Ras signaling is essential for skin development.

Proliferation in the epidermis is a tightly controlled process. During skin development, epidermis formation and hair follicle morphogenesis crucially depend on the regulated balance between proliferation and differentiation. Here we deleted all three Ras loci (H-Ras, N-Ras and K-Ras) from keratinocytes in vitro as well as specifically from the epidermis in mice using a K5Cre strain. Upon Ras elimination, keratinocytes ceased proliferation and entered into senescence without any signs of apoptosis induction. Constitutive activation of the mitogen-activated protein kinase pathway was able to partially rescue the proliferative defects. In mice, Ras signaling was essential for proper development of the epidermis and hair follicles. Deletion of the three Ras loci during epidermis formation in mouse embryos caused a dramatic decrease in proliferation, resulting in a substantially thinner epidermis and delayed appearance of differentiation markers. We could not detect apoptotic or senescent cells in these embryos suggesting that loss of Ras protein expression only leads to severe hypoproliferation. These observations provide genetic evidence for an essential role of Ras proteins in the control of keratinocyte and epidermal proliferation.

Decreased responsiveness of gluconeogenesis to the modulation by sulfonylureas in hepatocytes isolated from obese (fa/fa) Zucker rats.

The influence of the hypoglycemic agent glipizide (0-100 microM) on the rate of gluconeogenesis from lactate, as well as on the levels of fructose 2,6-bisphosphate, has been investigated in hepatocytes isolated from genetically obese (fa/fa) Zucker rats and from their corresponding lean (Fa/-) littermates. As compared to lean rat hepatocytes, liver cells isolated from obese animals showed a lower rate of basal gluconeogenesis (0.9 +/- 0.2 vs 5.4 +/- 0.5 micromol of lactate converted to glucose/g cell x 30 min, n=4) and higher levels of fructose 2,6-bisphosphate (11.5 +/- 1.0 vs 5.9 +/- 0.4 nmol/g cell, n=8-9). In lean rat hepatocytes, the presence of glipizide in the incubation medium caused a dose-dependent inhibition of the rate of lactate conversion to glucose (maximal inhibition=46%; EC50 value=26 microM), and simultaneously raised the cellular content of fructose-2,6-bisphosphate (maximal increment=40%; EC50 value=10 microM). In contrast, in hepatocytes isolated from obese rats, the inhibition of gluconeogenesis and the increment in fructose-2,6-bisphosphate levels elicited by glipizide were significantly reduced (maximal effects of 22 and 13%, respectively). Similarly, the activation of glycogen phosphorylase and the increase in hexose 6-phosphate levels in response to glipizide were less marked in obese rat hepatocytes than in liver cells isolated from lean animals. These results demonstrate that the efficacy of sulfonylureas as inhibitors of hepatic gluconeogenesis is reduced in the genetically obese (fa/fa) Zucker rat.

Casein kinase 2 inactivation by Mg2+, Mn2+ and Co2+ ions.

Mg2+ as well as Mn2+, and Co2+, which may substitute Mg2+ in the mental ion requirement of casein kinase 2 (Gatica et al., FEBS Lett: 315:173-173, 1993), have been repeatedly reported to display an optimal concentration at which activity of casein kinase 2 is maximal. As far as we know this intriguing property has always been observed with casein as substrate. This phosphoprotein is not the natural substrate of the enzyme, and it is well known that it binds divalent metal ions, which provoke the aggregation and precipitation of the protein. Since an optimal concentration of metal ion might have a regulatory role, we have examined if it is a consequence of the particular properties of casein, or it is an inherent property of the enzyme, extensive to other substrates. We have used the type II regulatory subunit of protein kinase A which is a physiological substrate of the enzyme, and the peptide RRREEETEEE as a specific substrate. No optimal concentration of Mg2+ is observed when these two substrates are used. The results explain, however, why that optimum is observed with casein. Although low concentration of Mn2+, and Co2+ render about 25% of the maximal activity found with Mg2+, they inactivate the enzyme almost fully at concentrations at which Mg2+ yield the maximal activity.

Impairment of the modulation by glucose of hepatic gluconeogenesis in the genetically obese (fa/fa) Zucker rat.

Genetically obese (fa/fa) Zucker rats show oral glucose intolerance, an alteration that has been attributed at least in part to an impaired suppression of hepatic glucose output after the ingestion of glucose. In this work, we studied the influence of different concentrations of glucose (2.5-30 mM) on gluconeogenesis from a mixture of [14C]lactate-pyruvate as well as on fructose 2,6-bisphosphate levels, pyruvate kinase activity, and flux through the reaction catalyzed by 6-phosphofructo-1-kinase, in hepatocytes isolated from fed obese (fa/fa) or lean (Fa/-) rats. In hepatocytes isolated from lean rats, incubation with increasing concentrations of glucose caused a dose-dependent inhibition of gluconeogenesis (5.02 +/- 0.54 and 1.82 +/- 0.33 mumol lactate converted to glucose/g cells.20 min in hepatocytes incubated in the presence of 2.5 and 30 mM glucose, respectively; n = 4 experiments; P < 0.01) together with a significant elevation of the fructose 2,6-bisphosphate content and a stimulation of the flux through 6-phosphofructo-1-kinase reaction. Glucose also provoked a dose-dependent activation of pyruvate kinase in the absence of changes in the cellular concentration of cAMP. In liver cells from obese animals, gluconeogenesis was not significantly modified by raising the glucose concentration in the incubation medium (1.26 +/- 0.11 and 0.83 +/- 0.14 mumol lactate converted to glucose/g cells.20 min in hepatocytes incubated with 2.5 and 30 mM glucose, respectively; n = 4 experiments; P = 0.11) despite significant increases in both fructose 2,6-bisphosphate levels and flux through the 6-phosphofructo-1-kinase reaction. In these cells, pyruvate kinase was only slightly activated by high glucose concentrations. These results indicate that, unlike fructose 2,6-bisphosphate levels and flux through the 6-phosphofructo-1-kinase reaction, hepatic gluconeogenesis is unresponsive to high glucose concentrations in genetically obese (fa/fa) rats.

Decreased responsiveness of basal gluconeogenesis to insulin action in hepatocytes isolated from genetically obese (fa/fa) Zucker rats.

In vivo studies have demonstrated that hepatic glucose production is poorly responsive to insulin in genetically obese Zucker rats. In this work, we have investigated the modulation by insulin of basal gluconeogenesis, fructose 2,6-bisphosphate levels, and pyruvate kinase and 6-phosphofructo 2-kinase activities in hepatocytes isolated from fed obese (fa/fa) or lean (Fa/-) rats. Gluconeogenesis was estimated by the conversion of a mixture of [14C]lactate-pyruvate to [14C]glucose. Basal gluconeogenesis was significantly reduced in hepatocytes isolated from obese rats compared to that measured in hepatocytes from lean animals (0.63 +/- 0.09 vs. 1.47 +/- 0.05 mumol lactate converted to glucose/g cells.20 min; n = 3-4; P < 0.001). In hepatocytes isolated from lean rats, insulin, without affecting the cellular cAMP concentration, caused a dose-dependent inhibition of the rate of gluconeogenesis, which was accompanied by a significant increase in fructose 2,6-bisphosphate levels and activation of both pyruvate kinase and 6-phosphofructo 2-kinase. In contrast, in hepatocytes isolated from obese (fa/fa) rats, neither basal gluconeogenesis nor any of the other metabolic parameters mentioned were significantly modified by insulin, even when assayed at high hormonal concentrations (10 nM). These results demonstrate a lack of responsiveness of hepatic gluconeogenesis to short term insulin action in genetically obese (fa/fa) rats.

A polylysine-induced aggregation of substrate accompanies the stimulation of casein kinase II by polylysine.

Casein kinase II (CK-II) activation by polylysine parallels an aggregation of substrates promoted by the polycation. CK-II is known to be stimulated by basic polypeptides and polyamines. The mechanism by which this stimulation takes place, however, is not yet fully understood. Here we show that, in the usual CK-II assay, polylysine induces the aggregation of casein. This aggregation has been monitored by turbidimetry, electron microscopy and gel filtration. The polylysine-concentration-dependence of the casein aggregation parallels the polylysine-concentration-dependence of the enzyme stimulation. In the presence of polylysine the enzyme is incorporated into the casein aggregates promoted by the polycation, thus supporting the view that this substrate aggregation is directly related to the mechanism of CK-II stimulation. Preliminary results show that a similar parallelism occurs with other natural substrates of the enzyme. The physiological meaning of this substrate aggregation, and its possible relation to other polylysine-stimulated enzymes and polylysine-aggregated proteins, are discussed.

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro.

Dipyridamole activates in vitro type II cAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of cAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A1 receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883-888, 1989) might be mediated through a direct action--an activation--on the catalytic subunit of a cAMP-dependent protein kinase.