**3.2 Regulation of carbohydrate selection by AMPK in the hypothalamus**

## *3.2.1 Fasting increases carbohydrate preference in mice*

Preferential consumption of high-fat foods among multiple palatable diets has increased worldwide and given rise to a high prevalence of metabolic syndrome, coronary heart disease, diabetes, and cancer [52]. The availability of highly palatable diets such as high-fat and high-sucrose diets promotes overfeeding in humans, with social stress often inducing "carbohydrate craving" [53, 54]. Although macronutrient components are associated with cardiometabolic health, aging, and longevity [55], the mechanisms for macronutrient intake have remained elusive.

The consumption of protein and essential amino acids is tightly controlled in animals. Previous studies revealed that forced feeding of animals with a low-protein diet increases total calorie intake, because of compensatory feeding for essential

#### **Figure 7.**

*Role of AMPK in leptin-induced suppression of feeding and activation of fatty acid oxidation in skeletal muscle. Leptin increases fatty acid oxidation in skeletal muscle through activation of AMPK both directly via the leptin receptor expressed in this tissue as well as indirectly through the hypothalamus-sympathetic nervous system. Leptin also inhibits food intake through suppression of AMPK in the hypothalamus.*

amino acid intake [56]. In contrast, rodents reject diets that lack even a single essential amino acid [57]. They sense such a deficiency within the first hour of feeding [58–60], and this sensing of essential amino acids is independent of taste and smell [59, 61]. Although the regulation of essential amino acid intake had been thought to involve the action of the kinase GCN2 (general control nonderepressible 2) in the piriform cortex [58, 60], the mechanism remains unclear, given that GCN2 knockout mice were recently found to be still capable of sensing a deficiency of essential amino acids [62].

Selection of an HCD increases under certain physiological and pathological conditions [10, 63, 64]. In humans, carbohydrate craving is often induced by stressful life events and mood disturbances [53, 54]. Rodents increase selection of an HCD and reduce that of an HFD during 24-h refeeding after fasting [10]. Injection of NPY or dynorphin A into the brain of mice increases selection of carbohydrate and fat, respectively [64, 65]. Furthermore, pharmacological agents that induce glucoprivic cues promote carbohydrate intake [66]. Feeding with a low-carbohydrate diet also results in an increase in total calorie consumption as a compensatory response to maintain carbohydrate intake [56]. These observations suggest that animals including rodents as well as humans have a "carbohydrate-specific appetite" that increases carbohydrate intake over a basally preferable diet such as an HFD.

The regulation of macronutrient intake is relatively weaker than that of water intake in water-deprived animals [67]. Intake of carbohydrate versus fat is strongly influenced by the basal preference of animals and other factors [56]. Indeed, most natural sources of carbohydrate have little sweet taste, and most rodents choose a basally preferred HFD over an HCD. Therefore, macronutrient selection is affected by feeding paradigms. The presentation of a single diet (single-diet approach) often leads to the incorrect conclusions in studies of macronutrient selection [9]. This disadvantage is particularly important if the amount of a specific nutrient in the diet is suboptimal. The single-diet approach revealed that 24-h fasting increased intake of an HFD (and of total calories) to a greater extent than that of an HCD [10]. In contrast, when mice were presented with both a highly palatable HFD and HCD simultaneously in the two-diet choice approach during refeeding after a 24-h fast, they reduced their intake of the HFD and increased that of the HCD [10]. Total calorie intake did not change between that in the two-diet choice and single-diet approaches.

The increase in HFD and total calorie intake that was apparent during refeeding after fasting with the single-diet approach is likely due to a compensatory response to increase carbohydrate intake. We found that refeeding with the HCD alone after fasting rapidly decreased the plasma concentration of ketone bodies than that with the HFD alone [10]. Furthermore, when the fasted mice were pair-fed with the same number of calories of the HFD as they consumed when presented with the HCD, the plasma level of ketone bodies did not decrease. Plasma ketone body levels were then negatively correlated with the amount of carbohydrate intake in this experiment [10]. These results suggested that mice increase carbohydrate intake in the two-diet choice approach during refeeding after fasting as a means to rapidly improve whole body metabolism [68].

### *3.2.2 AMPK-regulated CRH neurons control carbohydrate selection*

We recently showed that specific hypothalamic neurons increase selection of carbohydrate over fat in mice [10]. We thus found that a subset of CRH-positive neurons in the PVH that are regulated by AMPK regulate the selection of carbohydrate over a basally preferred HFD during refeeding for 24 h after food deprivation (**Figure 8**).

**13**

as an HFD.

**Figure 8.**

*levels.*

*Neural Control of Homeostatic Feeding and Food Selection*

is likely associated with carbohydrate feeding.

As mentioned above (Section 3.1), AMPK in the ARC plays an important role in food intake. However, AMPK activity in the PVH was also found to change during fasting and refeeding in mice [7, 10]. The activation of AMPK in the PVH was suppressed by refeeding with lab chow or an HCD for 3 h. In contrast, suppression of AMPK activity in the PVH was small after refeeding with an HFD. Immunohistofluorescence analysis showed that 24 h-fasting increased the level of AMPK phosphorylation preferentially in CRH neurons present in the rostral region of the PVH, and it decreased after refeeding with lab chow or an HCD for 3 h [10]. These results suggested that AMPK activity in a subset of CRH neurons in the PVH

*AMPK-regulated CRH neurons constitute a subpopulation of CRH neurons in the PVH that increases selection of an HCD over an HFD in a manner dependent on CPT1c. AMPK-regulated CRH neurons in the PVH are preferentially activated by fasting in a manner dependent on the AMPK-CPT1c axis. Activation of these neurons is sufficient and necessary for fasting-induced selection of an HCD over an HFD. The activated AMPK phosphorylates and thereby inhibits the activity of ACC, resulting in a reduction in the amount of malonyl-CoA and consequent increase in CPT1c activity. CPT1c, which is expressed in the ER and mitochondria, mediates an increase in the intracellular free Ca2+ concentration that results in neuronal activation and thereby promotes carbohydrate selection. Carbohydrate feeding after fasting results in a lowering of plasma ketone body* 

To examine the role of AMPK in PVH neurons in the regulation of food intake, we expressed an active (CA) form of the kinase [69] in PVH neurons of C57BL/6 J mice with the use of a lentivirus containing the synapsin gene promoter [10]. When the CA-AMPK mice were fed on lab chow, they increased body weight as a result of increased food intake. In contrast, when CA-AMPK mice were fed an HFD, they did not show hyperphagia or develop obesity. We performed a two-diet choice experiment with an HCD and an HFD that contained equal amounts of protein, micronutrients, and other constituents [10]. CA-AMPK mice chose the HCD, whereas control mice chose the HFD, with total calorie intake being similar for both groups of mice. Similar results were obtained with different combinations of diets derived from different nutrient sources. These observations suggested that AMPK in PVH neurons increases selection of an HCD over a basally preferable diet, such

We examined whether activation of AMPK in the PVH is necessary for the fasting-induced increase in HCD selection [10]. Decrease of AMPK expression in the PVH by expression of a short hairpin RNA (shRNA) specific for AMPK with the use of an adeno-associated virus suppressed the fasting-induced increase in

*DOI: http://dx.doi.org/10.5772/intechopen.93413*

*Neural Control of Homeostatic Feeding and Food Selection DOI: http://dx.doi.org/10.5772/intechopen.93413*

#### **Figure 8.**

*New Insights into Metabolic Syndrome*

amino acids [62].

amino acid intake [56]. In contrast, rodents reject diets that lack even a single essential amino acid [57]. They sense such a deficiency within the first hour of feeding [58–60], and this sensing of essential amino acids is independent of taste and smell [59, 61]. Although the regulation of essential amino acid intake had been thought to involve the action of the kinase GCN2 (general control nonderepressible 2) in the piriform cortex [58, 60], the mechanism remains unclear, given that GCN2 knockout mice were recently found to be still capable of sensing a deficiency of essential

Selection of an HCD increases under certain physiological and pathological conditions [10, 63, 64]. In humans, carbohydrate craving is often induced by stressful life events and mood disturbances [53, 54]. Rodents increase selection of an HCD and reduce that of an HFD during 24-h refeeding after fasting [10]. Injection of NPY or dynorphin A into the brain of mice increases selection of carbohydrate and fat, respectively [64, 65]. Furthermore, pharmacological agents that induce glucoprivic cues promote carbohydrate intake [66]. Feeding with a low-carbohydrate diet also results in an increase in total calorie consumption as a compensatory response to maintain carbohydrate intake [56]. These observations suggest that animals including rodents as well as humans have a "carbohydrate-specific appetite" that increases carbohydrate intake over a basally preferable diet such as an HFD.

The regulation of macronutrient intake is relatively weaker than that of water intake in water-deprived animals [67]. Intake of carbohydrate versus fat is strongly influenced by the basal preference of animals and other factors [56]. Indeed, most natural sources of carbohydrate have little sweet taste, and most rodents choose a basally preferred HFD over an HCD. Therefore, macronutrient selection is affected by feeding paradigms. The presentation of a single diet (single-diet approach) often leads to the incorrect conclusions in studies of macronutrient selection [9]. This disadvantage is particularly important if the amount of a specific nutrient in the diet is suboptimal. The single-diet approach revealed that 24-h fasting increased intake of an HFD (and of total calories) to a greater extent than that of an HCD [10]. In contrast, when mice were presented with both a highly palatable HFD and HCD simultaneously in the two-diet choice approach during refeeding after a 24-h fast, they reduced their intake of the HFD and increased that of the HCD [10]. Total calorie intake did not change between that in the two-diet choice and single-diet

The increase in HFD and total calorie intake that was apparent during refeeding after fasting with the single-diet approach is likely due to a compensatory response to increase carbohydrate intake. We found that refeeding with the HCD alone after fasting rapidly decreased the plasma concentration of ketone bodies than that with the HFD alone [10]. Furthermore, when the fasted mice were pair-fed with the same number of calories of the HFD as they consumed when presented with the HCD, the plasma level of ketone bodies did not decrease. Plasma ketone body levels were then negatively correlated with the amount of carbohydrate intake in this experiment [10]. These results suggested that mice increase carbohydrate intake in the two-diet choice approach during refeeding after fasting as a means to rapidly

We recently showed that specific hypothalamic neurons increase selection of carbohydrate over fat in mice [10]. We thus found that a subset of CRH-positive neurons in the PVH that are regulated by AMPK regulate the selection of carbohydrate over a basally preferred HFD during refeeding for 24 h after food deprivation

**12**

(**Figure 8**).

approaches.

improve whole body metabolism [68].

*3.2.2 AMPK-regulated CRH neurons control carbohydrate selection*

*AMPK-regulated CRH neurons constitute a subpopulation of CRH neurons in the PVH that increases selection of an HCD over an HFD in a manner dependent on CPT1c. AMPK-regulated CRH neurons in the PVH are preferentially activated by fasting in a manner dependent on the AMPK-CPT1c axis. Activation of these neurons is sufficient and necessary for fasting-induced selection of an HCD over an HFD. The activated AMPK phosphorylates and thereby inhibits the activity of ACC, resulting in a reduction in the amount of malonyl-CoA and consequent increase in CPT1c activity. CPT1c, which is expressed in the ER and mitochondria, mediates an increase in the intracellular free Ca2+ concentration that results in neuronal activation and thereby promotes carbohydrate selection. Carbohydrate feeding after fasting results in a lowering of plasma ketone body levels.*

As mentioned above (Section 3.1), AMPK in the ARC plays an important role in food intake. However, AMPK activity in the PVH was also found to change during fasting and refeeding in mice [7, 10]. The activation of AMPK in the PVH was suppressed by refeeding with lab chow or an HCD for 3 h. In contrast, suppression of AMPK activity in the PVH was small after refeeding with an HFD. Immunohistofluorescence analysis showed that 24 h-fasting increased the level of AMPK phosphorylation preferentially in CRH neurons present in the rostral region of the PVH, and it decreased after refeeding with lab chow or an HCD for 3 h [10]. These results suggested that AMPK activity in a subset of CRH neurons in the PVH is likely associated with carbohydrate feeding.

To examine the role of AMPK in PVH neurons in the regulation of food intake, we expressed an active (CA) form of the kinase [69] in PVH neurons of C57BL/6 J mice with the use of a lentivirus containing the synapsin gene promoter [10]. When the CA-AMPK mice were fed on lab chow, they increased body weight as a result of increased food intake. In contrast, when CA-AMPK mice were fed an HFD, they did not show hyperphagia or develop obesity. We performed a two-diet choice experiment with an HCD and an HFD that contained equal amounts of protein, micronutrients, and other constituents [10]. CA-AMPK mice chose the HCD, whereas control mice chose the HFD, with total calorie intake being similar for both groups of mice. Similar results were obtained with different combinations of diets derived from different nutrient sources. These observations suggested that AMPK in PVH neurons increases selection of an HCD over a basally preferable diet, such as an HFD.

We examined whether activation of AMPK in the PVH is necessary for the fasting-induced increase in HCD selection [10]. Decrease of AMPK expression in the PVH by expression of a short hairpin RNA (shRNA) specific for AMPK with the use of an adeno-associated virus suppressed the fasting-induced increase in

HCD selection and decrease in HFD selection in the two-diet choice approach. Intracerebroventricular (i.c.v.) injection of glucose, which inhibits AMPK activity in the PVH [7], also suppressed the fasting-induced increase in HCD selection without affecting total calorie intake. These results thus suggested that AMPK in the PVH is required for the fasting-induced increase in HCD selection.

We examined primarily responsible neurons in the PVH for regulation of selection between an HCD and an HFD by injecting various neuropeptides or cytokines that are expressed in the PVH into either the PVH or the lateral ventricle. Among the agents tested, injection of only CRH into the PVH increased selection of an HCD and reduced that of an HFD in the two-diet choice approach [10]. In contrast, injection of a CRH receptor 1 (corticotropin-releasing factor receptor 1, CRFR1) antagonist into the PVH suppressed the fasting-induced increase in HCD selection and decrease in HFD selection. CRFR1 is expressed at a high level in the PVH [70], and CRFR1-expressing neurons in this nucleus include glutamatergic and GABAergic neurons and do not express CRH, vasopressin, oxytocin, or TRH [70, 71]. CRFR1-expressing neurons in the PVH control brain areas that regulate food intake and sweet taste sensing [39, 68, 70, 72].

Suppression of CRH expression in the PVH with a specific shRNA inhibited the fasting-induced increase in HCD selection and decrease in HFD selection. Furthermore, it also blunted the effect of CA-AMPK expression in PVH neurons on HCD selection. Examination of the effects of activation and inhibition of CRH neurons in the PVH with the use of DREADD technology on selection between an HFD and an HCD in the two-diet choice approach also showed that these neurons are sufficient and necessary for the fasting-induced increase in carbohydrate selection [10].

Food selection has been reported to be affected by plasma corticosterone level [73]. We examined whether AMPK-regulated CRH neurons in the PVH might influence HCD selection through change in plasma corticosterone level [10]. Expression of CA-AMPK in PVH neurons did not affect plasma corticosterone levels, although it increased selection of an HCD. In contrast, activation of CRH neurons in the PVH by DREADD technology increased the plasma corticosterone level, whereas inhibition of these neurons by the same approach suppressed the fasting-induced increase in this parameter. Plasma corticosterone is thus unlikely to be a primary mediator of the change in food selection, although it might be necessary to control food intake [73]. AMPK-regulated CRH neurons in the rostral portion of the PVH appear to be distinct from the CRH neurons that regulate the hypothalamic-pituitaryadrenal axis.

CRH is known to be an anorexic neuropeptide. The i.c.v. injection of CRH thus attenuates total calorie intake in mice in the two-diet choice approach with an HCD and an HFD [10]. Activation of CRH neurons in the PVH with DREADD technology also decreased total calorie intake for the initial 3-h feeding but did not change after the 24-h feeding. In contrast, injection of CRH in the PVH did not change total calorie intake in mice in the two-diet choice approach. Thus, a subset of CRH neurons in the rostral part of the PVH that regulates CRFR1 neurons in the PVH may regulate selection of an HCD and an HFD, whereas another group of CRH neurons in this nucleus inhibits total calorie intake.

Diuretic hormone 44 (Dh44), the *Drosophila* ortholog of mammalian CRH, regulates the selection of nutritive sugars such as D-glucose, but not to that of nonnutritive sugars such as L-glucose [74]. Fasted wild-type flies initially choose the sweeter L-glucose before switching to D-glucose during refeeding in a twodiet choice paradigm. By contrast, Dh44 mutants choose the sweeter L-glucose but fail to increase the preference for D-glucose. Dh44 neurons do not regulate

**15**

*Neural Control of Homeostatic Feeding and Food Selection*

total consumption of food. Thus, CRH-dependent carbohydrate selection is likely

We next examined whether AMPK and its downstream target CPT1c, the neuronal isoform of CPT1, in CRH neurons of the PVH regulate selection of an HCD versus an HFD in the two-diet choice approach [10]. Expression of CA-AMPK specifically in CRH neurons of the PVH was sufficient to increase selection of an HCD and reduce that of an HFD. In contrast, expression of shRNA specific for AMPK or for CPT1c in these neurons attenuated the fasting-induced increase in HCD selection. Expression of an shRNA specific for AMPK in CRH neurons also inhibited the increase in HCD selection induced by expression of CA-AMPK in the PVH. Furthermore, expression of an AMPK shRNA in these neurons also resulted in downregulation of the amount of CPT1c mRNA but not that of CPT1a mRNA in the PVH. AMPK had been shown to regulate the abundance of CPT1c mRNA [75]. These findings suggested that the AMPK-CPT1c axis in CRH neurons of the PVH is necessary for the fasting-induced increase in the selection of an HCD over an HFD

CPT1c is localized to both the endoplasmic reticulum (ER) [76] and mitochondria [77], both of which contribute to intracellular Ca2+ signaling in a cooperative manner [78]. We found that the AMPK activator AICAR (5-aminoimidazole-4 carboxamide-1-β-D-ribofuranoside) increased the intracellular Ca2+ concentration in CRH neurons isolated from the PVH [10], and this effect was attenuated by the CPT1 inhibitor etomoxir. The shRNA-mediated depletion of AMPK or CPT1c in these neurons also blocked the Ca2+ response to AICAR, indicating that activation of the AMPK-CPT1c axis increases the intracellular Ca2+ concentration in CRH neurons of the PVH. These results suggested that activation of the AMPK-CPT1c system leads to increase synaptic activity in these neurons and in HCD selection by triggering an increase in intracellular Ca2+ concentration in CRH neurons. Activation of AMPK in these neurons inhibits ACC, decreases the abundance of malonyl-CoA and activates CPT1c. It also increases expression of the CPT1c gene

AMPK activity is regulated by the glycolytic pathway as well as by cellular energy level (Section 3.1) [4, 5, 44]. Increased glucose levels in the brain inhibit AMPK activity in the PVH and ARC [7]. The i.c.v. injection of glucose in mice also inhibited the fasting-induced selection of an HCD in the two-diet choice approach [10]. The glucose-induced inhibition of AMPK may result in increased ACC activity, an increase in the amount of malonyl-CoA, and consequent inhibition of CPT1c activity in AMPK-regulated CRH neurons. The AMPK-CPT1c system may thus act

Homeostatic regulation of feeding is essential for maintenance of total energy balance and whole-body metabolism. NPY/AgRP neurons and POMC neurons in the ARC are the most important neurons in the homeostatic regulation of feeding. New technologies have revealed that the activity of NPY/AgRP neurons changes in response to feeding and fasting. Of note, the activity of these neurons declines rapidly after the onset and before the completion of feeding behavior. Their activity also changes in response to the smell or anticipation of food. The neuronal activity thus appears to be correlated with food values. The evidence suggests that NPY/ AgRP neurons are regulated by anticipatory stimuli related to food reward as well as

as a glucose sensor in AMPK-regulated CRH neurons of the PVH.

*DOI: http://dx.doi.org/10.5772/intechopen.93413*

conserved from insects to rodents.

(**Figure 8**).

(**Figure 8**).

**4. Concluding remarks**

by energy and nutrient levels in the body.

*New Insights into Metabolic Syndrome*

HCD selection and decrease in HFD selection in the two-diet choice approach. Intracerebroventricular (i.c.v.) injection of glucose, which inhibits AMPK activity in the PVH [7], also suppressed the fasting-induced increase in HCD selection without affecting total calorie intake. These results thus suggested that AMPK in the PVH is

We examined primarily responsible neurons in the PVH for regulation of selection between an HCD and an HFD by injecting various neuropeptides or cytokines that are expressed in the PVH into either the PVH or the lateral ventricle. Among the agents tested, injection of only CRH into the PVH increased selection of an HCD and reduced that of an HFD in the two-diet choice approach [10]. In contrast, injection of a CRH receptor 1 (corticotropin-releasing factor receptor 1, CRFR1) antagonist into the PVH suppressed the fasting-induced increase in HCD selection and decrease in HFD selection. CRFR1 is expressed at a high level in the PVH [70], and CRFR1-expressing neurons in this nucleus include glutamatergic and GABAergic neurons and do not express CRH, vasopressin, oxytocin, or TRH [70, 71]. CRFR1-expressing neurons in the PVH control brain areas that regulate

Suppression of CRH expression in the PVH with a specific shRNA inhibited the fasting-induced increase in HCD selection and decrease in HFD selection. Furthermore, it also blunted the effect of CA-AMPK expression in PVH neurons on HCD selection. Examination of the effects of activation and inhibition of CRH neurons in the PVH with the use of DREADD technology on selection between an HFD and an HCD in the two-diet choice approach also showed that these neurons are sufficient and necessary for the fasting-induced increase in carbohydrate

Food selection has been reported to be affected by plasma corticosterone level [73]. We examined whether AMPK-regulated CRH neurons in the PVH might influence HCD selection through change in plasma corticosterone level [10]. Expression of CA-AMPK in PVH neurons did not affect plasma corticosterone levels, although it increased selection of an HCD. In contrast, activation of CRH neurons in the PVH by DREADD technology increased the plasma corticosterone level, whereas inhibition of these neurons by the same approach suppressed the fasting-induced increase in this parameter. Plasma corticosterone is thus unlikely to be a primary mediator of the change in food selection, although it might be necessary to control food intake [73]. AMPK-regulated CRH neurons in the rostral portion of the PVH appear to be distinct from the CRH neurons that regulate the hypothalamic-pituitary-

CRH is known to be an anorexic neuropeptide. The i.c.v. injection of CRH thus attenuates total calorie intake in mice in the two-diet choice approach with an HCD and an HFD [10]. Activation of CRH neurons in the PVH with DREADD technology also decreased total calorie intake for the initial 3-h feeding but did not change after the 24-h feeding. In contrast, injection of CRH in the PVH did not change total calorie intake in mice in the two-diet choice approach. Thus, a subset of CRH neurons in the rostral part of the PVH that regulates CRFR1 neurons in the PVH may regulate selection of an HCD and an HFD, whereas another group of CRH neurons

Diuretic hormone 44 (Dh44), the *Drosophila* ortholog of mammalian CRH, regulates the selection of nutritive sugars such as D-glucose, but not to that of nonnutritive sugars such as L-glucose [74]. Fasted wild-type flies initially choose the sweeter L-glucose before switching to D-glucose during refeeding in a twodiet choice paradigm. By contrast, Dh44 mutants choose the sweeter L-glucose but fail to increase the preference for D-glucose. Dh44 neurons do not regulate

required for the fasting-induced increase in HCD selection.

food intake and sweet taste sensing [39, 68, 70, 72].

selection [10].

adrenal axis.

in this nucleus inhibits total calorie intake.

**14**

total consumption of food. Thus, CRH-dependent carbohydrate selection is likely conserved from insects to rodents.

We next examined whether AMPK and its downstream target CPT1c, the neuronal isoform of CPT1, in CRH neurons of the PVH regulate selection of an HCD versus an HFD in the two-diet choice approach [10]. Expression of CA-AMPK specifically in CRH neurons of the PVH was sufficient to increase selection of an HCD and reduce that of an HFD. In contrast, expression of shRNA specific for AMPK or for CPT1c in these neurons attenuated the fasting-induced increase in HCD selection. Expression of an shRNA specific for AMPK in CRH neurons also inhibited the increase in HCD selection induced by expression of CA-AMPK in the PVH. Furthermore, expression of an AMPK shRNA in these neurons also resulted in downregulation of the amount of CPT1c mRNA but not that of CPT1a mRNA in the PVH. AMPK had been shown to regulate the abundance of CPT1c mRNA [75]. These findings suggested that the AMPK-CPT1c axis in CRH neurons of the PVH is necessary for the fasting-induced increase in the selection of an HCD over an HFD (**Figure 8**).

CPT1c is localized to both the endoplasmic reticulum (ER) [76] and mitochondria [77], both of which contribute to intracellular Ca2+ signaling in a cooperative manner [78]. We found that the AMPK activator AICAR (5-aminoimidazole-4 carboxamide-1-β-D-ribofuranoside) increased the intracellular Ca2+ concentration in CRH neurons isolated from the PVH [10], and this effect was attenuated by the CPT1 inhibitor etomoxir. The shRNA-mediated depletion of AMPK or CPT1c in these neurons also blocked the Ca2+ response to AICAR, indicating that activation of the AMPK-CPT1c axis increases the intracellular Ca2+ concentration in CRH neurons of the PVH. These results suggested that activation of the AMPK-CPT1c system leads to increase synaptic activity in these neurons and in HCD selection by triggering an increase in intracellular Ca2+ concentration in CRH neurons. Activation of AMPK in these neurons inhibits ACC, decreases the abundance of malonyl-CoA and activates CPT1c. It also increases expression of the CPT1c gene (**Figure 8**).

AMPK activity is regulated by the glycolytic pathway as well as by cellular energy level (Section 3.1) [4, 5, 44]. Increased glucose levels in the brain inhibit AMPK activity in the PVH and ARC [7]. The i.c.v. injection of glucose in mice also inhibited the fasting-induced selection of an HCD in the two-diet choice approach [10]. The glucose-induced inhibition of AMPK may result in increased ACC activity, an increase in the amount of malonyl-CoA, and consequent inhibition of CPT1c activity in AMPK-regulated CRH neurons. The AMPK-CPT1c system may thus act as a glucose sensor in AMPK-regulated CRH neurons of the PVH.
