Glucose (Glc) metabolism protects cells against oxidant injury. By virtue of their central position in both Glc uptake and utilization, hexokinases (HKs) are ideally suited to contribute to these effects. Compatible with this hypothesis, endogenous HK activity correlates inversely with injury susceptibility in individual renal cell types. We recently reported that ectopic HK expression mimics the anti-apoptotic effects of growth factors in cultured fibroblasts, but anti-apoptotic roles for HKs have not been examined in other cell types or in a cellular injury model. We therefore evaluated HK overexpression for the ability to mitigate acute oxidant-induced cell death in an established epithelial cell culture injury model. In parallel, we examined salutary heparinbinding epidermal growth factor (EGF)-like growth factor (HB-EGF) treatment for the ability to 1) increase endogenous HK activity and 2) mimic the protective effects of ectopic HK expression. Both HK overexpression and HB-EGF increased Glc-phosphorylating capacity and metabolism, and these changes were associated with markedly reduced susceptibility to acute oxidantinduced apoptosis. The uniform Glc dependence of these effects suggests an important adaptive role for Glc metabolism, and for HK activity in particular, in the promotion of epithelial cell survival. These findings also support the contention that HKs contribute to the protective effects of growth factors. Glucose (Glc)1 metabolism plays a critical role in the protection of a variety of cell types against oxidant-induced cell death (1-9). The mechanisms underlying these cytoprotective effects are poorly understood, but contributions by both altered energy metabolism and cellular redox status have been suggested. Hexokinases (HKs) play a central role in both the uptake and utilization of Glc by catalyzing the first committed step of its metabolism, the phosphorylation of Glc to yield Glc 6-phosphate. By this mechanism, HKs maintain the downhill concentration gradient that permits facilitated Glc entry into cells. They also initiate all major pathways of Glc utilization and are ideally positioned to influence metabolic flux through both the glycolytic and the pentose phosphate pathways. Glycolytic flux is intimately linked to cellular energy metabolism, whereas pentose phosphate pathway flux plays a pivotal role in maintaining the cellular redox state. In principle, therefore, HK activity may directly contribute to the salutary effects associated with Glc metabolism in the setting of oxidative stress.

The prototypical extracellular phospholipid mediator, lysophosphatidic acid (LPA), exhibits growth factor-like properties and represents an important survival factor in serum. This potent mesangial cell mitogen is increased in conditions associated with glomerular injury. It is also a known activator of the classic mitogen-activated protein kinase (MAPK) pathway, which plays an important role in the regulation of mesangial cell hexokinase (HK) activity. To better understand the mechanisms coupling metabolism to injury, we examined the ability of LPA to regulate HK activity and expression in cultured murine mesangial cells. LPA increased total HK activity in a concentration- and time-dependent manner, with maximal increases of >50% observed within 12 h of exposure to LPA concentrations > or =25 microM (apparent ED(50) 2 microM). These effects were associated with increased extracellular signal-regulated kinase (ERK) activity and were prevented by the pharmacological inhibition of either MAPK/ERK kinase or protein kinase C (PKC). Increased HK activity was also associated with increased glucose (Glc) utilization and lactate accumulation, as well as selectively increased HKII isoform abundance. The ability of exogenous LPA to increase HK activity was both Ca2+ independent and pertussis toxin insensitive and was mimicked by LPA-generating phospholipase A2. We conclude that LPA constitutes a novel lipid regulator of mesangial cell HK activity and Glc metabolism. This regulation requires sequential activation of both Ca2+-independent PKC and the classic MAPK pathway and culminates in increased HKII abundance. These previously unrecognized metabolic consequences of LPA stimulation have both physiological and pathophysiological implications. They also suggest a novel mechanism whereby metabolism may be coupled to cellular injury via extracellular lipid mediators.

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.

Mesangial cell hexokinase (HK) activity is increased by a diverse array of factors that share both an association with pathological conditions and a common requirement for classic MAPK pathway activation. To better understand the relationship between glucose (Glc) metabolism and injury and to indirectly test the hypothesis that these changes constitute a general adaptive response to insult, we have sought to identify and characterize injury-associated factors that couple to mesangial cell HK regulation. Proinflammatory interleukin-1 (IL-1) cytokines activate the MAPK pathway and have known salutary effects in this cell type. We therefore examined their ability to influence mesangial cell HK activity, Glc utilization, MAPK pathway activation, and individual HK isoform abundance. IL-1beta increased HK activity in both a time- and concentration-dependent manner: activity increased maximally by approximately 50% between 12 and 24 h with an apparent EC(50) of 3 pM. IL-1alpha mimicked, but did not augment, the effects of IL-1beta. Specific IL-1 receptor antagonism and selective MAPK/ERK kinase or upstream Ras inhibition prevented these increases, whereas PKC inhibition did not. Changes in HK activity were associated with both increased Glc metabolism and selective increases in HKII isoform abundance. We conclude that IL-1 cytokines can regulate cellular Glc phosphorylating capacity via an IL-1 receptor-, Ras-, and classic MAPK pathway-mediated increase in HKII abundance. These findings suggest a novel, previously undescribed mechanism whereby metabolism may be coupled to inflammation and injury.

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.