Treatment of rats with growth hormone (GH; 1 mg/kg sc) twice daily over 2.5 days did not alter fasting plasma glucose or glucose tolerance but increased fasting plasma insulin levels 65% and peak insulin response to a glucose load 35% over controls, indicating the development of insulin resistance. Studies on partially purified insulin receptors from soleus muscles showed that GH increased the abundance of insulin receptor beta-subunits by 48% as measured by immunoblotting. Despite this increase, GH abolished the increase in autophosphorylation of the insulin receptor beta-subunit in response to physiological hyperinsulinemia and diminished by 28% the response to supraphysiological hyperinsulinemia. Similarly, insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) was decreased 25% by GH, but the abundance of IRS-1 was not affected. Studies on rats pretreated with streptozotocin suggested that the effects of GH are direct and not secondary to GH-induced hyperinsulinemia. GH decreased basal GLUT-1 abundance in the low-density microsome and plasma membrane fractions of epididymal adipocytes by 50 and 42%, respectively, but decreased basal GLUT-4 abundance only in the low-density microsome fraction by 24%. Despite these alterations, the abundance of both transporters in the plasma membrane fraction of adipocytes incubated with 0.1 U insulin/ml was not diminished by GH.

Activities of Akt1, Akt2, and Akt3 kinases and glucose uptake in hindlimb muscles of the rat in vivo were investigated. The rats were studied either after intravenous injection of 0.1 U of insulin or during exercise induced by stimulating calf muscles electrically at 1 contraction/s. Akt kinases were immunoprecipitated from supernatants of muscle homogenates. Glucose uptake by muscles in vivo was assessed by cellular accumulation of 2-deoxy-d-[1,2-3H(N)]glucose. Administration of insulin resulted in rapid activation of Akt1 kinase, with peak activity observed 5 min after insulin injection. Soleus muscle, a slow-twitch muscle, and plantaris muscle, a fast-twitch muscle, differed in their content of Akt1 kinase and in their response to insulin. Soleus muscle exhibited a 105% higher abundance of Akt1 kinase, a 101% higher insulin-stimulated activity of Akt1 kinase, and 83% higher insulin-stimulated 2-deoxyglucose uptake compared with plantaris muscle. Additionally, insulin administration increased the activities of Akt1, Akt2, and Akt3 kinases in calf muscles and caused a sevenfold augmentation in 2-deoxyglucose uptake by these muscles. In contrast, the exercised calf muscles exhibited an increase in Akt1 kinase activity at 5, 15, and 25 min of exercise but no change in activities of Akt2 and Akt3 isoforms, and the 2-deoxyglucose uptake by calf muscles exercised for 25 min was 11-fold higher compared with muscles of resting rats. The data demonstrate that 1) there is a close, direct correlation between the magnitude of insulin-stimulated activity of Akt1 kinase and the level of glucose uptake in muscles with different fiber populations, 2) insulin activates three isoforms of Akt kinase in skeletal muscle, and 3) exercise in vivo is associated with activation of Akt1 but not Akt2 and Akt3 kinases in contracting muscles.

Sciatic nerve in one hindlimb of rats was sectioned, and animals were investigated 3, 6, 9, and 24 h and 3, 8, and 17 days after surgery. Cellular uptakes of 2-deoxy-D-glucose (DG) and alpha-aminoisobutyric acid (AIB) by soleus, plantaris, and gastrocnemius muscles of the denervated and contralateral sham hindlimb were compared in vivo. Effects of insulin on DG and AIB uptakes by soleus and plantaris muscles, but not gastrocnemius muscles, were reduced 20-58% as early as 3-6 h after denervation. Three to 17 days after denervation, soleus muscle did not respond to insulin stimulation of DG uptake, whereas plantaris and gastrocnemius muscles responded, but the insulin-induced increment in DG uptake was reduced 24-68% compared with the sham muscles. In contrast, none of the denervated muscles increased AIB uptake in response to insulin stimulation 3-17 days after surgery. The levels of creatine phosphate and ATP in calf muscles frozen in situ 3 days after denervation were increased 7 and 9%, respectively, and those of ADP, AMP, glucose-6-phosphate, and lactate were unchanged compared with sham muscles. It is concluded that muscle denervation results in a rapid development of insulin resistance, the effect of denervation depends on muscle fiber population, and insulin resistance of denervated muscles is not due to a cellular energy deficit.

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The effects of sphingomyelinase, phosphorylcholine, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide) and sphingosine on basal and insulin-stimulated cellular accumulation of 2-deoxy--glucose in rat soleus muscles were investigated. Preincubation of muscles with sphingomyelinase (100 or 200 m-units\ml) for 1 or 2 h augmented basal 2-deoxyglucose uptake by 29-91 %, and that at 0.1 and 1.0 munit of insulin\ml by 32-82 % and 19-25 % respectively compared with control muscles studied at the same insulin concentrations. The sphingomyelinase-induced increase in basal and insulin-stimulated 2-deoxyglucose uptake was inhibited by 91 % by 70 µM cytochalasin B, suggesting that it involves glucose transporters. Sphingomyelinase had no effect on the cellular accumulation of -glucose, which is not transported by glucose transporters. The sphingomyelinase-induced increase in 2-deoxyglucose uptake could not be reproduced by preincubating the muscles with 50 µM phosphorylcholine, 50 µM C2-ceramide or 50 µM C6-ceramide. Preincubation of muscles with 50 µM sphingosine augmented basal 2-deoxyglucose transport by 32 %,