Clonidine Prevents Insulin Resistance and Hypertension in Obese Dogs

Methods

Thirteen adult mongrel dogs (6 males and 7 females) were trained to stand quietly in a padded sling and were surgically instrumented with 1 ascending aortic and 2 right atrial catheters. The dogs recovered for 3 weeks before baseline measurements were made. Dogs were randomly assigned to either a high-fat-diet, no-clonidine group (n=6) that received the control diet (1 can of dog food [Ken-L-Ration]) for 2 weeks followed by 6 weeks of a high fat diet consisting of 0.8 kg of cooked beef fat in addition to the regular diet² or a high-fat-diet, clonidine group (n=7) that received 0.3 mg clonidine PO BID initiated 1 week before the start of the high fat diet and continued with the fat diet for an additional 6 weeks. (Figure 1⇓) All dogs received vitamin supplements (VAL Syrup; Fort Dodge Laboratories) and antibiotics throughout the entire study. Dogs were housed in air-conditioned cages and fed between 1:00 and 3:00 pm each day. Blood pressure, heart rate, and body weight were measured daily. Cardiac output, plasma glucose, and insulin were measured twice a week. All measurements were made between 8:00 and 11:00 am and before the daily feeding. All the procedures in this study were in accordance with the University of Minnesota, Northwestern University, and University of Michigan guidelines on animal experimentation.

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Figure 1.

Schematic representation of the study design of the project.

Results

Euglycemic Clamp Data

During the euglycemic clamp studies, the steady-state blood glucose concentration in all dogs averaged ∼5.2 mmol/L and did not differ from the fasting concentration at any insulin infusion rate. The coefficient of variation of glucose level at each insulin plateau was<5%.

To characterize the ability of clonidine treatment to alter the insulin-mediated glucose uptake relation that occurs in dogs fed a high fat diet, we measured insulin-mediated glucose uptake dose-response curves before and after the high fat diet. Basal rates of whole body glucose uptake (hepatic glucose production) were measured on 4 dogs in the clonidine group and 4 dogs in the no-clonidine group. The high fat diet did not significantly change basal rates of whole body glucose uptake in either group of dogs (24±6 μmol · kg ⁻¹ · min⁻¹ at week 0 versus 22±8 μmol · kg⁻¹ · min⁻¹ at week 6 in the no-clonidine group and 26±8 μmol · kg⁻¹ · min⁻¹ at week 0 versus 25±9 μmol · kg⁻¹ · min⁻¹ at week 6 in the clonidine group). Throughout the study, the 1–mU · kg⁻¹ · min⁻¹ insulin dose completely suppressed hepatic glucose output in both groups of dogs. During increasing insulin dose rates, whole body glucose uptake increased in a sigmoidal fashion (Figure 2⇓). In the no-clonidine group, the high fat diet was associated with a shift in the glucose uptake curve to the right, and the rate of maximal whole body glucose uptake was significantly decreased (P<0.001) (Table 2⇓). Compared with the control, pre–high fat period, the insulin concentration expected to produce a half-maximal response in glucose uptake (insulin ED₅₀ dose) was 50% (P<0.01) higher at 1 week and 85% (P<0.001) higher at 6 weeks of the fat diet. In contrast to the no-clonidine group, the clonidine-treated group did not experience any change in the glucose uptake curve during the 6 weeks of the high fat diet. After 6 weeks of the high fat diet, the clonidine group had both maximal glucose uptake and the insulin concentration expected to produce a half-maximal response in glucose uptake (insulin ED₅₀ dose), which were unchanged from values obtained in this control period. (Table 2⇓) In addition, 1 week of clonidine treatment without the high fat diet (week −1 versus week 0) also did not change the insulin-mediated glucose uptake curve. Finally, despite the infusion of the same amount of insulin per kilogram of weight in both groups of dogs, only the group of dogs that did not receive clonidine experienced, when fed the high fat diet, an increase over time in the plateau insulin values during the multiple-dose clamps (Table 2⇓).

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Figure 2.

Rates of whole body insulin-mediated glucose uptake (M) determined during euglycemic clamp studies over a wide range of steady-state insulin concentrations (□, week 0; ▵, week 1 of fat diet; X, week 6 of fat diet) in dogs fed a high fat diet with and without clonidine treatment. Weight gain resulted in a shift to the right and a decrease in the maximal rate of M (P<0.001) in dogs not treated with clonidine; however, if the dogs were treated with clonidine, weight gain did not alter the dose-response relation.

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Table 2.

Maximal and ED₅₀ Values for Insulin-Mediated Glucose Uptake, Basal Maximal and ED₅₀ Values for Cardiac Output, and Plateau Insulin Concentrations in High-Fat Clonidine-Fed and High Fat Non–Clonidine-Fed Dogs

In the no-clonidine group, the high fat diet was associated with an increase in resting cardiac output (P<0.01), whereas in the clonidine-treated group, the high fat diet resulted in no significant increase in cardiac output (P>0.1) (Table 1⇑). In both groups of dogs, before starting the high fat diet, euglycemic hyperinsulinemia caused a dose-dependent increase in cardiac output (Figure 3⇓). In the no-clonidine group, feeding the high fat diet resulted in rightward shift of the effect of insulin to increase cardiac output and a reduced maximal insulin-stimulated cardiac output (P<0.05) (Table 2⇑). After 6 weeks of the fat diet, euglycemic hyperinsulinemia caused virtually no increase in cardiac output. However, in the clonidine group, the high fat diet did not change the ability of insulin to increase cardiac output. After 6 weeks of the high fat diet, euglycemic hyperinsulinemia in clonidine-treated dogs still resulted in a 37% increase in cardiac output.

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Figure 3.

Insulin-induced changes in cardiac output were determined during euglycemic clamp studies over a wide range of steady-state insulin concentrations (□, week 0; ▵, week 1 of fat diet; X, week 6 of fat diet) in dogs fed a high fat diet with and without clonidine. In the absence of clonidine, weight gain resulted in an increase in basal cardiac output and a shift to the right of the effect of insulin to increase cardiac output (P<0.01); however, with clonidine treatment, weight gain was not associated with either an increase in cardiac output or a shift in the effect of insulin to increase cardiac output.

In both groups of dogs, before starting the high fat diet, the euglycemic clamp resulted in a 6±2 mm Hg decrease in arterial pressure and a 7±3 bpm increase in rate. In the no-clonidine group, after 6 weeks of a high fat diet, euglycemic hyperinsulinemia did not cause either a decrease in arterial pressure or an increase in heart rate; however, in the clonidine group, the high fat diet euglycemic hyperinsulinemia was still associated with a 5±2 mm Hg decrease in pressure and a 5±3 increase in heart rate.

Discussion

The present study demonstrates that clonidine, an antihypertensive agent that lowers blood pressure through stimulation of central α₂-receptors and, to a lesser degree, the I₁ imidazole receptors, prevented both the hypertension and insulin resistance associated with weight gain in the dog. Our finding that clonidine prevented hypertension in the obese dog model is consist with the report of Hall et al⁷ They demonstrated that combined α- and β-adrenergic blockade reduced arterial pressure to a much greater extent in obese than in normal dogs. We believe the mechanism whereby clonidine treatment prevented the increase in blood pressure associated with weight gain is due to central inhibition of the peripheral sympathic nervous system.¹³ Because we did not directly measure sympathetic activity, further studies are necessary to directly prove that clonidine inhibited peripheral sympathetic activity. The failure of weight gain to induce tachycardia in the clonidine-treated dogs is consistent with the known central nervous system action of clonidine. In addition, the failure of clonidine-treated dogs to increase their cardiac output in association with weight gain is consistent with a reduced degree of renal salt and water retention. Kassab et al¹⁴ have shown that bilateral renal denervation prevents the hypertension and sodium retention associated with obesity in the dog. We believe that clonidine treatment could have prevented the high fat diet–induced activation of the renal efferent sympathetic nerves. Further studies that measure in detail the sodium and fluid balance in clonidine-treated dogs fed a high fat diet will be necessary to more directly answer whether clonidine does prevent sodium and fluid retention in obese dogs.

Perhaps the more important and new finding in the present study is that clonidine also prevented the insulin resistance associated with obesity in the dog. Using the multiple insulin dose euglycemic clamp, we demonstrated that clonidine prevents the reduced sensitivity and responsiveness of the insulin-mediated glucose uptake dose-response curve that is associated with weight gain (Figure 2⇑, Table 2⇑). It has been speculated that insulin resistance (ie, resistance to the ability of insulin to stimulate glucose uptake) is the common metabolic abnormality connecting obesity, hypertension, and increased sympathetic nervous system activity. This hypothesis is supported by numerous reports that document a relation between insulin resistance and hypertension.^{1 2 3} However, the present study supports the concept, originally proposed by Julius et al,¹⁵ that central nervous system–induced sympathetic activation, not insulin resistance or hyperinsulinemia, is the metabolic link that connects obesity to hypertension.

Our finding that clonidine treatment prevented the insulin resistance associated with weight gain in the dog is consistent with other reports. Giugliano et al¹⁶ reported that in 20 hypertensive patients with non–insulin-dependent diabetes mellitus, clonidine treatment was associated with an improvement in insulin sensitivity in peripheral tissues. Using the glucose clamp, these investigators found that clonidine significantly improved overall glucose metabolism and that this improvement was accompanied by increases in both oxidative and nonoxidative glucose metabolism. Other central acting antihypertensive drugs also have been reported to improve insulin resistance. Moxonidine, a highly selective I₁ imidazole receptor agonist with weak central α₂-receptor effects, has been shown to prevent the insulin resistance, hyperinsulinemia, and hypertension in rats fed a fructose-enriched diet¹⁷ and to reduce blood pressure, reduce triglycerides, and improve glucose tolerance in obese spontaneously hypertensive rats.¹⁸ However, in their fructose-fed rate model, Hwang et al¹⁹ failed to show a beneficial effect of clonidine in preventing insulin resistance despite demonstrating that clonidine did prevent the increase in blood pressure. The acute administration of clonidine has been shown to induce hyperglycemia and impaired glucose tolerance in rats and humans.²⁰ However, chronic clonidine treatment does not appear to exert such effects.²¹

The present study was not designed to determine the mechanism whereby clonidine prevents the development of insulin resistance that occurs in dogs fed a high fat diet. However, because of previous studies and our present study design, some inferences can be made into the mechanism of insulin resistance. Insulin-mediated glucose uptake is determined both by the ability of insulin to stimulate glucose extraction at the level of tissues and cells and by the rate of glucose and insulin delivery (blood flow). Thus, the relative contributions of tissue and blood flow actions of insulin will determine the overall rate of glucose uptake (ie, degree of insulin resistance). Baron et al²² observed that the reduced rate of insulin-mediated glucose uptake that occurs in non–insulin-dependent diabetes mellitus, obesity, and hypertension may be due in large part to an impairment in the action of insulin to increase skeletal muscle blood flow. In the present and other studies,¹ we have demonstrated that obese dogs have a reduced ability of euglycemic hyperinsulinemia to increase cardiac output (Figure 3⇑). Because clonidine treatment enabled euglycemic hyperinsulinemia to cause a dose-dependent increase in cardiac output even after the dogs gained weight, we believe that the increased blood flow responses to insulin may have contributed to the improvement in insulin resistance with clonidine. Vollenweider et al²³ demonstrated that insulin resistance in obese subjects is associated, in skeletal muscle, with a specific impairment of sympathetic neural and vasodilatory responsiveness to hyperinsulinemia. The impairment of insulin to increase skeletal muscle blood flow in obesity and in non–insulin-dependent diabetes is speculated to be related to an abnormality in the nitric oxide system.²⁴ Insulin is known to interact with the sympathetic nervous system at the vascular level, predominantly through the α₂-adrenergic pathway.²⁵ Lacolley et al²⁶ demonstrated that the sympathetic nervous system plays an important role in modulating the synthesis, release, or both of vascular nitric oxide. Thus, it is possible that clonidine treatment of dogs fed a high fat diet may have prevented insulin resistance by blocking the sympathetically mediated reduced vasodilator responsiveness to insulin known to occur in obesity.

Besides the affect of the sympathetic nervous system on glucose uptake by reducing blood flow, there is evidence that the sympathetic nervous system can directly influence the cellular uptake of glucose. Takahashi et al²⁷ demonstrated in the rat that ventromedial hypothalamic stimulation can alter peripheral glucose uptake at the cellular level. Catecholamine treatment of rat adipocytes has also been demonstrated to reduce the tyrosine kinase activity of the insulin receptor.²⁸ Finally, because clonidine is known to suppress free fatty acid levels,²⁹ it is possible that glucose uptake in our obese dogs was improved by increasing oxidative glucose metabolism.

In summary, the results of the present study document that clonidine treatment of dogs fed a high fat diet blocks the development of both hypertension and insulin resistance even though the dogs still gained weight. Further studies will be necessary to better clarify how clonidine is able to dissociate weight gain from both hypertension and insulin resistance.