Central melanocortin receptors regulate insulin action (original) (raw)
We aimed to generate bidirectional changes in the activity of hypothalamic melanocortin receptors in order to examine their impact on body composition and insulin action independent of changes in food intake. However, treatment with the potent antagonist of melanocortin receptors, SHU9119, increased cumulative food intake (∼70%) (Table 1). This confirms the observation of Fan et al. (17), which showed that hypothalamic melanocortinergic neurons exert a tonic inhibitory effect on feeding behavior. To delineate whether this pathway plays a role in the regulation of insulin action and carbohydrate metabolism that is independent of its anorectic effects, we pair-fed rats treated with either vehicle, SHU9119, or α-MSH. Plasma leptin (Table 2) and insulin levels were moderately increased (∼2 fold; P < 0.01) in rats receiving SHU9119 and fed ad libitum, while they were modestly decreased (∼30%) in rats receiving α-MSH, compared with vehicle. Finally, plasma corticosterone levels were similar in all experimental groups (data not shown).
Plasma concentrations of glucose, insulin, leptin, and free fatty acids during the insulin clamp studies
Body composition was assessed by measuring the distribution space of body water with 3H2O and by postmortem dissection of fat depots (13). The modest changes in body weight in rats treated with α-MSH and SHU9119 were largely accounted for by changes in fat mass (Table 1). While fat-free mass was not markedly affected in any of the experimental groups, it tended to be lower in rats treated with SHU9119 and pair-fed (Table 1). Importantly, administration of α-MSH caused a selective decrease (∼50%; P < 0.01 vs. vehicle) in intra-abdominal adiposity (sum of omental, epididymal, and perirenal fat depots) (Figure 3, a and b), while subcutaneous fat mass was only increased in rats treated with SHU9119 when fed ad libitum (Figure 3c). These results indicate that α-MSH, in addition to its known effects on food intake and whole body fat mass, has selective actions on visceral or intra-abdominal adiposity. Since leptin exerts a similar action on fat distribution (13), it is likely that leptin activation of the melanocortin pathway (22) partly accounts for this effect. The relative distribution of body fat between subcutaneous and visceral sites determines the metabolic impact of adiposity and intra-abdominal adiposity is a risk factor for diabetes mellitus, arteriosclerosis, and mortality (23, 24). Our results suggest that the neural melanocortin pathway could play a major role in the regulation of fat distribution.
Effect of α-MSH and SHU9119 administration on subcutaneous and visceral fat. (a) Weight of epidydimal, omental, and perirenal fat depots in the four experimental groups. (b) Total visceral fat mass represents the sum of omental, epididymal, and perirenal fat depots. (c) Subcutaneous fat mass was estimated by subtracting the total visceral fat from the whole body fat mass. Of note, administration of α-MSH selectively decreased the size of all visceral fat depots compared with SHU-PF, despite similar food intake, weight changes, and subcutaneous fat mass. Values represent mean ± SE. *P < 0.01 vs. vehicle.
We next examined whether altering the activity of neural melanocortin receptors led to changes in the in vivo actions of insulin. Insulin action on carbohydrate metabolism can be assessed in conscious rats using a combination of the insulin (3 mU/kg/min) clamp and tracer dilution techniques (13, 19, 20). The plasma glucose, free fatty acids, and insulin concentrations during the insulin clamp studies were similar in the four groups (Table 2). During physiologic hyperinsulinemia (plasma insulin ∼450 pM), the rate of glucose infusion required to maintain the plasma glucose concentration at basal levels was markedly increased (56%) by α-MSH and decreased (32%) by SHU9119 (Figure 4a). Thus, modulation of central melanocortinergic neurons results in dramatic changes in insulin’s ability to promote glucose disposal. The two major effects of insulin in vivo are to stimulate the uptake of glucose into peripheral tissues (mostly in skeletal muscle and adipose tissue) and to diminish the production of glucose by the liver. The rate of tissue glucose uptake in α-MSH (27.8 ± 1.7 mg/kg/min) was 28% higher than in vehicle (21.9 ± 1.4; P < 0.01) (Figure 3b). Conversely, the rate of glucose uptake was decreased (18%) in rats treated with SHU9119 (18.2 ± 1.4 mg/kg/min) compared with vehicle (Figure 4b). Similarly, α-MSH markedly enhanced the action of insulin in inhibiting glucose production (Figure 3c) (1.3 ± 0.6 vs. 5.0 ± 0.3 mg/kg/min; P < 0.01). By contrast, SHU9119 diminished insulin’s effect on glucose production (8.2 ± 0.9 mg/kg/min; P < 0.01). The effect of insulin on glucose production can also be expressed as percentage of inhibition from basal levels (Figure 4d). Insulin inhibited glucose production by 90% ± 5% in α-MSH, 56% ± 3% in vehicle, and 34% ± 4% in SHU9119. During the insulin clamp studies, 54–61% of the changes in glucose metabolism (rate of glucose infusion) induced by either α-MSH or SHU9119 were due to changes in glucose disposal and 39–46% were due to changes in glucose production. Thus, bidirectional modulation of central melanocortin receptors regulates both hepatic and peripheral insulin sensitivity. Since the metabolic effects of α-MSH may be mediated by way of either type 3 or 4 melanocortin receptors, we next examined whether a selective decrease in hypothalamic MCR4 expression is sufficient to negate the effects of intracerebroventricular α-MSH on fat distribution and insulin action. Intracerebroventricular delivery of ODN antisense has been shown previously to be effective in decreasing the expression of other central receptors (25). Here the intracerebroventricular infusion of ODN antisense to MCR4 generated a consistent decrease (∼50%) in hypothalamic MCR4 protein as assessed by Western blot analysis (Figure 5, a–c). It should be noted that this decrease in whole hypothalamus is likely to underestimate the extent of the change in MCR4 protein in the hypothalamic nuclei surrounding the third cerebral ventricle. Intracerebroventricular administration of scrambled ODN did not alter the effect of α-MSH on intra-abdominal fat mass (decreased to 1.8 ± 0.3 g). However, the concomitant administration of ODN antisense to MCR4 blunted the effect of α-MSH on intra-abdominal fat (3.8 ± 0.5 g). Our results suggest that the neural melanocortin pathway regulates fat distribution largely by its stimulation of MCR4 in the hypothalamus.
Role of the melanocortin pathway in the regulation of insulin action on peripheral glucose uptake and production. (a) During insulin clamp studies, the rate of glucose infusion was markedly increased by α-MSH and markedly decreased by SHU9119. (c) Insulin action on glucose uptake (Rd) was significantly enhanced by α-MSH and decreased by SHU9119. (b) During insulin clamp studies, the rate of glucose production (GP) was lower in rats treated with α-MSH and higher in rats receiving SHU9119, compared with vehicle. (d) The inhibition of GP in response to physiological hyperinsulinemia was markedly increased by α-MSH and markedly decreased by SHU9119. Values represent mean ± SE. *P < 0.01 vs. vehicle. GIR, glucose infusion rate.
The MCR4 mediates the effect of α-MSH on hepatic insulin action. (a) Western blot of MCR4 in hypothalamus of rats receiving intracerebroventricular infusions of either scrambled ODN (SCR; lanes 1 and 3) or MCR4 antisense (AS; lanes 2 and 4) for 7 days. Representative blots are displayed. The identity of the 43-kDa band was confirmed by peptide competition. Addition of a specific MCR4 peptide eliminated this band (lanes 3 and 4), but failed to alter a nonspecific band. (b) Intracerebroventricular administration of MCR4 antisense decreased hypothalamic MCR4 protein, while insulin receptor IR-β and MCR3 proteins were not affected. (c) Quantitation of hypothalamic Western blots: intracerebroventricular administration of MCR4 antisense led to a consistent, approximately 50% decrease in hypothalamic MCR4 protein. (d) During insulin clamp studies, the rate of glucose disappearance (Rd) was increased in rats treated with α-MSH with scrambled ODN; however, α-MSH failed to increase Rd in rats receiving MCR4 antisense. (e) During insulin clamp studies, the rate of glucose production (GP) was lower in rats treated with α-MSH with scrambled ODN; however, α-MSH failed to decrease GP in rats receiving MCR4 antisense. Values represent mean ± SE. *P < 0.01 vs. vehicle.
We next examined whether decreasing the abundance of MCR4 in the hypothalamus alters the effect of the neural melanocortin pathway on in vivo insulin action. During physiologic hyperinsulinemia, the rate of tissue glucose uptake was increased in rats receiving α-MSH and scrambled ODN (Figure 5d). Conversely, α-MSH failed to increase the rate of glucose uptake in rats pretreated with ODN antisense to MCR4 (Figure 5c). Similarly, α-MSH with scrambled ODN markedly enhanced the action of insulin in inhibiting glucose production (Figure 5e). By contrast, intracerebroventricular administration of ODN antisense to MCR4 negated the effect of α-MSH on glucose production. Overall, these experiments support the notion that α-MSH exerts its metabolic effects largely by hypothalamic MCR4.
To investigate potential mechanism(s) by which α-MSH enhances hepatic insulin action, we measured the tyrosine phosphorylation of the two major substrates of the insulin receptor, IRS-1 and IRS-2, in the liver. In liver samples obtained at the completion of the insulin clamp studies, IRS-1 and IRS-2 proteins were present in similar amounts in all groups (Figure 6). However, tyrosine phosphorylation of IRS-1 (71%; P < 0.01) and IRS-2 (98%; P < 0.01) were markedly enhanced with α-MSH compared with vehicle (Figure 6). Treatment with SHU9119 resulted in a modest decrease in IRS-1 and IRS-2 phosphorylation, which did not achieve statistical significance.
Effect of α-MSH on hepatic insulin signaling. Liver samples were immunoprecipitated with anti–IRS-1 and anti–IRS-2 Ab’s and then immunoblotted with anti-phosphotyrosine and anti–IRS1/2 Ab’s. IRS-1 and IRS-2 proteins were not affected by treatment with α-MSH. By contrast, tyrosine phosphorylation of both insulin receptor substrates was markedly increased following α-MSH treatment. Values represent mean ± SE. *P < 0.01 vs. vehicle.