVN) of the hypothalamus (Lu et al., 2011; Ryan et al., 2011), an area which receives inputs from nutrient-sensing cells from other hypothalamic areas (including the lateral hypothalamus), and its output modulates peripheral metabolism. To increase our understanding of the link between obesity, diabetes and hypothalamic physiology we looked for direct, acute effects of the TZD rosiglitazone in cells of the PVN in acute brain slices from rats. PVN neurones were located and directly targeted with a glass electrode to record their electrical activity in the whole-cell configuration, as described (González et al., 2008). Healthy recordings were with spike peak above 20 mV and series resistance below 20 mOhm. Cells were tested for their response to rosiglitazone applied in the bath (n = 14, 20 µM). Different PVN cells show different electrical properties that can be readily assessed during electrophysiological recordings, and these properties relate to the phenotype of the various neurones in the PVN (Hoffman et al., 1991). For example, so-called type I neurones are thought to be neuroendocrine magnocellular cells, whereas type II neurones belong to the parvocellular division of the PVN (Luther and Tasker, 2000). With the cells grouped according to their electrical patterns it was found that 8 (of 10) type II and 2 (of 4) type I cells responded to rosiglitazone, which suggests that type II (putative parvocellular) cells are more susceptible than type II (putative magnocellular) neurones to the effects of the drug. In addition to these, five PVN cells were also tested for their response to rosiglitazone while kept in tetrodotoxin to inhibit action potential-dependent synaptic transmission. These cells all showed a small but slightly significant decrease in membrane potential upon application of rosiglitazone. This indicated that direct effects of rosiglitazone were inhibitory, whereas indirect effects may be either excitatory or inhibitory.
In addition to the experiments summarised, an in silico model has also been implemented with a view to making use of additional tools to complement experimental data. For this, a single-compartmental model of a hypothalamic cell (Roper et al. 2003) was implemented using NEURON simulation environment (see http://www.neuron.yale.edu/neuron/ online for further details). The model was capable of reproducing the main biophysical properties of the cells recorded in vitro.
Objective 1.2.2
Cellular bioenergetics and glucose-sensing in MCH neurones of the lateral hypothalamus
In most glucose-sensing cells glucose is taken up and metabolised, and the products of this metabolism are the effector molecules that trigger the corresponding cellular responses. By contrast, few glucose-sensing neurones do not require glucose metabolism and / or uptake to respond to changes in the concentration of the carbohydrate. The cellular mechanisms used by MCH neurones to detect variations in glucose were investigated using fluorescence microscopy imaging in acute slice preparations from MCH-green fluorescent protein (GFP) mice. Confocal fluorescence microscopy was used to detect alterations in cellular physiology and mitochondrial bioenergetics in response to glucose (15 mM). A specific fast dye to detect alterations in mitochondrial membrane potential, TMRM, an indicator of altered substrate supply and mitochondrial respiration, was used. In a separate approach, the fellow has furthermore established organotypic hypothalamic slice cultures from MCH-GFP expressing mice. MCH-GFP positive neurons in the hypothalamus were found to take up significant amounts of TMRM in response to 15 mM glucose, indicating a coupling of energy metabolism and glucose uptake.
Objective 1.2.3
Effects of a high-fat diet on glucose-sensing neurones of the lateral hypothalamus
Both glucose-excited and glucose-inhibited neurones in the hypothalamus contribute to keep energy balance in order, and disrupting either group of cells may lead to disorders of metabolism. Moreover, changes in diet have a direct impact in some neurones of the hypothalamus linked to regulation of metabolism, e.g. a high fat diet triggered apoptosis in some nutrient-sensitive cells of the arcuate nucleus in the rat hypothalamus which resulted in diabetes (Moraes et al., PLoS One 4:e5045, 2009). Apoptosis is triggered by permeabilisation of the mitochondrial membrane, leading to caspase activation. Caspases cleave multiple cellular substrates leading to nucleases activation and deoxyribonucleic acid (DNA) cleavage. The techniques used included stereotactic neuron counts, detection of apoptosis in MCH neurons in situ using labelling of cleaved DNA with the TUNEL technique, and detection of active caspase subunits. Db/db mice which lack the long form of the leptin receptor were used. db/db mice are obese, hyperglycaemic and non-fertile, and they reach blood glucose concentrations of at least 200 mg / dL at 8 weeks of age (Hummel et al. 1966). For controls, db heterozygotes (db / lean) were used. Mice were fed high-fat (60 cal% fat, Research Diets Inc.,) or control diet (18 cal% fat, Teklad,) for 4, 8 and 12 weeks and the number of MCH and orexin (ORX)-positive neurons in the lateral hypothalamus was compared. In none of the timepoints did the amount of MCH or ORX-positive neurons differ from those found in the control group. Further studies on evaluating apoptosis were therefore not necessary to be performed. The amount of MCH-positive cells differed over time (p < 0.01 between week 4 and 8, n = 10, p < 0.01 between week 8 and 12, n = 10) but the same difference in cell numbers were observed in both control and high-fat fed group.
In conclusion, electrophysiological experiments showed that rosiglitazone has acute effects in the PVN, and that these effects are both direct (inhibitory only) and indirect (excitatory and inhibitory). The complex nature of these responses suggests a complex interaction of metabolism-related signals in the hypothalamus. More experiments will be required to offer a better perspective of these interactions and to explain the ionic mechanisms involved. Furthermore, the fellow has acquired novel techniques through the establishment of organotypic hypothalamic slice cultures and confocal image analysis, using MCH-GFP expressing, transgenic mice. In vivo, a high-fat diet did not change the constitution of MCH and ORX-positive cells in the neurones of the lateral hypothalamus, indicating that there is no plasticity in terms of cell number adjustments during obese and (pre-)diabetic states in the models investigated as originally hypothesised.
References
Wild S., Roglic G., Green A., Sicree R. and King H. (2004). Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27, 1047-1053.
Zimmet P., Alberti K. G. and Shaw J. (2001). Global and societal implications of the diabetes epidemic. Nature 414, 782-787
Alberti K. G. M. M., Zimmet P. and Shaw J. (2006). Metabolic syndrome-a new world-wide definition. A consensus state-ment from the International Diabetes Federation. Diabet Med 23, 469-480
Levin B. E. (2006). Metabolic sensing neurons and the control of energy homeostasis. Physiol Behav 89, 486-489
Lu, M.; Sarruf, D. A.; Talukdar, S.; Sharma, S.; Li, P.; Bandyopadhyay, G.; Nalbandian, S.; Fan, W.; Gayen, J. R.; Mahata, S. K.; Webster, N. J.; Schwartz, M. W. and Olefsky, J. M. (2011), 'Brain PPAR-# promotes obesity and is required for the insulin-sensitising effect of thiazolidinediones.', Nat Med 17(5), 618-622
González, J. A.; Jensen, L. T.; Fugger, L. and Burdakov, D. (2008), 'Metabolism-independent sugar sensing in central orexin neurons.', Diabetes 57(10), 2569-2576
Luther, J. A. and Tasker, J. G. (2000), 'Voltage-gated currents distinguish parvocellular from magnocellular neurones in the rat hypothalamic paraventricular nucleus.', J Physiol 523 Pt 1, 193-209
Roper, P.; Callaway, J.; Shevchenko, T.; Teruyama, R. and Armstrong, W. (2003), 'AHP's, HAP's and DAP's: how potassium currents regulate the excitability of rat supraoptic neurones.', J Comput Neurosci 15(3), 367-389
Moraes, J. C.; Coope, A.; Morari, J.; Cintra, D. E.; Roman, E. A.; Pauli, J. R.; Romanatto, T.; Carvalheira, J. B.; Oliveira, A. L. R.; Saad, M. J. & Velloso, L. A. (2009), 'High-fat diet induces apoptosis of hypothalamic neurons.', PLoS One 4(4), e5045
Hummel K. P., Dickie M. M. and Coleman D. L. (1966). Diabetes, a new mutation in the mouse. Science 153, 1127.
Objective 1.2.1
Glucose-sensing mechanisms in glucose-excited neurones of the lateral hypothalamus
In the hypothalamus, specific groups of neurones react to physiological changes in extracellular glucose, but the mechanisms used to sense glucose are different across different neuronal phenotypes. Here, we investigated the mechanisms allowing cells of the lateral hypothalamus that express melanin-concentrating hormone (MCH) to detect variations in glucose. To identify MCH-expressing cells, a transgenic mouse that expresses green fluorescent protein in MCH cells was used as well as in vitro experiments using electrophysiological and pharmacological tools.
A class of drugs (thiazolidinediones, (TZD)) that are widely used in the treatment of type 2 diabetes for their insulin-sensitising properties induce weight gain as a side effect. This has been shown to result from neuronal actions of TZDs, in particular its effects on the Paraventricular nucleus (PVN) of the hypothalamus (Lu et al., 2011; Ryan et al., 2011), an area which receives inputs from nutrient-sensing cells from other hypothalamic areas (including the lateral hypothalamus), and its output modulates peripheral metabolism. To increase our understanding of the link between obesity, diabetes and hypothalamic physiology we looked for direct, acute effects of the TZD rosiglitazone in cells of the PVN in acute brain slices from rats. PVN neurones were located and directly targeted with a glass electrode to record their electrical activity in the whole-cell configuration, as described (González et al., 2008). Healthy recordings were with spike peak above 20 mV and series resistance below 20 mOhm. Cells were tested for their response to rosiglitazone applied in the bath (n = 14, 20 µM). Different PVN cells show different electrical properties that can be readily assessed during electrophysiological recordings, and these properties relate to the phenotype of the various neurones in the PVN (Hoffman et al., 1991). For example, so-called type I neurones are thought to be neuroendocrine magnocellular cells, whereas type II neurones belong to the parvocellular division of the PVN (Luther and Tasker, 2000). With the cells grouped according to their electrical patterns it was found that 8 (of 10) type II and 2 (of 4) type I cells responded to rosiglitazone, which suggests that type II (putative parvocellular) cells are more susceptible than type II (putative magnocellular) neurones to the effects of the drug. In addition to these, five PVN cells were also tested for their response to rosiglitazone while kept in tetrodotoxin to inhibit action potential-dependent synaptic transmission. These cells all showed a small but slightly significant decrease in membrane potential upon application of rosiglitazone. This indicated that direct effects of rosiglitazone were inhibitory, whereas indirect effects may be either excitatory or inhibitory.
In addition to the experiments summarised, an in silico model has also been implemented with a view to making use of additional tools to complement experimental data. For this, a single-compartmental model of a hypothalamic cell (Roper et al. 2003) was implemented using NEURON simulation environment (see http://www.neuron.yale.edu/neuron/ online for further details). The model was capable of reproducing the main biophysical properties of the cells recorded in vitro.
Objective 1.2.2
Cellular bioenergetics and glucose-sensing in MCH neurones of the lateral hypothalamus
In most glucose-sensing cells glucose is taken up and metabolised, and the products of this metabolism are the effector molecules that trigger the corresponding cellular responses. By contrast, few glucose-sensing neurones do not require glucose metabolism and / or uptake to respond to changes in the concentration of the carbohydrate. The cellular mechanisms used by MCH neurones to detect variations in glucose were investigated using fluorescence microscopy imaging in acute slice preparations from MCH-green fluorescent protein (GFP) mice. Confocal fluorescence microscopy was used to detect alterations in cellular physiology and mitochondrial bioenergetics in response to glucose (15 mM). A specific fast dye to detect alterations in mitochondrial membrane potential, TMRM, an indicator of altered substrate supply and mitochondrial respiration, was used. In a separate approach, the fellow has furthermore established organotypic hypothalamic slice cultures from MCH-GFP expressing mice. MCH-GFP positive neurons in the hypothalamus were found to take up significant amounts of TMRM in response to 15 mM glucose, indicating a coupling of energy metabolism and glucose uptake.
Objective 1.2.3
Effects of a high-fat diet on glucose-sensing neurones of the lateral hypothalamus
Both glucose-excited and glucose-inhibited neurones in the hypothalamus contribute to keep energy balance in order, and disrupting either group of cells may lead to disorders of metabolism. Moreover, changes in diet have a direct impact in some neurones of the hypothalamus linked to regulation of metabolism, e.g. a high fat diet triggered apoptosis in some nutrient-sensitive cells of the arcuate nucleus in the rat hypothalamus which resulted in diabetes (Moraes et al., PLoS One 4:e5045, 2009). Apoptosis is triggered by permeabilisation of the mitochondrial membrane, leading to caspase activation. Caspases cleave multiple cellular substrates leading to nucleases activation and deoxyribonucleic acid (DNA) cleavage. The techniques used included stereotactic neuron counts, detection of apoptosis in MCH neurons in situ using labelling of cleaved DNA with the TUNEL technique, and detection of active caspase subunits. Db/db mice which lack the long form of the leptin receptor were used. db/db mice are obese, hyperglycaemic and non-fertile, and they reach blood glucose concentrations of at least 200 mg / dL at 8 weeks of age (Hummel et al. 1966). For controls, db heterozygotes (db / lean) were used. Mice were fed high-fat (60 cal% fat, Research Diets Inc.,) or control diet (18 cal% fat, Teklad,) for 4, 8 and 12 weeks and the number of MCH and orexin (ORX)-positive neurons in the lateral hypothalamus was compared. In none of the timepoints did the amount of MCH or ORX-positive neurons differ from those found in the control group. Further studies on evaluating apoptosis were therefore not necessary to be performed. The amount of MCH-positive cells differed over time (p < 0.01 between week 4 and 8, n = 10, p < 0.01 between week 8 and 12, n = 10) but the same difference in cell numbers were observed in both control and high-fat fed group.
In conclusion, electrophysiological experiments showed that rosiglitazone has acute effects in the PVN, and that these effects are both direct (inhibitory only) and indirect (excitatory and inhibitory). The complex nature of these responses suggests a complex interaction of metabolism-related signals in the hypothalamus. More experiments will be required to offer a better perspective of these interactions and to explain the ionic mechanisms involved. Furthermore, the fellow has acquired novel techniques through the establishment of organotypic hypothalamic slice cultures and confocal image analysis, using MCH-GFP expressing, transgenic mice. In vivo, a high-fat diet did not change the constitution of MCH and ORX-positive cells in the neurones of the lateral hypothalamus, indicating that there is no plasticity in terms of cell number adjustments during obese and (pre-)diabetic states in the models investigated as originally hypothesised.
References
Wild S., Roglic G., Green A., Sicree R. and King H. (2004). Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27, 1047-1053.
Zimmet P., Alberti K. G. and Shaw J. (2001). Global and societal implications of the diabetes epidemic. Nature 414, 782-787
Alberti K. G. M. M., Zimmet P. and Shaw J. (2006). Metabolic syndrome-a new world-wide definition. A consensus state-ment from the International Diabetes Federation. Diabet Med 23, 469-480
Levin B. E. (2006). Metabolic sensing neurons and the control of energy homeostasis. Physiol Behav 89, 486-489
Lu, M.; Sarruf, D. A.; Talukdar, S.; Sharma, S.; Li, P.; Bandyopadhyay, G.; Nalbandian, S.; Fan, W.; Gayen, J. R.; Mahata, S. K.; Webster, N. J.; Schwartz, M. W. and Olefsky, J. M. (2011), 'Brain PPAR-# promotes obesity and is required for the insulin-sensitising effect of thiazolidinediones.', Nat Med 17(5), 618-622
González, J. A.; Jensen, L. T.; Fugger, L. and Burdakov, D. (2008), 'Metabolism-independent sugar sensing in central orexin neurons.', Diabetes 57(10), 2569-2576
Luther, J. A. and Tasker, J. G. (2000), 'Voltage-gated currents distinguish parvocellular from magnocellular neurones in the rat hypothalamic paraventricular nucleus.', J Physiol 523 Pt 1, 193-209
Roper, P.; Callaway, J.; Shevchenko, T.; Teruyama, R. and Armstrong, W. (2003), 'AHP's, HAP's and DAP's: how potassium currents regulate the excitability of rat supraoptic neurones.', J Comput Neurosci 15(3), 367-389
Moraes, J. C.; Coope, A.; Morari, J.; Cintra, D. E.; Roman, E. A.; Pauli, J. R.; Romanatto, T.; Carvalheira, J. B.; Oliveira, A. L. R.; Saad, M. J. & Velloso, L. A. (2009), 'High-fat diet induces apoptosis of hypothalamic neurons.', PLoS One 4(4), e5045
Hummel K. P., Dickie M. M. and Coleman D. L. (1966). Diabetes, a new mutation in the mouse. Science 153, 1127.