Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment

Abstract

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.

Hepatic Acetyl CoA Links Adipose Tissue Inflammation to Hepatic Insulin Resistance and Type 2 Diabetes

“…While hepatic acetyl CoA has been long known to modulate pyruvate carboxylase activity in vitro (Barritt et al, 1966; Cazzulo and Stoppani, 1968; Cooper and Benedict, 1966; Keech and Utter, 1963; Krebs et al, 1965), methodological limitations stemming from acetyl CoA’s low hepatocellular concentrations and rapid degradation ex vivo have prevented its measurement in vivo. Additionally, Chisholm et al were not able to demonstrate any direct effect of insulin on pyruvate carboxylase activity in isolated hepatocytes (Chisholm et al, 1983), begging the question of whether a hepatocyte-autonomous process is primarily responsible for insulin-mediated suppression of PC activity and hepatic glucose production.…”

Effect of treatment of rats with dexamethasone in vivo on gluconeogenesis and metabolite compartmentation in subsequently isolated hepatocytes

“…Fed male Sprague-Dawley rats weighing 180-240g were injected with dexamethasone acetate (15 ,imol/100g)or0.9% NaCl intraperitoneally 2or 3 h before the preparation of the hepatocytes as indicated. After the appropriate time, the animals were anaesthetized with sodium pentobarbital (60mg/kg), and hepatocytes were prepared as described previously (Chisholm et al, 1983). Batches of collagenase were carefully screened in terms of metabolic function and Trypan Blue exclusion, and only those producing cells of which over 85% excluded the dye were used.…”

“…Citrate was assayed as described by Passonneau & Brown (1974), malate as described by Goldberg & Passonneau (1974), pyruvate by the method of Passonneau & Lowry (1974), aspartate by the method of Williamson & Corkey (1969) and phosphoenolpyruvate by the method of Czok & Lamprecht (1974). Other metabolites were measured as described previously (Chisholm et al, 1983).…”

Adrenergic regulation of gluconeogenesis: possible involvement of two mechanisms of signal transduction in alpha 1-adrenergic action.

“…Our hypothesis is schematically represented in Fig. 1 and is based mainly on the following findings: (t) metabolic effects due to a1-adrenergic activation are clearly observed in cells incubated in the absence of extracellular calcium and even in calcium-depleted hepatocytes, whereas those of the vasopressor peptides are abolished (9,14,20); (ih) hypothyroidism markedly diminishes the metabolic effects of vasopressin and angiotensin II but not those due to a1-adrenergic activation (11, 12); (iii) insulin reduces the stimulation of glycogenolysis due to a1-adrenergic activation but not that produced by vasopressin or angiotensin II (13,16,17,20); (iv) the inhibitory action of insulin on a1-adrenergic actions is markedly magnified in calciumdepleted hepatocytes and in hepatocytes from hypothyroid rats (13,16,20); (v) in hepatocytes from adrenalectomized rats the metabolic effects due to a1-adrenergic amines became dependent on the presence of extracellular calcium (14, 23)-i.e., a1-adrenergic actions resemble those of vasopressin or angiotensin II; (vi) we have recently observed that cycloheximide, which stimulates hepatic metabolism through an a1-adrenergic mechanism (24), mimics the actions of epinephrine in an insulin-insensitive calcium-dependent fashion and that the action of cycloheximide is observed in hepatocytes from control and adrenalectomized rats but not in cells from hypothyroid animals (25). Thus, in summary, our model suggests that a1-adrenergic effects are mediated through two pathways: one of them also shared by vasopressin and angiotensin II, modulated by thyroid status, calciumdependent, insulin-insensitive, and possibly involving phosphoinositide turnover and calcium in its mechanism of transduction; and another pathway, not shared with the vasopressor peptides, modulated by glucocorticoids, calcium-independent, insulin-sensitive, and mediated through unknown second messenger(s) (see Fig.…”

“…During the last 4 years we (9)(10)(11)(12)(13)(14) and others (15)(16)(17)(18)(19)(20)(21)(22) have observed differences between the action of the vasopressor peptides and those due to a1-adrenergic activation. These differences led us to propose the possible existence of two mechanisms of signal transduction for a1-adrenergic action in the liver cell (9)(10)(11)(12)(13)(14).…”