Serine-palmitoyl transferase activity in cultured human keratinocytes

Sphingolipids comprise approximately 25% of the stratum corneum lipids and are considered critical constituents of the epidermal permeability barrier. Whether sphingoid base structures are synthesized in the epidermis or whether they are derived from circulating or dermal sources is not known. We report here the initial characterization of serine-palmitoyl transferase (EC 2.3.1.50; SPT), the rate-limiting enzyme in the synthesis of sphingolipids, from cultured human neonatal keratinocytes. Subcellular fractionation studies demonstrated that 79% of the total cellular SPT activity was associated with the microsomes. The specific activity of keratinocyte SPT was 270 +/- 20 pmol/min per mg of microsomal protein, a level significantly higher than activities reported in other tissues. Keratinocyte SPT showed an apparent Km for L-serine of 0.40 (+/- 0.04 mM, with an alkaline pH optimum (8.2 +/- 0.4). Keratinocyte SPT utilizes palmitoyl-CoA preferentially over other saturated or unsaturated acyl-CoA substrates; increasing acyl-CoA chain lengths above C16 by one or two carbons was less detrimental to activity than similar decrements in chain length. Finally, the mechanism-based inhibitors L-cycloserine and beta-chloro-L-alanine, demonstrated potent inhibition of keratinocyte SPT activity, with 50% inhibitory concentrations of approximately 3.0 and 25 microM, respectively. In summary, we have found that cultured human neonatal keratinocytes contain unusually high levels of serine-palmitoyl transferase activity, and that the substrate specificity of keratinocyte SPT may determine the base composition of epidermal sphingolipids.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Ventilatory effects of impaired glial function in a brain stem chemoreceptor region in the conscious rat.

Glia are thought to regulate ion homeostasis, including extracellular pH; however, their role in modulating central CO2 chemosensitivity is unclear. Using a push-pull cannula in chronically instrumented and conscious rats, we administered a glial toxin, fluorocitrate (FC; 1 mM) into the retrotrapezoid nucleus (RTN), a putative chemosensitive site, during normocapnia and hypercapnia. FC exposure significantly increased expired minute ventilation (VE) to a value 38% above the control level during normocapnia. During hypercapnia, FC also significantly increased both breathing frequency and expired VE. During FC administration, maximal ventilation was achieved at approximately 4% CO2, compared with 8-10% CO2 during control hypercapnic trials. RTN perfusion of control solutions had little effect on any ventilatory measures (VE, tidal volume, or breathing frequency) during normocapnic or hypercapnic conditions. We conclude that unilateral impairment of glial function in the RTN of the conscious rat results in stimulation of respiratory output.     

Apolipoprotein [a] genotype influences isoform dominance pattern differently in African Americans and Caucasians

Plasma lipoprotein [a] (Lp[a]) concentrations are inversely associated with, and largely determined by, apolipoprotein [a] (apo[a]) gene size, a highly polymorphic trait. We studied if, within an individual, the smaller apo[a] isoform always dominated, whether there was interaction between the two alleles, and whether these features differed between Caucasians and African Americans. We determined apo[a] gene sizes, apo[a] protein sizes and relative amounts, and plasma Lp[a] levels in 430 individuals (263 Caucasians and 167 African Americans). Of the 397 heterozygotes with at least one detectable apo[a] isoform (238 Caucasians and 159 African Americans), the larger allele dominated in 28% of Caucasians and 23% of African Americans, while the smaller allele dominated in 56% of Caucasians and 45% of African Americans. In Caucasians, dominance of the smaller allele increased with Lp[a] levels, from 44% at Lp[a] < or = 30 nM to 81% at Lp[a] >100 nM (P < 0.0001). Dominance by the smaller allele increased with increasing size of the larger allele in both groups but with the smaller allele only in African Americans. There was no interaction between apo[a] alleles within genotypes; one apo[a] isoform level was not associated with the other isoform level, and isoform levels were not affected by the difference in size. More of the dominance pattern was explained by Lp[a] level and apo[a] genotype in African Americans than in Caucasians (29% vs. 13%). Thus, genotype influences isoform-specific Lp[a] levels and dominance patterns differently in African Americans and in Caucasians.     

Treatment with high-dose simvastatin reduces secretion of apolipoprotein B-lipoproteins in patients with diabetic dyslipidemia

HMG-CoA reductase inhibitors (statins) are effective lipid-altering drugs for the treatment of dyslipidemia in patients with type 2 diabetes mellitus. We conducted a randomized, double-blind, placebo-controlled, crossover design trial to determine the effects of simvastatin, 80 mg/day, on plasma lipid and lipoprotein levels and on the metabolism of apolipoprotein B (apoB) in VLDL, intermediate density lipoprotein (IDL), and LDL and of triglycerides (TGs) in VLDL. Simvastatin therapy decreased TG, cholesterol, and apoB significantly in VLDL, IDL, and LDL. These effects were associated with reduced production of LDL-apoB, mainly as a result of reduced secretion of apoB-lipoproteins directly into the LDL density range. Statin therapy also reduced hepatic production of VLDL-TG. There were no effects of simvastatin on the fractional catabolic rates of VLDL-apoB or -TG or LDL-apoB. The basis for decreased VLDL-TG secretion during simvastatin treatment is not clear, but recent studies suggest that statins may activate peroxisomal proliferator-activated receptor alpha (PPARalpha). Activation of PPARalpha could lead to increased hepatic oxidation of fatty acids and less synthesis of TG for VLDL assembly.     

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT₁ receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT₁ receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28^(1.32)–Ile-53^(1.57) fragment of TMD1 and Leu-143^(4.40)–Phe-170^(4.67) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C^(1.34)-AT₁ and T33C^(1.37)-AT₁ and TMD4 mutant V169C^(4.66)-AT₁. Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT₁) background, we found that V169C^(4.66)-AT₁ remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C^(4.64)-AT₁ was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30^(1.34) and Thr-33^(1.37), as well as residues Arg-167^(4.64) and Val-169^(4.66) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT₁ receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Effects of a 1,3-diacylglycerol oil-enriched diet on postprandial lipemia in people with insulin resistance

Postprandial hypertriglyceridemia is common in individuals with insulin resistance, and diets enriched in 1,3-diacylglycerol (DAG) may reduce postprandial plasma triglycerides (PPTGs). We enrolled 25 insulin-resistant, nondiabetic individuals in a double-blind, randomized crossover trial to test the acute and chronic effects of a DAG-enriched diet on PPTG. Participants received either DAG or triacylglycerol (TAG) oil, in food products, for 5 weeks. Fasting lipids, and two separate postprandial tests, one with DAG oil and one with TAG oil, were performed at the end of each 5 week diet period. We found no acute or chronic effects of DAG oil on PPTG. Thus, neither the DAG oil PPTG (h/mg/dl) on a chronic TAG diet [area under the curve (AUC) = 503 +/- 439] nor the TAG oil PPTG on a chronic DAG diet (AUC = 517 +/- 638) was different from the TAG oil PPTG on a chronic TAG diet (AUC = 565 +/- 362). Five weeks of a DAG-enriched diet had no acute or chronic effects on PPTG in insulin-resistant individuals. We suggest further studies to evaluate the effects of DAG on individuals with low and high TG levels.