Abstract
The anorectic anx/anx mouse exhibits a mitochondrial complex I dysfunction that is related to aberrant expression of hypothalamic neuropeptides and transmitters regulating food intake. Hypothalamic activity, i.e. neuronal firing and transmitter release, is dependent on glucose utilization and energy metabolism. To better understand the role of hypothalamic activity in anorexia, we assessed carbohydrate and high-energy phosphate metabolism, in vivo and in vitro, in the anx/anx hypothalamus. In the fasted state, hypothalamic glucose uptake in the anx/anx mouse was reduced by ~50% of that seen in wild-type (wt) mice (P < 0.05). Under basal conditions, anx/anx hypothalamus ATP and glucose 6-P contents were similar to those in wt hypothalamus, whereas phosphocreatine was elevated (~2-fold; P < 0.001) and lactate was reduced (~35%; P < 0.001). The anx/anx hypothalamus had elevated total AMPK (~25%; P < 0.05) and GLUT4 (~60%; P < 0.01) protein contents, whereas GLUT1 and GLUT3 were similar to that of wt hypothalamus. Interestingly, the activation state of AMPK (ratio of phosphorylated AMPK/total AMPK) was significantly decreased in hypothalamus of the anx/anx mouse (~60% of that in wt; P < 0.05). Finally, during metabolic stress (ischemia), accumulation of lactate (measure of glycolysis) and IMP and AMP (breakdown products of ATP) were ~50% lower in anx/anx vs wt hypothalamus. These data demonstrate that carbohydrate and high-energy phosphate utilization in the anx/anx hypothalamus are diminished under basal and stress conditions. The decrease in hypothalamic metabolism may contribute to the anorectic behavior of the anx/anx mouse, i.e. its inability to regulate food intake in accordance with energy status.
Introduction
Neuronal networks crucial for the regulation of food intake, i.e. satiety and hunger, are located mainly in the hypothalamus. In particular, two populations of neurons in the mediobasal part of the hypothalamus, the arcuate nucleus (Arc), are fundamental in this regulation. These are the agouti-gene-related protein (AGRP) and neuropeptide Y (NPY) expressing neurons that are orexigenic, and the proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) expressing populations that are anorexigenic (Grill & Kaplan 2002, Hillebrand et al. 2002, Morton et al. 2006, Schwartz 2006). Additionally, hypothalamic AMP-dependent protein kinase (AMPK), a sensor of cellular metabolism that responds to changes in energy status (i.e., contents of high energy phosphates), has been implicated in control of feeding, being activated during starvation/food deprivation and inactivated during feeding (Minokoshi et al. 2004, Kahn et al. 2005, Andrews et al. 2008, Ronnett et al. 2009).
Based on its role in the regulation of food intake, the hypothalamus has been extensively studied in an animal model of anorexia, the anx/anx mouse (Son et al. 1994, Broberger et al. 1997, 1998, 1999, Johansen et al. 2000, Fetissov et al. 2005, Lachuer et al. 2005, Mercader et al. 2008a, Nilsson et al. 2008, 2011, Lindfors et al. 2011). The anx mutation arose spontaneously at the Jackson Laboratory in the mid-seventies. The anx/anx mouse is indistinguishable from its normal siblings, i.e. homozygote and heterozygote wild-type (wt) mice, at birth. However, during the first postnatal weeks, they gradually decrease their food intake, develop starvation and emaciation. By three weeks of age, they weigh approximately half as much as wt sibling mice and die around or shortly after this early point in life (Maltais et al. 1984). The serum level of the food intake regulating hormone leptin is reduced in the anx/anx mice (Johansen et al. 2000). Reduced leptin should in accordance to the energy homeostatic mechanisms give rise to increased hunger signaling (Hillebrand et al. 2002, Morton et al. 2006, Schwartz 2006), thus indicating an intrinsic hypothalamic problem in the anx/anx mice underlying their paradoxical response to negative energy balance. Abnormal expression and immunohistochemical labeling pattern for several neuropeptide/transmitter systems in the hypothalamus have been documented (Son et al. 1994, Broberger et al. 1997, 1998, 1999, Johansen et al. 2000, 2007, Fetissov et al. 2005, Nilsson et al. 2008). Additionally, signs of hypothalamic inflammation and degeneration have been shown (Lachuer et al. 2005, Mercader et al. 2008b, Nilsson et al. 2008, 2011). Microglia cells of a morphology indicative of an activated state are dramatically increased exclusively in the hypothalamic AGRP system of these mice, starting from postnatal day 12–15. The activated microglia are present solely in close vicinity to, and occasionally even embracing, the AGRP-expressing neuronal cell bodies in the arcuate nucleus, as well as in the projection areas of these same neurons, e.g. in the paraventricular nucleus of the hypothalamus, the lateral hypothalamic area, the medial preoptic region and the bed nuclei of stria terminalis. Interestingly, the occurrence of activated microglia coincides also in time with a reduction of AGRP-positive neuronal fibers in these animals (Nilsson et al. 2008). Microarray studies have revealed an inflammatory profile in the hypothalamus of these mice (Lachuer et al. 2005, Mercader et al. 2008a). Expression of a marker of axonal degeneration, i.e. activated caspase 6, has been documented in the hypothalamic food intake-regulating systems (Nilsson et al. 2011). The phenotypes of the anx/anx mice are also associated with mitochondrial dysfunction, in particular, lower levels and capacity of complex I (CI) in the oxidative phosphorylation system, which is associated with increased levels of reactive oxygen species (ROS) in the hypothalamus (Lindfors et al. 2011).
Firing of neurons in the central nervous system, including the hypothalamus, is dependent on the rate of carbohydrate utilization (Silver & Erecinska 1998, Gilbert et al. 2006, Lutas et al. 2014), which therefore can be used as a marker of neural activity. Based on the described aberrancies in the hypothalamus of the anx/anx mouse, and their paradoxical response to negative energy balance, we examined whether there are alterations in hypothalamic metabolism of these mice.
Materials and methods
Animals
The local ethical committee (Stockholms norra djurförsöksetiska nämnd) approved all experiments involving animals. Heterozygous anx breeding pairs (B6C3Fe–a/a–anx A/+a) originally obtained from the Jackson Laboratory were used to set up an intercross. Genotyping was performed using simple sequence-length polymorphism markers mapped to the sub-chromosomal region where the anx mutation is situated (Maltais et al. 1984, Lindfors et al. 2011). anx/anx mice and their wt siblings were housed at room temperature (22°C) with a 12:12-h light–darkness cycle and had free access to food and water, via milk from the mothers. Female and male mice were killed by decapitation. Anesthesia was not administered as it would interfere with the metabolism (Toyama et al. 2004). Killing occurred at postnatal day (P) 19–21 in the fed state, unless stated otherwise. The average weight (±s.e.) for anx/anx and wt mice was 4.6 ± 0.2 and 7.2 ± 0.3 g (P < 0.001), respectively.
Spectrophotometric analysis of respiratory chain enzyme activities in hypothalamic tissue
For analysis of respiratory chain subunits, mice were decapitated and the hypothalamus was rapidly dissected out and kept on ice until analyzed (approximately 30 min). Three animals from each genotype were pooled for the analysis, and five different such pools per genotype were analyzed. The respiratory chain enzyme activities were determined in isolated mitochondria from the tissue as previously described (Wibom et al. 2002), with the exception that a buffer containing 10 mM Tris–HCl, 250 mM sucrose, 3 mM EGTA and 4 mg/mL BSA, pH 7.7 was used for the homogenization procedure.
Spectrophotometric analysis of citrate synthase activity in the hypothalamic tissue
For quantitative analysis of the amount of mitochondria in anx/anx vs wt hypothalamus, we used a mitochondrial marker, citrate synthase (CS) (Alp et al. 1976). anx/anx and wt animals (n = 5 per genotype) were decapitated at P19, and the hypothalamus was rapidly dissected out, frozen in isopentane and kept at −70°C until further processed.
After thawing, the tissue was homogenized in a solution containing Triton X-100 (0.05% vol/vol), 50 mmol/L KH2PO4 and 1 mmol/L EDTA, pH 7.5. The homogenate was spun in a microcentrifuge for a minute, and CS activity was determined in the supernatant as previously described (Alp et al. 1976). The assay was performed on an automated photometer (Konelab T20 xti, Thermo Scientific).
Ex vivo autoradiography analysis of hypothalamic glucose uptake
For analysis of glucose uptake, mice (n = 5 per genotype) were fasted for 5–6 h prior to intraperitoneal injection of [3H]-2-deoxy-glucose (0.3 mCi/mL of sterile saline, 10 mL/kg body weight). Forty-five min after injection (sufficient time for adequate glucose uptake to occur (Wong et al. 2011)), mice were decapitated, and brain and liver were rapidly dissected out and frozen in ice-cold isopentane. The tissues were stored at −80°C. Blood glucose concentration was measured at the time of killing using an Accu-Chek blood glucose analyzer (Roche Diagnostics). Serum glucose values were calculated by multiplying the blood values by 1.15 (Frank et al. 2012) to estimate glucose-specific activity in serum (see below). Brain tissue was cryosectioned (14 µm) and exposed to phosphor imaging plates (Fujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan). Quantitative analysis of plates was performed using Multi Gauge 3.2 phosphorimager software (Fujifilm) as described elsewhere (Miyamoto et al. 2000, Waldron et al. 2015).
The radioactivity in serum was measured with a scintillation counter by adding 5 µL serum to 90 µL ddH2O, and then, 10 mL scintillation fluid (Emulsifier safe, Perkin Elmer). The glucose uptake into hypothalamus, motor cortex and thalamus and liver was calculated by the following formula: (µmol glucose per mL serum/dpm per mL serum) × dpm/g tissue = µmol glucose/45 min/g tissue) as described elsewhere (Beglopoulos et al. 2016).
Western blot analysis of AMPK and GLUTs in hypothalamic tissue
For Western blot, animals were decapitated and the hypothalamus was rapidly dissected out, frozen in isopentane and stored at −70°C until further processed. 7–10 animals per genotype were used for this analysis. The hypothalamic tissue was homogenized in lysis buffer (20 mM HEPES, pH 7.4, 40 mM KCl, 1 mM dithiothreitol, 0.3% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), 1 mM phenyl-methylsulfonyl fluoride, 1 mg/mL leupeptin, 1 mg/mL pepstatin, and 1 mM EDTA). The lysates were centrifuged at 700 g for 15 min, 4°C, and proteins were measured in supernatant by the Bradford assay (Bio-Rad). 30 µg total protein were loaded onto NuPAGE 4–12% gels (Invitrogen). Proteins were transferred onto Immobilon-FL membranes (Millipore). Membranes were blocked in 3% BSA and incubated overnight at 4°C with the following primary antibodies: total AMP-activated protein kinase (AMPK-T, 1:1000, Cell Signaling Technology), phosphorylated AMPK (AMPK-P, 1:1000, Cell Signaling Technology), glucose transporter (GLUT) 4 (1:5000, Chemicon/Millipore) GLUT1 (1:5000, Chemicon/Millipore) and GLUT3 (combination of two antisera directed against different epitopes of GLUT3, 1:250, The Human Protein Atlas, Stockholm, Sweden). The membranes were then incubated for 1 h in the darkness with secondary antibody (donkey anti-rabbit at 1:20,000 dilution) and were scanned with an Odyssey infra-red imager (LI-COR Biosciences, Lincoln, NE, USA) after washing. Band intensities were quantified by ImageJ and normalized with the intensities of bands after incubation with anti-beta-actin (1:2500, Abcam).
Analysis of metabolites and ATP turnover in hypothalamic tissue
Animals were divided into two groups and killed by decapitation. For one group of animals, representing the basal condition, the whole head was immediately frozen in liquid N2, freeze-dried and the hypothalamus was dissected out. In another group of animals, representing the ischemic/stress condition, the hypothalamus was dissected out immediately after decapitation and then rapidly frozen in liquid N2 (a process taking approximately 1 min) and subsequently freeze-dried. Ice-cold 0.5 M perchloric acid was added to the freeze-dried hypothalamus, kept in an ice bath for 20 min and then centrifuged at 10,000 g at 4°C. The supernatant was neutralized with 2.2 M KHCO3 and centrifuged as described previously. The latter supernatant was assayed for phosphocreatine (PCr), creatine (Cr), ATP, glucose 6-P and lactate with enzymatic techniques (changes in NAD(P)H) adapted for fluorometry (Lowry 1972), as well as assayed for ATP, ADP, AMP and IMP by high-perfomance liquid chromatography (HPLC) (Schweinsberg & Loo 1980). To adjust for admixture of non-hypothalamic tissue (e.g. blood), metabolite values were divided by the sum of PCr + Cr (total Cr) and then multiplied by the mean total Cr content for the whole material. Total Cr did not differ significantly between groups under any condition studied (data not shown) and averaged 41.9 ± 2.0 µmol/g dry weight for the whole material. Anaerobic ATP turnover was calculated from the following equation: −2ΔATP − ΔPCr + (1.5Δ lactate) (Lowry et al. 1964, Katz 1988), where Δ refers to the mean difference between ischemia and basal values.
Statistical analysis
Statistically significant (P < 0.05) differences between groups were determined with the unpaired two-tailed t test. All values are presented as means ± s.e.m.
Results
Respiratory chain enzyme activities in the anx/anx hypothalamus
To assess the mitochondrial capacity of the anx/anx hypothalamus, we measured the enzymatic activity of the different complexes (C) in the respiratory chain in isolated mitochondria and found small but significant decreases in activities of CI (NADH:coenzyme Q reductase) (0.33 ± 0.01 vs 0.38 ± 0.01 units/unit CS, P = 0.01) and of CI + III (NADH:cytochrome c reductase) (1.99 ± 0.02 vs 2.27 ± 0.06 units/unit CS, P = 0.003) in anx/anx vs wt hypothalamus. No differences were seen in CII (succinate dehydrogenase) (0.21 ± 0.01 vs 0.22 ± 0.00 units/unit CS, P = 0.23), CII + III, (succinate:cytochrome c reductase) (0.55 ± 0.01 vs 0.57 ± 0.01 units/unit CS, P = 0.11) or CIV (cytochrome c oxidase) (2.77 ± 0.08 vs 2.85 ± 0.09 units/unit CS, P = 0.54). No difference in total tissue CS activity was seen when comparing anx/anx and wt hypothalamus (7.6 ± 0.6 vs 7.4 ± 0.4 µmol/min/g, respectively, P = 0.8), indicating similar levels of mitochondria in the two groups. Thus, we confirm the previously documented mitochondrial CI dysfunction in the anx/anx mouse, which cannot be explained by a difference in mitochondrial content (Lindfors et al. 2011).
Metabolites in the anx/anx hypothalamus
In the basal state, anx/anx and wt hypothalamus exhibited similar contents of ATP and glucose 6-P, whereas PCr was significantly higher and lactate was significantly lower in anx/anx vs wt hypothalamus (Table 1). One minute of ischemia (as explained in the Materials and methods section) resulted in marked depletion of ATP and PCr, and accumulation of lactate, in both anx/anx and wt hypothalamus (Table 1). However, ATP and PCr were significantly higher, whereas lactate was significantly lower, in the tissue from anx/anx vs wt after ischemia (Table 1). Particularly evident were the markedly smaller accumulations of AMP and IMP (breakdown products of ATP) in the ischemic hypothalamus of the anx/anx mouse (Fig. 1). IMP is a particularly sensitive indicator of net ATP degradation (Sahlin & Katz 1989, Spencer & Katz 1991). The ATP/AMP ratio was significantly increased in anx/anx vs wt hypothalamus in the ischemic condition (Fig. 1E). It could be argued that the dissection of the hypothalamus after decapitation prior to freezing (ischemic condition) significantly influenced the metabolite values during ischemia. This is, however, unlikely as the absolute values for ATP, PCr, lactate and AMP after ischemia in the present study were very similar to values reported earlier for mouse whole brain and cerebrum one min after decapitation (assuming a wet/dry ratio of 4.3) (Lowry et al. 1964, Lin et al. 1993). Further, metabolite levels in the basal state were also similar in the present study to those in whole brain and cerebrum (Lowry et al. 1964, Lin et al. 1993).
Metabolites in the anx/anx hypothalamus. Metabolites in the anx/anx and wt mouse hypothalamus under basal conditions and after 1 min of ischemia analyzed by HPLC. (A) IMP; (B) ADP; (C) ATP; (D) AMP and (E) ATP/AMP. Values are means ± s.e.m., n = 3–7 hypothalami per genotype. ***P < 0.001 between groups (unpaired t-test).
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0383
Metabolites in the anx/anx hypothalamus in basal state and one minute of ischemia.
| PCr | Cr | ATP | Lactate | G6P | |
|---|---|---|---|---|---|
| Basal state | |||||
| wt | 6.5 ± 0.4 | 35.0 ± 0.4 | 7.8 ± 0.3 | 16.5 ± 1.2 | 0.32 ± 0.08 |
| anx/anx | 12.2 ± 1.0*** | 29.2 ± 1.0*** | 9.0 ± 0.8 | 5.6 ± 1.3*** | 0.34 ± 0.06 |
| Ischemia | |||||
| wt | 0.6 ± 0.1 | 41.9 ± 0.1 | 1.1 ± 0.2 | 37.4 ± 0.4 | 0.27 ± 0.01 |
| anx/anx | 1.6 ± 0.4* | 40.9 ± 0.4* | 2.2 ± 0.4* | 19.3 ± 1.4*** | 0.26 ± 0.08 |
Values are means ± s.e.m. and were obtained by enzymatic analyses; n = 5–9 hypothalami per genotype. Values are given in µmol/g dry weight.
*P < 0.05; ***P < 0.001 (unpaired t-test).
G6P, glucose 6-P; PCr, phosphocreatine.
AMPK and GLUTs in anx/anx hypothalamus
AMPK-T and GLUT4 proteins were significantly elevated (~25% and ~60%, respectively) in anx/anx compared to those in wt hypothalamus, whereas AMPK-P, GLUT1 and GLUT3 were similar between groups (Fig. 2A). The ratio AMPK-P/AMPK-T was decreased by ~40% in anx/anx hypothalamus (Fig. 2B).
Protein expression of AMPK and GLUTs in the anx/anx hypothalamus. (A) Relative protein levels from Western blot analysis of AMPK, AMPK-P, GLUT4, GLUT1 and GLUT3; (B) ratio of AMPK-P:AMPK-T in the hypothalamus of the anx/anx and wt mouse. Values are means ± s.e.m., n = 6–10 hypothalami per genotype. All values are normalized to the levels of beta-actin. *P < 0.05; ***P < 0.001 (unpaired t-test).
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0383
Carbohydrate utilization in the anx/anx mouse
The higher content of PCr and lower degree of AMPK activation (AMPK-P/AMPK-T) suggested a lower degree of metabolism in the anx/anx hypothalamus in the basal condition. To directly examine this possibility, we assessed the rate of glucose uptake in hypothalamus of conscious anx/anx mice. Blood glucose concentration in the fasted state averaged 5.6 ± 0.6 mM in anx/anx and 7.9 ± 0.6 mM in wt mice (P = 0.04), which agrees with our recent findings for both groups under comparable conditions (Lindfors et al. 2015). In the fasted state, serum insulin does not differ significantly between groups (Lindfors et al. 2015). Glucose uptake was significantly reduced in the hypothalamus of anx/anx compared to that in wt mice (Fig. 3). The lower glucose uptake was observed in other areas of the brain, as well as in the liver (Fig. 3). To assess whether hypometabolism also occurred under stress conditions, carbohydrate and energy metabolism were quantified in ischemic hypothalamus (~1 min of ischemia). Under this condition, glycolysis (lactate accumulation) was reduced by almost 40%, whereas anaerobic ATP turnover was ~10% lower in the anx/anx group (Table 2). Thus, carbohydrate utilization was diminished under both resting and stress conditions in the anx/anx hypothalamus. It should be noted that the term carbohydrate utilization refers to glycolysis from utilization of intracellular glucose and glycogen that are trapped in the cell during ischemia (no significant glucose transport under this condition), as demonstrated elsewhere (Lowry et al. 1964), and glucose uptake under basal conditions, which is assumed to reflect glycolysis as well.
Glucose uptake in the anx/anx hypothalamus. (A) Glucose uptake in the hypothalamus (Hypo), thalamus (Thala), motor cortex (MC) and liver of the anx/anx and wt mouse, measured by injections with [3H]-2-deoxy-glucose and subsequent ex vivo radiography, as shown in coronal sections from illustrative samples of (B) the anx/anx and (C) wt mouse. Values are means ± s.e.m., n = 5 animals per genotype. *P < 0.05, ***P < 0.005 (unpaired t-test).
Citation: Journal of Endocrinology 233, 1; 10.1530/JOE-16-0383
ATP turnover in the anx/anx hypothalamus.
| −2ΔATP | −ΔPCr | +1.5ΔLactate | ATP turnover (µmol/g dry weight) | |
|---|---|---|---|---|
| wt | 13.4 | 5.8 | 31.4 | 50.6 |
| anx/anx | 13.7 | 10.6 | 20.5 | 44.8 |
Values are calculated from the mean values shown in Table 1. ΔATP, ΔPCr, and ΔLactate values are given as µmol/g dry weight. ATP turnover = −2ΔATP − ΔPCr + (1.5ΔLactate).
Discussion
The major finding of the present study is that the anorexia of the anx/anx mouse is associated with a lower rate of hypothalamic glucose utilization.
Diseases associated with mitochondrial dysfunction (in brain and skeletal muscle) are generally associated with a stressed metabolic profile, as judged by lower levels of phosphocreatine and elevated levels of lactate as well as increased rates of glucose uptake (Wredenberg et al. 2006, Jeppesen et al. 2007, Milone & Wong 2013). These metabolic responses occur, presumably, to ensure set point levels of ATP. In some cases, however, mitochondrial dysfunction has been associated with reduced glucose uptake and hypometabolism, e.g. in epilepsy and Alzheimer’s disease (Chandrasekaran et al. 1996, Tenney et al. 2014). This is similar to what we see in the anx/anx hypothalamus in the present study. The hypothalamic metabolism is evidenced by a lower glucose uptake rate and decreased lactate content, as well as elevated PCr content in the basal state. Furthermore, the ratio of AMPK-P/AMPK-T is decreased in the basal state, which is consistent with lower metabolic stress in the anx/anx hypothalamus. This is somewhat reminiscent of the hypometabolic state associated with hibernation (Healy et al. 2011) and likely reflects lower neural activity, as seen, for example, in Alzheimer’s disease (Cunnane et al. 2011).
Different neuronal populations respond differently to metabolic stress (Schreiber & Baudry 1995), and this has been attributed to the subtype of ATP-sensitive potassium channel (K-ATP) expressed. A subtype of K-ATP channel consisting of Kir6.2 and SUR1 subunits, expressed, e.g., by dopaminergic neurons in substantia nigra, becomes activated by mitochondrial CI dysfunction, leading to ceased electrical activity, hyperpolarization and reduced firing (Liss et al. 1999). Kir6.2/SUR1 K-ATP channels are expressed by hypothalamic neurons i.e. the POMC/CART and AGRP/NPY neurons, and by a few other cell populations including the pancreatic beta-cells and brain stem neurons (Miki et al. 2001, Ibrahim et al. 2003, van den Top et al. 2007). High amounts of energy are required for firing of action potentials and neurotransmitter release, and mechanisms that inhibit such activities would conserve energy during conditions of negative energy balance (Attwell & Laughlin 2001, Sengupta et al. 2010). Uncontrolled generation of ROS in NPY/AGRP neurons can also cause diminished firing of these neurons, thus resulting in a reduced orexigenic drive (Andrews et al. 2008, Horvath et al. 2009).
The marked difference in glucose uptake between anx/anx and wt mice can be attributed to several factors. First, the higher concentration of blood glucose in wt mice can result in a higher mass action effect and therefore a higher glucose uptake. The difference in blood glucose between groups in the present study is ~2 mM. Noteworthy is that large increases in blood glucose can be associated with significant reductions in mouse brain glucose utilization (Vannucci et al. 1997, Toyama et al. 2004) and, conversely, chronic hypoglycemia can result in increased brain glucose uptake (McCall et al. 1986). Indeed, in general, chronic hyperglycemia decreases and chronic hypoglycemia increases brain glucose uptake (McCall 1992). Further, mass action should be most significant under conditions where extracellular glucose concentration is below the Km for the glucose transporter. In brain, the major glucose transport proteins are considered to be GLUT1 and GLUT3 (Simpson et al. 2007, Zhang et al. 2014). The Km of these transporters for glucose is generally in the 1–3 mM range (Augustin 2010, Thorens & Mueckler 2010), i.e., lower than the blood glucose concentration in anx/anx and wt mice. Therefore, it is unlikely that the lower blood glucose concentration is primarily responsible for the marked decrease in glucose uptake in anx/anx mice.
An alternative possibility is that there is a decreased availability of GLUT proteins in anx/anx mice. However, neither GLUT1 nor GLUT3 protein levels are decreased in the anx/anx hypothalamus. Consistent with our results is the finding that the decreased brain glucose uptake in hyperglycemic db/db mice is also not associated with decreases in brain GLUT1 or GLUT3 protein levels (Vannucci et al. 1997). Indeed, under normoglycemic conditions in adult normal animals, the levels of glucose transporter proteins are not considered to limit brain glucose utilization (Vannucci et al. 1997).
Finally, intracellular variables related to metabolic rate may explain the differences in glucose uptake. It is well documented that the metabolic rate/energy turnover is a key factor in determining glucose transport and utilization in cells. This is particularly clear in skeletal muscle where glucose uptake can increase up to 50-fold during maximal exercise (Katz et al. 1986), and the same principle holds for neurons (see introduction). Thus, the lower rate of glucose uptake in anx/anx mice may primarily be derived from a lower metabolic rate. The metabolic profile of the anx/anx hypothalamus is consistent with this explanation (increased PCr, decreased lactate content and lower AMPK-P/AMPK-T). The question is how a lower metabolic rate would directly affect glucose transport. One possibility is via diminished activation of AMPK, resulting in less translocation of GLUT4 to the plasma membrane, which has been shown in skeletal muscle (Kurth-Kraczek et al. 1999). Although hypothalamic AMPK has been implicated in food intake (Minokoshi et al. 2004, Kahn et al. 2005) to our knowledge, evidence for a role for AMPK in control of glucose transport in hypothalamus has not been reported. Alternatively, a block in glycolysis (for example at phosphofructokinase) could result in the inhibition of glucose phosphorylation (Randle et al. 1964, Katz et al. 1986, Hue & Taegtmeyer 2009), resulting in an accumulation of glucose in the brain. This would decrease the concentration gradient for glucose and thereby decrease glucose uptake. Indeed, measurements in db/db mouse brain show that the decreased glucose utilization is associated with a marked increase in brain glucose levels, resulting in a decrease of about 30% in the plasma/brain glucose ratio (Vannucci et al. 1997). Conversely, the increased brain glucose uptake observed during chronic hypoglycemia is associated with a marked decrease in the brain glucose levels (McCall et al. 1986). Thus, the decreased glucose uptake in anx/anx hypothalamus likely stems from a hypometabolic state, possibly owing to an inhibition of glycolysis. However, this awaits experimental confirmation.
Interestingly, AMPK-T and GLUT4 protein expressions are both elevated in the anx/anx hypothalamus. Such adaptations have been seen in skeletal muscle in response to increases in metabolic stress, such as chronic exercise (Richter & Ruderman 2009, Richter & Hargreaves 2013). Thus, the elevated expression of the two proteins in the presence of hypometabolism is unexpected, and the mechanism for this is not clear at present.
The lower AMPK-P/AMPK-T ratio observed in the anx/anx hypothalamus compared to wt is indicative of lower AMPK activation. AMPK is not only of importance in regulating cellular energy status (as discussed previously) but is also implicated in whole body energy homeostasis and hypothalamic systems regulating food intake (Claret et al. 2007). Noteworthy is that the activity of AMPK (as judged by increase in the AMPK-P/AMPK-T ratio) has been shown to increase during fasting to enhance feeding (Kahn et al. 2005) and decrease after refeeding (Ronnett et al. 2009), and lack of AMPK expression in the hypothalamus results in reduced food intake and body weight (Minokoshi et al. 2004). Although AMPK-T was elevated in the anx/anx hypothalamus, the activation level of the enzyme was decreased in the basal state, and this could contribute to the anorexia of the anx/anx mouse.
We conclude that hypothalamic metabolism is reduced in the anorectic anx/anx mouse and that this may contribute to their inability to regulate food intake according to energy status and the premature death of these animals. A potential mechanism underlying this phenomena involving mitochondrial CI dysfunction and a specific subtype of K-ATP channel is discussed previously, but further studies are needed to identify the exact mechanism for the hypometabolism. Further, it is not known whether, or to what extent, activation of microglia, neuroinflammation, neurodegeneration and peripheral hormones (e.g., leptin) contribute to the hypometabolism. Additional research is also required to examine the basis for the reduced glucose uptake in other brain regions, i.e. motor cortex and thalamus. It also remains to be determined if hypometabolism pertains to the whole hypothalamus or to specific hypothalamic cells, i.e. neurons or glia, or even specific neuronal populations such as the orexigenic NPY/AGRP neurons and other populations expressing the specific K-ATP channel. Nevertheless, in view of the findings of the present study, we feel that imaging investigations of hypothalamic activity in humans with anorectic conditions, e.g. anorexia nervosa, are warranted.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
The work presented in this paper was supported by funding from the Karolinska Institutet (KI), the Karolinska University Hospital, Hjärnfonden/Swedish Brain Foundation, Drottning Silvias Jubileums Fond, and the following Foundations: Magnus Bergvall, Tore Nilsson, Thuring, Mjölkdroppen, Märtha Lundqvist and Eva and Oscar Ahrén.
Author contribution statement
The study was designed by J E J, A K, M Sc, R W, M Sv, J H and I A K N. Experiments were performed and analyzed by C L, U B, M Sv, J E J, R W and I A K N. The manuscript was written by C L, U B, A K and I A K N and was edited by all authors.
Acknowledgements
The authors thank Vahid Edrisi, Åsa Södergren and Siv Eriksson for their invaluable help with the laboratory work and Dr Jan Mulder (The Human Protein Atlas, Science for Life Labs/Karolinska Institutet) for generously providing the GLUT3 antisera.
References
Alp PR, Newsholme EA & Zammit VA 1976 Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates. Biochemical Journal 154 689–700. (doi:10.1042/bj1540689)
Andrews ZB, Liu ZW, Walllingford N, Erion DM, Borok E, Friedman JM, Tschop MH, Shanabrough M, Cline G & Shulman GI et al. 2008 UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals. Nature 454 846–851. (doi:10.1038/nature07181)
Attwell D & Laughlin SB 2001 An energy budget for signaling in the grey matter of the brain. Journal of Cerebral Blood Flow and Metabolism 21 1133–1145. (doi:10.1097/00004647-200110000-00001)
Augustin R 2010 The protein family of glucose transport facilitators: it’s not only about glucose after all. IUBMB Life 62 315–333. (doi.org/10.1002/iub.315)
Beglopoulos V, Tulloch J, Roe AD, Daumas S, Ferrington L, Watson R, Fan Z, Hyman BT, Kelly PA & Bard F et al. 2016 Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice. Nature Communications 7 11761. (doi:10.1038/ncomms11761)
Broberger C, Johansen J, Schalling M & Hokfelt T 1997 Hypothalamic neurohistochemistry of the murine anorexia (anx/anx) mutation: altered processing of neuropeptide Y in the arcuate nucleus. Journal of Comparative Neurology 387 124–135. (doi:10.1002/(SICI)1096-9861(19971013)387:1<124::AID-CNE10>3.0.CO;2-U)
Broberger C, Johansen J, Johansson C, Schalling M & Hokfelt T 1998 The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. PNAS 95 15043–15048. (doi:10.1073/pnas.95.25.15043)
Broberger C, Johansen J, Brismar H, Johansson C, Schalling M & Hokfelt T 1999 Changes in neuropeptide Y receptors and pro-opiomelanocortin in the anorexia (anx/anx) mouse hypothalamus. Journal of Neuroscience 19 7130–7139.
Chandrasekaran K, Hatanpaa K, Brady DR & Rapoport SI 1996 Evidence for physiological down-regulation of brain oxidative phosphorylation in Alzheimer’s disease. Experimental Neurology 142 80–88. (doi:10.1006/exnr.1996.0180)
Claret M, Smith MA, Batterham RL, Selman C, Choudhury AI, Fryer LG, Clements M, Al-Qassab H, Heffron H & Xu AW et al. 2007 AMPK is essential for energy homeostasis regulation and glucose sensing by POMC and AgRP neurons. Journal of Clinical Investigation 117 2325–2336. (doi:10.1172/JCI31516)
Cunnane S, Nugent S, Roy M, Courchesne-Loyer A, Croteau E, Tremblay S, Castellano A, Pifferi F, Bocti C & Paquet N et al. 2011 Brain fuel metabolism, aging, and Alzheimer’s disease. Nutrition 27 3–20. (doi:10.1016/j.nut.2010.07.021)
Fetissov SO, Bergstrom U, Johansen JE, Hokfelt T, Schalling M & Ranscht B 2005 Alterations of arcuate nucleus neuropeptidergic development in contactin-deficient mice: comparison with anorexia and food-deprived mice. European Journal of Neuroscience 22 3217–3228. (doi:10.1111/j.1460-9568.2005.04513.x)
Frank EA, Shubha MC & D’Souza CJ 2012 Blood glucose determination: plasma or serum? Journal of Clinical Laboratory Analysis 26 317–320. (doi:10.1002/jcla.21524)
Gilbert E, Tang JM, Ludvig N & Bergold PJ 2006 Elevated lactate suppresses neuronal firing in vivo and inhibits glucose metabolism in hippocampal slice cultures. Brain Research 1117 213–223. (doi:10.1016/j.brainres.2006.07.107)
Grill HJ & Kaplan JM 2002 The neuroanatomical axis for control of energy balance. Frontiers in Neuroendocrinology 23 2–40. (doi:10.1006/frne.2001.0224)
Healy JE, Gearhart CN, Bateman JL, Handa RJ & Florant GL 2011 AMPK and ACC change with fasting and physiological condition in euthermic and hibernating golden-mantled ground squirrels (Callospermophilus lateralis). Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology 159 322–331. (doi:10.1016/j.cbpa.2011.03.026)
Hillebrand JJ, de Wied D & Adan RA 2002 Neuropeptides, food intake and body weight regulation: a hypothalamic focus. Peptides 23 2283–2306. (doi:10.1016/S0196-9781(02)00269-3)
Horvath TL, Andrews ZB & Diano S 2009 Fuel utilization by hypothalamic neurons: roles for ROS. Trends in Endocrinology and Metabolism 20 78–87. (doi:10.1016/j.tem.2008.10.003)
Hue L & Taegtmeyer H 2009 The randle cycle revisited: a new head for an old hat. American Journal of Physiology: Endocrinology and Metabolism 297 E578–E591. (doi.org/10.1152/ajpendo.00093.2009)
Ibrahim N, Bosch MA, Smart JL, Qiu J, Rubinstein M, Ronnekleiv OK, Low MJ & Kelly MJ 2003 Hypothalamic proopiomelanocortin neurons are glucose responsive and express K(ATP) channels. Endocrinology 144 1331–1340. (doi:10.1210/en.2002-221033)
Jeppesen TD, Quistorff B, Wibrand F & Vissing J 2007 31P-MRS of skeletal muscle is not a sensitive diagnostic test for mitochondrial myopathy. Journal of Neurology 254 29–37. (doi:10.1007/s00415-006-0229-5)
Johansen JE, Broberger C, Lavebratt C, Johansson C, Kuhar MJ, Hokfelt T & Schalling M 2000 Hypothalamic CART and serum leptin levels are reduced in the anorectic (anx/anx) mouse. Brain Research: Molecular Brain Research 84 97–105. (doi:10.1016/S0169-328X(00)00228-X)
Johansen JE, Fetissov SO, Bergstrom U, Nilsson I, Fay C, Ranscht B, Hokfelt T & Schalling M 2007 Evidence for hypothalamic dysregulation in mouse models of anorexia as well as in humans. Physiology and Behavior 92 278–282. (doi:10.1016/j.physbeh.2007.05.019)
Kahn BB, Alquier T, Carling D & Hardie DG 2005 AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism 1 15–25. (doi:10.1016/j.cmet.2004.12.003)
Katz A 1988 G-1,6-P2, glycolysis, and energy metabolism during circulatory occlusion in human skeletal muscle. American Journal of Physiology 255 C140–C144.
Katz A, Broberg S, Sahlin K & Wahren J 1986 Leg glucose uptake during maximal dynamic exercise in humans. American Journal of Physiology 251 E65–E70.
Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ & Winder WW 1999 5′ AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48 1667–1671. (doi:10.2337/diabetes.48.8.1667)
Lachuer J, Ouyang L, Legras C, Del Rio J & Barlow C 2005 Gene expression profiling reveals an inflammatory process in the anx/anx mutant mice. Brain Research: Molecular Brain Research 139 372–376. (doi:10.1016/j.molbrainres.2005.06.003)
Lin TA, Zhang JP & Sun GY 1993 Metabolism of inositol 1,4,5-trisphosphate in mouse brain due to decapitation ischemic insult: effects of acute lithium administration and temporal relationship to diacylglycerols, free fatty acids and energy metabolites. Brain Research 606 200–206. (doi:10.1016/0006-8993(93)90985-V)
Lindfors C, Nilsson IA, Garcia-Roves PM, Zuberi AR, Karimi M, Donahue LR, Roopenian DC, Mulder J, Uhlen M & Ekstrom TJ et al. 2011 Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse. PNAS 108 18108–18113. (doi:10.1073/pnas.1114863108)
Lindfors C, Katz A, Selander L, Johansen JE, Marconi G, Schalling M, Hokfelt T, Berggren PO, Zaitsev S & Nilsson IA 2015 Glucose intolerance and pancreatic beta-cell dysfunction in the anorectic anx/anx mouse. American Journal of Physiology: Endocrinology and Metabolism 309 E418–E427. (doi:10.1152/ajpendo.00081.2015)
Liss B, Bruns R & Roeper J 1999 Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons. EMBO Journal 18 833–846. (doi:10.1093/emboj/18.4.833)
Lowry OH 1972 A Flexible System of Enzymatic Analysis. Orlando, FL, USA: Academic Press.
Lowry OH, Passonneau JV, Hasselberger FX & Schulz DW 1964 Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. Journal of Biological Chemistry 239 18–30.
Lutas A, Birnbaumer L & Yellen G 2014 Metabolism regulates the spontaneous firing of substantia nigra pars reticulata neurons via KATP and nonselective cation channels. Journal of Neuroscience 34 16336–16347. (doi:10.1523/JNEUROSCI.1357-14.2014)
Maltais LJ, Lane PW & Beamer WG 1984 Anorexia, a recessive mutation causing starvation in preweanling mice. Journal of Heredity 75 468–472.
McCall AL 1992 The impact of diabetes on the CNS. Diabetes 41 557–570. (doi:10.2337/diab.41.5.557)
McCall AL, Fixman LB, Fleming N, Tornheim K, Chick W & Ruderman NB 1986 Chronic hypoglycemia increases brain glucose transport. American Journal of Physiology 251 E442–E447.
Mercader JM, Lozano JJ, Sumoy L, Dierssen M, Visa J, Gratacos M & Estivill X 2008a Hypothalamus transcriptome profile suggests an anorexia-cachexia syndrome in the anx/anx mouse model. Physiological Genomics 35 341–350. (doi:10.1152/physiolgenomics.90255.2008)
Mercader JM, Saus E, Aguera Z, Bayes M, Boni C, Carreras A, Cellini E, de Cid R, Dierssen M & Escaramis G et al. 2008b Association of NTRK3 and its interaction with NGF suggest an altered cross-regulation of the neurotrophin signaling pathway in eating disorders. Human Molecular Genetics 17 1234–1244. (doi:10.1093/hmg/ddn013)
Miki T, Liss B, Minami K, Shiuchi T, Saraya A, Kashima Y, Horiuchi M, Ashcroft F, Minokoshi Y & Roeper J et al. 2001 ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis. Nature Neuroscience 4 507–512.
Milone M & Wong LJ 2013 Diagnosis of mitochondrial myopathies. Molecular Genetics and Metabolism 110 35–41. (doi:10.1016/j.ymgme.2013.07.007)
Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P & Birnbaum MJ et al. 2004 AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428 569–574. (doi:10.1038/nature02440)
Miyamoto S, Leipzig JN, Lieberman JA & Duncan GE 2000 Effects of ketamine, MK-801, and amphetamine on regional brain 2-deoxyglucose uptake in freely moving mice. Neuropsychopharmacology 22 400–412. (doi:10.1016/S0893-133X(99)00127-X)
Morton GJ, Cummings DE, Baskin DG, Barsh GS & Schwartz MW 2006 Central nervous system control of food intake and body weight. Nature 443 289–295. (doi:10.1038/nature05026)
Nilsson I, Lindfors C, Fetissov SO, Hokfelt T & Johansen JE 2008 Aberrant agouti-related protein system in the hypothalamus of the anx/anx mouse is associated with activation of microglia. Journal of Comparative Neurology 507 1128–1140. (doi:10.1002/cne.21599)
Nilsson IA, Thams S, Lindfors C, Bergstrand A, Cullheim S, Hokfelt T & Johansen JE 2011 Evidence of hypothalamic degeneration in the anorectic anx/anx mouse. Glia 59 45–57. (doi:10.1002/glia.21075)
Randle PJ, Newsholme EA & Garland PB 1964 Regulation of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles. Biochemical Journal 93 652–665. (doi:10.1042/bj0930652)
Richter EA & Hargreaves M 2013 Exercise, GLUT4, and skeletal muscle glucose uptake. Physiological Reviews 93 993–1017. (doi:10.1152/physrev.00038.2012)
Richter EA & Ruderman NB 2009 AMPK and the biochemistry of exercise: implications for human health and disease. Biochemical Journal 418 261–275. (doi:10.1042/BJ20082055)
Ronnett GV, Ramamurthy S, Kleman AM, Landree LE & Aja S 2009 AMPK in the brain: its roles in energy balance and neuroprotection. Journal of Neurochemistry 109 (Supplement 1) 17–23. (doi:10.1111/j.1471-4159.2009.05916.x)
Sahlin K & Katz A 1989 Hypoxaemia increases the accumulation of inosine monophosphate (IMP) in human skeletal muscle during submaximal exercise. Acta Physiologica Scandinavica 136 199–203. (doi:10.1111/j.1748-1716.1989.tb08653.x)
Schreiber SS & Baudry M 1995 Selective neuronal vulnerability in the hippocampus – a role for gene expression? Trends in Neuroscience 18 446–451. (doi:10.1016/0166-2236(95)94495-Q)
Schwartz MW 2006 Central nervous system regulation of food intake. Obesity 14 (Supplement 1) 1S–8S. (doi:10.1038/oby.2006.275)
Schweinsberg PD & Loo TL 1980 Simultaneous analysis of ATP, ADP, AMP, and other purines in human erythrocytes by high-performance liquid chromatography. Journal of Chromatography 181 103–107. (doi:10.1016/S0378-4347(00)81276-1)
Sengupta B, Stemmler M, Laughlin SB & Niven JE 2010 Action potential energy efficiency varies among neuron types in vertebrates and invertebrates. PLoS Computational Biology 6 e1000840. (doi:10.1371/journal.pcbi.1000840)
Silver IA & Erecinska M 1998 Glucose-induced intracellular ion changes in sugar-sensitive hypothalamic neurons. Journal of Neurophysiology 79 1733–1745.
Simpson IA, Carruthers A & Vannucci SJ 2007 Supply and demand in cerebral energy metabolism: the role of nutrient transporters. Journal of Cerebral Blood Flow and Metabolism 27 1766–1791. (doi:10.1038/sj.jcbfm.9600521)
Son JH, Baker H, Park DH & Joh TH 1994 Drastic and selective hyperinnervation of central serotonergic neurons in a lethal neurodevelopmental mouse mutant, Anorexia (anx). Brain Research: Molecular Brain Research 25 129–134. (doi:10.1016/0169-328X(94)90287-9)
Spencer MK & Katz A 1991 Role of glycogen in control of glycolysis and IMP formation in human muscle during exercise. American Journal of Physiology 260 E859–E864.
Tenney JR, Rozhkov L, Horn P, Miles L & Miles MV 2014 Cerebral glucose hypometabolism is associated with mitochondrial dysfunction in patients with intractable epilepsy and cortical dysplasia. Epilepsia 55 1415–1422. (doi:10.1111/epi.12731)
Thorens B & Mueckler M 2010 Glucose transporters in the 21st Century. American Journal of Physiology: Endocrinology and Metabolism 298 E141–E145. (doi.org/10.1152/ajpendo.00712.2009)
Toyama H, Ichise M, Liow JS, Modell KJ, Vines DC, Esaki T, Cook M, Seidel J, Sokoloff L & Green MV et al. 2004 Absolute quantification of regional cerebral glucose utilization in mice by 18F-FDG small animal PET scanning and 2-14C-DG autoradiography. Journal of Nuclear Medicine 45 1398–1405.
van den Top M, Lyons DJ, Lee K, Coderre E, Renaud LP & Spanswick D 2007 Pharmacological and molecular characterization of ATP-sensitive K(+) conductances in CART and NPY/AgRP expressing neurons of the hypothalamic arcuate nucleus. Neuroscience 144 815–824. (doi:10.1016/j.neuroscience.2006.09.059)
Vannucci SJ, Gibbs EM & Simpson IA 1997 Glucose utilization and glucose transporter proteins GLUT-1 and GLUT-3 in brains of diabetic (db/db) mice. American Journal of Physiology 272 E267–E274.
Waldron AM, Wintmolders C, Bottelbergs A, Kelley JB, Schmidt ME, Stroobants S, Langlois X & Staelens S 2015 In vivo molecular neuroimaging of glucose utilization and its association with fibrillar amyloid-beta load in aged APPPS1-21 mice. Alzheimer’s Research and Therapy 7 76. (doi.org/10.1186/s13195-015-0158-6)
Wibom R, Hagenfeldt L & von Dobeln U 2002 Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples. Analytical Biochemistry 311 139–151. (doi:10.1016/S0003-2697(02)00424-4)
Wong KP, Sha W, Zhang X & Huang SC 2011 Effects of administration route, dietary condition, and blood glucose level on kinetics and uptake of 18F-FDG in mice. Journal of Nuclear Medicine 52 800–807. (doi:10.2967/jnumed.110.085092)
Wredenberg A, Freyer C, Sandstrom ME, Katz A, Wibom R, Westerblad H & Larsson NG 2006 Respiratory chain dysfunction in skeletal muscle does not cause insulin resistance. Biochemical and Biophysical Research Communications 350 202–207. (doi:10.1016/j.bbrc.2006.09.029)
Zhang S, Zuo W, Guo XF, He WB & Chen NH 2014 Cerebral glucose transporter: the possible therapeutic target for ischemic stroke. Neurochemistry International 70 22–29. (doi:10.1016/j.neuint.2014.03.007)
