Regulation of HMG-CoA reductase, HSL and ACAT expression and activity in testicular cholesterol metabolism in mink and in mouse following experimental genetic deletion

Introduction: L'homéostasie du cholestérol est indispensable à la synthèse de la testostérone dans le tissu interstitiel et la production de gamètes mâles fertiles dans les tubules séminifères. Les facteurs enzymatiques contribuent au maintien de cet équilibre intracellulaire du cholestérol. L'absence d'un ou de plusieurs enzymes telles que la HMG-CoA réductase, la HSL et l'ACAT-1 a été associée à l'infertilité masculine. Toutefois, les facteurs enzymatiques qui contribuent au maintien de l'équilibre intra-tissulaire du cholestérol n'ont pas été étudiés. Cette étude a pour but de tester l'hypothèse que le maintien des taux de cholestérol compatibles avec la spermatogenèse nécessite une coordination de la fonction intracellulaire des enzymes HMG-CoA réductase, ACAT1 et ACAT2 et la HSL. Méthodes: Nous avons analysé l'expression de l’ARNm et de la protéine de ces enzymes dans les fractions enrichies en tubules séminifères (STf) de vison durant le développement postnatal et le cycle reproductif annuel et dans les fractions enrichies en tissu interstitiel (ITf) et de STf durant le développement postnatal chez la souris. Nous avons développé deux nouvelles techniques pour la mesure de l'activité enzymatique de la HMG-CoA réductase et de celle de l'ACAT1 et ACAT2. En outre, l'immunohistochimie a été utilisée pour localiser les enzymes dans le testicule. Enfin, les souris génétiquement déficientes en HSL, en SR-BI et en CD36 ont été utilisées pour élucider la contribution de la HMG-CoA réductase, l'ACAT1 et l'ACAT2 et la HSL à l'homéostasie du cholestérol. Résultats: 1) HMG-CoA réductase: (Vison) La variation du taux d’expression de l’ARNm de la HMG-CoA réductase était corrélée à celle de l'isoforme de 90 kDa de la protéine HMG-CoA réductase durant le développement postnatal et chez l'adulte durant le cycle reproductif saisonnier. L'activité enzymatique de la HMG-CoA réductase augmentait de façon concomitante avec le taux protéinique pour atteindre son niveau le plus élevé à 240 jours (3.6411e-7 mol/min/μg de protéines) au cours du développement et en Février (1.2132e-6 mol/min/μg de protéines) durant le cycle reproductif chez l’adulte. (Souris), Les niveaux d'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase étaient maximales à 42 jours. A l'opposé, le taux protéinique diminuait au cours du développement. 2) HSL: (Vison), l'expression de la protéine de 90 kDa de la HSL était élevée à 180- et 240 jours après la naissance, ainsi qu'en Janvier durant le cycle saisonnier chez l'adulte. L'activité enzymatique de la HSL augmentait durant le développement pour atteindre un pic à 270 jours (36,45 nM/min/μg). Chez l'adulte, l'activité enzymatique de la HSL était maximale en Février. (Souris) Le niveau d’expression de l'ARNm de la HSL augmentait significativement à 21-, 28- et 35 jours après la naissance concomitamment avec le taux d'expression protéinique. L'activité enzymatique de la HSL était maximale à 42 jours suivie d'une baisse significative chez l'adulte. 3) ACAT-1 et ACAT-2: Le présent rapport est le premier à identifier l’expression de l'ACAT-1 et de l'ACAT-2 dans les STf de visons et de souris. (Vison) L'activité enzymatique de l'ACAT-2 était maximale à la complétion du développement à 270 jour (1190.00 CPMB/200 μg de protéines) et en janvier (2643 CPMB/200 μg de protéines) chez l'adulte. En revanche, l'activité enzymatique de l'ACAT-1 piquait à 90 jours et en août respectivement durant le développement et chez l'adulte. (Souris) Les niveaux d'expression de l'ARNm et la protéine de l'ACAT-1 diminuait au cours du développement. Le taux de l'ARNm de l'ACAT-2, à l’opposé du taux protéinique, augmentait au cours du développement. L'activité enzymatique de l'ACAT-1 diminuait au cours du développement tandis que celle de l'ACAT-2 augmentait pour atteindre son niveau maximal à 42 jours. 4) Souris HSL-/ -: Le taux d’expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase diminuaient significativement dans les STf de souris HSL-/- comparés aux souris HSL+/+. Par contre, les taux de l'ARNm et les niveaux des activités enzymatiques de l'ACAT-1 et de l'ACAT-2 étaient significativement plus élevés dans les STf de souris HSL-/- comparés aux souris HSL+/+ 5) Souris SR-BI-/-: L'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase et de l'ACAT-1 étaient plus basses dans les STf de souris SR-BI-/- comparées aux souris SR-BI+/+. A l'opposé, le taux d'expression de l'ARNm et l'activité enzymatique de la HSL étaient augmentées chez les souris SR-BI-/- comparées aux souris SR-BI+/+. 6) Souris CD36-/-: L'expression de l'ARNm et l'activité enzymatique de la HMG-CoA réductase et de l'ACAT-2 étaient significativement plus faibles tandis que celles de la HSL et de l'ACAT-1 étaient inchangées dans les STf de souris CD36-/- comparées aux souris CD36+/+. Conclusion: Nos résultats suggèrent que: 1) L'activité enzymatique de la HMG-CoA réductase et de la HSL sont associées à l'activité spermatogénétique et que ces activités ne seraient pas régulées au niveau transcriptionnel. 2) L'ACAT-1 et de l'ACAT-2 sont exprimées dans des cellules différentes au sein des tubules séminifères, suggérant des fonctions distinctes pour ces deux isoformes: l'estérification du cholestérol libre dans les cellules germinales pour l'ACAT-1 et l'efflux du cholestérol en excès dans les cellules de Sertoli au cours de la spermatogenèse pour l'ACAT-2. 3) La suppression génétique de la HSL diminuait la HMG-CoA réductase et augmentait les deux isoformes de l'ACAT, suggérant que ces enzymes jouent un rôle critique dans le métabolisme du cholestérol intratubulaire. 4) La suppression génétique des transporteurs sélectifs de cholestérol SR-BI et CD36 affecte l'expression (ARNm et protéine) et l'activité des enzymes HMG-CoA réductase, HSL, ACAT-1 et ACAT-2, suggérant l'existence d’un effet compensatoire entre facteurs enzymatiques et non-enzymatiques du métabolisme du cholestérol dans les fractions tubulaires. Ensemble, les résultats de notre étude suggèrent que les enzymes impliquées dans la régulation du cholestérol intratubulaire agissent de concert avec les transporteurs sélectifs de cholestérol dans le but de maintenir l'homéostasie du cholestérol intra-tissulaire du testicule.

Cholesteryl ester storage disease. Report of a case.

Cholesteryl ester storage disease (CESD) is a rare disorder of familial incidence characterized by the accumulation of cholesteryl ester and triglycerides in the liver, intestine and bone marrow. Until now only 21 cases have been reported in the literature. We present a 9 months old girl presenting with increased abdominal girth. She had normal liver function tests and increased cholesterol and triglycerides serum levels. The liver biopsy showed many cholesterol cristals seen as needle shaped cristals under polarized light. This is the youngest patient being diagnosed clinically in the literature.

High cholesterol intake modifies chylomicron metabolism in normolipidemic young men

Whether the consumption of egg yolk, which has a very high cholesterol content without excess saturated fats, has deleterious effects on lipid metabolism is controversial. Absorbed dietary cholesterol enters the bloodstream as chylomicrons, but the effects of regular consumption of large amounts of cholesterol on the metabolism of this lipoprotein have not been explored even though the accumulation of chylomicron remnants is associated with coronary artery disease (CAD). We investigated the effects of high dietary cholesterol on chylomicron metabolism in normolipidemic, healthy young men. The plasma kinetics of a chylomicron-like emulsion, doubly-labeled with 14C-cholesteryl ester ( 14C-CE) and 3H-triolein ( 3H-TG) were assessed in 25 men (17-22 y old, BMI 24.1 ± 3.4 kg/m 2). One group (n = 13) consumed 174 ± 41 mg cholesterol/d and no egg yolk. The other group (n = 12) consumed 3 whole eggs/d for a total cholesterol intake of 804 ± 40 mg/d. The nutritional composition of diets was the same for both groups, including total lipids and saturated fat, which comprised 25 and 7%, respectively, of energy intake. Serum LDL and HDL cholesterol and apoprotein B concentrations were higher in the group consuming the high-cholesterol diet (P < 0.05), but serum triacylglycerol, apo AI, and lipoprotein (a) did not differ between the 2 groups. The fractional clearance rate (FCR) of the 14C-CE emulsion, obtained by compartmental analysis, was 52% slower in the high-cholesterol than in the low-cholesterol group (P < 0.001); the 3H-TG FCR did not differ between the groups. Finally, we concluded that high cholesterol intakes increase the residence time of chylomicron remnants, as indicated by the 14C-CE kinetics, which may have undesirable effects related to the development of CAD. © 2006 American Society for Nutrition.

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

Plasma lipid and lipoprotein responses during exercise

The purpose of this study was to examine the effect of prolonged exercise oil plasma lipid and lipoprotein concentrations and to identify caloric time-points where changes occurred. Eleven active male Subjects ran oil a treadmill at 70%,, of maximal fitness (VO2max) and expended 6 278.7 kilojoules (Kj) energy (1500 kcal). Blood samples were obtained at the 4185.8 Kj (1000 kcal) time-point during exercise and at each additional 418.6 Kj (100 kcal) expenditure until 6278.7 Kj was expended. After correcting for plasma volume changes, decreases in low-density lipoprotein cholesterol (LDL-C) were observed during exercise at time-points corresponding to 4604.4 and 5441.5 Kj (1100 and 1300 kcal) of energy expenditure, and immediately after exercise. Total cholesterol concentrations decreased significantly at exercise kilojoule expenditures of 4604.4, 5441.5 and 5860.1 (1100, 1300 and 1400 kcal). There were also exercise induced increases in high-density lipoprotein cholesterol (HDL-C) and HDL2-C concentrations immediately after exercise. Although acute lipid and lipoprotein changes are typically reported in the days following exercise, the Current data indicate that some lipoprotein concentrations change during acute exercise. Our data suggest that a threshold of exercise may be necessary to change lipoproteins during exercise. Future work Should identify potential mechanisms (lipoprotein lipase, cholesterol ester transport protein, LDL uptake) that alter lipoprotein concentrations during prolonged exercise.

Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

Background: In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. Methods: Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. Results and discussion: The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy. Conclusions: The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.

Atherosclerosis: Viewing the problem from a different perspective including possible treatment options

This paper proposes that atherosclerosis is initiated by a signaling event that deposits calcium hydroxyapatite (Ca-HAP). This event is preceded by a loss of mechanical structure in the arterial wall. After Ca-HAP has been deposited, it is unlikely that it will be reabsorbed because the solubility product constant (K sp) is very small, and the large stores of Ca +2 and PO 4-3 in the bones oppose any attempts to dissolve Ca-HAP by decreasing the common ions. The hydroxide ion (OH -) of Ca-HAP can be displaced in nature by fluoride (F -) and carbonate (CO 3-2) ions, and it is proposed that anions associated with cholesterol ester hydrolysis and, in very small quantities, the enolate of 7-ketocholesterol could also displace the OH -of Ca-HAP, forming an ionic bond. The free energy of hydration of Ca-HAP at 310 K is most likely negative, and the ionic radii of the anions associated with the hydrolysis of cholesterol ester are compatible with the substitution. Furthermore, examination of the pathology of atherosclerotic lesions by Raman and NMR spectroscopy and confocal microscopy supports deposition of Ca-HAP associated with cholesterol. Investigating the affinity of intermediates of cholesterol hydrolysis for Ca-HAP compared to lipoproteins such as HDL, LDL, and VLDL using isothermic titration calorimetry could add proof of this concept and may lead to the development of a new class of medications targeted at the deposition of cholesterol within Ca-HAP. Treatment of acute ischemic events as a consequence of atherosclerosis with denitrogenation and oxygenation is discussed. © the author(s), publisher and licensee Libertas Academica Ltd.

EAE cerebrospinal fluid augments in vitro phagocytosis and metabolism of CNS myelin by macrophages.

Previous studies from this laboratory have shown that CNS myelin is phagocytized and metabolized by cultured rat macrophages to a much larger extent when myelin is pretreated with serum containing antibodies to myelin constituents than when it is left untreated or pretreated with non-specific serum. In this study the effect of cerebrospinal fluid (CSF) from rabbits with experimental allergic encephalomyelitis (EAE) in promoting myelin phagocytosis was examined. Fourteen rabbits were immunized with purified myelin in Freund's complete adjuvant, seven of which developed clinical EAE symptoms. Serum and CSF were collected from EAE and control rabbits, and the CSF was centrifuged to remove cells. Sera and CSF from these rabbits and from Freund's adjuvant-immunized controls and untreated controls were measured for IgG content by radial diffusion assay, their myelin antibody characteristics were analyzed by immunoblots, and the ability of these serum and CSF samples to promote myelin phagocytosis when used for myelin opsonization was examined. The ability of a CSF sample to enhance radioactive myelin uptake and phagocytosis by cultured macrophages as measured by the appearance of radioactive cholesterol ester was linearly proportional to its total IgG titer, and correlated approximately both with clinical symptoms of the animal and the presence of antibody against the myelin constituents myelin basic protein, proteolipid protein, and galactocerebroside. The cholesterol esterification activities of EAE sera correlated to a lesser extent with IgG levels and clinical symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of anti-myelin antibodies in EAE and their possible role in demyelination.

Experimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7-95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance of antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10-95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26-27 days after immunization but remained significantly elevated up to 95 days post-immunization compared to the activity of serum from the Freund's adjuvant-injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol-fast blue stained spinal cords up to 60 days post-immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.

Lipoprotein composition in HNF1A-MODY: Differentiating between HNF1A-MODY and Type 2 diabetes.

INTRODUCTION:

The young-onset diabetes seen in HNF1A-MODY is often misdiagnosed as Type 2 diabetes. Type 2 diabetes, unlike HNF1A-MODY, is associated with insulin resistance and a characteristic dyslipidaemia. We aimed to compare the lipid profiles in HNF1A-MODY, Type 2 diabetes and control subjects and to determine if lipids can be used to aid the differential diagnosis of diabetes sub-type.
METHODS:

1) 14 subjects in each group (HNF1A-MODY, Type 2 diabetes and controls) were matched for gender and BMI. Fasting lipid profiles and HDL lipid constituents were compared in the 3 groups. 2) HDL-cholesterol was assessed in a further 267 patients with HNF1A-MODY and 297 patients with a diagnosis of Type 2 diabetes to determine its discriminative value.

RESULTS:

1) In HNF1A-MODY subjects, plasma-triglycerides were lower (1.36 vs. 1.93 mmol/l, p = 0.07) and plasma-HDL-cholesterol was higher than in subjects with Type 2 diabetes (1.47 vs. 1.15 mmol/l, p = 0.0008), but was similar to controls. Furthermore, in the isolated HDL; HDL-phospholipid and HDL-cholesterol ester content were higher in HNF1A-MODY, than in Type 2 diabetes (1.59 vs. 1.33 mmol/L, p = 0.04 and 1.10 vs. 0.83 mmol/L, p = 0.019, respectively), but were similar to controls (1.59 vs. 1.45 mmol/L, p = 0.35 and 1.10 vs. 1.21 mmol/L, p = 0.19, respectively). 2) A plasma-HDL-cholesterol > 1.12 mmol/L was 75% sensitive and 64% specific (ROC AUC = 0.76) at discriminating HNF1A-MODY from Type 2 diabetes.

CONCLUSION:

The plasma-lipid profiles of HNF1A-MODY and the lipid constituents of HDL are similar to non-diabetic controls. However, HDL-cholesterol was higher in HNF1A-MODY than in Type 2 diabetes and could be used as a biomarker to aid in the identification of patients with HNF1A-MODY.

High-density lipoprotein subfractions display proatherogenic properties in overweight and obese children

BACKGROUND: In adults, obesity-driven inflammation can lead to increased cardiovascular disease (CVD). However, information regarding childhood obesity and its inflammatory sequelae is less well defined. Serum amyloid-A (SAA) is an inflammatory molecule that rapidly associates with high-density lipoproteins (HDLs) and renders them dysfunctional. Therefore, SAA may be a useful biomarker to identify increased CVD potential in overweight and obese children.

METHODS: Young Hearts 2000 is a cross-sectional cohort study in which 92 children who were obese were matched for age and sex with 92 overweight and 92 lean children. HDL2 and HDL3 (HDL2&3) were isolated from plasma by a three-step rapid-ultracentrifugation procedure. SAA was measured in serum and HDL2&3 by an enzyme-linked immunosorbent assay procedure, and the activities of cholesterol ester transfer protein (CETP) and lecithin cholesteryl acyltransferase (LCAT) were measured by fluorimetric assays.

RESULTS: Trends across the groups indicated that SAA increased in serum and HDL2&3 as BMI increased, as did HDL2-CETP and HDL2-LCAT activities.

CONCLUSION: These results have provided evidence that overweight and obese children are exposed to an inflammatory milieu that impacts the antiatherogenic properties of HDL and that could increase CVD risk. This supports the concept that it is important to target childhood obesity to help minimize future cardiovascular events.

Serum amyloid A-related inflammation is lowered by increased fruit and vegetable intake, while high-sensitive C-reactive protein, IL-6 and E-selectin remain unresponsive

The present study assessed whether increased fruit and vegetable (F&V) intake reduced the concentrations of the inflammatory marker serum amyloid A (SAA) in serum, HDL2 and HDL3 and whether the latter reduction influenced any of the functional properties of these HDL subfractions. The present study utilised samples from two previous studies: (1) the FAVRIT (Fruit and Vegetable Randomised Intervention Trial) study - hypertensive subjects (systolic blood pressure (BP) range 140-190 mmHg; diastolic BP range 90-110 mmHg) were randomised to receive a 1-, 3- or 6-portion F&V/d intervention for 8 weeks, and (2) the ADIT (Ageing and Dietary Intervention Trial) study - older subjects (65-85 years) were randomised to receive a 2- or 5-portion F&V/d intervention for 16 weeks. HDL2 and HDL3 were isolated by rapid ultracentrifugation. Measurements included the following: serum high-sensitive C-reactive protein (hsCRP) by an immunoturbidimetric assay; serum IL-6 and E-selectin and serum-, HDL2- and HDL3-SAA by ELISA procedures; serum-, HDL2- and HDL3-cholesterol ester transfer protein (CETP) activity by a fluorometric assay. Although the concentrations of hsCRP, IL-6 and E-selectin were unaffected by increasing F&V intake in both studies (P>0·05 for all comparisons), those of SAA in HDL3 decreased in the FAVRIT cohort (P= 0·049) and those in HDL2 and HDL3 decreased in the ADIT cohort (P= 0·035 and 0·032), which was accompanied by a decrease in the activity of CETP in HDL3 in the FAVRIT cohort (P= 0·010) and in HDL2 in the ADIT cohort (P= 0·030). These results indicate that SAA responds to increased F&V intake, while other inflammatory markers remain unresponsive, and this leads to changes in HDL2 and HDL3, which may influence their antiatherogenic potential. Overall, the present study provides tangible evidence of the effectiveness of increased F&V intake, which may be of use to health policy makers and the general public.

Evidence of a possible role for Lys-gamma 3-MSH in the regulation of adipocyte function

Lys-gamma 3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma 3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma 3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma 3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma 3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma 3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyper-phosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma 3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.