Elsevier

Life Sciences

Volume 82, Issues 25–26, 20 June 2008, Pages 1262-1271
Life Sciences

Citrate diminishes hypothalamic acetyl-CoA carboxylase phosphorylation and modulates satiety signals and hepatic mechanisms involved in glucose homeostasis in rats

https://doi.org/10.1016/j.lfs.2008.04.015Get rights and content

Abstract

The hypothalamic AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway is known to play an important role in the control of food intake and energy expenditure. Here, we hypothesize that citrate, an intermediate metabolite, activates hypothalamic ACC and is involved in the control of energy mobilization. Initially, we showed that ICV citrate injection decreased food intake and diminished weight gain significantly when compared to control and pair-fed group results. In addition, we showed that intracerebroventricular (ICV) injection of citrate diminished (80% of control) the phosphorylation of ACC, an important AMPK substrate. Furthermore, citrate treatment inhibited (75% of control) hypothalamic AMPK phosphorylation during fasting. In addition to its central effect, ICV citrate injection led to low blood glucose levels during glucose tolerance test (GTT) and high glucose uptake during hyperglycemic–euglycemic clamp. Accordingly, liver glycogen content was higher in animals given citrate (ICV) than in the control group (23.3 ± 2.5 vs. 2.7 ± 0.5 μg mL 1 mg 1, respectively). Interestingly, liver AMPK phosphorylation was reduced (80%) by the citrate treatment. The pharmacological blockade of β3-adrenergic receptor (SR 59230A) blocked the effect of ICV citrate and citrate plus insulin on liver AMPK phosphorylation. Consistently with these results, rats treated with citrate (ICV) presented improved insulin signal transduction in liver, skeletal muscle, and epididymal fat pad. Similar results were obtained by hypothalamic administration of ARA-A, a competitive inhibitor of AMPK. Our results suggest that the citrate produced by mitochondria may modulate ACC phosphorylation in the hypothalamus, controlling food intake and coordinating a multiorgan network that controls glucose homeostasis and energy uptake through the adrenergic system.

Introduction

Enzyme AMP-activated protein kinase (AMPK) acts as a cell energetic status sensor (Kahn et al., 2005, Hardie et al., 2006). It responds to and integrates nutrient and hormonal signals involved in energetic homeostasis, thus modulating cellular function according to energy availability. Once activated, AMPK inhibits fatty acid biosynthesis through the phosphorylation of acetyl-CoA carboxylase (ACC) at its Ser79 residue. Whenever active, ACC catalyzes the formation of malonyl-CoA, which allosterically inhibits the entrance of long-chain acyl-CoA into the mitochondria to undergo β-oxidation for energy production (Long and Zierath, 2006). Initial studies have explored the multiple facets of AMPK activity in cells of peripheral tissues, especially in skeletal muscle (Fisher et al., 2002), where it is known to promote glucose uptake through insulin-independent mechanism, and in the liver, where it plays a role in the control of glucose production during fasting (Koo et al., 2005).

Some recent studies have evaluated the roles of AMPK in the control of hypothalamic functions. In the hypothalamus, AMPK is highly activated during fasting or in the presence of orexigenic peptides such as ghrelin and AGRP (Hardie, 1989, Kahn et al., 2005, Lee et al., 2005). After feeding or following the intracerebroventricular (ICV) administration of leptin and insulin, AMPK is inhibited (Minokoshi et al., 2004, Kim and Lee, 2005, Lee et al., 2005). Once activated in the hypothalamus, AMPK drives to an orexigenic response, which is attenuated by feeding and by the subsequent increase in leptin and insulin levels. Thus, in the hypothalamus, AMPK participates in the complex network that controls the feeding and fasting cycles (Long and Zierath, 2006). Recent studies have suggested that besides its direct role in energy acquisition by feeding control, hypothalamic AMPK might also play an indirect role in the control of peripheral energy stores by controlling the hypothalamus-sympathetic nervous system axis (Kahn et al., 2005, Xue and Kahn, 2006). For example, the administration of leptin into ventromedial hypothalamus increased heart, brown adipose tissue (BAT), and skeletal muscle glucose uptake through the mediation of a β-adrenergic mechanism (Haque et al., 1999). Furthermore, it has been demonstrated that systemic and intracerebroventricular administration of leptin increases sympathetic activity to BAT and other peripheral tissues (Collins et al., 1996, Haynes et al., 1997) and increases insulin sensitivity (Barzilai et al., 1997, Shi et al., 1998). As well as leptin, other nutrient factors such as pyruvate, glucose, and synthetic compounds (AICAR and sodium azide) are capable of modulating hypothalamic AMPK and restoring ATP levels in neuronal cells (Lee et al., 2005).

Recently, Roman et al. (2005) showed that citrate promotes satiety signal when injected into the hypothalamus. Citrate is an intermediate metabolite produced in the mitochondria in the citric acid cycle. It is transported to the cytoplasm from mitochondria and used in lipid biosynthesis in response to an increase in the ATP/AMP ratio. Recently, a novel sodium-coupled citrate transporter (NaCT) has been described in mouse brain (Inoue et al., 2002). It is expressed in most brain neurons. Furthermore, citrate is a positive allosteric effector of the acetyl-CoA carboxylase activity and a negative modulator of phosphofructokinase, a key glycolysis pathway enzyme. Supported by the fact that leptin, insulin, glucose, and pyruvate inhibit AMPK, promote satiety signal, and increase peripheral glucose uptake, it was interesting to study the role of citrate in the neuronal and peripheral mechanisms of energy acquisition and disposal. In addition, the identification of the target protein would be interesting for the development of new drugs that act in the hypothalamus, as they could modulate satiety and glucose homeostasis.

To address this issue, we performed a series of experiments in rats after citrate intracerebroventricular injection treatment. To obtain high hypothalamic AMPK activity, the animals were fasted. Using this approach, we demonstrated that citrate can diminish hypothalamic AMPK/ACC phosphorylation and reduce food intake. It can also increase glucose uptake and glycogen level in the liver and reduce liver AMPK phosphorylation. Besides, upon central administration, citrate can improve insulin signal transduction in the liver and is delivered, at least in part, by sympathetic β-adrenergic signals.

Section snippets

Experimental animals and surgical procedures

Male Wistar Hannover rats (12 wk old, 250–280 g) from the Universidade Estadual de Campinas animal breeding center were used in all experiments. The rats were maintained at room temperature (25 °C) and in 12:12-h light–dark cycles with free access to water and chow, unless otherwise indicated. The rats were chronically instrumented with an intracerebroventricular (ICV) cannula and kept under controlled temperature and light–dark conditions (07:00–19:00 h) in individual metabolic cages (Beiramar

Food intake and body weight

Since intracellular citrate is known to control ACC activity and malonyl-CoA levels, we decided to investigate whether this metabolite could affect food intake and weight gain. A protocol of experiments was adopted for this investigation. In this protocol, citrate (20 nmol) was administered daily and the cumulative food intake (24 h) in the control group and the citrate group was evaluated during the next 8 days (treatment phase). As can be observed in Fig. 1A, the group that received citrate

Discussion

In the present study, we evaluated the role played by the hypothalamic ACC/AMPK pathway in the control of whole-body glucose homeostasis. To warrant the biological relevance of the phenomena studied herein, we decided to employ physiological means to modulate AMPK/ACC phosphorylation. To stimulate hypothalamic AMPK, the rats were submitted to fasting. Food deprivation increases neuronal levels of AMP while reducing ATP levels. The increased AMP/ATP ratio is one of the most important

Acknowledgements

This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP and CNPq. We thank Mr. Luiz Janeri, Mr. Józimo Ferreria, and Mr. Márcio Alves da Cruz for their technical assistance and Mr. Laerte J. Silva for the English language editing.

References (49)

  • SongX. et al.

    Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4

    The Journal of Biological Chemistry

    (2001)
  • TaoJ. et al.

    Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo

    The Journal of Biological Chemistry

    (2001)
  • ThampyK.G. et al.

    Regulation of acetyl-coenzyme A carboxylase. II. Effect of fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme

    The Journal of Biological Chemistry

    (1988)
  • BarzilaiN. et al.

    Leptin selectively decreases visceral adiposity and enhances insulin action

    The Journal of Clinical Investigation

    (1997)
  • ChaS.H. et al.

    Inhibition of hypothalamic fatty acid synthase triggers rapid activation of fatty acid oxidation in skeletal muscle

    Proceedings of the National Academy of Sciences of the United States of America

    (2005)
  • ChenJ.F. et al.

    Conditional, tissue-specific expression of Q205L G alpha i2 in vivo mimics insulin action

    Journal of molecular medicine

    (1997)
  • CollinsS. et al.

    Role of leptin in fat regulation

    Nature

    (1996)
  • CombsT.P. et al.

    Endogenous glucose production is inhibited by the adipose-derived protein Acrp30

    The Journal of Clinical Investigation

    (2001)
  • Eldar-FinkelmanH. et al.

    Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

    Proceedings of the National Academy of Sciences of the United States of America

    (1997)
  • FediucS. et al.

    Inhibition of insulin-stimulated glycogen synthesis by 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside-induced adenosine 5¢-monophosphate-activated protein kinase activation: interactions with Akt, glycogen synthase kinase 3-3alpha/beta, and glycogen synthase in isolated rat soleus muscle

    Endocrinology

    (2006)
  • FisherJ.S. et al.

    Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin

    American Journal of Physiology. Endocrinology and Metabolism

    (2002)
  • GaoS. et al.

    Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

    Proceedings of the National Academy of Sciences of the United States of America

    (2007)
  • HaqueM.S. et al.

    Role of the sympathetic nervous system and insulin in enhancing glucose uptake in peripheral tissues after intrahypothalamic injection of leptin in rats

    Diabetes

    (1999)
  • HardieD.G.

    Regulation of fatty acid synthesis via phosphorylation of acetyl-CoA carboxylase

    Progress in Lipid Research

    (1989)
  • Cited by (28)

    • Energy expenditure and caloric targets during continuous renal replacement therapy under regional citrate anticoagulation. A viewpoint

      2020, Clinical Nutrition
      Citation Excerpt :

      To escape this closed circle, the metabolites of citrate have to be incorporated as α-ketoglutarate or acetoacetate into proteinogenesis, as citrate and then acetyl coenzyme A into lipogenesis, and/or as malate or oxaloacetate into gluconeogenesis (Fig. 2) [36]. Citrate is also involved in the hypothalamic control of glucose homeostasis, modulates cellular fatty oxidation, and improves insulin signal transduction [35,37]. In summary, citrate anticoagulation during CRRT enhances anabolic processes that prevent accumulation of citrate and other metabolites in the Krebs cycle.

    • Mitochondrial dysfunction, AMPK activation and peroxisomal metabolism: A coherent scenario for non-canonical 3-methylglutaconic acidurias

      2020, Biochimie
      Citation Excerpt :

      This notion needs to be shaded by the fact that cytoplasmic import of citrate (from mitochondrial origin) should be operational to aliment cytosolic production of acetyl-CoA. As a function of its cytoplasmic concentration, citrate may, however, partially reverse AMPK-driven inhibition of acetyl-CoA carboxylases, and vice-versa AMPK activation reducing allosteric activation of the carboxylase by citrate [332–335]. Ultimately, operationality under AMPK activation of ATP:citrate lyase activity may limit a local rise in citrate and hence may contribute to maintain an optimal AMPK-driven switching down of acetyl-CoA carboxylase(s), being aware that if ATP citrate lyase is not inactivated by AMPK, it may interact with the catalytic subunit of the kinase and inhibit its activity [336].

    • Fatty acid sensing in the gut and the hypothalamus: In vivo and in vitro perspectives

      2014, Molecular and Cellular Endocrinology
      Citation Excerpt :

      Acetyl-CoA is a precursor from which malonyl-CoA is enzymatically derived by acetyl-CoA carboxylase (ACC), and within neurons, acetyl-CoA may be generated from citrate or from pyruvate (the latter via pyruvate dehydrogenase (PDH)). Citrate, a metabolite produced in the mitochondria during the citric acid cycle, has allosteric regulation of hypothalamic ACC activity (Cesquini et al., 2008) and thus regulates the formation of malonyl-CoA. Indeed, central administration of citrate has been demonstrated to affect glucose homeostasis by improving peripheral insulin sensitivity during conditions of a hyperinsulinemic–euglycemic clamp by promoting glucose uptake and hepatic glycogen synthesis (Cesquini et al., 2008).

    View all citing articles on Scopus
    View full text