Sub-chronic administration of the 11beta-HSD1 inhibitor, carbenoxolone, improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.     

Obesity causes very low density lipoprotein clearance defects in low-density lipoprotein receptor-deficient mice

We have reported that obese leptin-deficient mice (ob/ob) lacking the low-density lipoprotein receptor (LDLR(-/-)) develop severe hyperlipidemia and spontaneous atherosclerosis. In the present study, we show that obese leptin receptor-deficient mice (db/db) lacking LDLR have a similar phenotype, even in the presence of elevated plasma leptin levels. We investigated the mechanism for the hyperlipidemia in obese LDLR(-/-) mice by comparing lipoprotein production and clearance rates in C57BL/6, ob/ob, LDLR(-/-) and ob/ob;LDLR(-/-) mice. Hepatic triglyceride production rates were equally increased ( approximately 1.4-fold, P<.05) in both LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob mice. LDL clearance was decreased ( approximately 1.3- fold, P<.01) to a similar extent in LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob controls. While VLDL clearance was delayed in LDLR(-/-) compared to C57BL/6 and ob/ob mice (2-fold, P<.001), this delay was exaggerated in ob/ob;LDLR(-/-) mice (3.8-fold, P<001). The VLDL clearance defects were due to decreased hepatic uptake compared to C57BL/6 (54% and 26% for LDLR(-/-) and ob/ob;LDLR(-/-), respectively, P<.001). When VLDL was collected from C57BL/6, ob/ob, LDLR(-/-), and ob/ob;LDLR(-/-) donors and injected into LDLR(-/-) recipient mice, counts remaining in the liver were 1.4-fold elevated in mice receiving LDLR(-/-) VLDL and 2-fold increased in mice receiving ob/ob;LDLR(-/-) VLDL compared to controls receiving C57BL/6 VLDL (P<.01). Thus, the increase in plasma lipoproteins in ob/ob;LDLR(-/-) mice is caused by delayed VLDL clearance. This appears to be due to defects in both the liver and the lipoproteins themselves in these obese mice.     

Obesity potentiates development of fatty liver and insulin resistance, but not atherosclerosis, in high-fat diet-fed agouti LDLR-deficient mice

Obesity is increasing at an alarming rate, and its related disorders are placing a considerable strain on our healthcare system. Although they are not always coincident, obesity is often accompanied by hyperlipidemia. Both obesity and hyperlipidemia are independently associated with atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and insulin resistance (IR). Thus, we sought to determine the relative contributions of obesity and hyperlipidemia to these associated pathologies. Obese agouti (A(y)/a) mice and their littermate controls (a/a) were placed on an LDL receptor (LDLR)(-/-) background. At 4 mo of age, mice were either maintained on chow diet (CD) or placed on Western diet (WD) for 12 wk. These genetic and dietary manipulations yielded four experimental groups: 1) lean, a/a;LDLR(-/-)CD; 2) genetic-induced obesity (GIO), A(y)/a;LDLR(-/-)CD; 3) diet-induced obesity (DIO), a/a;LDLR(-/-)WD; and 4) genetic- plus diet-induced obesity (GIO/DIO), A(y)/a;LDLR(-/-)WD. Lipoprotein profiles revealed increased VLDL and LDL particles in WD-fed mice compared with CD-fed controls. The hyperlipidemia present in this mouse model was the result of both increased hepatic triglyceride production and delayed lipoprotein clearance from the plasma. Both WD-fed groups exhibited similar levels of atherosclerotic lesion area, with increased obesity in the GIO/DIO group having no impact on atherogenesis. However, the severe obesity in the GIO/DIO group did aggravate NAFLD and IR. These findings suggest that, although obesity and hyperlipidemia exert individual pathological effects, the combination of the two has the potential to exert an additive effect on NAFLD and IR but not atherosclerosis in this mouse model.     

Effect of voluntary exercise on peripheral tissue glucocorticoid receptor content and the expression and activity of 11beta-HSD1 in the Syrian hamster.

Recent findings indicate that elevated levels of glucocorticoids (GC), governed by the expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and GC receptors (GR), in visceral adipose tissue and skeletal muscle lead to increased insulin resistance and the metabolic syndrome. Paradoxically, evidence indicates that aerobic exercise attenuates the development of the metabolic syndrome even though it stimulates acute increases in circulating GC levels. To investigate the hypothesis that training alters peripheral GC action to maintain insulin sensitivity, young male hamsters were randomly divided into sedentary (S) and trained (T) groups (n = 8 in each). The T group had 24-h access to running wheels over 4 wk of study. In muscle, T hamsters had lower 11beta-HSD1 protein expression (19.2 +/- 1.40 vs. 22.2 +/- 0.96 optical density, P < 0.05), similar 11beta-HSD1 enzyme activity (0.9 +/- 0.27% vs. 1.1 +/- 0.26), and lower GR protein expression (9.7 +/- 1.86 vs. 15.1 +/- 1.78 optical density, P < 0.01) than S hamsters. In liver, 11beta-HSD1 protein expression tended to be lower in T compared with S (19.2 +/- 0.56 vs. 21.4 +/- 1.05, P = 0.07), whereas both enzyme activity and GR protein expression were similar. In contrast, visceral adipose tissue contained approximately 2.7-fold higher 11beta-HSD1 enzyme activity in T compared with S (12.9 +/- 3.3 vs. 4.8 +/- 1.5% conversion, P < 0.05) but was considerably smaller in mass (0.24 +/- 0.02 vs. 0.71 +/- 0.06 g). Thus the intracellular adaptation of GC regulators to exercise is tissue specific, resulting in decreases in GC action in skeletal muscle and increases in GC action in visceral fat. These adaptations may have important implications in explaining the protective effects of aerobic exercise on insulin resistance and other symptoms of the metabolic syndrome.     

LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice

The very low density lipoprotein receptor (VLDLR) has been proposed to play a role in the delivery of fatty acids to peripheral tissues. However, despite reduced adipose tissue mass in VLDLR-deficient (VLDLR(-)(/-)) mice, this has been difficult to substantiate. In the present study, VLDLR-deficient and VLDLR-overexpressing (PVL) mice were cross-bred onto a low density lipoprotein receptor knockout (LDLR(-)(/-)) background to study the VLDLR under conditions of relatively high serum VLDL and triglyceride levels. Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. When kept on a chow diet, a period of prolonged fasting revealed a significant increase in serum triglyceride levels in VLDLR(-)(/-); LDLR(-)(/-) mice (2.3-fold) as compared with LDLR(-)(/-) controls. This could not be attributed to altered apolipoprotein B and VLDL triglyceride production rates. Furthermore, no major differences in nascent VLDL triglyceride content were found between VLDLR(-)(/-); LDLR(-)(/-) mice and LDLR(-)(/-) controls. However, the triglyceride content of circulating VLDL of VLDLR(-)(/-); LDLR(-)(/-) mice (63%) was relatively high as compared with LDLR(-)(/-) controls (49%). These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides.In conclusion, under conditions of LDLR deficiency in combination with high fat feeding or prolonged fasting, the effect of the VLDLR on VLDL triglyceride metabolism was revealed.     

Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11β-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11β-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.     

MRNA and enzyme activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 are elevated in C57BL/KsJ-db/db mice.

To evaluate the importance of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in insulin resistant diabetic C57BL/KsJ-db/db mice, we measured the activity and mRNA level of 11beta-HSD1 in the liver of db/db mice and their heterozygote litter mates, db/+m mice. The blood glucose, plasma insulin, and corticosterone levels of db/db mice were significantly higher than those of db/+m mice. Despite hyperinsulinemia, the activity level of this enzyme was significantly higher in db/db mice, and the mRNA level of hepatic 11beta-HSD1 was also significantly higher in db/db mice. Since hepatic 11beta-HSD1 in vivo mainly functions as 11-keto-reductase and does not work as 11beta-oxidase, these results suggest that the rate of hepatic conversion of 11-dehydrocorticosterone to corticosterone is increased in db/db mice, resulting in higher glucocorticoid activity in the liver. The increased hepatic corticosterone concentration due to the elevation of 11beta-HSD1 and high plasma corticosterone concentration may antagonize the action of insulin and cause insulin resistance. These findings have a potentially important implication for relationships between increased hepatic 11beta-HSD1 and insulin resistance in db/db mice. The present paper is the first to demonstrate the increased activities and mRNA level of hepatic 11beta-HSD1 in db/db mice.     

Angiotensin II increases adipose angiotensinogen expression

In addition to the well-defined contribution of the liver, adipose tissue has been recognized as an important source of angiotensinogen (AGT). The purpose of this study was to define the angiotensin II (ANG II) receptors involved in regulation of adipose AGT and the relationship of this control to systemic AGT and/or angiotensin peptide concentrations. In LDL receptor-deficient (LDLR(-/-)) male mice, adipose mRNA abundance of AGT was 68% of that in liver, and adipose mRNA abundance of the angiotensin type 1a (AT(1a)) receptor (AT(1a)R) was 38% of that in liver, whereas mRNA abundance of the angiotensin type 2 (AT(2)) receptor (AT(2)R) was 57% greater in adipose tissue than in liver. AGT and angiotensin peptide concentrations were decreased in plasma of AT(1a)R-deficient (AT(1a)R(-/-)) mice and were paralleled by reductions in AGT expression in liver. In contrast, adipose AGT mRNA abundance was unaltered in AT(1a)R(-/-) mice. AT(2)R(-/-) mice exhibited elevated plasma angiotensin peptide concentrations and marked elevations in adipose AGT and AT(1a)R mRNA abundance. Increases in adipose AGT mRNA abundance in AT(2)R(-/-) mice were abolished by losartan. In contrast, liver AGT and AT(1a)R mRNA abundance were unaltered in AT(2)R(-/-) mice. Infusion of ANG II for 28 days into LDLR(-/-) mice markedly increased adipose AGT and AT(1a)R mRNA but did not alter liver AGT and AT(1a)R mRNA. These results demonstrate that differential mRNA abundance of AT(1a)/AT(2) receptors in adipose tissue vs. liver contributes to tissue-specific ANG II-mediated regulation of AGT. Chronic infusion of ANG II robustly stimulated AT(1a)R and AGT mRNA abundance in adipose tissue, suggesting that adipose tissue serves as a primary contributor to the activated systemic renin-angiotensin system.     

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR(-/-) mice were lethally irradiated and reconstituted with bone marrow from either apoE(-/-) or apoE(+/+) mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR(-/-) mice with apoE(-/-) bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001). Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR(-/-) mice.     

Glycosylphosphatidylinositol-specific phospholipase D influences triglyceride-rich lipoprotein metabolism

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a minor HDL-associated protein. Because many minor HDL-associated proteins exchange between different lipoprotein classes during the postprandial state and are also involved in triglyceride (TG) metabolism, we hypothesized that GPI-PLD may play a role in the metabolism of TG-rich lipoproteins. To test this hypothesis, we examined the distribution of GPI-PLD among lipoprotein classes during a fat tolerance test in C57BL/6 and LDL receptor-deficient (LDLR(-/-)) mice fed either a chow or high-fructose diet. In the fasting state in wild-type mice fed a chow diet, GPI-PLD was only present in HDL, whereas in LDLR(-/-) mice GPI-PLD was present in HDL and intermediate-density lipoproteins (IDL)/LDL. During the fat tolerance test, there was no change in total serum GPI-PLD levels in either model; however, a significant amount of GPI-PLD appeared in both VLDL (0.5-1% of total GPI-PLD) and IDL/LDL (5-10% of total GPI-PLD) in both models. The high-fructose diet increased both fasting and postprandial TG and serum GPI-PLD levels in both strains as well as the amount of GPI-PLD in VLDL. To determine whether GPI-PLD plays a direct role in TG metabolism, we increased liver GPI-PLD expression in C57BL/6 mice by adenovirus-mediated gene transfer, which resulted in a sevenfold increase in serum GPI-PLD levels. This change was associated with an increase in fasting (30%) and postprandial TG (50%) and a twofold reduction in TG-rich lipoprotein catabolism compared with saline or control adenovirus-treated mice. These studies demonstrate that GPI-PLD affects serum TG levels by altering catabolism of TG-rich lipoproteins.     

Local amplification of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase type 1 promotes macrophage phagocytosis of apoptotic leukocytes

Glucocorticoids promote macrophage phagocytosis of leukocytes undergoing apoptosis. Prereceptor metabolism of glucocorticoids by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) modulates cellular steroid action. 11beta-HSD type 1 amplifies intracellular levels of active glucocorticoids in mice by reactivating corticosterone from inert 11-dehydrocorticosterone in cells expressing the enzyme. In this study we describe the rapid (within 3 h) induction of 11beta-HSD activity in cells elicited in the peritoneum by a single thioglycolate injection in mice. Levels remained high in peritoneal cells until resolution. In vitro experiments on mouse macrophages demonstrated that treatment with inert 11-dehydrocorticosterone for 24 h increased phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11beta-HSD1, as 11beta-HSD1 mRNA, but not 11beta-HSD2 mRNA, was expressed in these cells; 11-dehydrocorticosterone was ineffective in promoting phagocytosis by Hsd11b1(-/-) macrophages, and carbenoxolone, an 11beta-HSD inhibitor, prevented the increase in phagocytosis elicited in wild-type macrophages by 11-dehydrocorticosterone. Importantly, as experimental peritonitis progressed, clearance of apoptotic neutrophils was delayed in Hsd11b1(-/-) mice. These data point to an early role for 11beta-HSD1 in promoting the rapid clearance of apoptotic cells during the resolution of inflammation and indicate a novel target for therapy.     

Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet

We investigated the mechanisms that lead to combined hyperlipidemia in transgenic mice that overexpress human apolipoprotein (apo) A-II (line 11.1). The 11.1 transgenic mice develop pronounced hypertriglyceridemia, and a moderate increase in free fatty acid (FFA) and plasma cholesterol, especially when fed a high-fat/high-cholesterol diet. Post-heparin plasma lipoprotein lipase and hepatic lipase activities (using artificial or natural autologous substrates), the decay of plasma triglycerides with fasting, and the fractional catabolic rate of the radiolabeled VLDL-triglyceride (both fasting and postprandial) were similar in 11. 1 transgenic mice and in control mice. In contrast, a 2.5-fold increase in hepatic VLDL-triglyceride production was observed in 11. 1 transgenic mice in a period of 2 h in which blood lipolysis was inhibited. This increased synthesis of hepatic VLDL-triglyceride used preformed FFA rather than FFA of de novo hepatic synthesis. The 11.1 transgenic mice also presented reduced epididymal/parametrial white adipose tissue weight (1.5-fold), increased rate of epididymal/parametrial hormone-sensitive lipase-mediated lipolysis (1.2-fold) and an increase in cholesterol and, especially, in triglyceride liver content, suggesting an enhanced mobilization of fat as the source of preformed FFA reaching the liver. Increased plasma FFA was reverted by insulin, demonstrating that 11.1 transgenic mice are not insulin resistant. We conclude that the overexpression of human apoA-II in transgenic mice induces combined hyperlipidemia through an increase in VLDL production. These mice will be useful in the study of molecular mechanisms that regulate the overproduction of VLDL, a situation of major pathophysiological interest since it is the basic mechanism underlying familial combined hyperlipidemia.     

Diabetic pregnancy in rats leads to impaired glucose metabolism in offspring involving tissue-specific dysregulation of 11beta-hydroxysteroid dehydrogenase type 1 expression

Population-based studies have shown that the offspring of diabetic mothers have an increased risk of developing obesity, insulin resistance, type 2 diabetes and hypertension in later life. To investigate mechanism for the high incidence of metabolic diseases in the offspring of diabetic mothers, we focused on the tissue-specific glucocorticoid regulation by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and studied offspring born to streptozotocin-induced diabetic rats. The body weights of newborn rats from diabetic mothers were heavier than those from control mothers. Offspring born to diabetic mothers demonstrated insulin resistance and mild glucose intolerance after glucose loading at 10 weeks and showed significantly increased 11beta-HSD1 mRNA and enzyme activity in adipose tissue at 12 weeks of age without obvious obesity. Hepatic 11beta-HSD1 mRNA was also elevated. We propose that the 11beta-HSD1 in adipose tissue and liver may play a key role in the development of metabolic syndrome in the offspring of diabetic mothers. Tissue-specific glucocorticoid dysregulation provides a candidate mechanism for the high incidence of metabolic diseases in the offspring of diabetic mothers. Therefore early analyses before apparent obesity are needed to elucidate the molecular mechanisms that may be programmed during the fetal period.     

Adipose tissue intracrinology: potential importance of local androgen/estrogen metabolism in the regulation of adiposity.

The present article summarizes some of the studies available on steroid hormone conversion through the specific expression of steroidogenic enzymes in adipose tissue (adipose tissue intracrinology) and discusses the potential impact of local adipose tissue steroid metabolism on the regulation of adipocyte function and other metabolic parameters. Several studies have demonstrated significant steroid hormone uptake and conversion by adipose tissues from various body sites and in various cell fractions. Activities and/or mRNAs of aromatase, 3beta-hydroxysteroid dehydrogenase (HSD), 3alpha-HSD, 11beta-HSD, 17beta-HSD, 7alpha-hydroxylase, 17alpha-hydroxylase, 5alpha-reductase and UDP-glucuronosyltransferase 2B15 have been detected in adipose tissue or adipose cells. These studies have demonstrated potentially important roles for these enzymes in obesity, central fat accumulation, and the metabolic syndrome. Future studies on adipose tissue intracrinology will contribute further to our understanding of steroid action in adipocytes.     

Loss of small heterodimer partner expression in the liver protects against dyslipidemia

Multiple studies suggest increased conversion of cholesterol to bile acids by cholesterol 7alpha-hydroxylase (CYP7A1) protects against dyslipidemia and atherosclerosis. CYP7A1 expression is repressed by the sequential activity of two nuclear hormone receptors, farnesoid X receptor (FXR) and small heterodimer partner (SHP). Here we demonstrate 129 strain SHP(-/-) mice are protected against hypercholesterolemia resulting from either a cholesterol/cholic acid (chol/CA) diet or from hypothyroidism. In a mixed 129-C57Bl/6 background, LDLR(-/-) and LDLR(-/-)SHP(-/-) mice had nearly identical elevations in hepatic cholesterol content and repression of cholesterol regulated genes when fed a Western diet. However, the LDLR(-/-)SHP(-/-) mice had greatly reduced elevations in serum VLDL and LDL cholesterol levels and triglyceride (TG) levels as compared with LDLR(-/-) mice. Additionally, the hepatic inflammation produced by the Western diet in the LDLR(-/-) mice was abolished in the LDLR(-/-)SHP(-/-) mice. CYP7A1 expression was induced 10-fold by the Western diet in the LDLR(-/-)SHP(-/-) mice but not in the LDLR(-/-) mice. Finally, hepatocyte-specific deletion of SHP expression was also protective against dyslipidemia induced by either a chol/CA diet or by hypothyroidism. While no antagonist ligands have yet been identified for SHP, these results suggest selective inhibition of hepatic SHP expression may provide protection against dyslipidemia.