Regulation Of Hormone-sensitive Lipase Research Articles

Hsa_circ_0021205 enhances lipolysis via regulating miR-195-5p/HSL axis and drives malignant progression of glioblastoma

Abnormal lipid metabolism is an essential hallmark of glioblastoma. Hormone sensitive lipase (HSL), an important rate-limiting enzyme contributed to lipolysis, which was involved in aberrant lipolysis of glioblastoma, however, its definite roles and the relevant regulatory pathway have not been fully elucidated. Our investigations disclosed high expression of HSL in glioblastoma. Knock-down of HSL restrained proliferation, migration, and invasion of glioblastoma cells while adding to FAs could significantly rescue the inhibitory effect of si-HSL on tumor cells. Overexpression of HSL further promoted tumor cell proliferation and invasion. Bioinformatics analysis and dual-luciferase reporter assay were performed to predict and verify the regulatory role of ncRNAs on HSL. Mechanistically, hsa_circ_0021205 regulated HSL expression by sponging miR-195-5p, which further promoted lipolysis and drove the malignant progression of glioblastoma. Besides, hsa_circ_0021205/miR-195-5p/HSL axis activated the epithelial-mesenchymal transition (EMT) signaling pathway. These findings suggested that hsa_circ_0021205 promoted tumorigenesis of glioblastoma through regulation of HSL, and targeting hsa_circ_0021205/miR-195-5p/HSL axis can serve as a promising new strategy against glioblastoma.

KRAS Controls Pancreatic Cancer Cell Lipid Metabolism and Invasive Potential through the Lipase HSL

Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone-sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS-HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis. SIGNIFICANCE: KRAS-dependent regulation of HSL biases cells towards lipid storage for subsequent utilization during invasion of pancreatic cancer cells, representing a potential target for therapeutic intervention.See related commentary by Man et al., p. 4886.

Abstract PR09: KRas modulates pancreatic cancer cell metabolism and invasive potential through the lipase HSL

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 9%, due in part to its robust metastatic capability. Metastatic invasion is a particularly energy-intensive cellular process; thus, novel therapeutic strategies could include targeting the energy source for tumor cell invasion and migration. Excess lipids, present due to increased fatty acid synthesis or obesity, could provide a fuel source for cancer progression and metastasis. Neutral lipids are stored in organelles called lipid droplets (LDs). How LDs contribute to PDAC tumor cell invasion and metastasis remains unknown. We hypothesize that PDAC cells can store excess lipid to drive metastatic invasion. Indeed, introduction of excess exogenous fatty acids augments LD production and accumulation in PDAC cells and leads to enhanced PDAC cell motility and invasion in vitro. LDs are metabolized by the concerted action of lipases. Importantly, we have found that treatment of tumor cells with lipase inhibitors blocks tumor cell invasion, indicating that utilization of stored lipids via lipase-mediated LD breakdown is required for invasive migration. Hormone sensitive lipase (HSL) is a lipolytic enzyme, best characterized in adipose tissue, that is critical to the breakdown and utilization of LDs in vitro and in vivo. We have made the surprising observation that oncogenic KRas downregulates expression of HSL in pancreatic cancer cells, which shifts the cells towards lipid storage. In PDAC, mutational activation of the oncogene KRas occurs in 95% of tumors and contributes to metabolic rewiring of tumor cells. Our data suggest that the metabolic shift induced by mutant KRas is comprised of decreased LD utilization via downregulation of HSL. Indeed, re-expression of HSL shifts tumor cell metabolism towards elevated oxidative phosphorylation. Importantly, KRas-mutant cells still require the catalytic activity of HSL for tumor cell invasion, even though HSL expression is downregulated. Using redox ratio imaging, we have found that migratory cells selectively utilize oxidative phosphorylation during the process of migration to metabolize stored lipid droplets and fuel invasive migration. Finally, depletion of LD stores via overexpression of HSL blocks tumor cell invasion in vitro and metastasis in mice, even in the context of oncogenic KRas. These data indicate that regulation of HSL is important for both lipid storage and maintaining cancer cell invasiveness. Therefore, we propose a novel mechanism by which oncogenic KRas mediates HSL activity in a tunable manner, thereby altering the metabolism of PDAC cells and leading to increased metastatic ability. This abstract is also being presented as Poster B47. Citation Format: Cody Rozeveld, Ryan Schulze, Lizhi Zhang, Gina L Razidlo. KRas modulates pancreatic cancer cell metabolism and invasive potential through the lipase HSL [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR09.

Redox regulation of hormone sensitive lipase: Potential role in the mechanism of MEHP-induced stimulation of basal steroid synthesis in MA-10 Leydig cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0–300 μM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.

Structural and functional analyses of a bacterial homologue of hormone-sensitive lipase from a metagenomic library

Intracellular mobilization of fatty acids from triacylglycerols in mammalian adipose tissues proceeds through a series of lipolytic reactions. Among the enzymes involved, hormone-sensitive lipase (HSL) is noteworthy for its central role in energy homeostasis and the pathogenic role played by its dysregulation. By virtue of its broad substrate specificity, HSL may also serve as an industrial biocatalyst. In a previous report, Est25, a bacterial homologue of HSL, was identified from a metagenomic library by functional screening. Here, the crystal structure of Est25 is reported at 1.49 Å resolution; it exhibits an α/β-hydrolase fold consisting of a central β-sheet enclosed by α-helices on both sides. The structural features of the cap domain, the substrate-binding pocket and the dimeric interface of Est25, together with biochemical and biophysical studies including native PAGE, mass spectrometry, dynamic light scattering, gel filtration and enzyme assays, could provide a basis for understanding the properties and regulation of hormone-sensitive lipase (HSL). The increased stability of cross-linked Est25 aggregates (CLEA-Est25) and their potential for extensive reuse support the application of this preparation as a biocatalyst in biotransformation processes.

Insulin increases macrophage triglyceride accumulation under diabetic conditions through the down regulation of hormone sensitive lipase and adipose triglyceride lipase

Diabetes mellitus (DM) is a major risk factor for the development of atherosclerosis, and high-serum levels of insulin are strongly associated with type 2 DM. Atherosclerosis is characterized by lipid-laden macrophage foam cell formations, which contain substantial amount of cholesterol and triglycerides (TG). This study analyzed for the first time, the effects of insulin on TG metabolism in macrophages under normal and diabetic conditions. Mouse peritoneal macrophages from C57BL6 mice were cultured under normal (5 mM) or high (diabetic condition, 25 mM) glucose concentration, with or without insulin, followed by the assessment of TGs metabolism in these cells. Under diabetic condition, insulin increased TG accumulation in macrophages by 100%, decreased cellular TG degradation by 21%, and increased C-reactive protein levels in macrophages by 83%. Insulin decreased hormone-sensitive lipase mRNA and protein expression by 28 and 60%, respectively, and adipose TG lipase (ATGL) protein expression by 36%, with no significant reduction in ATGL mRNA levels. The inhibition of insulin-mediated phosphorylation, and the addition of cyclic adenosine 3'5'-monoposphate, abolished the insulin-mediated inhibition of TGs degradation in cells. Insulin increases macrophage TGs accumulation only under diabetic conditions, suggesting that impaired glycemic control in diabetic patients treated with insulin may contribute to foam cell formations and enhanced inflammation in macrophages.

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise

Women have been shown to use more intramuscular triacylglycerol (IMTG) during exercise than men. To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women and eight men performed bicycle exercise (90 min, 60% Vo(2peak)), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P < 0.001) and net IMTG decrease during exercise (P < 0.01) were higher in women than in men and correlated significantly (r = 0.72, P = 0.001). Muscle HSL mRNA (80%, P = 0.11) and protein content (50%, P < 0.05) were higher in women than in men. HSL total activity increased during exercise (47%, P < 0.05) but did not differ between sexes. Accordingly, HSL specific activity (HSL activity per HSL protein content) increased during exercise (62%, P < 0.05) and was generally higher in men than in women (82%, P < 0.05). A similar pattern was observed for HSL Ser(659) phosphorylation, suggesting a role in regulation of HSL activity. Likewise, plasma epinephrine increased during exercise (P < 0.05) and was higher in men than in women during the end of the exercise bout (P < 0.05). We conclude that, although HSL expression and Ser(659) phosphorylation in skeletal muscle during exercise is sex specific, total muscle HSL activity measured in vitro was similar between sexes. The higher basal IMTG content in women compared with men is therefore the best candidate to explain the higher IMTG net hydrolysis during exercise in women.

Muscle triacylglycerol and hormone-sensitive lipase activity in untrained and trained human muscles

During exercise, triacylglycerol (TG) is recruited in skeletal muscles. We hypothesized that both muscle hormone-sensitive lipase (HSL) activity and TG recruitment would be higher in trained than in untrained subjects in response to prolonged exercise. Healthy male subjects (26 +/- 1 years, body moss index 23.3 +/- 0.5 kg m(-2)), either untrained (N = 8, VO(2max) 3.8 +/- 0.2 l min(-1)) or trained (N = 8, VO(2max) 5.1 +/- 0.1 l min(-1)), were studied. Before and after 3-h exercise (58 +/- 1% VO(2max)), a biopsy was taken. Muscle citrate synthase (32 +/- 2 vs. 47 +/- 6 mumol g(-1) min(-1) d.w.) and beta-hydroxy-acyl-CoA-dehydrogenase (38 +/- 3 vs. 52 +/- 5 mumol g(-1) min(-1) d.w.) activities were lower in untrained than in trained subjects (p < 0.05). Throughout the exercise, fat oxidation was higher in trained than in untrained subjects (p < 0.05). Muscle HSL activity was similar at rest (0.72 +/- 0.08 and 0.74 +/- 0.03 mU mg(-1) protein) and after exercise (0.71 +/- 0.1 and 0.68 +/- 0.03 mU mg(-1) protein) in untrained and trained subjects. At rest, the chemically determined muscle TG content (37 +/- 8 and 26 +/- 5 mmol g(-1) d.w.) was similar (p > 0.05), and after exercise it was unchanged in untrained and lower (p < 0.05) in trained subjects (41 +/- 9 and 10 +/- 2 mmol g((1) d.w.). Determined histochemically, TG was decreased (p < 0.05) after exercise in type I and II fibres. Depletion of TG was not different between fibre types in untrained, but tended to be higher (p = 0.07) in type I compared with type II fibres in trained muscles. In conclusion, HSL activity is similar in untrained and trained skeletal muscles both before and after prolonged exercise. However, the tendency to higher muscle TG recruitment during exercise in the trained subjects suggests a difference in the regulation of HSL or other lipases during exercise in trained compared with untrained subjects.

Regulation of hormone-sensitive lipase in islets

An unique isoform of hormone-sensitive lipase (HSL) is expressed in β-cells. Recent findings suggest that HSL could be involved in the regulation of glucose stimulated insulin secretion (GSIS), however, these findings are controversial. To test the hypothesis that HSL is involved in control of normal GSIS via changes in its expression and/or activity in response to stimuli, we examined the effects of free fatty acid (FFA) loading and glucagon like peptide-1 (GLP-1) stimulation on the regulation of HSL expression and activity. With prolonged FFA loading, there was increased expression of β-cell HSL and increased HSL hydrolytic activity in clonal β-cells. Short-term treatment with GLP-1 increased HSL activity without changing the expression of the β-cell isoform of HSL. Basal insulin secretion was increased, whereas GLP-1 potentiation of GSIS was decreased in islets isolated from HSL−/− mice, as compared to islets from wild type mice. Furthermore, using PancChip 2.2 cDNA microarrays (NIDDK consortium), the gene expression profile in the islets of HSL−/− mice was compared with wild type mice. Results showed changes in several metabolic pathways due to changes in lipid homeostasis caused by inactivation of HSL. Quantitative PCR for selected genes also revealed changes in genes that are related to insulin secretion, such as UCP-2. Therefore, these results suggest that the β-cell isoform of HSL is involved in maintaining lipid homeostasis in islets and contributes to the proper control of GSIS.

Hormone-sensitive lipase in skeletal muscle: regulatory mechanisms.

The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone-sensitive lipase (HSL), which controls TG breakdown in adipose tissue. Isolated rat muscle as well as exercising humans were studied. The presence of HSL was demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying. The content of HSL varies between fibre types, being higher in oxidative than in glycolytic fibres. Analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by presence of anti-HSL antibody during analysis. Moreover, immunoprecipitation with affinity-purified anti-HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta-adrenergic activation of protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659 and Ser660. Contraction probably also enhances muscle-HSL activity by phosphorylation, because the contraction-induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction-induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser600. So, phosphorylation of different sites may explain that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases the contraction-mediated, but diminishes the adrenaline mediated HSL activation in muscle. The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.

HORMONE SENSITIVE LIPASE ACTIVITY AND FATTY ACYL-COA CONTENT IN HUMAN SKELETAL MUSCLE DURING PROLONGED EXERCISE

Hormone sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG) but HSL regulation is poorly understood in skeletal muscle. PURPOSE To measure human skeletal muscle HSL activity and several putative regulators of HSL at rest and during prolonged moderate intensity exercise. METHODS Healthy male volunteers (n = 7; 21 ± years, 77 ± kg) cycled for 120 min at 60% peak pulmonary oxygen uptake. Muscle, venous blood and respiratory gas samples were obtained at rest and 10, 60 and 120 min of exercise. RESULTS HSL activity increased from resting levels by 10 min of exercise (2.09 ± 0.19 to 2.56 ± 0.22 mmol/min/kg dm, P < 0.05), increased further by 60 min (3.1227 ± 0. mmol/min/kg dm, P < 0.05) and decreased to near-resting rates after 120 min of cycling. Skeletal muscle LCFA CoA increased (P < 0.05) above rest by 60 min (15.9 ± 3.0 to 50.4 ± 7.9 mmol/kg dm) and increased further by 120 min. Estimated free AMP increased (P < 0.05) from rest to 60 min, and was ∼20-fold greater than rest by 120 min. Epinephrine was increased above rest (P < 0.05) at 60 (1.47 ± 0.15 nM) and 120 min (4.87 ± 0.76 nM) of exercise. Insulin concentrations decreased rapidly and were lower than rest by 10 min and continued to decrease throughout exercise. CONCLUSION HSL activity was increased above rest by 10 min, increased further by 60 min and decreased to near-resting values by 120 min. The increased HSL activity from 10–60 min was associated with the stimulating effect of increased epinephrine and decreased insulin levels. After 120 min, the decreased HSL activity was associated with the proposed inhibitory effects of increased free AMP. The LCFA CoA accumulation in the second hour of exercise may have reduced flux through HSL and accounted for the reduction in IMTG utilization previously observed late in prolonged exercise. Supported by NSERC and CIHR, Canada.

Hormone-sensitive Lipase (HSL) Expression and Regulation By Epinephrine and Exercise in Skeletal Muscle

Triacylglycerol (TG) is stored in lipid droplets in the cytoplasm of skeletal muscle. The energy content of the TG depot is higher than the energy content of the muscle glycogen depot. The enzymatic regulation of intracellular TG hydrolysis in skeletal muscle has not been elucidated. Therefore, we investigated the expression and the regulation of hormone-sensitive lipase (HSL) in skeletal muscle. This enzyme is a neutral lipase and known as the rate-limiting enzyme of intracellular TG hydrolysis in adipose tissue. The total and the activated form of the neutral lipase are referred to as MOME and TO, respectively. In isolated rat skeletal muscle fibers, the presence of HSL was demonstrated by Western blotting. The expression of HSL was correlated to fiber type, being higher in oxidative than in glycolytic fibers. In incubated soleus and extensor digitorum longus (EDL) muscles stimulation with epinephrine or electrically induced contractions increased neutral lipase activity against triolein (TO), but not against a diacylglycerol analogue (MOME). Glycogen phosphorylase activity increased in parallel with TO activity. No measurable increase in muscle homogenate TO activity existed in the presence of an anti-HSL antibody. The effect of epinephrine could be blocked by propanolol and mimicked by incubation of a crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase. The effect of contractions was transient as TO activity declined to basal levels after 10 min of electrical stimulation. Indicating involvement of protein kinase C the effect of contractions was abolished by Calphostin C. Okadaic acid doubled the contraction-mediated increase in TO activity, whereas the increase was reversed by phosphatase treatment. The effects of epinephrine and contractions were partially additive. In rats training increased epinephrine-stimulated TO activity and HSL concentration in adipose tissue but not in muscle. In humans, at the end of 60 min of exercise muscle, TO activity was increased in healthy, but not in adrenalectomized, subjects. In conclusion, HSL is present in skeletal muscle and can be activated by phosphorylation by both epinephrine and muscle contractions. In addition, HSL and glycogen phosphorylase are stimulated in parallel in muscle indicating simultaneous activation of triacylglycerol and glycogen breakdown.

The regulation of HSL and LPL expression by DHT and flutamide in human subcutaneous adipose tissue

Clinical observations suggest a role for testosterone in the accumulation of central adiposity and with an associated increased risk of disease. To date, no human study has analysed the role of dihydrotestosterone (DHT) on adipose tissue mass regulation in vitro. This study investigated the role of DHT and androgen receptors (AR) in the regulation of lipolysis and lipogenesis by examining the key enzymes hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) respectively. Isolated abdominal subcutaneous adipocytes (Scad) (n = 15) were treated with either DHT (10(-7)-10(-9) m), an antiandrogen, flutamide (FLT: 10(-7)-10(-9) m) or a combination of DHT (10(-7)-10(-9) m) with FLT (10(-8) m). Relative protein expression of HSL, LPL and AR was determined. In Scad, DHT inhibited HSL expression maximally at 10(-9) m (0.7 +/- 0.4**; p < 0.01**) compared with control (control: 1.0 +/- (s.e.m.) 0.0), whereas LPL protein expression was stimulated at DHT10(-9) m (2.22 +/- 0.48*; p < 0.05*). Glycerol release assay results correlated with HSL expression data. LPL expression was reduced at all doses with combinations of DHT + FLT compared with DHT alone. Androgen receptor expression studies showed an inverse correlation with DHT, whereas DHT + FLT reduced AR expression. These studies indicate that DHT may alter HSL and LPL expression, whereas only LPL expression appears mediated by AR. These findings suggest a physiological role for DHT in the control of adipose tissue mass in women, and indicate that androgens may also play an important role in regulating lipid metabolism.

Regulation of hormone-sensitive lipase in streptozotocin-induced diabetic rats

Insulin deficiency as seen in insulin-dependent diabetes mellitus causes an activation of lipolysis in adipose tissue that results in hydrolysis of stored triglycerides and release of large amounts of fatty acids into the plasma, leading to diabetic ketoacidosis (DKA). Hormone-sensitive lipase (HSL) is thought to be the rate-limiting enzyme of lipolysis in adipose tissue. This study was designed to examine the effects of insulin deficiency on the regulation of HSL in isolated adipocytes. Insulin deficiency was induced by a single dose of streptozotocin. After 8 days, some animals were treated with insulin, and all animals were killed 10 days after induction of insulin deficiency. Compared with levels in control rats, 10 days of insulin deficiency increased HSL activity twofold ( P < .05), as assayed for neutral cholesterol esterase activity, and insulin treatment returned HSL activity to normal. HSL protein was increased twofold ( P < .05) in streptozotocin-induced diabetic rats, as estimated by immunoblotting, but remained elevated after insulin treatment. Levels of HSL mRNA assessed by Northern blot analysis also increased twofold ( P < .01) in adipose cells isolated from streptozotocin-induced diabetic rats, and remained elevated after insulin treatment. In conclusion, our studies suggest that 10 days of insulin deficiency increases HSL expression via pretranslational mechanisms and short-term insulin treatment returns HSL activity to normal via posttranslational mechanisms in adipose tissue.

Regulation of hormone-sensitive lipase during fasting

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. The activity of HSL is thought to be primarily regulated by phosphorylation-dephosphorylation reactions. Although FFA levels are elevated during fasting, it has been difficult to demonstrate an increase in HSL activity with fasting. The current studies were undertaken to explore directly the regulation of HSL expression in adipose tissue in the rat during fasting. Rats were fasted for periods up to 5 days and HSL activity, HSL immunoreactive protein, and HSL mRNA levels were measured both in intact epididymal adipose tissue and in isolated adipose cells. Fasting caused a progressive decline in total body weight and the weight of epididymal fat pads, whereas adipose cell size decreased approximately 50% after 2 days of fasting. Serum FFA levels approximately doubled within 1 day of fasting and remained elevated thereafter. Basal lipolysis, measured as glycerol release, did not increase until 2 days of fasting. HSL activity remained relatively unchanged until 3 days of fasting when it was increased twofold after 3-5 days of fasting. Likewise, HSL immunoreactive protein and HSL mRNA levels increased twofold after 3-5 days of fasting. Thus HSL activity appears to be regulated by pretranslational mechanisms during prolonged fasting. However, increases in FFA flux during short-term fasting appear to involve either post-translational control of HSL or the regulation of other enzymes.