Reijo Käkelä

Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry

Phospholipids (PL) in cerebral cortex from patients with infantile (INCL or CLN1) and juvenile (JNCL or CLN3) forms of neuronal ceroid‐lipofuscinosis (NCL) and controls were analysed by normal phase HPLC and on‐line electrospray ionization ion‐trap mass spectrometric detection (LC‐ESI‐MS). The method provided quantitative data on numerous molecular species of different PL classes, which are not achieved by using the conventional chromatographic methods. Compared with the controls, the INCL brains contained proportionally more phosphatidylcholine (PC), and less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Different molecular species of PC, PE, PS, phosphatidylinositol and sphingomyelin were quantified using multiple internal PL standards that differed in fatty acyl chain length and thus allowed correction for chain length dependency of instrument response. In INCL cortex, which had lost 65% of the normal PL content, the proportions of polyunsaturated molecular species, especially the PS and PE that contained docosahexaenoic acid (22:6n‐3), were dramatically decreased. The membranes may have adapted to this alteration by increasing the proportions of PL molecules substituted with monounsaturated and short‐chain fatty acids. Lysobisphosphatidic acid was highly elevated in the INCL brain and consisted mostly of polyunsaturated species. It is possible that changes in the composition of PL membranes accelerate progression of INCL by altering signalling and membrane trafficking in neurons.

SITE-SPECIFIC FATTY ACID COMPOSITION IN ADIPOSE TISSUES OF SEVERAL NORTHERN AQUATIC AND TERRESTRIAL MAMMALS

Abstract Site-specific differences in fatty acid compositions (by gas-liquid chromatography) were compared in aquatic, semiaquatic and terrestrial mammals: the ringed seals ( Phoca hispida hispida and P. h. botnica ), otter ( Lutra lutra ), raccoon dog ( Nyctereutes procyonoides ), brown bear ( Ursus arctos ) and grey wolf ( Canis lupus ). In addition, we briefly discuss our earlier results for the Canadian beaver ( Castor canadensis ) and muskrat ( Ondatra zibethicus ). In both aquatic and terrestrial species, large amounts of Δ9-monounsaturated fatty acids (MUFAs) and small amounts of saturated fatty acids and exogenous long-chain MUFAs were found in the cold tissues of the extremities. In seals, the poikilothermic outer blubber had these characteristics and differed from the inner blubber. On the other hand, the subcutaneous and inner fat depots of the coated semiaquatic and terrestrial mammals were uniform. In the bare extremities, however, these mammals also had an excess of A9-MUFAs. The degree of Δ9-desaturation in the outer blubber of the seals was significantly correlated with age. The excess of Δ9-MUFAs in the bare extremities of land mammals increased the overall double bond content of these tissues compared with the inner depots. In contrast, due to the large amounts of dietary polyunsaturated fatty acids, this was not found in the aquatic and semiaquatic species. The observed site-specific differences are discussed as possible inherited evolutionary adaptations to low temperature of the tissues.

Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P(3) and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.

PNPLA3 mediates hepatocyte triacylglycerol remodeling

The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3WT) and PNPLA3I148M on HuH7 cell lipidome after [13C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [13C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3WT and PNPLA3I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3WT associated in PCA with newly synthesized [13C]TAGs, particularly 52:1 and 50:1, while PNPLA3I148M associated with similar preexisting TAGs. PNPLA3WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3I148M. PNPLA3I148M localized more extensively to lipid droplets (LDs) than PNPLA3WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3I148M.

VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications

Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.

Stratification, Composition, and Function of Marine Mammal Blubber: The Ecology of Fatty Acids in Marine Mammals

This study of vertical fatty acid profiles, based on analysis of 58 fatty acids sampled at 3‐mm intervals throughout the blubber column of a model marine mammal, the ringed seal (Pusa hispida), revealed three chemically distinct layers. The average depths of the outer and inner layers were quite consistent (∼1.5 and ∼1 cm, respectively). Consequently, the middle layer varied greatly in thickness, from being virtually absent in the thinnest animals to 2.5 cm thick in the fattest. The relative consistencies of the thickness and composition of the layers as well as the nature of the fatty acids making up each layer support the generally assumed function of the various layers: (1) the outer layer is primarily structural and thermoregulatory, (2) the inner layer is metabolically active with a fatty acid composition that is strongly affected by recent/ongoing lipid mobilization/deposition, and (3) the middle layer is a storage site that contracts and expands with food availability/consumption. The remarkable dynamics of the middle layer along with the discrete pattern of stratification found in the vertical fatty acid profiles have important implications for methodological sampling design for studies of foraging ecology and toxicology based on analyses of blubber of marine mammals.

Mass spectrometric analysis reveals changes in phospholipid, neutral sphingolipid and sulfatide molecular species in progressive epilepsy with mental retardation, EPMR, brain: a case study

Progressive epilepsy with mental retardation, EPMR, belongs to a group of inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The CLN8 gene that underlies EPMR encodes a novel transmembrane protein that localizes to the endoplasmic reticulum (ER) and ER–Golgi intermediate compartment. Recently, CLN8 was linked to a large eukaryotic protein family of TLC (TRAM, Lag1, CLN8) domain homologues with postulated functions in lipid synthesis, transport or sensing. By using liquid chromatography/mass spectrometry we analysed molecular species of major phosholipid and simple sphingolipid classes from cerebral samples of two EPMR patients representing a progressive and advanced state of the disease. The progressive state brain showed reduced levels of ceramide, galactosyl‐ and lactosylceramide and sulfatide as well as a decrease in long fatty acyl chain containing molecular species within these classes. Among glycerophospholipid classes, an increase in species containing polyunsaturated acyl chains was detected especially in phosphatidylserines and phosphatidylethanolamines. By contrast, saturated and monounsaturated species were overrepresented among phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol classes in the advanced state sample. The observed changes in brain sphingo‐ and phospholipid molecular profiles may result in altered membrane stability, lipid peroxidation, vesicular trafficking or neurotransmission and thus may contribute to the progression of the molecular pathogenesis of EPMR.

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho‐ and glycolipids in the brains of ctsd−/− and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd−/− and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd−/− mice. Of particular note, a 20‐fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd−/− mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd−/− mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho‐ and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.

Effects of nickel chloride on reproduction of the rat and possible antagonistic role of selenium

Nickel (10-100 ppm added as NiCl2) was studied to determine its effects on reproduction of Wistar rats. In nine experimental groups, females, males or both were exposed to nickel in drinking water. In one female group and one male group, the drinking water was also supplemented with 0.3 ppm selenium (added as Na2SeO3). Breeding success and the growth and viability of pups were recorded. Nickel, copper and zinc concentrations in kidneys, liver and skin (with fur) of the females, males and pups were determined with an atomic absorption spectrophotometer. In addition, histology of the male testes (from control and nickel-exposed groups) was studied. The female exposures started 14, 28 or 100 days before copulation and continued during pregnancy and lactation. When the males were exposed (for 28 or 42 days before copulation), NiCl2 reduced both the number of pregnancies and the number of pups born. In the testes, NiCl2 induced shrinkage of the seminiferous tubules, which seemed to close some of the tubules. In the tubules, NiCl2 decreased the number of basal spermatogonia. When the females or both parents were exposed to NiCl2, pup mortality during lactation was high. However, when the females were drinking NiCl2 supplemented with selenium, all the pups survived and development of the total mass of the litters was even better than in the control group. In the same way, in males, selenium supplementation of the drinking water protected those pups that were born; but fertility was lower than with the control treatment. In the tissues studied, nickel accumulated most in the kidneys and then in the liver and skin. In each type of organ, there was a clear dose response relationship. In the pups, in particular, selenium (given to the females) increased the amount of nickel in tissues compared with corresponding administration of nickel without selenium. In summary, selenium seemed to counteract the deleterious effects of NiCl2 on the reproduction of rats.

Vitamins A1 (Retinol), A2 (3,4-Didehydroretinol) and E (α-Tocopherol) in the Liver and Blubber of Lacustrine and Marine Ringed Seals (Phoca hispida sp.)

Concentrations of vitamins A1 (retinol), A2 (3,4-didehydroretinol) and E (alpha-tocopherol) in the liver and blubber of ringed seals from Lake Saimaa (Phoca hispida saimensis), Lake Ladoga (P. h. ladogensis), the Baltic Sea (P. h. botnica) and Spitsbergen (P. h. hispida) were determined by high-performance liquid chromatography. The freshwater seals had much lower levels of vitamin A1 but higher levels of vitamin A2 than the marine seals. The concentrations of vitamin E in the livers of the subspecies studied were high compared with earlier reports of seals, but the ranges were large. The livers of the marine seals contained more vitamin E than the livers of the freshwater seals, but the levels in the blubber were uniform in all populations, except in old specimens from the Baltic. The differences between the freshwater and marine seals are suggested to be due mainly to diet. The ratios of A1 to A2 in the liver and blubber and in the fish diet were similar for the marine seals and for the freshwater seals (but differed in the marine and freshwater populations), which suggests no great differences in the absorption, transport and metabolism of the two analogues. Blubber was an important storage site for the vitamins studied, and age-dependent increases were detected, especially for vitamin E. In the 2-month to 2-year-old ringed seals of Lake Saimaa, however, the vitamin E concentration in the blubber was not affected by age.