Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 50, Num. 4, 2002, pp. 452-458

Neurology India, Vol. 50, No. 4, Dec, 2002, pp. 452-458

Hypothalamic Digoxin and Regulation of Body Mass Index

A. Ravi Kumar, P.A. Kurup*

Department of Neurology, Medical College Hospital and Metabolic Disorders Research Centre*, Trivandrum, Kerala, India.
Correspondence to : Dr. P.A. Kurup, Gouri Sadan, T.C.4/1525, North of Cliff House, Kattu Road, Kowdiar Trivandrum, Kerala, India.

Accepted for publication : 3rd August, 2001.

Code Number: ni020121

Summary

The hypothalamus produces digoxin, an endogenous membrane Na⁺-K⁺ ATPase inhibitor and regulator of neurotransmission. Digoxin being a steroidal glycoside, is synthesised by the isoprenoid pathway. In view of the reports of elevated digoxin levels in metabolic syndrome X with high body mass index, the isoprenoid pathway mediated biochemical cascade was assessed in individuals with high and low body mass index. It was also assessed in individuals with differing hemispheric dominance to find out the relationship between digoxin status, body mass index and hemispheric dominance. The isoprenoid pathway metabolites, tryptophan / tyrosine catabolic patterns and membrane composition were assessed. In individuals with high body mass index an upregulated isoprenoid pathway with increased HMG CoA reductase activity, serum digoxin and dolichol levels and low ubiquinone levels were observed. The RBC membrane Na⁺-K⁺ ATPase activity and serum magnesium levels were decreased. The tyrosine catabolites (dopamine, morphine, epinephrine and norepinephrine) were reduced and the tryptophan catabolites (serotonin, quinolinic acid, strychnine and nicotine) were increased. There was an increase in membrane cholesterol : phospholipid ratio and a reduction in membrane glycoconjugates in individuals with high body mass index. The reverse patterns were seen in individuals with low body mass index. The patterns in individuals with high body mass index and low body mass index correlated with right hemispheric dominance and left hemispheric dominance respectively. Hemispheric dominance and digoxin status regulates the differential metabolic pattern observed in individuals with high and low body mass index.

Key words : Digoxin, Dolichol, Ubiquinone, Body mass index, Hemispheric dominance.

Introduction

Metabolic syndrome X is associated with trunkal obesity and high body mass index. Increased secretion of an endogenous digoxin like factor (EDLF) has been shown in syndrome X.¹ EDLF is secreted by the hypothalamus and functions as the endogenous regulator of membrane sodium-potassium ATPase and synaptic neurotransmission.^2,3 Studies from this laboratory have demonstrated that the EDLF is chemically the steroidal glycoside digoxin, and that it is synthesised by the isoprenoid pathway.^4,5 The isoprenoid pathway also synthesises three other metabolites important in cellular regulation - dolichol, ubiquinone and cholesterol. We have clinically documented a group of individuals with low body mass index. It was therefore considered pertinent to study the isoprenoid pathway related biochemical cascade - digoxin, dolichol and ubiquinone levels, neurotransmitter patterns and membrane composition in individuals with high and low body mass index. Since hypothalamic digoxin can regulate neuronal transmission the isoprenoid pathway and its related cascade was assessed in individuals with right hemispheric, left hemispheric and bihemispheric dominance to find out the correlation between hemispheric dominance, metabolic status and body mass index.

Material and Methods

The study was designed as a case control study. Four sets of population free from any systemic illness were included in the study : (i) 15 male individuals aged between 20-30 years with trunkal obesity and high body mass index (body mass index of 30-39.9 kg/m²). (ii) 15 male individuals aged between 20-30 years with low body mass index (less than 18.5 kg/m²) (iii) 15 male individuals aged between 20-30 years with normal body mass index (between 18.5 to 24.9 kg/m²) were chosen as control. (iv) 15 male individuals aged between 20-30 years each with right hemispheric, bihemispheric and left hemispheric dominance chosen by the dichotic listening test. The survey was conducted over the period 1999-2000 and it was a house to house survey and involved a population of 2000. The individuals were chosen by random sampling. None of the subjects studied was under any medication. All subjects chosen for the study were non-smokers (active or passive). Blood sample was collected in fasting state in citrate tubes. RBCs were separated within one hour of collection of blood for the estimation of membrane Na⁺-K⁺ ATPase. Serum was used for the analysis of various parameters. All chemicals used in this study were obtained from M/s.Sigma Chemicals, USA. Activity of HMG CoA reductase of the serum was determined by the method of Rao and Ramakrishnan by determining the ratio of HMG CoA to mevalonate.⁶ RBC Na⁺-K⁺ ATPase activity of the erythrocyte membrane was tested by the procedure described by Wallach and Kamat.⁷ Digoxin in the serum was determined by the procedure described by Arun et al,⁴ and ubiquinone and dolichol by method described by Palmer et al.⁸ Magnesium in the serum was estimated by atomic absorption spectrophotometry. Tryptophan, tyrosine, serotonin and catecholamines were estimated as described in Methods of biochemical analysis.⁹ Quinolinic acid content of serum was estimated by HPLC (C18 column micro Bondapak^TM 4.6 x 150 mm), solvent system 0.01 M acetate buffer (pH3.0) and methanol (6:4), flow rate 1.0 ml/minute and detection UV 250 nm). Morphine, strychnine and nicotine were estimated by the method described by Arun et al.¹⁰ The procedures used for the estimation of total and individual GAG, and carbohydrate components of glycoproteins are as described in Methods in biochemical analysis.⁹ Phospholipid were estimated by the method of Zilversmits and Davis¹¹ and cholesterol was estimated by using commercial kits supplied by Sigma Chemicals, USA. Statistical analysis was done by student's 't' test.

Results

The results showed that serum HMG CoA reductase activity, digoxin, cholesterol and dolichol levels were increased and RBC membrane Na⁺-K⁺ ATPase activity, serum ubiquinone and magnesium were reduced in individuals with high body mass index and right hemispheric dominance. The blood glucose values were significantly higher in individuals with high body mass index and right hemispheric dominance. The results showed that serum HMG CoA reductase activity, digoxin, cholesterol and dolichol levels were decreased and RBC membrane Na⁺-K⁺ ATPase activity, serum ubiquinone and magnesium were increased in individuals with low body mass index and left hemispheric dominance. The blood glucose values were significantly lower in individuals with low body mass index and left hemispheric dominance (Table I).

The concentration of tryptophan and its catabolites (serotonin, quinolinic acid, nicotine and strychnine) was found to be higher in the serum of individuals with high body mass index and right hemispheric dominance while that of tyrosine and its catabolites (epinephrine, norepinephrine, dopamine and morphine) was lower. The reverse patterns were obtained in individuals with low body mass index and left hemispheric dominance (Table I and II).

The cholesterol : phospholipid ratio of the RBC membrane was increased in individuals with high body mass index. The concentration of total GAG, hexose and fucose of glycoprotein was decreased in the RBC membrane in individuals with high body mass index. The reverse patterns were obtained in individuals with low body mass index (Table III).

Discussion

There was significant inhibition of the RBC membrane Na⁺-K⁺ ATPase in individuals with high body mass index. There is a close correlation between elevated digoxin levels and reduced RBC membrane Na⁺-K⁺ ATPase activity. There is increased digoxin synthesis in individuals with high body mass index as evidenced by increased HMG CoA reductase activity, the rate limiting enzyme of the isoprenoid pathway. The upregulation of the isoprenoid pathway is also indicated by the increase in the other isoprenoidal metabolites - cholesterol and dolichol in individuals with high body mass index. Studies in our laboratory using¹⁴C labeled acetate has demonstrated that digoxin is synthesised by the isoprenoid pathway.⁵ The inhibition of membrane Na⁺-K⁺ ATPase by digoxin is known to cause an increase in intracellular calcium resulting from increased Na⁺-Ca⁺⁺ exchange,¹⁹ which by displacing magnesium from its binding sites, causes a decrease in the functional availability of magnesium.¹² This causes decreased mitochondrial ATP formation, which along with low magnesium can cause further inhibition of membrane Na⁺-K⁺ ATPase, since ATP-magnesium complex is the actual substrate for this reaction. There is thus a progressive inhibition of membrane Na⁺-K⁺ ATPase activity first triggered by digoxin.¹² Serum magnesium in individuals with high body mass index was found to be reduced. On the other hand in individuals with low body mass index the reverse patterns were obtained. There was decreased digoxin synthesis and consequent stimulation of membrane Na⁺-K⁺ ATPase, resulting in decreased intracellular calcium / increased intracellular magnesium stores. There was reduced digoxin synthesis as indicated by a decrease in the activity of HMG CoA reductase, the rate limiting enzyme of the isoprenoid pathway. Serum magnesium was elevated in individuals with low body mass index.

There was an increase in tryptophan and its catabolites (serotonin, quinolinic acid, nicotine and strychnine) and a reduction in tyrosine and its catabolites (epinephrine, norepinephrine, dopamine and morphine) in the serum of individuals with high body mass index which could be due to the fact that digoxin can regulate neutral aminoacid transport system, with a preferential promotion of tryptophan transport over tyrosine.³ The intestinal absorption and renal tubular re-absorption of tryptophan is preferentially upregulated over tyrosine. This results in increased loss of tyrosine in the stool and in the urine. Therefore the serum levels of tryptophan is elevated and tyrosine reduced in individuals with hyperdigoxinemia and high body mass index. The decrease in membrane Na⁺-K⁺ ATPase activity in individuals with high body mass index could be due to the fact that the hyperpolarising neurotransmitters (dopamine morphine and noradrenaline) are reduced and the depolarising neuroactive compounds (serotonin, strychnine, nicotine and quinolinic acid) are increased. The Schizoid neurotransmitter pattern of reduced dopamine, noradrenaline and morphine and increased serotonin, strychnine and nicotine is common to individuals with high body mass index and schizophrenia and could predispose to its development.¹³ In the presence of hypomagnesemia, the magnesium block on the NMDA receptor is removed leading to NMDA excitotoxicity.¹⁴ The increase in positive modulators of the NMDA receptor - serotonin, quinolinic acid and strychnine would also lead to increased glutamatergic transmission. Thus, in individuals with high body mass index with hyperdigoxinemic state there is upregulated serotoninergic, cholinergic and glutamatergic transmission and downregulated dopaminergic, glycinergic, adrenergic and noradrenergic transmission. On the other hand the reverse patterns were obtained in patients with low body mass index with a hypodigoxinemia induced increase in tyrosine catabolites (dopamine, epinephrine, norepinephrine and morphine) over tryptophan catabolites (serotonin, quinolinic acid, strychnine and nicotine) contributing to membrane Na⁺-K⁺ ATPase stimulation. Serum tyrosine levels are elevated compared to serum tryptophan levels in individuals with low body mass index. Hypermagnesemia could also inhibit NMDA transmission.¹⁴ The decrease in positive modulators of the NMDA receptor - serotonin, quinolinic acid and strychnine can down regulate glutamatergic transmission in individuals with low body mass index. Low serotonin is associated with psychological states of depression and obsessive compulsive disorder which could predispose to the development of low body mass index.¹³ Thus in the low body mass index state there is upregulated dopaminergic, noradrenergic, adrenergic and morphinergic transmission and down regulated serotoninergic, cholinergic and glutamatergic transmission. There are no previous reports correlating body mass index with neurotransmitter patterns.

The biochemical patterns in individuals with a high body mass index correlated well with right hemispheric dominance, which is associated with an upregulated isoprenoid pathway and hyperdigoxinemia. The biochemical patterns in individuals with a low body mass index correlated well with left hemispheric dominance, which is associated with a downregulated isoprenoid pathway and hypodigoxinemia. Hemispheric dominance may play a vital role in determining body mass index, metabolic status and risk for vascular thrombosis. It could also modulate insulin resistance and development of non insulin dependent diabetes mellitus. There are no previous reports relating hemispheric dominance to metabolic syndrome X.

Magnesium is required as a co-factor for cell membrane glucose transport.¹⁵ Hypomagnesemia can lead on to defective cell membrane transport of glucose. Alteration in cellular membrane composition reported here can also inhibit the membrane transport of glucose. Increased intracellular calcium can activate the G-protein coupled signal transduction of the contrainsulin hormones (growth hormone and glucagon) leading to hyperglycemia. Magnesium translocation appears to be an early event in insulin action.¹⁵ Decrease in intracellular magnesium can block the phosphorylation reactions involved in protein tyrosine kinase receptor activity leading to insulin resistance.¹⁶ Alteration in cell membrane composition reported here can also modulate the insulin receptor leading on to insulin resistance. Decrease in intracellular magnesium can lead on to inhibition of the glycolytic pathway. Increase in intracellular calcium can open up the mitochondrial PT pore and block oxidative phosphorylation.¹⁷ This leads to defective glucose utilisation and contributes to the development of non-insulin dependent diabetes mellitus, common in individuals with high body mass index. Increase in beta cell calcium can contribute to increased insulin release from beta cells and hyperinsulinemia.¹⁸ Hypomagnesemia has been reported to markedly increased glucose stimulated insulin secretion by the perfused pancreas.¹⁸ The blood glucose levels were high in individuals with high body mass index. Decreased intracellular magnesium can produce dysfunction of lipoprotein lipase producing defective catabolism of triglycerides rich lipoproteins and hypertriglyceridemia.¹⁹ In hypomagnesemia lecithin cholesterol acyl transferase (LCAT) is defective and there is reduced formation of cholesterol esters in HDL.¹⁹ This results in reduced HDL cholesterol described in individuals with high body mass index. Magnesium deficiency has also been reported to increase LDL cholesterol levels.¹⁹ Nicotine administration has also been reported to produce significant changes in lipid metabolism.²⁰ There is increased tissue cholesterogenesis, decreased hepatic degradation of cholesterol and increased triglycerides synthesis. The uptake of circulating triglyceride rich lipoprotein is decreased as revealed by decreased activity of extra hepatic lipoprotein lipase. Plasma LCAT activity is also reduced on nicotine administration. HDL cholesterol is decreased while the LDL-VLDL cholesterol is increased. Serum cholesterol levels were high in individuals with high body mass index. The absence of morphine in individuals with high body mass index is also significant. Morphine has been reported to have an effect on glucose metabolism²¹ by increasing glucagon secretion, modulating insulin release from the beta cell and also independently through opioid / alpha-adrenergic receptor stimulation. These patterns are reversed in individuals with low body mass index. In individuals with low body mass index, intracellular hypermagnesemia consequent to membrane sodium potassium ATPase stimulation can promote glucose transport in to the cell, increase the activity of insulin receptor, promote mitochondrial oxidative phosphorylation and increase glucose utilisation. Blood glucose levels were low in individuals with low body mass index. Increase in intracellular magnesium can promote lipoprotein lipase activity promoting triglyceride catabolism and increase the efficiency of mitochondrial beta oxidation of fatty acids. The LCAT activity is increased while the HMG CoA reductase activity and cholesterol synthesis are decreased in intracellular hypermagnesemia. Serum cholesterol levels were low in individuals with low body mass index. Increase in morphine levels in individuals with low body mass index can promote glucose utilisation. Thus membrane Na⁺-K⁺ ATPase activity can modulate the insulin receptor activity and influence lipid and carbohydrate metabolism, important in the regulation of body mass index. Membrane Na⁺-K⁺ ATPase activity can also modulate the intracellular magnesium / calcium concentration within the vascular smooth muscle cell. In individuals with high body mass index membrane Na⁺-K⁺ ATPase inhibition can lead to depletion of intracellular magnesium contributing to vasospasm. Increase in intracellular calcium can increase the G-protein coupled activity of platelet activating factor and thrombin receptor contributing to thrombosis. This could relate membrane Na⁺-K⁺ ATPase inhibition and high body mass index with vascular thrombosis. The reverse holds good for low body mass index where there is a decreased predilection for vascular thrombosis. The upregulation of isoprenoid pathway in individuals with high body mass index can lead to increased cholesterol synthesis and magnesium deficiency can inhibit phospholipid synthesis leading to increased membrane cholesterol : phospholipid ratio. The concentration of total GAG, hexose and fucose content of glycoprotein decreased in the RBC membrane and increased in the plasma suggesting their reduced incorporation into the membrane and defective membrane formation. This is a consequence of defective membrane trafficking which depends upon GTPases and lipid kinases requiring magnesium as a cofactor and are defective in magnesium deficiency. The change in membrane structure produced by alteration in glycoconjugates and cholesterol : phospholipid ratio can produce changes in the conformation of membrane Na⁺-K⁺ ATPase resulting in further membrane Na⁺-K⁺ ATPase inhibition. The opposite patterns with hypermagnesemia induced decreased cholesterol synthesis, increased phospholipid synthesis and decreased membrane cholesterol : phospholipid ratio is noticed in individuals with low body mass index. Also the membrane glycoconjugates are increased and plasma glycoconjugates decreased owing to increased activity of trafficking enzymes consequent to hypermagnesemia. This leads on to further membrane Na⁺-K⁺ ATPase stimulation in individuals with low body mass index. Alteration in membrane structure can affect the transport of glucose in to the cell as well as modulate the function of the insulin receptor contributing insulin resistance.

The concentration of ubiquinone decreased significantly in individuals with high body mass index which may be the result of low tyrosine levels, reported in these individuals, consequent to digoxin's effect in preferentially promoting tryptophan transport over tyrosine.³ The aromatic ring portion of ubiquinone is derived from tyrosine. The other tyrosine metabolites - dopamine, epinephrine, norepinephrine and morphine - are also reduced in individuals with high body mass index. Ubiquinone is a important component of the mitochondrial electron transport chain, and its deficiency leads to mitochondrial oxidative phosphorylation defects, incomplete reduction of oxygen and generation of superoxide ion which produces lipid peroxidation. Ubiquinone deficiency also leads to reduced free radical scavenging. Thus there is decreased efficiency of mitochondrial oxidative phosphorylation in individuals with high body mass index. Increased generation of free radicals can oxidise LDL contributing to atherosclerosis described in metabolic syndrome X and in individuals with high body mass index. The patterns are reversed in individuals with low body mass index. The concentration of ubiquinone increased significantly in individuals with low body mass index which may be the result of increased tyrosine levels, consequent to digoxin deficiency.⁷ The other tyrosine metabolites - epinephrine, norepinephrine, dopamine and morphine - are also increased in individuals with low body mass index. There is improved mitochondrial function and efficiency leading to reduced free radical generation. Ubiquinone excess also leads to increased free radical scavenging. Decreased free radical mediated LDL oxidation can result in decreased incidence of vascular disease.

Thus body mass index depends on hemispheric dominance and alterations in the isoprenoid pathway. In individuals with high body mass index there is chemical right hemispheric dominance with an upregulated isoprenoid pathway, hyperdigoxinemia, increased tryptophan catabolism over tyrosine and decreased efficiency of mitochondrial oxidative phosphorylation. In individuals with low body mass index there is chemical left hemispheric dominance with an downregulated isoprenoid pathway, hypodigoxinemia, decreased tryptophan catabolism over tyrosine and increased efficiency of mitochondrial oxidative phosphorylation.

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