Discovery and pharmacological characterization of SAR707 as novel and selective small molecule inhibitor of stearoyl-CoA desaturase (SCD1).

Stearoyl-CoA desaturase (SCD1) is linked to the pathogenesis of obesity, dyslipidemia and type 2 diabetes. It is the rate-limiting enzyme in the synthesis of monounsaturated 16:1 n-7 and 18:1 n-9 fatty acyl-CoAs and catalyzes an essential part of lipogenesis. Here, we describe the identification, in vitro properties and in vivo efficacy of a novel class of heterocyclic small molecule hexahydro-pyrrolopyrrole SCD1 inhibitors. SAR707, a compound representative for the series, was optimized to high in vitro potency, selectivity and favorable overall properties in enzymatic and cellular assays. In vivo, this compound reduced serum desaturation index, decreased body weight gain and improved lipid parameters and blood glucose levels of obese Zucker diabetic fatty rats treated for 4 weeks in a chronic study. In parallel, fissures of the eye lid, alopecia and inflammation of the skin were observed from day 11 on in all animals treated with the same metabolically active dose. In summary, we described in vitro and in vivo properties of a novel, potent and selective SCD1 inhibitor that improved body weight, blood glucose and triglycerides in an animal model of obesity, type 2 diabetes and dyslipidemia. However, the favorable in vivo properties of systemic SCD1 inhibition shown in our study were accompanied by dose-dependently occurring adverse target-related effects observed in skin. Thus, systemic SCD1 inhibition by small molecules might therefore not represent a feasible approach for the treatment of chronic metabolic diseases.

Upregulation of lipid synthesis in small rat adipocytes by microvesicle-associated CD73 from large adipocytes.

Filling-up lipid stores is critical for size increase of mammalian adipocytes. The glycosylphosphatidylinositol (GPI)-anchored protein, CD73, is released from adipocytes into microvesicles in response to the lipogenic stimuli, palmitate, the antidiabetic sulfonylurea drug glimepiride, phosphoinositolglycans (PIG), and H(2)O(2). Upon incubation of microvesicles with adipocytes, CD73 is translocated to cytoplasmic lipid droplets (LD) and esterification is upregulated. The role of CD73-harboring microvesicles in coordinating esterification between differently sized adipocytes was studied here. Populations consisting of either small or large or of both small and large isolated rat adipocytes as well as native adipose tissue pieces from young and old rats were incubated with or depleted of endogenous microvesicles and analyzed for translocation of CD73 and esterification in response to the lipogenic stimuli. Large adipocytes exhibited higher and lower efficacy in releasing CD73 into microvesicles and in translocating CD73 to LD, respectively, compared to small adipocytes. Populations consisting of both small and large adipocytes were more active in esterification in response to the lipogenic stimuli than either small or large adipocytes. With both adipocytes and adipose tissue pieces from young rats esterification stimulation by the lipogenic stimuli was abrogated by depletion of CD73-harboring microvesicles from the incubation medium and interstitial spaces, respectively. In conclusion, stimulus-induced lipid synthesis between differently sized adipocytes is controlled by the release of microvesicle-associated CD73 from large cells and its subsequent translocation to LD of small cells. This information transfer via microvesicles harboring GPI-anchored proteins may shift the burden of triacylglycerol storage from large to small adipocytes.

Microvesicles released from rat adipocytes and harboring glycosylphosphatidylinositol-anchored proteins transfer RNA stimulating lipid synthesis.

Small microvesicles, such as microparticles and exosomes, have been demonstrated to transfer proteins and nucleic acids from a variety of donor to acceptor cells with corresponding (patho)physiological consequences. Recently the in vitro transfer of glycosylphosphatidylinositol (GPI)-anchored proteins from microvesicles released from large rat adipocytes to intracellular lipid droplets (LDs) of small adipocytes has been shown to be upregulated by physiological (palmitate, H(2)O(2)) and pharmacological (anti-diabetic sulfonylurea drug glimepiride) stimuli and to increase the esterification into as well as to reduce the release of fatty acids from triacylglycerol. Here microvesicles derived from (preferentially large) rat adipocytes or plasma and harboring the GPI-anchored proteins, Gce1 and CD73, were demonstrated to contain specific transcripts and microRNAs that are both transferred into and expressed in acceptor adipocytes and are involved in the upregulation of lipogenesis and cell size. The transferred transcripts were specific for fatty acid esterification (glycerol-3-phosphate acyltransferase-3, diacylglycerol acyltransferase-2), lipid droplet biogenesis (FSP27, caveolin-1) and adipokines (leptin, adiponectin). The transfer and lipogenic activity were more efficient for small rather than large acceptor adipocytes and significantly upregulated by palmitate, glimepiride and H(2)O(2). Together the data suggest that microvesicles released from large adipocytes stimulate lipid storage in small adipocytes by mediating horizontal transfer of lipogenic information which is encoded by relevant (micro)RNA and GPI-anchored protein species. Paracrine and endocrine regulation of lipid storage and, in parallel, cell size of white adipocytes by specific (micro)RNAs in GPI-anchored protein-harboring microvesicles may represent a novel target for interference with metabolic diseases, such as obesity and metabolic syndrome.

Glycosylphosphatidylinositol-anchored proteins coordinate lipolysis inhibition between large and small adipocytes.

In response to palmitate, the antidiabetic sulfonylurea drug glimepiride, phosphoinositoglycans, or H(2)O(2), the release of the glycosylphosphatidylinositol-anchored and cyclic adenosine monophosphate-degrading phosphodiesterase Gce1 from adipocytes into small vesicles (adiposomes) and its translocation from adiposomes to cytoplasmic lipid droplets (LD) of adipocytes have been reported. Here the role of Gce1-harboring adiposomes in coordinating lipolysis between differently sized adipocytes was studied. Separate or mixed populations of isolated epididymal rat adipocytes of small and large size and native adipose tissue pieces from young and old rats were incubated with exogenous adiposomes or depleted of endogenous adiposomes and then analyzed for translocation of Gce1 and lipolysis in response to above antilipolytic stimuli. Large compared with small adipocytes are more efficient in releasing Gce1 into adiposomes but less efficient in translocating Gce1 from adiposomes to LDs. Maximal lipolysis inhibition by above antilipolytic stimuli, but not by insulin, was observed with mixed populations of small and large adipocytes (1:1 to 1:2) rather than with separate populations. In mixed adipocyte populations and adipose tissue pieces from young, but not old, rats, lipolysis inhibition by above antilipolytic stimuli, but not by insulin, was dependent on the function of Gce1-harboring adiposomes. Inhibition of lipolysis in rat adipose tissue in response to palmitate, glimepiride, and H(2)O(2) is coordinated via the release of adiposome-associated and glycosylphosphatidylinositol-anchored Gce1 from large "donor" adipocytes and their subsequent translocation to the LDs of small "acceptor" adipocytes. This transfer of antilipolytic information may be of pathophysiologic relevance.

Lipid storage in large and small rat adipocytes by vesicle-associated glycosylphosphatidylinositol-anchored proteins.

Adipose tissue mass in mammals expands by increasing the average cell volume and/or total number of the adipocytes. Upregulated lipid storage in fully differentiated adipocytes resulting in their enlargement is well documented and thought to be a critical mechanism for the expansion of adipose tissue depots during the growth of both lean and obese animals and human beings. A novel molecular mechanism for the regulation of lipid storage and cell size in rat adipocytes was recently elucidated for the physiological stimuli, palmitate and H(2)O(2), and the antidiabetic sulfonylurea drug, glimepiride. It encompasses (1) the release of small vesicles, so-called adiposomes, harboring the glycosylphosphatidylinositol -anchored (c)AMP-degrading phosphodiesterase Gce1 and 5'-nucleotidase CD73 from donor adipocytes, (2) the transfer of the adiposomes and their interaction with detergent-insoluble glycolipid-enriched microdomains of the plasma membrane of acceptor adipocytes, (3) the translocation of Gce1 and CD73 from the adiposomes to the intracellular lipid droplets of the acceptor adipocytes, and (4) the degradation of (c)AMP at the lipid droplet surface zone by Gce1 and CD73 in the acceptor adipocytes, leading to the upregulation of the esterification of fatty acids into triacylglycerol s and the downregulation of their release from triacylglycerols. This mechanism may provide novel strategies for the therapy of metabolic diseases, such as type 2 diabetes and obesity.

Reversal of inflammation-induced impairment of glucose uptake in adipocytes by direct effect of CB1 antagonism on adipose tissue macrophages.

Macrophage infiltration into adipose tissue (AT-MP) is thought to induce insulin resistance and diabetes in obesity. Here, we investigated the effect of the antiobesity drug SR141716 (a CB1 antagonist) on macrophage-mediated inhibition of insulin signaling in adipocytes. THP1 macrophages (THP1) were stimulated in vitro with lipopolysaccharide (LPS) and SR141716 or vehicle. The resulting conditioned medium (CM) was analyzed and incubated on human adipocytes. CM from LPS-stimulated THP1 inhibited insulin-induced AKT phosphorylation in adipocytes, in contrast to CM from nonactivated THP1. Moreover, it contained higher concentrations of tumor necrosis factor-α (TNFα) and lower levels of the anti-inflammatory cytokine IL-10. SR141716 reduced TNFα production and increased IL-10 secretion, resulting in a rescue of insulin signaling in adipocytes. To confirm these findings in vivo, AT-MP CM from cafeteria diet-fed or Zucker diabetic fatty (ZDF) rats that had received SR141716 for 3 weeks were isolated, analyzed, and incubated with adipocytes. Cafeteria diet induced macrophage-mediated inhibition of insulin signaling in adipocytes. Interestingly, SR141716 rescued insulin-induced glucose uptake in adipocytes. Finally, AT-MP CM from obese ZDF rats inhibited insulin-stimulated glucose uptake in adipocytes in contrast to AT-MP CM from lean ZDF rats. After treatment with SR141716, AT-MP CM rescued insulin-induced glucose uptake in adipocytes. In summary, our data indicate that CB1 receptor antagonism in macrophages modified their cytokine production and improved the insulin responsiveness of adipocytes that had been incubated with macrophage CM. Thus, SR141716 ameliorated adipose tissue insulin resistance by direct action on AT-MP demonstrating a novel peripheral mode of action of CB1 antagonism.

Inhibition of lipolysis by adiposomes containing glycosylphosphatidylinositol-anchored Gce1 protein in rat adipocytes.

Small membrane vesicles released from large adipocytes and harbouring the glycosylphosphatidylinositol-anchored (GPI-) AMP-degrading protein CD73 have previously been demonstrated to stimulate the signal-induced esterification of free fatty acids into neutral lipids suggesting a role of these so-called adiposomes (ADIP) in the paracrine regulation of lipid metabolism in the adipose tissue. Here the involvement of another constituent GPI-protein of ADIP, the cAMP-degrading protein Gce1 in the signal-induced inhibition of lipolysis was investigated in primary rat adipocytes. Incubation of small, and to a lower degree, large adipocytes with ADIP inhibited lipolysis and increased its sensitivity toward inhibition by H(2)O(2), the anti-diabetic drug glimepiride and palmitate. This was accompanied by the transfer of Gce1 from the ADIP to detergent-insoluble glycolipid-enriched plasma membrane microdomains (DIGs) and its subsequent translocation to cytoplasmic lipid droplets (LD) of the acceptor adipocytes. The translocation from DIGs to LD rather than the transfer from ADIP to DIGs of Gce1 was stimulated by H(2)O(2) > glimepiride > palmitate. Both transfer and translocation led to salt- and carbonate-resistant association of Gce1 with DIGs and LD, respectively, and relied on the structural integrity of the DIGs and GPI anchor of Gce1. In conclusion, the trafficking of GPI-proteins from ADIP of donor adipocytes via DIGs to LD of acceptor adipocytes mediates paracrine regulation of lipolysis within adipose tissue.

Characterization of adenosine-A1 receptor-mediated antilipolysis in rats by tissue microdialysis, 1H-spectroscopy, and glucose clamp studies.

Increased supply of fatty acids to muscle and liver is causally involved in the insulin resistance syndrome. Using a tissue microdialysis technique in Wistar and Zucker fatty (ZF) rats, we determined tissue glycerol levels as a marker of lipolysis in gastrocnemius muscle (gMT), subcutaneous adipose (SAT), and visceral adipose tissue (VAT) as well as the reduction of plasma free fatty acids, glycerol, and triglycerides caused by the antilipolysis-specific adenosine-A1 receptor agonist (ARA). In Wistar and ZF rats, ARA significantly lowered dialysate glycerol levels in SAT, VAT, and gMT. Whereas in SAT and VAT the decrease in dialysate glycerol indicated adipocytic antilipolysis, this decrease in gMT was not caused by a direct effect of ARA on intramyocellular lipolysis, as demonstrated by the lack of inhibition of the protein kinase A activity ratio in gMT. In addition, no differences of the fed-starved-refed dynamics of intramyocellular triglyceride levels compared with untreated controls were measured by in vivo (1)H-spectroscopy, excluding any adenylate cyclase-independent antilipolysis in muscle. Treatment with ARA resulted in pronounced reductions of plasma free fatty acids, glycerol, and triglycerides. Furthermore, in ZF rats, ARA treatment caused an immediate improvement of peripheral insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp technique.

Muscle-type specific intramyocellular and hepatic lipid metabolism during starvation in wistar rats.

The physiological dynamics of intramyocellular lipids (IMCLs) in different muscle types and of hepatocellular lipids (HepCLs) are still uncertain. The dynamics of IMCLs in the soleus, tibialis anterior, and extensor digitorum longus (EDL) muscles and HepCL during fed, 12- to 72-h starved, and refed conditions were measured in vivo by (1)H-magnetic resonance spectroscopy (MRS) in Wistar rats. Despite significant elevations of free fatty acids (FFAs) during starvation, HepCLs and IMCLs in soleus remained constant. In tibialis anterior and EDL, however, IMCLs increased significantly by 170 and 450% after 72 h of starvation, respectively. After refeeding, elevated IMCLs dropped immediately in both muscles. Total muscle long-chain acyl-CoAs (LCACoAs) remained constant during the study period. Hepatic palmitoleoyl-CoA (C16:1) decreased significantly during starvation while total hepatic LCACoAs increased significantly. Consistent with constant values for FFAs, HepCLs, IMCLs, and muscle LCACoAs from 12-72 h of starvation, insulin sensitivity did not change. We conclude that during starvation-induced adipocytic lipolysis, oxidative muscles dispose elevated FFAs by oxidation, while nonoxidative ones neutralize FFAs by reesterification. Both mechanisms might prevent impairment of insulin signaling by maintaining low levels of LCACoAs. Hepatic palmitoleoyl-CoA might have a special role in lipid metabolism due to its unique dynamic profile during starvation.