Investigations on the role of insulin and scorpion antivenom in scorpion envenoming syndrome

ORIGINAL PAPER

Investigations on the role of insulin and scorpion antivenom in scorpion envenoming syndrome

K. Radha Krishna Murthy; Dubey As; M. Zare Abbas; L. Haghnazari

Department of Physiology, Seth G.S.Medical College and K.E.M.Hospital, Parel, Mumbai 400 012, India

^{Correspondence}

ABSTRACT

Acute myocardiopathy in alloxan treated experimental dogs and rabbits was induced by subcutaneous (SQ) injection of scorpion venom from Mesobuthus tamulus concanesis, Pocock. Envenoming resulted in an initial transient hypertension (180-320 mm Hg.) followed by hypotension. Simultaneous administration of venom and species-specific scorpion antivenom (SAV) prevented hypertension and hypotension. Hypotension did not occur when SAV was given 60 min after envenoming. Blood glucose, triglycerides, cholesterol, amylase, insulin, glucagon, cortisol, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, red blood cell (RBC) count, hemoglobin (Hb), 2,3-diphosphoglycerate (2,3-DPG), and glutathione levels were increased 60 and 90 min after envenoming. Total white blood cell (WBC) count was reduced 60 min and increased 90 min after envenoming. Simultaneous administration of venom and SAV did not alter Hb, MCHC, and packed cell volume (PCV) levels, or ECG, and cardiovascular, biochemical, metabolic, and hormonal changes. Hematological parameters were reversed when SAV was given 30 and 60 min after envenoming. PCV, Hb, and MCHC values returned to normal 120 min after SAV. Alloxan-treated dogs showed increased blood glucose, cholesterol, glucagon, cortisol levels; reduced glycogen content of liver, cardiac and skeletal muscles; and reduced insulin levels and insulin/ glucagon ratio (I/G ratio). Envenoming in the alloxan pre-treated dogs further increased these levels and reduced tissue glycogen content, insulin levels, and I/G ratio. Administration of 4 U of insulin to alloxan pre-treated envenomed rabbits caused a biochemical and clinical improvement and increased glycogen content of all tissues in comparison with the values from those administered with SAV to alloxan pre-treated envenomed animals. SAV administration to envenomed alloxan pre-treated rabbits did not cause clinical or biochemical improvement. Severe scorpion envenoming causes an autonomic storm with a massive release of catecholamines and other counter-regulatory hormones; changes in insulin secretion resulting in fuel energy deficits producing multi-system-organ-failure (MSOF); and death. Administration of either insulin or SAV (through the release of insulin) appears to be the physiological basis for the control of the metabolic support to control the adverse effects triggered by counter-regulatory hormones.

Keywords:Mesobuthus tamulus concanesis, Pocock, insulin therapy, serotherapy, alloxan, tissue glycogen, blood pressure, hematological changes.

INTRODUCTION

Scorpion envenoming is common in tropical and subtropical regions, especially in Latin America, China, India, the Middle East, and North, Central and South Africa (1,2,4-6,10,16-20,22-24,26-28,36-64). An estimated number of 100,000 to 200,000 envenomations in Mexico; 24,000 to 45,000 scorpion stings in Algeria, Tunisia, Saudi Arabia and many other countries occur annually. Venomous scorpion infested regions comprise the majority of underdeveloped and developing countries. Much information is available in medicine textbooks about the differences between venomous and non-venomous snakes and management of snakebite, whereas these very texts dismiss scorpion-envenoming syndrome in a few sentences. No effort is made by the international scientific community to develop treatment protocols for scorpion envenoming syndrome or explain the pathophysiological effects of the venom. Scorpion stings and the resulting large number of deaths of the victims is not a major concern in these less developed countries because scientific and medical research is not a priority, which might account for lack of data. Use of certain drugs such as hydralazine, prazosin, and nifedipine in scorpion envenoming are claimed to reduce mortality. However, selection of these drugs is not based on a deeper understanding of the mechanisms of action of scorpion venom (19,24,26,27).

Metabolic and ECG changes induced by experimental scorpion envenoming have been reversed by the administration of either insulin alone (54,74) or insulin along with an alpha-blocker (43). Insulin administration reversed the hemodynamic changes and pulmonary edema in children stung by scorpion in South and Western India (57,70). Recent studies utilized insulin therapy in critically ill patients (7,11,40) and confirmed our hypothesis of the new uses of insulin (43,54,57,70).

Serotherapy for scorpion envenoming syndrome was the subject of much controversy. While the value of antivenom after snakebites was never questioned, opinions differed in case of scorpion stings. Many investigators consider antivenom the only specific treatment (1,10-12,19,20,24,26,27,36,37,39,40,42-48,50-57). Others, however, questioned the effectiveness of the antivenom in preventing and abolishing cardiovascular manifestations of human envenoming (6,19,22,23,59,60,63). We demonstrate the role of insulin and antivenom in scorpion envenoming syndrome.

MATERIALS AND METHODS

Lyophilized crude venom from the scorpion Mesobuthus tamulus concanesis, Pocock (earlier called Buthus tamulus) was purchased from the Haffkine Institute, Mumbai. SAV was produced at the Haffkine Biopharmaceutical Corporation Ltd., Mumbai. The whole contents of each SAV vial was reconstituted by the addition of 10 ml of water; 10 ml of reconstituted SAV can neutralize 12-18 mg of crude venom.

Forty-eight dogs (weight 8+2 kg) and 32 rabbits (weight 1.5 to 2.0 kg) were used in this study. All animals received food and water at libidum. After an overnight fast of 12-14 hours, the dogs were anesthetized with IV thiopentone sodium (35mg/kg). The experimental animals were randomly divided into eleven groups as described below.

Scorpion venom (3.5-mg/kg) was given subcutaneously (SQ.) to Groups 1, 2, 9, 10, and 11, while 3-mg/kg venom was given to Groups 3, 4, 5, and 6. SAV was given 0, 30, and 60 min after venom injection to Groups 4, 5, and 6, respectively.

Alloxan was given IV. Group 2 received 40 mg/kg of alloxan; Groups 9, 10 ,and 11 received 125 mg/kg of alloxan. These animals received scorpion venom 72 hours after alloxan treatment. Group 10 were given 2.5 ml of reconstituted SAV one hour after envenoming; Group 11 were given 4 Units of IV insulin 60 min after envenoming.

Group 7 did not receive alloxan and served as control. The remaining animals received alloxan and were divided into five groups as shown below.

Group 7 = control group of rabbits.

Group 8 = these rabbits were given alloxan.

Group 9 = the rabbits in this group were treated with alloxan as mentioned above for Group 8. Seventy-two hours later, the alloxan treated rabbits were injected with scorpion venom.

Group 10 = these rabbits were treated with alloxan and subsequently injected with scorpion venom as described for Group 9. SAV (2.5 ml) was given to these animals IV one hour after venom injection.

Group 11 = this group was injected with scorpion venom after alloxan treatment as described for Group 9. Four Units of crystalline insulin was given 60 min after venom injection.

Blood collection

Blood was collected from the dogs and rabbits as mentioned below.

Dogs

Group 1: before venom injection, 60, and 90 min after envenoming.

Group 2: before alloxan administration (control), 72 hours after alloxan treatment, and 60 min after venom injection.

Groups 3-6: blood was collected before venom injection , and thereafter, every 30 min for 2 hours after SAV.

Rabbits

Group 7: these rabbits served as control; they did not receive alloxan, scorpion venom, SAV, or insulin.

Group 8: blood collected was used to assess effects of alloxan treatment.

Group 9: blood was collected 1 hour after envenoming.

Group 10: blood was collected 1 hour after SAV administration.

Group 11: blood was collected 1 hour after insulin administration.

Blood samples were processed for blood sugar (8), triglycerides, cholesterol (67), alkaline phosphatase, amylase, glucose 6-phosphate dehydrogenase (G6PD), glutathione, LDH, 2,3 DPG (Miles India Ltd.), hematocrit, Hb, RBC, WBC, and platelet count (14,72). R I A kits from Diagnostic Products Corporation, Los Angeles, were used to measure insulin, glucagons, and cortisol levels.

Limb Lead II ECG was recorded continuously in Group 6 before and after venom, and up to 120 min after SAV. Mean arterial blood pressure was recorded continuously before and after venom, and up to 120 minutes after SAV in Groups 3, 4, 5, and 6.

Tissue sample collection

The rabbits were sacrificed by stunning. Tissue samples from liver, atria, ventricle, and skeletal muscles (rectus abdominis and gastrocnemius) were processed for glycogen content by the phenol-sulfuric acid method (33).

Statistical Analysis

The results were analyzed using Student ‘t’ test and paired Student ‘t’ test (3). Comparisons were made between groups.

RESULTS

Envenoming resulted in fasciculations, clonus and tetany-like contractions, thick ropy saliva dribbling from the mouth, lacrimation, nasal secretions, frequent micturition, and defecation. All animals in Groups 1 and 3 died between 120 to 150 min following envenoming. There were no deaths in Groups 4, 5, and 6 until sacrifice.

Changes in ECG

The following ECG changes were observed after envenoming in Group 6: axis, RR intervals, extra systoles (ventricular and supra ventricular), appearance of Q wave, and many other abnormal ECG patterns. All ECG changes after SAV administration initially reduced in frequency and finally disappeared. Normal sinus rhythm was sustained at the end of 120 min of SAV.

Changes in mean arterial blood pressure

Group 3 showed an initial transient hypertension (180-320 mm Hg.) that lasted from the 28th to the 40th min after envenoming. Hypotension was observed between 50 to 75 min following envenoming. There was a further decrease in pressure and the animals died between 120-150 min after envenoming.

Simultaneous administration of venom and SAV in Group 4 did not cause changes in blood pressure. The animals did not manifest initial transient hypertension followed by hypotension but maintained normal blood pressure after SAV. Group 6 animals had initial transient hypertension but did not manifest hypotension. Pressure returned to normal levels after SAV and was maintained until sacrifice.

Changes in biochemical and hormonal parameters

Figure 1 shows the changes in biochemical and hormonal parameters due to envenoming in Group 1. Increased levels of blood glucose, triglycerides, cholesterol, and amylase were seen 60 and 90 min after envenoming. There was an increase in insulin, glucagon, and cortisol levels; and I/G ratio 60 and 90 min after envenoming.

Figure 1 - Click to relarge

Figure 2 shows changes in the hematological parameters due to envenoming in Group 1. A rise in hematocrit, MCV, MCH, MCHC, platelet count, RBC count, hemoglobin, 2,3 diphosphoglycerate, and glutathione levels was observed 60 and 90 min after envenoming. Total WBC count was reduced at 60 min and increased at 90 min after envenoming.

Figure 2 - Click to relarge

Figure 3 shows the effects of envenoming on blood sugar, cholesterol, alkaline phosphatase, amylase, insulin, glucagons and cortisol, and insulin/ glucagon ratio in alloxan-treated Group 2. Administration of alloxan increased blood glucose, cholesterol, alkaline phosphatase, glucagon, and cortisol levels; reduced insulin levels; and the I/G ratio became 0.16. Envenoming in alloxan-treated Group 2 animals further increased blood sugar, cholesterol, amylase, glucagons, and cortisol levels; a further decrease in insulin levels; and the I/G ratio became 0.078.

Figure 3 - Click to relarge

Figure 4 shows the effect of simultaneous administration of venom and SAV on Hb, PCV, plasma Hb, and MCHC levels in Group 3. Hb, MCHC, and PCV levels did not change 60, 90, and 120 min after simultaneous administration of venom and SAV. Hb and MCHC levels were increased only at 30 min.

Figure 4 - Click to relarge

Figure 5 shows the effect of simultaneous administration of venom and SAV on blood sugar, free fatty aids (FFA), triglycerides, and insulin levels. Very little change was seen in these levels at 30 min. SAV reversed the values back to normal. SAV increased triglycerides levels.

Figure 5 - Click to relarge

Figure 6 shows the effect of SAV administration 30 min after envenoming on Hb, plasma Hb, PCV, and MCHC in Group 5. Increased plasma Hb, PCV, Hb, and MCHC were observed 30 min after envenoming. A further increase was observed in these levels 60 min after envenoming; SAV reduced these levels. PCV, Hb, and MCHC values returned to normal 120 min after SAV administration. However, plasma Hb continued to show an increase even 120 min after SAV.

Figure 6 - Click to relarge

Figure 7 shows the effect of SAV administration 60 min after envenoming on Hb, plasma Hb, PCV, and MCHC in Group 6. An increase in plasma Hb, PCV, Hb, and MCHC was seen 30 and 60 min after envenoming; SAV reduced these levels. These levels returned to normal 120 min after SAV. Hb and plasma Hb levels were increased in Groups 4, 5, and 6 after envenoming. SAV caused the values to return to control (before venom injection) levels. PCV and MCHC levels were increased after venom injection in Groups 5 and 6. At the end of 120 min, SAV reduced these values to less than pre-venom injection levels in Groups 5 and 4. Simultaneous administration of venom and SAV did not change PCV and MCHC in Group 4, except for an increase in MCHC at 30 minutes.

Figure 7 - Click to relarge

Figure 8 shows the effect of administration of either SAV or insulin on hormonal and other biochemical parameters in alloxan-treated envenomed rabbits (Groups 7, 8, 9, 10, and 11). Alloxan increased blood glucose, triglycerides, and cortisol; reduced insulin, glucagon, and cholesterol levels; and I/G ratio.

Figure 8 - Click to relarge

Figure 9 shows the effect of administration of either SAV or insulin on tissue glycogen content in alloxan-treated envenomed and other rabbits (Groups 7, 8, 9, 10, and 11 rabbits). The following changes were observed:

Group 8: alloxan reduced tissue glycogen content.

Group 9: envenoming in the alloxan pre-treated animals caused further reduction in glycogen content of all tissues.

Group 10: the alloxan pre-treated scorpion envenomed animals received SAV one hour after venom injection. SAV did not change glycogen content of the liver, atria, ventricle, rectus abdominis, and gastrocnemius compared to values obtained from Group 9. These values, however, were significantly different from Group 7.

Group 11: the alloxan pre-treated scorpion envenomed animals received 4 Units of insulin one hour after venom injection. An increase in glycogen content was observed in the liver, atrium, ventricle, rectus abdominis, and gastrocnemius, respectively, in comparison to the values obtained from alloxan pre-treated scorpion envenomed animals (Group 9).

Insulin treatment showed an increase in glycogen content in the liver, atrium, ventricle, rectus abdominis, and gastrocnemius, respectively, compared to the values obtained in alloxan pre-treated envenomed animals administered with antivenom.

Figures 10 and 11 show the scorpions Mesobuthus tamulus concanesis and Heterometrous wroughtoni.

Figure 10.
Mesobuthus tamulus concanesis, Pocock.

Figure 11.
Heterometrous (Chersonesometrus) wroughtoni, Pocock.

DISCUSSION

Scorpion envenoming is a common medical hazard in all developing countries (1,2,4-6,10,16,19,22-24,26-28,36-39,41-57,59,60,62,63,64,66,70). The killer Indian red scorpion Mesobuthus tamulus concanesis, Pocock of the Buthidae family and the black scorpion Heterometrus (Chersonesometrus) wroughtoni, Pocock are shown in Figures 10 and 11, respectively. Severe envenoming by scorpions of the Buthidae family includes cardiac, respiratory, hematological, and other dysfunctions causing multi-system-organ-failure (MSOF) and death.

Initial Transient Hypertension

Initial transient hypertension (180-320 mm Hg.) from the 28th to the 40th min after envenoming was observed in this study. Scorpion venoms cause pronounced hypertension (1,20,60,61,70). The combined actions of alpha, beta neurotoxins and potassium channel toxins present in the venom might be responsible for the hypertensive response potentiation (24,26,27).

Hypotension in scorpion envenoming

Hypotension was observed between 50 to 75 min after envenoming. Hypotension is a serious medical problem in severe envenoming (20,22-24,26,27,36-57,59-64,70).

Hemodynamic effects following scorpion envenoming

Increased hematocrit, MCV, MCH, MCHC, platelet count, RBC count, and Hb levels were observed 60 and 90 min after envenoming. WBC count was reduced 60 min and increased 90 min after envenoming (Figure 2). Plasma Hb, PCV, Hb, and MCHC levels increased 30 min and 60 min after envenoming in Groups 5 and 6 (Figures 6 and 7). Adrenergic receptor stimulation could be the reason for these hematological changes (Figures 2, 6, and 7) (23,24,26,27,60,61,63).

Scorpion envenoming and hemoconcentration

RBC count, PCV, and Hb are increased after venom injection in Group 3, 5, and 6 (Figures 2, 4, and 7). This could be explained by increased angiotensin II secretion (43). Increased sympathetic activity causes increased renin release by direct stimulation of juxtaglomerular cells (18). Subsequent increase in angiotensin II secretion enhances ongoing sympathetic nerve output by direct action on the brainstem and by blunting of baroreceptor mechanisms (18,22,23,32).

Angiotensin II produces a significant decrease in blood volume by shift of fluid from intravascular to extravascular compartment, leading to peripheral circulatory failure and pulmonary edema (22,23). Adrenergic receptor stimulation of the spleen induces an increase in venous blood flow and increased hematocrit (24,26,27).

Significance of an increased plasma hemoglobin levels

Envenoming increased plasma hemoglobin levels. Increased plasma Hb indicates hemolysis, and the highest levels are found in intravascular hemolysis (48). The venom of certain snakes, such as the cobra, cause RBC destruction. Cobra venom releases an enzyme, phosphatidase A; this converts lecithin to lysolecithin, a powerful hemolytic and cytolytic substance. Since lecithin is present in erythrocytes and all cells, the introduction of the venom into the body stimulates production of hemolytic substance. When the hemolysis rate is excessive, the plasma extra corpuscular hemoglobin cannot be converted into bilirubin as quickly as it is released and hemoglobinuria may occur. When plasma Hb concentration exceeds the hemoglobin binding capacity and kidney tubular capacity, the excess free plasma Hb will be filtered and excreted in the urine. This could be the cause of hemolysis associated with hematuria due to scorpion envenoming (58).

Significance of increased MCHC

Envenoming increased MCHC in Groups 4, 5, and 6 (Figures 2, 4, 6, and 7). Under no conditions, the value for MCHC is increased unless the blood is hemolyzed (14). Thus, increased MCHC in scorpion envenoming indicates hemolysis.

Significance of increased WBC Count

Increased WBC count was observed after envenoming (Figure 2). Arrhythmias, conduction defects, ischemia, and infarction suggestive of myocardial infarction (MI) also occur in envenoming (4,5,36,37,39,41,45-47,49-57,66,70). Increased WBC count occurs after MI. A striking neutrophil leucocytosis was found in children after scorpion envenoming. This effect is due to the release of catecholamines by the toxin (1,2,4,5,10,16,19,20,22-24,26-28,36,37,39,40-57,59,60,62-64,66,70). Leucocytosis may be a response to tissue necrosis, increased secretion of cortisol, or both (Figures 1, 3, and 8).

Significance of increased 2,3 DPG

Increased 2,3 DPG was observed after envenoming (Figure 2). Hematocrit often increases due to hemoconcentration after MI. Hb affinity for oxygen is reduced and the P50 is increased in patients with MI complicated by left ventricular failure or cardiogenic shock. Increase in P50 results from increased levels of erythrocyte 2,3 DPG. This could be an important compensatory mechanism responsible for oxygen release from oxyhemoglobin (Figures 1, 3, and 8).

Role of Hb and 2,3 DPG

When 2,3 DPG concentration is high, affinity to O2 is less and dissociation is more. Tissue hypoxia has an important effect on the erythrocyte 2,3 DPG levels. Hypoxia favors an increase in 2,3 DPG levels, thus enhancing O2 unloading in tissues (13).

Scorpion envenoming and acidosis

Envenoming caused progressive respiratory and metabolic acidosis (49). Acid-base abnormalities would act cumulatively to other factors in depressing cardio-circulatory function (63). Lactic acidosis is produced by infusion of adrenaline (26,29,35). Scorpion envenoming syndrome is characterized by a massive release of catecholamines, which stimulate increased lactate production by creating anaerobic conditions through peripheral vasoconstriction. Cold and clammy extremities in scorpion sting victims would support this mechanism (6,57,69).

Glycolysis and lactic acidosis

Glucose or glycogen oxidation to pyruvate and lactate is called glycolysis. Erythrocytes, nervous tissues, and skeletal muscle derive energy mainly from glycolysis. Tissues that function under hypoxic circumstances will produce lactic acid causing local acidosis.

The experimental animals frequently showed skeletal muscle fasciculations, clonus and tetany (15,36,37,39,40-47,49-57). Shock, hemorrhage, and anoxia are common in scorpion sting victims. These could cause ‘acid gain acidosis’ due to endogenous lactic acid production and lactic acidosis.

Glutathione in scorpion envenoming syndrome

In this study, increased glutathione levels were observed (Figure 2). Very little is known about the role of circulating glutathione levels in scorpion envenoming. Glutathione is an important reducing agent in tissues. Oxidized glutathione (GSSG) is harmful to the tissues, especially to RBC. Glutathione (GSSG) is converted into glutathione (GSH), which is required for the integrity of RBC membrane. Glutathione helps in insulin catabolism and degradation as a co-enzyme with liver glutathione-insulin-transhydrogenase (13). Many SH group containing enzymes are also protected by glutathione against oxidation of their SH groups. Glutathione is also required as a ‘co-enzyme’ for enzyme glyoxylase, which converts methylglyoxal to lactic acid (13).

Catecholamines have been known to produce oxidative stress. Adrenaline causes glycogenolysis and lactic acidosis. Stress and hypoxia are known to divert glucose to pentose phosphate pathway, which produces NADDPH. Over-production of NADDPH may be utilized either for fatty acid synthesis or for reduction of glutathione disulfide to reduced glutathione. Adrenaline activates glutathione peroxidase in heart, liver, and kidney.

It is interesting to speculate that glutathione converts methylglyoxal to lactic acid through intramolecular oxidation-reduction by acting as a co-enzyme for the enzyme glyoxylase. It is speculated that the activities of glyoxylase and glutathione reductase might account for lactic acidosis in scorpion envenoming.

Glycolysis regulation

Insulin enhances synthesis of key enzymes responsible for glycolysis. Insulin antagonizes the effects of glucocorticoids and glucagon in stimulating key enzymes responsible for gluconeogenesis. Epinephrine and glucagon inhibit glycolysis. Change in the hormonal milieu in this study could be responsible for increased glycolysis and lactic acidosis (49,63).

Mature erythrocytes maintain a high steady state concentration of 2,3 DPG produced by a diversion in glycolytic pathway "Re Pa Port-Leubering Cycle or Shunt" (RLC or RLS). Erythrocytes utilize more glucose than is requires for maintaining cellular energy. RLC or RLS provides a mechanism to dissipate excess energy (13).

High hematocrit levels and insulin resistance in scorpion envenoming

High hematocrit levels may be often associated with insulin resistance. Increased plasma insulin levels are associated with an increased transcapillary escape rate of albumin and reduced plasma volume. This may explain the association between hematocrit, hyperinsulinemia, and insulin sensitivity (21,34,73).

Hormonal and biochemical disturbances in scorpion envenoming

ECG changes, cardiovascular manifestations, and metabolic disturbances in scorpion envenoming could be due to actions of catecholamines, angiotensin II, insulin deficiency, and many other changes. These may cause increased myocardial oxygen consumption by increased FFA. Increased FFA alter the function of platelets and, increase the tendency for intravascular thrombosis and disseminated intravascular coagulation (2,4,20,36,37,39,41-46,49-57). These factors could contribute for hypoinsulinemia, hyperinsulinemia, and insulin resistance causing multi-system-organ-failure (MSOF), adult respiratory distress syndrome (ARDS), pulmonary edema, and death.

Effect of envenoming on carbohydrate metabolism producing hyperglycemia

Hyperglycemia (Figures 1, 3, 5, and 8) accompanied by changes in insulin secretion, and elevated glucagon and cortisol levels are observed (Figures 1 and 8). Hyperglycemia could be due to increased secretion of catecholamines, glucagon, cortisol, thyroid hormones, and changes in insulin secretion (1,2,4-6,13,16,20,22-24,26-28,37,39,42,45,46,49-51,53-56,59,60,66,70).

Glucose toxicity in scorpion envenoming

Hyperglycemia associated with insulin resistance is common in critically ill patients. It has been reported that pronounced hyperglycemia might lead to complications (7). In virtually all tissues except the brain, glucose, at a fixed insulin concentration, promotes its own utilization in a concentration-dependent manner. The superiority of insulin in stimulating glucose oxidation seems to be explained by an anti-lipolytic effect. Even a small increment in serum insulin concentration promptly suppresses lipolysis and consequently the use of FFA for energy production, which in turn, enhances glucose oxidation. In contrast, glucose per se is unable to suppress lipolysis (30). Hyperglycemia may cause a generalized desensitization of all cells in the body. In muscles and adipocytes this would be reflected by a defect in insulin action, whereas at the Langerhans islet beta cell level, it results in impaired insulin secretion (30).

Scorpion envenoming and cholesterol

Scorpion envenoming increased blood cholesterol levels 60 min after venom injection (Figure 1).

Increased free fatty acid (FFA) levels in scorpion envenoming syndrome

Scorpion envenoming increased FFA along with a reduction in triglycerides levels (Figure 5) (41,46,49,53,54,56). Increased FFA levels could be due to increased secretion of catecholamines, glucagon, thyroid hormones, cortisol levels, and changes in insulin secretion (1,2,4-6,13,16,20,22-24,26-28,37,39,42,45,46,49,50,51,53-56,59,60,66,70).

Effect of increased FFA on heart in scorpion envenoming syndrome

Utilization of increased amounts of circulating FFA increases oxygen consumption. This could aggravate the ischemic injury to myocardium, predisposing to arrhythmias and heart failure. Elevated FFA also increases susceptibility of the ventricles to the disorganized electrical behavior and produces ectopic beats in the vulnerable period of cardiac cycle (35,41,45,46,50,51,55,56,58,65). Under pathological conditions, high FFA levels produce inhibition of Na⁺ - K⁺ ATPase activity and sarcolemmal defects (35,36,51,55). Increased FFA, by altering the function of platelets, may increase the tendency for intravascular thrombus and result in disseminated intravascular coagulation (D I C) (53).

Insulin levels in scorpion envenoming

Scorpion envenoming altered insulin secretion (Figures 1, 3, 5, and 8). Insulin levels are either inhibited or elevated after envenoming (24,26,27,37,42,49,51,53,54,55).

Insulin/glucagon ratio in scorpion envenoming

Scorpion envenoming altered I/G ratio (Figures 1, 3, and 8). This may be more important than the individual hormone levels (61). A high I/G ratio produces an anabolic state with more nutrient incorporation into peripheral tissues. High ratios indicate that carbohydrates are the predominant energy source (Figure 1). Low I/G ratios indicate that a catabolic state is produced in which nutrients are mobilized. Scorpion envenoming caused a low I/G ratio (45) (Figures 3 and 8).

What is Hyperinsulinemia?

Hyperinsulinemia is said to exist when plasma insulin levels are inappropriate for the blood glucose estimated simultaneously. ‘True hyperinsulinemia’ is the most appropriate term when insulin levels are elevated with normal glucose levels, while high insulin levels that occur with elevated blood glucose may be referred as ‘insulin resistance’ (34). Elevated insulin levels are observed 30 min after venom injection. Short-term hyperglycemia can induce insulin resistance (7,25,34,65,69,73).

Insulin-resistance state in scorpion venom envenoming

The relationship between insulin resistance, plasma insulin levels, and glucose intolerance is mediated to a significant degree by changes in ambient plasma FFA concentrations (34,58,59,65,69). Plasma FFA levels can be suppressed by relatively small increments in insulin concentration. Consequently, elevation of circulating FFA concentration can be prevented if large amounts of insulin can be secreted. If hyperinsulinemia cannot be maintained, plasma FFA concentration will result in increased hepatic glucose production. The observation of hyperglycemia or euglycemia in the face of concomitant hyperinsulinemia suggests an insulin-resistant state (34,58,59,65,69) in scorpion envenoming.

Effects of an acute increase in epinephrine and cortisol levels on carbohydrate metabolism during insulin deficiency

Scorpion envenoming increases epinephrine, glucagons, and cortisol levels (1,20,22,23,24,26,27,28,48,59,63). In diabetic patients, plasma epinephrine and cortisol levels increase during diabetic ketoacidosis. An acute rise in plasma epinephrine level causes a transient increase in hepatic glucose production and a sustained decrease in glucose clearance with persistent hyperglycemia. Glucagon and cortisol modify lactate gluconeogenesis (7,9,25,34,45,65).

Hemodynamic abnormalities in short-term insulin deficiency

In diabetic ketoacidosis, simultaneous relative insulin deficiency and excessive secretion of counter-regulatory hormones lead to magnified lipolysis and beta oxidation of FFA with a parallel hepatic overproduction and peripheral underutilization of ketone bodies with clinical characteristics of drowsiness, weak pulse, and low blood pressure. Similar clinical manifestations are usually observed in scorpion sting victims (1,6,10,19,20,22-24,26,27,47,59,60,62,63,66,70).

Role of insulin as a potent antithrombotic hormone

Insulin probably has a preventive role in the development of cardiovascular complications and is independent of its well-known hypoglycemic effect. Platelets are aggregated in the presence of aggregating agents like ADP, l-epinephrine, collagen, thrombin, or platelet-activating factor. Platelet aggregation is an important event in the life saving process of blood coagulation. On the other hand, hyper-aggregation of platelets and their adhesion at injury site leads to thrombogenesis. During aggregation, prostaglandin G2 and prostaglandin H2 act as platelet aggregating agents. Thromboxane A2 cause substantial constriction of the blood vessel. This induces further blockade of circulation and aggravates the ischemic condition. Platelet aggregation is inhibited by several humoral factors including prostacyclin, blood coagulation factor Xa, and endothelial-derived relaxing factor - nitric oxide (NO). Insulin infusion results in platelet aggregation inhibition, which may lead to beneficial effects for thrombosis prevention. During the episode of an acute cardiac ischemia, platelets are hyperactive and resistant to the inhibitory effect of prostacyclin. Treatment of platelets with physiological amounts of insulin causes an increase of prostacyclin receptor numbers on the platelet surface, returning them to normal levels, and thereby restoring sensitivity of these platelets. Furthermore, prostacyclin synthesis is stimulated when endothelial cells are exposed to physiological amounts of insulin (29,31). Scorpion sting victims may die from respiratory failure unrelated to pulmonary edema and secondary to brain hemorrhage, thrombosis, or ischemia (1,2,4-6,10,16,20,22-24,26-28,36-57,59-64,67,68,70).

Alloxan

Alloxan induces chemical diabetes destroying the Langerhans islet beta cells. Alloxan administration reduced insulin and increased cortisol levels in dogs and rabbits; it increased glucagon secretion in dogs after envenoming. However, glucagon levels were reduced in rabbits. These changes caused hyperglycemia and other metabolic effects (Figures 3, 8, and 9).

After envenoming, alloxan-treated rabbits showed further reductions in insulin and glucagons, and rise in cortisol levels. These changes indicated the contribution made by insulin lack and excess cortisol secretion in the genesis of hyperglycemia and other metabolic effects.

Alloxan, scorpion envenoming, and triglycerides

Alloxan increased triglyceride levels and envenoming further increased triglyceride levels in the alloxan pre-treated rabbits (Figure 3).

Alloxan, scorpion envenoming, and cholesterol

Alloxan increased cholesterol levels and envenoming further increased cholesterol levels in the alloxan pre-treated dogs (Figure 3). Alloxan did not increase cholesterol levels in rabbits (Figure 8). Venom injection in alloxan pre-treated rabbits did not cause any considerable increase in cholesterol levels (Figure 8).

Alloxan, scorpion envenoming, and tissue glycogen

Reduction in glycogen content of liver and cardiac and skeletal muscles in alloxan-treated envenomed rabbits could be due to a reduction in circulating insulin levels and release of counter-regulatory hormones (Figure 9). SAV did not change glycogen content of these tissues (Figure 9). SAV administration in alloxan-treated envenomed animals would not cause further rise in insulin secretion since beta cells are already destroyed. This could be the possible mechanism, which explained the lack of difference in glycogen content after SAV to alloxan-treated scorpion envenomed Groups 8, 9, and 10.

Alloxan, scorpion envenoming, tissue glycogen, and insulin treatment

Insulin administration increased tissue glycogen content in alloxan-treated scorpion envenomed animals (Figure 9). This could be due to the direct action of administered insulin in promoting glycogenesis in muscle, adipose tissue, and liver.

Glycogenesis is an anabolic process and glycogen availability may be an important independent determinant of cardiac function. Elevated glycogen content in the heart partially protects it against mechanical deterioration in anoxia (58). Tissue glycogen content showed a massive increase in liver, skeletal muscle, and cardiac muscle after insulin administration in experimental scorpion envenoming (49, 54).

Scorpion envenoming and SAV

Treatment of scorpion envenoming is a difficult problem. Is SAV effective? Sofer et al concluded that after envenoming heart and circulation are rapidly affected by toxins or other substances released by venom, which do not respond to antivenom (59,60,63). Although SAV given before venom injection almost totally prevented cardiac effects, SAV given after venom did not abolish them (20,24,26,27). Fekri et al found no evidence of beneficial effects of antivenom administration (19).

Effect of SAV on blood pressure

In this study, all animals died within 120-150 min after envenoming. There were no changes in blood pressure when SAV was given along with venom. Blood pressure returned to normal from hypertensive or hypotensive levels when SAV was given 30 or 60 min, respectively, after envenoming. Thereafter, blood pressure remained normal.

Effect of SAV on biochemical and hematological changes

Simultaneous administration of venom and SAV caused very little changes in blood sugar and FFA levels. There was an increase in triglyceride levels accompanied by insulin (Figure 5). MCHC and plasma Hb increased after simultaneous administration of venom and SAV. SAV did not change cholesterol levels (Figure 8).

Hb, plasma Hb, PCV, and MCHC increased 30 min following envenoming in Group 5. A further rise in Hb, plasma Hb, PCV, and MCHC occurred 60 min after envenoming. SAV arrested this tendency. PCV, Hb, and MCHC values returned to control values 120 min after SAV (Figure 6). However, plasma Hb remained high (Figure 6).

Hb, plasma Hb, PCV, and MCHC levels increased 30 and 60 min after envenoming in Group 6 (Figure 7). SAV reversed these levels (Figure 7).

SAV neutralized venom-induced hyperglycemia and lipolysis (Figure 5) and reversed them to euglycemia and lipogenesis (Figures 5, 6, and 7). If SAV did not inhibit the catecholamine-mediated toxicity that suppressed the release of insulin secretion, then we would have observed only the arrest of further rise in the products of glycogenolysis and lipolysis. Attainment of near normal blood glucose levels and lipogenesis after SAV indicated that SAV effectively neutralized the inhibiting actions of catecholamines on insulin secretion. Thus, SAV essentially acted through insulin release.

Alloxan, scorpion envenoming, tissue glycogen, and SAV administration

If SAV essentially acted through insulin release and reversed metabolic, hematological, cardiovascular, and ECG changes then it is expected that SAV might not be effective in the envenomed animals after alloxan treatment. This is due to alloxan destroying the endocrine pancreas beta cells (Group 10, Figures 8 and 9).

Alloxan, scorpion envenoming, tissue glycogen, and insulin administration

The alloxan pre-treated scorpion envenomed rabbits after insulin injection (Group 11, Figures 8 and 9) showed clinical improvement, glycogenesis in liver, atria, ventricle, and skeletal muscles, along with a reduction in blood, sugar, and triglycerides (Group 10, Figures 8 and 9).

Serotherapy in scorpion envenomation

Specificity, dose, and antivenom potency could be potential causes of lack of effectiveness of scorpion antivenom. Antigenic similarity between the venoms of scorpions of the Buthidae family is suggested to explain the fact that any antivenom given after sting by any scorpion of this species protects against the venom of others (19). Severity of envenoming is related to venom concentration (24,26,27). SAV should be administered IV (20) and maintain the acid-base-fluid-electrolyte balance (51,55). SAV should be given because of slow elimination half-life of scorpion venom (24,26,27). SAV will neutralize the circulating venom and that being absorbed from the sting site into circulation.

CONCLUSIONS

We have highlighted the physiological basis of the role of insulin in scorpion envenoming syndrome and the probable mechanisms in the genesis of hormonal, electrocardiographic, hematological, and cardiovascular changes. We have also highlighted the role of insulin as a therapeutic agent in scorpion envenoming syndrome either directly or indirectly after SAV administration. Literature highlightes the role of insulin therapy in critically ill patients (7,10,42,46,48,53,55,67,71).

ACKNOWLEDGEMENTS

We thank the Indian Council of Medical Research for financing the Task-Force Research Project No. 46/3/90-B M S –I. This work was carried out in the Department of Physiology, Lokmanya Tilak Municipal Medical College & Lokmanya Tilak Municipal Hospital, Sion, Mumbai 400 022.

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Received February 14, 2002

Accepted June 4, 2002

S.E. = Standard error of mean difference, I/G ratio = insulin/glucagon ratio, P = * < 0.05, ** < 0.01, *** < 0.001, I/G ratio is calculated considering the values of insulin and glucagon levels as 100% in the control (before venom).

Figure 1. Effect of scorpion Mesobuthus tamulus concanesis, Pocock venom (3.5 mg/kg) on blood sugar (mg/dl), triglycerides (mg/dl), cholesterol (mg/dl), alkaline phosphatase (I.U./L), amylase (I.U./L), glucagon (pg/ml), insulin (µU/ml), cortisol (µg/dl), and I/G ratio in dogs (n=12) (mean + S.E.D.).

n

= umber of aimals, S.E. = Stadard error of mea differece, P = * < 0.05, ** < 0.01, *** < 0.001, MCV = mea corpuscular volume, MCH = mea corpuscular hemoglobi, MCHC = mea corpuscular hemoglobi cocetratio 2,3-DPG = 2,3-diphosphoglycerate.

Figure 2. Effect of scorpion Mesobuthus tamulus concanesis, Pocock venom (3.5 mg/kg) on hematocrit (%), glutathione (mg%), MCV (cu.micron), MCH (mgm), MCHC (%), total platelet count (x1000/mm³), RBC count (millions/mm³), total WBC count (x1000/mm³), hemoglobin (gm%), and 2,3-DPG (µmol/gm Hb) in Dogs (n=12) (mean + S.E.D.)

S.E. = Standard error of mean difference, P = * < 0.05, ** < 0.01, *** < 0.001, I/G ratio is calculated considering the values of insulin and glucagon levels as 100% in the control (before venom).

Figure 3. Effect of administration of scorpion Mesobuthus tamulus concanesis, Pocock venom (3.5 mg/kg) on blood sugar (mg/dl), glucagon (pg/ml), cholesterol (mg/dl), alkaline phosphatase (I.U./L), amylase (I.U./L), insulin (µU/ml), cortisol (µg/dl), and I/G ratio in alloxan-treated dogs (mean + S.E.).

Figure 4. Effect of simultaneous administration of venom (SQ) and antivenom (I.V.) on dog blood plasma Hb, PCV, Hb, and MCHC in Group 3 dogs (mean + S.E.D.) (n=9).

S.E.D = Standard error of mean difference, n = number of animals, P* < 0.05, P** < 0.02, P*** < 0.01, P****< 0.001. Comparison of results for biostatistical analysis: before venom and antivenom with all other values.

Figure 5. Effect of simultaneous administration of venom (SQ) and antivenom (I.V.) on blood sugar, free fatty acids, insulin, and triglycerides in Group 3 dogs (mean + S.E.D.) (n=9).

S.E.D. = Standard error of mean difference, n = number of animals, P* < 0.05, P** < 0.02, P*** < 0.01, P****< 0.001. Comparison of results for biostatistical analysis: before venom with 30 min after venom; and 30 min after venom with all other values after antivenom.

Figure 6. Effect of antivenom administration 30 min after venom injection on blood plasma Hb, PCV, Hb, and MCHC in Group 5 dogs (mean + S.E.D.) (n=7).

S.E.D. = Standard error of mean difference, n = number of animals, P* < 0.05, P** < 0.02, P*** < 0.01, P****< 0.001. Comparison of results for biostatistical analysis: before venom with 30 min and 60 min after venom; and 60 min after venom with all other values after antivenom.

Figure 7. Effect of antivenom administration 60 min after venom injection on blood plasma Hb, PCV, Hb, and MCHC in Group 6 dogs (mean + S.E.D.) (n=8).

S.E. = Standard error, n = number of animals, P* < 0.05, ** < 0.01, *** < 0.001. I/G ratio is calculated considering the values of insulin and glucagon levels as 100% in control group (Group 7).

Figure 8. Effect of administration of either scorpion antivenom or insulin on hormonal and other biochemical investigations in alloxan-treated envenomed rabbits (mean + S.E.).

Publication Dates

• Publication in this collection
  09 Dec 2003
• Date of issue
  2003

History

• Received
  14 Feb 2002
• Accepted
  04 June 2002