Philip N. Howles

The central melanocortin system directly controls peripheral lipid metabolism

Disruptions of the melanocortin signaling system have been linked to obesity. We investigated a possible role of the central nervous melanocortin system (CNS-Mcr) in the control of adiposity through effects on nutrient partitioning and cellular lipid metabolism independent of nutrient intake. We report that pharmacological inhibition of melanocortin receptors (Mcr) in rats and genetic disruption of Mc4r in mice directly and potently promoted lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue (WAT), while increased CNS-Mcr signaling triggered lipid mobilization. These effects were independent of food intake and preceded changes in adiposity. In addition, decreased CNS-Mcr signaling promoted increased insulin sensitivity and glucose uptake in WAT while decreasing glucose utilization in muscle and brown adipose tissue. Such CNS control of peripheral nutrient partitioning depended on sympathetic nervous system function and was enhanced by synergistic effects on liver triglyceride synthesis. Our findings offer an explanation for enhanced adiposity resulting from decreased melanocortin signaling, even in the absence of hyperphagia, and are consistent with feeding-independent changes in substrate utilization as reflected by respiratory quotient, which is increased with chronic Mcr blockade in rodents and in humans with loss-of-function mutations in MC4R. We also reveal molecular underpinnings for direct control of the CNS-Mcr over lipid metabolism. These results suggest ways to design more efficient pharmacological methods for controlling adiposity.

Carboxyl ester lipase: structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis

Carboxyl ester lipase (CEL), previously named cholesterol esterase or bile salt-stimulated (or dependent) lipase, is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, tri-, di-, and mono-acylglycerols, phospholipids, lysophospholipids, and ceramide. The active site catalytic triad of serine-histidine-aspartate is centrally located within the enzyme structure and is partially covered by a surface loop. The carboxyl terminus of the protein regulates enzymatic activity by forming hydrogen bonds with the surface loop to partially shield the active site. Bile salt binding to the loop domain frees the active site for accessibility by water-insoluble substrates. CEL is synthesized primarily in the pancreas and lactating mammary gland, but the enzyme is also expressed in liver, macrophages, and in the vessel wall. In the gastrointestinal tract, CEL serves as a compensatory protein to other lipolytic enzymes for complete digestion and absorption of lipid nutrients. Importantly, CEL also participates in chylomicron assembly and secretion, in a mechanism mediated through its ceramide hydrolytic activity. Cell culture studies suggest a role for CEL in lipoprotein metabolism and oxidized LDL-induced atherosclerosis. Thus, this enzyme, which has a wide substrate reactivity and diffuse anatomic distribution, may have multiple functions in lipid and lipoprotein metabolism, and atherosclerosis.

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Reduced absorption of saturated fatty acids and resistance to diet-induced obesity and diabetes by ezetimibe-treated and Npc1l1 / mice

The impact of NPC1L1 and ezetimibe on cholesterol absorption are well documented. However, their potential consequences relative to absorption and metabolism of other nutrients have been only minimally investigated. Thus studies were undertaken to investigate the possible effects of this protein and drug on fat absorption, weight gain, and glucose metabolism by using Npc1l1(-/-) and ezetimibe-treated mice fed control and high-fat, high-sucrose diets. Results show that lack of NPC1L1 or treatment with ezetimibe reduces weight gain when animals are fed a diabetogenic diet. This resistance to diet-induced obesity results, at least in part, from significantly reduced absorption of dietary saturated fatty acids, particularly stearate and palmitate, since food intake did not differ between groups. Expression analysis showed less fatty acid transport protein 4 (FATP4) in intestinal scrapings of Npc1l1(-/-) and ezetimibe-treated mice, suggesting an important role for FATP4 in intestinal absorption of long-chain fatty acids. Concomitant with resistance to weight gain, lack of NPC1L1 or treatment with ezetimibe also conferred protection against diet-induced hyperglycemia and insulin resistance. These unexpected beneficial results may be clinically important, given the focus on NPC1L1 as a target for the treatment of hypercholesterolemia.

Development and Physiological Regulation of Intestinal Lipid Absorption. III. Intestinal transporters and cholesterol absorption

Intestinal cholesterol absorption is modulated by transport proteins in enterocytes. Cholesterol uptake from intestinal lumen requires several proteins on apical brush-border membranes, including Niemann-Pick C1-like 1 (NPC1L1), scavenger receptor B-I, and CD36, whereas two ATP-binding cassette half transporters, ABCG5 and ABCG8, on apical membranes work together for cholesterol efflux back to the intestinal lumen to limit cholesterol absorption. NPC1L1 is essential for cholesterol absorption, but its function as a cell surface transporter or an intracellular cholesterol transport protein needs clarification. Another ATP transporter, ABCA1, is present in the basolateral membrane to mediate HDL secretion from enterocytes.

Ginsenoside Rb1 reduces fatty liver by activating AMP-activated protein kinase in obese rats

Ginsenoside Rb1 (Rb1), a natural compound extracted from ginseng, exerts anti-obesity activity and improves insulin sensitivity in high-fat diet (HFD)-induced obese rats. The objective of the current study was to evaluate the protective effect of Rb1 on fatty liver in HFD-induced obese rats and to elucidate underlying mechanisms. After chronic intraperitoneal administration, Rb1 (10 mg/kg) significantly ameliorated hepatic fat accumulation in HFD-induced obese rats, as demonstrated by reduced liver weight, hepatic triglyceride content, and histological evaluation of liver sections by hematoxylin and eosin and Oil Red O staining. Using primary cultured rat hepatic cells, we found that the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase 1 (CPT1), a key enzyme in fatty acid β-oxidation, were significantly elevated in Rb1-treated hepatocytes compared with those of vehicle-treated cells. HPLC analysis revealed that Rb1 increased the cellular AMP/ATP ratio, which is associated with elevated activation of hepatic AMP-activated protein kinase (AMPK) and phosphorylated acetyl-CoA carboxylase. Consistent with the activation of AMPK, Rb1 stimulated the expression of genes encoding fatty acid oxidative enzymes and proteins, and suppressed the expression of genes encoding enzymes or proteins that function in lipogenesis, assessed by quantitative PCR. We conclude that Rb1 has a potent ability to reduce hepatic fat accumulation and might be useful as a therapeutic agent for fatty liver disorder.

Pancreatic Triglyceride Lipase Deficiency Minimally Affects Dietary Fat Absorption but Dramatically Decreases Dietary Cholesterol Absorption in Mice

This study generated pancreatic triglyceride lipase (PTL)-null mice to test the hypothesis that PTL-mediated hydrolysis of dietary triglyceride is necessary for efficient dietary cholesterol absorption. The PTL–/– mice grew normally and displayed similar body weight as their PTL+/+ littermates. Plasma lipid levels between animals of various PTL genotypes were similar when they were maintained on either a basal low fat diet or a western-type high fat/high cholesterol diet. Although the lack of a functional PTL delayed fat absorption during the initial hour of feeding a bolus load of olive oil containing [3H]triolein and [14C]cholesterol, the rate of [3H]triolein absorption was similar between PTL+/+ and PTL–/– mice after the initial 1-h period. Importantly, comparison of fecal fat content revealed similar overall fat absorption efficiency between PTL+/+ and PTL–/– mice. In contrast, the PTL–/– mice displayed significant decrease in both the rate and the amount of cholesterol absorbed after a single meal. The plasma appearance of [14C]cholesterol was found to be 75% lower (p < 0.0005) in PTL–/– mice compared with PTL+/+ mice after 4 h. The total amount of [14C]cholesterol excreted in the feces was 45% higher (p < 0.0004) in PTL–/– mice compared with PTL+/+ mice over a 24-h period. These results indicate that the delayed fat digestion due to PTL deficiency results in a significant reduction in cholesterol absorption, although other enzymes in the digestive tract may compensate for the lack of PTL in PTL–/– mice in fat digestion and absorption.

Hepatic deficiency of low density lipoprotein receptor-related protein-1 reduces high density lipoprotein secretion and plasma levels in mice

The low density lipoprotein receptor-related protein-1 (LRP1) is known to serve as a chylomicron remnant receptor in the liver responsible for the binding and plasma clearance of apolipoprotein E-containing lipoproteins. Previous in vitro studies have provided evidence to suggest that LRP1 expression may also influence high density lipoprotein (HDL) metabolism. The current study showed that liver-specific LRP1 knock-out (hLrp1−/−) mice displayed lower fasting plasma HDL cholesterol levels when compared with hLrp1+/+ mice. Lecithin:cholesterol acyl transferase and hepatic lipase activities in plasma of hLrp1−/− mice were comparable with those observed in hLrp1+/+ mice, indicating that hepatic LRP1 inactivation does not influence plasma HDL remodeling. Plasma clearance of HDL particles and HDL-associated cholesteryl esters was also similar between hLrp1+/+ and hLrp1−/− mice. In contrast, HDL secretion from primary hepatocytes isolated from hLrp1−/− mice was significantly reduced when compared with that observed with hLrp1+/+ hepatocytes. Biotinylation of cell surface proteins revealed decreased surface localization of the ATP-binding cassette, subfamily A, member 1 (ABCA1) protein, but total cellular ABCA1 level was not changed in hLrp1−/− hepatocytes. Finally, hLrp1−/− hepatocytes displayed reduced binding capacity for extracellular cathepsin D, resulting in lower intracellular cathepsin D content and impairment of prosaposin activation, a process that is required for membrane translocation of ABCA1 to facilitate cholesterol efflux and HDL secretion. Taken together, these results documented that hepatic LRP1 participates in cellular activation of lysosomal enzymes and through this mechanism, indirectly modulates the production and plasma levels of HDL.

Apolipoprotein A-IV: a protein intimately involved in metabolism

The purpose of this review is to summarize our current understanding of the physiological roles of apoA-IV in metabolism, and to underscore the potential for apoA-IV to be a focus for new therapies aimed at the treatment of diabetes and obesity-related disorders. ApoA-IV is primarily synthesized by the small intestine, attached to chylomicrons by enterocytes, and secreted into intestinal lymph during fat absorption. In circulation, apoA-IV is associated with HDL and chylomicron remnants, but a large portion is lipoprotein free. Due to its anti-oxidative and anti-inflammatory properties, and because it can mediate reverse-cholesterol transport, proposed functions of circulating apoA-IV have been related to protection from cardiovascular disease. This review, however, focuses primarily on several properties of apoA-IV that impact other metabolic functions related to food intake, obesity, and diabetes. In addition to participating in triglyceride absorption, apoA-IV can act as an acute satiation factor through both peripheral and central routes of action. It also modulates glucose homeostasis through incretin-like effects on insulin secretion, and by moderating hepatic glucose production. While apoA-IV receptors remain to be conclusively identified, the latter modes of action suggest that this protein holds therapeutic promise for treating metabolic disease.

Carboxyl Ester Lipase Deficiency Exacerbates Dietary Lipid Absorption Abnormalities and Resistance to Diet-induced Obesity in Pancreatic Triglyceride Lipase Knockout Mice

This study evaluated the contributions of carboxyl ester lipase (CEL) and pancreatic triglyceride lipase (PTL) in lipid nutrient absorption. Results showed PTL deficiency has minimal effect on triacylglycerol (TAG) absorption under low fat dietary conditions. Interestingly, PTL–/– mice displayed significantly reduced TAG absorption compared with wild type mice under high fat/high cholesterol dietary conditions (80.1 ± 3.7 versus 91.5 ± 0.7%, p < 0.05). Net TAG absorption was reduced further to 61.1 ± 3.8% in mice lacking both PTL and CEL. Cholesterol absorption was 41% lower in PTL–/– mice compared with control mice (p < 0.05), but this difference was not exaggerated in PTL–/–, CEL–/– mice. Retinyl palmitate absorption was reduced by 45 and 60% in PTL–/– mice (p < 0.05) and PTL–/–, CEL–/– mice (p < 0.01), respectively. After 15 weeks of feeding, the high fat/high cholesterol diet, wild type, and CEL–/– mice gained ∼24 g of body weight. However, body weight gain was 6.2 and 8.6 g less (p < 0.01) in PTL–/– and PTL–/–, CEL–/– mice, respectively, despite their consumption of comparable amounts of the high fat/high cholesterol diet. The decrease body weight gain in PTL–/– and PTL–/–, CEL–/– mice was attributed to their absorption of fewer calories from the high fat/high cholesterol diet, thereby resulting in less fat mass accumulation than that observed in wild type and CEL–/– mice. Thus, this study documents that PTL and CEL serve complementary functions, working together to mediate the absorption of a major portion of dietary fat and fat-soluble vitamin esters. The reduced lipid absorption efficiency due to PTL and CEL inactivation also resulted in protection against diet-induced obesity.