Long-acting insulin analog detemir displays reduced effects on adipocyte differentiation of human subcutaneous and visceral adipose stem cells.

BACKGROUND AND AIMS: Since treatment with insulin detemir results in a lower weight gain compared to human insulin, we investigated whether detemir is associated with lower ability to promote adipogenesis and/or lipogenesis in human adipose stem cells (ASC). METHODS AND RESULTS: Human ASC isolated from both the subcutaneous and visceral adipose tissues were differentiated for 30 days in the presence of human insulin or insulin detemir. Nile Red and Oil-Red-O staining were used to quantify the rate of ASC conversion to adipocytes and lipid accumulation, respectively. mRNA expression levels of early genes, including Fos and Cebpb, as well as of lipogenic and adipogenic genes, were measured at various phases of differentiation by qRT-PCR. Activation of insulin signaling was assessed by immunoblotting. ASC isolated from subcutaneous and visceral adipose tissue were less differentiated when exposed to insulin detemir compared to human insulin, showing lower rates of adipocyte conversion, reduced triglyceride accumulation, and impaired expression of late-phase adipocyte marker genes, such as Pparg2, Slc2a4, Adipoq, and Cidec. However, no differences in activation of insulin receptor, Akt and Erk and induction of the early genes Fos and Cebpb were observed between insulin detemir and human insulin. CONCLUSION: Insulin detemir displays reduced induction of the Pparg2 adipocyte master gene and diminished effects on adipocyte differentiation and lipogenesis in human subcutaneous and visceral ASC, in spite of normal activation of proximal insulin signaling reactions. These characteristics of insulin detemir may be of potential relevance to its weight-sparing effects observed in the clinical setting.

Glucagon-like peptide-1 counteracts oxidative stress-dependent apoptosis of human cardiac progenitor cells by inhibiting the activation of the c-Jun N-terminal protein kinase signaling pathway.

Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H(2)O(2)). Phospho- and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9-39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H(2)O(2) induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H(2)O(2)-triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9-39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1.

Release of ischemia in paced rat Langendorff hearts by supply of L-carnitine: role of endogenous long-chain acylcarnitine.

Rat Langendorff hearts perfused with media that do not contain erythrocytes or fluorocarbon as oxygen carriers are borderline aerobic during 5 Hz pacing. This follows from the release of catabolic products measured: lactate, urate and Iysophosphatidyl-choline (IysoPC). Addition of L-carnitine to the perfusion medium reduced the level of these compounds, while the release of long-chain acylcarnitine (LCAC) increased. Previously, we found (Biochim Biophys Acta 847:62-66,1985) that micromolar LCAC protects membranes during reperfusion after ischemia. Therefore, the observed inverse relation between LCAC and the other compounds measured suggests that LCAC is the basis of an acute relief of imminent ischemia by carnitine addition. LCAC may be released from various cell types, including vascular endothelium, as demonstrated. The cationic amphiphilic nature of LCAC is responsible for protection of membrane functions in imminent ischemia.

High performance liquid chromatography of long-chain acylcarnitine and phospholipids in fatty acid turnover studies.

In this paper we describe a rapid, isocratic high performance liquid chromatography (HPLC) method for the study of radioactive fatty acid incorporation into complex lipids of human erythrocytes, which allows the simultaneous separation of the major phospholipid classes and long-chain acylcarnitines. The lipid extract of erythrocytes pulsed with radioactive fatty acids was injected into an HPLC system equipped with a silica column. The individual components eluted were monitored by ultraviolet absorption and radioactive emission. With respect to the UV profile, the radioactive profile showed an additional peak between phosphatidyl-choline and phosphatidylethanolamine, which was identified as long-chain acylcarnitine by different experimental approaches. The radioactivity recovered in the long-chain acylcarnitines contains essential information enabling definition of acyl trafficking in red cells.

Uptake and release of carnitine by vascular endothelium in culture; effects of protons and oxygen free radicals.

The present paper shows that cultured bovine endothelial cells can be labeled with 3H-carnitine by incubation. This process is slow and is uphill, requiring Na+/K+ ATPase activity. After 3 days incubation isotopic equilibrium is reached, when the cells contain about 0.5 mM (total) carnitine at a medium concentration of about 3 microM. The plasmamembrane barrier is rather resistant to acidosis and oxygen free radicals (OFR). The rate of carnitine release increases significantly only at pH below 5.8. At pH 6.0 the release of stored carnitine can be initiated by the addition of D- or L-lactate. OFR, generated by the addition of xanthine and xanthine oxidase, did not affect carnitine release. Both mild acidosis and OFR left plasmamembranes of endothelial cells intact as judged by the absence of lactate dehydrogenase loss from the cells. Therefore, the known increase of capillary permeability during ischemia and reperfusion may not be due to plasmalemmal disruption of individual endothelial cells, but to increase of inter-endothelial spaces.

Comparison of the effects of carnitine palmitoyltransferase-1 and -2 inhibitors on rat heart hypertrophy.

Rats treated orally for 21 days with aminocarnitine, an inhibitor of carnitine palmitoyltransferase-2 (CPT-2), do not show hypertrophy of the heart. This contrasts with the effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, that, according to the literature, cause hypertrophy. As CPT-1 and CPT-2 are both required for the oxidation of long-chain fatty acids in mitochondria, it can be concluded that inhibition of fatty acid oxidation per se is not responsible for cell growth, but rather the accumulation of a metabolite, probably long-chain acylcoenzyme A. CPT-1 and CPT-2 inhibitors cause different metabolic changes in the heart. Electron microscopy of hearts fixed 1 hour after Langendorff perfusion with the two types of inhibitors reveals some of these changes. Multilamellar vesicles were observed with aminocarnitine (CPT-2 inhibitor) but not with etomoxir (CPT-1 inhibitor). When both inhibitors were present, electron-dense spots adjacent to mitochondria were observed, possibly containing long-chain acylaminocarnitine.

Carnitine and cardiac interstitium.

An important part of (acyl)carnitine may be stored in interstitial spaces and the external surface of adjacent cells. A high concentration of carnitine in the direct vicinity of cells may enhance the synthesis and export of long-chain acylcarnitine. Long-chain acylcoenzyme A, from which long-chain acyl carnitine is formed, cannot penetrate intact cell membranes. During hypoperfusion or ischemia, when long-chain acylcoenzyme A accumulates due to hampered fatty acid oxidation, there is an increased formation of long-chain acyl carnitine which diffuses into the interstitium and adjacent vascular endothelial cells. Due to its lipophilic nature and net positive charge (limitation of carboxyl-group dissociation in ischemic acidosis), long-chain acyl carnitine may decrease the affinity of Ca2+ to the cell surface and prevent Ca2+ overload of cells. The advantage of carnitine over many other cationic amphiphiles in the protection of areas of ischemia is that long-chain acyl carnitine is formed and stored only in ischemic areas.

Anaplerotic effect of propionyl carnitine in rat heart mitochondria.

Carnitine and propionyl carnitine both increase production of 14CO2 from [1-14C]pyruvate but only the latter, in a process inhibited by malonate, increases oxygen uptake and 14CO2 evolution from [2-14C]pyruvate. Carnitine increases the rate of formation of acetyl carnitine from pyruvate to a greater extent than propionyl carnitine. These effects of carnitine and propionyl carnitine are not detectable in mitochondria (e.g., rat liver) deficient in carnitine acetyl transferase activity.

Regional distribution of ubiquinones and tocopherols in the mouse brain: lowest content of ubiquinols in the substantia nigra.

C57 black mice of 3 months of age were sacrificed, and their brain regionally dissected according to a protocol that strickly control for the death-freezing interval of each region. HPLC measurements of tocopherols and oxidized and reduced ubiquinones demonstrated significant regional variations. The substantia nigra had the lowest content of Q10 and a skewed ratio in favor of its oxidized form. Forebrain cholinergic nuclei had also more oxydized than reduced Q10 and in addition the lowest content of tocopherols. These findings suggest that nuclei that show neuronal depletion with age are the ones prone to oxidative stress.

Transient nigral ubiquinone depletion after single MPTP administration in mice.

The administration of a single injection of 30 mg/kg MPTP to mice produces at 1 hr, a transient significant decrease of the reduced ubiquinol Q10 in the substantia nigra that is normalized afterwards. This suggests a transient stress imposed by MPTP (or more likely MPP+) in the mitochondrial respiratory and/or oxidoreducing system, located in close proximity to the NADH system.