Cytosolic phosphoenolpyruvate carboxykinase as a cataplerotic pathway in the small intestine.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme that is highly expressed in the liver and kidney but is also expressed at lower levels in a variety of other tissues where it may play adjunct roles in fatty acid esterification, amino acid metabolism, and/or TCA cycle function. PEPCK is expressed in the enterocytes of the small intestine, but it is unclear whether it supports a gluconeogenic rate sufficient to affect glucose homeostasis. To examine potential roles of intestinal PEPCK, we generated an intestinal PEPCK knockout mouse. Deletion of intestinal PEPCK ablated ex vivo gluconeogenesis but did not significantly affect glycemia in chow, high-fat diet, or streptozotocin-treated mice. In contrast, postprandial triglyceride secretion from the intestine was attenuated in vivo, consistent with a role in fatty acid esterification. Intestinal amino acid profiles and 13C tracer appearance into these pools were significantly altered, indicating abnormal amino acid trafficking through the enterocyte. The data suggest that the predominant role of PEPCK in the small intestine of mice is not gluconeogenesis but rather to support nutrient processing, particularly with regard to lipids and amino acids. NEW & NOTEWORTHY The small intestine expresses gluconeogenic enzymes for unknown reasons. In addition to glucose synthesis, the nascent steps of this pathway can be used to support amino acid and lipid metabolisms. When phosphoenolpyruvate carboxykinase, an essential gluconeogenic enzyme, is knocked out of the small intestine of mice, glycemia is unaffected, but mice inefficiently absorb dietary lipid, have abnormal amino acid profiles, and inefficiently catabolize glutamine. Therefore, the initial steps of intestinal gluconeogenesis are used for processing dietary triglycerides and metabolizing amino acids but are not essential for maintaining blood glucose levels.

Hypoglycemic Effect of Combined Ghrelin and Glucagon Receptor Blockade.

Glucagon receptor (GcgR) blockade has been proposed as an alternative to insulin monotherapy for treating type 1 diabetes since deletion or inhibition of GcgRs corrects hyperglycemia in models of diabetes. The factors regulating glycemia in a setting devoid of insulin and glucagon function remain unclear but may include the hormone ghrelin. Not only is ghrelin release controlled by glucose but also ghrelin has many actions that can raise or reduce falls in blood glucose level. Here, we tested the hypothesis that ghrelin rises to prevent hypoglycemia in the absence of glucagon function. Both GcgR knockout (Gcgr-/-) mice and db/db mice that were administered GcgR monoclonal antibody displayed lower blood glucose levels accompanied by elevated plasma ghrelin levels. Although treatment with the pancreatic ß-cell toxin streptozotocin induced hyperglycemia and raised plasma ghrelin levels in wild-type mice, hyperglycemia was averted in similarly treated Gcgr-/- mice and the plasma ghrelin level was further increased. Notably, administration of a ghrelin receptor antagonist further reduced blood glucose levels into the markedly hypoglycemic range in overnight-fasted, streptozotocin-treated Gcgr-/- mice. A lowered blood glucose level also was observed in overnight-fasted, streptozotocin-treated ghrelin receptor-null mice that were administered GcgR monoclonal antibody. These data suggest that when glucagon activity is blocked in the setting of type 1 diabetes, the plasma ghrelin level rises, preventing hypoglycemia.

Cellular Aging Contributes to Failure of Cold-Induced Beige Adipocyte Formation in Old Mice and Humans.

Cold temperatures induce progenitor cells within white adipose tissue to form beige adipocytes that burn energy and generate heat; this is a potential anti-diabesity therapy. However, the potential to form cold-induced beige adipocytes declines with age. This creates a clinical roadblock to potential therapeutic use in older individuals, who constitute a large percentage of the obesity epidemic. Here we show that aging murine and human beige progenitor cells display a cellular aging, senescence-like phenotype that accounts for their age-dependent failure. Activating the senescence pathway, either genetically or pharmacologically, in young beige progenitors induces premature cellular senescence and blocks their potential to form cold-induced beige adipocytes. Conversely, genetically or pharmacologically reversing cellular aging by targeting the p38/MAPK-p16Ink4a pathway in aged mouse or human beige progenitor cells rejuvenates cold-induced beiging. This in turn increases glucose sensitivity. Collectively, these data indicate that anti-aging or senescence modalities could be a strategy to induce beiging, thereby improving metabolic health in aging humans.

Leptin Is Required for Glucose Homeostasis after Roux-en-Y Gastric Bypass in Mice.

BACKGROUND & AIMS: Leptin, the protein product of the ob gene, increases energy expenditure and reduces food intake, thereby promoting weight reduction. Leptin also regulates glucose homeostasis and hepatic insulin sensitivity via hypothalamic proopiomelanocortin neurons in mice. Roux-en-Y gastric bypass (RYGB) induces weight loss that is substantial and sustained despite reducing plasma leptin levels. In addition, patients who fail to undergo diabetes remission after RYGB are hypoletinemic compared to those who do and to lean controls. We have previously demonstrated that the beneficial effects of RYGB in mice require the melanocortin-4 receptor, a downstream effector of leptin action. Based on these observations, we hypothesized that leptin is required for sustained weight reduction and improved glucose homeostasis observed after RYGB. METHODS: To investigate this hypothesis, we performed RYGB or sham operations on leptin-deficient ob/ob mice maintained on regular chow. To investigate whether leptin is involved in post-RYGB weight maintenance, we challenged post-surgical mice with high fat diet. RESULTS: RYGB reduced total body weight, fat and lean mass and caused reduction in calorie intake in ob/ob mice. However, it failed to improve glucose tolerance, glucose-stimulated plasma insulin, insulin tolerance, and fasting plasma insulin. High fat diet eliminated the reduction in calorie intake observed after RYGB in ob/ob mice and promoted weight regain, although not to the same extent as in sham-operated mice. We conclude that leptin is required for the effects of RYGB on glucose homeostasis but not body weight or composition in mice. Our data also suggest that leptin may play a role in post-RYGB weight maintenance.

Altered ghrelin secretion in mice in response to diet-induced obesity and Roux-en-Y gastric bypass.

The current study examined potential mechanisms for altered circulating ghrelin levels observed in diet-induced obesity (DIO) and following weight loss resulting from Roux-en-Y gastric bypass (RYGB). We hypothesized that circulating ghrelin levels were altered in obesity and after weight loss through changes in ghrelin cell responsiveness to physiological cues. We confirmed lower ghrelin levels in DIO mice and demonstrated elevated ghrelin levels in mice 6 weeks post-RYGB. In both DIO and RYGB settings, these changes in ghrelin levels were associated with altered ghrelin cell responsiveness to two key physiological modulators of ghrelin secretion - glucose and norepinephrine. In DIO mice, increases in ghrelin cell density within both the stomach and duodenum and in somatostatin-immunoreactive D cell density in the duodenum were observed. Our findings provide new insights into the regulation of ghrelin secretion and its relation to circulating ghrelin within the contexts of obesity and weight loss.

Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin.

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreER(T2) transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.

Imaging cytoplasmic lipid droplets in enterocytes and assessing dietary fat absorption.

The primary function of the small intestine is digesting and absorbing nutrients from consumed food. Because of this, the small intestine is often thought of as a nutrient thoroughfare-enterocytes taking up nutrients on the apical side and then secreting nutrients from the basolateral side. The small intestine is not commonly thought of as a lipid storage organ; however, when meals and diets containing high amounts of fat are consumed, some dietary fat is stored in cytoplasmic lipid droplets (CLDs). The balance between storage and secretion of dietary fat by enterocytes is important in determining the physiological fate of dietary fat, including regulating blood lipid concentrations and energy balance. The existence of CLDs within enterocytes has likely evolved for three important physiological functions: (i) to allow the small intestine to efficiently absorb large amounts of energy dense fat, (ii) to control the rate of dietary fat entering circulation, and (iii) to alleviate lipotoxicity to enterocytes induced by high concentrations of free fatty acids, especially when a high fat meal is consumed. The purpose of this chapter is to provide methods for imaging CLDs in enterocytes and assessing different aspects of dietary fat absorption.

Ghrelin and eating behavior: evidence and insights from genetically-modified mouse models.

Ghrelin is an octanoylated peptide hormone, produced by endocrine cells of the stomach, which acts in the brain to increase food intake and body weight. Our understanding of the mechanisms underlying ghrelin's effects on eating behaviors has been greatly improved by the generation and study of several genetically manipulated mouse models. These models include mice overexpressing ghrelin and also mice with genetic deletion of ghrelin, the ghrelin receptor [the growth hormone secretagogue receptor (GHSR)] or the enzyme that post-translationally modifies ghrelin [ghrelin O-acyltransferase (GOAT)]. In addition, a GHSR-null mouse model in which GHSR transcription is globally blocked but can be cell-specifically reactivated in a Cre recombinase-mediated fashion has been generated. Here, we summarize findings obtained with these genetically manipulated mice, with the aim to highlight the significance of the ghrelin system in the regulation of both homeostatic and hedonic eating, including that occurring in the setting of chronic psychosocial stress.

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 overexpression enhances postprandial triglyceridemic response and exacerbates high fat diet-induced hepatic triacylglycerol storage.

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) is important in the cellular and physiological responses to dietary fat. To determine the effect of increased intestinal DGAT2 on cellular and physiological responses to acute and chronic dietary fat challenges, we generated mice with intestine-specific overexpression of DGAT2 and compared them with intestine-specific overexpression of DGAT1 and wild-type (WT) mice. We found that when intestinal DGAT2 is present in excess, triacylglycerol (TG) secretion from enterocytes is enhanced compared to WT mice; however, TG storage within enterocytes is similar compared to WT mice. We found that when intestinal DGAT2 is present in excess, mRNA levels of genes involved in fatty acid oxidation were reduced. This result suggests that reduced fatty acid oxidation may contribute to increased TG secretion by overexpression of DGAT2 in intestine. Furthermore, this enhanced supply of TG for secretion in Dgat2(Int) mice may be a significant contributing factor to the elevated fasting plasma TG and exacerbated hepatic TG storage in response to a chronic HFD. These results highlight that altering fatty acid and TG metabolism within enterocytes has the capacity to alter systemic delivery of dietary fat and may serve as an effective target for preventing and treating metabolic diseases such as hepatic steatosis.

Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Reduced triglyceride secretion in response to an acute dietary fat challenge in obese compared to lean mice.

Obesity results in abnormally high levels of triglyceride (TG) storage in tissues such as liver, heart, and muscle, which disrupts their normal functions. Recently, we found that lean mice challenged with high levels of dietary fat store TGs in cytoplasmic lipid droplets in the absorptive cells of the intestine, enterocytes, and that this storage increases and then decreases over time after an acute dietary fat challenge. The goal of this study was to investigate the effects of obesity on intestinal TG metabolism. More specifically we asked whether TG storage in and secretion from the intestine are altered in obesity. We investigated these questions in diet-induced obese (DIO) and leptin-deficient (ob/ob) mice. We found greater levels of TG storage in the intestine of DIO mice compared to lean mice in the fed state, but similar levels of TG storage after a 6-h fast. In addition, we found similar TG storage in the intestine of lean and DIO mice at multiple time points after an acute dietary fat challenge. Surprisingly, we found remarkably lower TG secretion from both DIO and ob/ob mice compared to lean controls in response to an acute dietary fat challenge. Furthermore, we found altered mRNA levels for genes involved in regulation of intestinal TG metabolism in lean and DIO mice at 6 h fasting and in response to an acute dietary fat challenge. More specifically, we found that many of the genes related to TG synthesis, chylomicron synthesis, TG storage, and lipolysis were induced in response to an acute dietary fat challenge in lean mice, but this induction was not observed in DIO mice. In fact, we found a significant decrease in intestinal mRNA levels of genes related to lipolysis and fatty acid oxidation in DIO mice in response to an acute dietary fat challenge. Our findings demonstrate altered TG handling by the small intestine of obese compared to lean mice.

Migration of MDA-MB-231 breast cancer cells depends on the availability of exogenous lipids and cholesterol esterification.

We previously described a lipid-accumulating phenotype of estrogen receptor negative (ER(-)) breast cancer cells exemplified by the MDA-MB-231 and MDA-MB-436 cell lines. These cells had more lipid droplets, a higher uptake of oleic acid and LDL, a higher ratio of cholesteryl ester (CE) to triacylglycerol (TAG), and higher expression of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) as compared to ER(+) MCF-7 breast cancer cells. LDL stimulated proliferation of ER-cells only, and proliferation was reduced by inhibition of ACAT. We hypothesized that storage of exogenous lipids would confer an energetic advantage. We tested this by depriving cells of exogenous lipids and measuring chemotactic migration, an energy-intensive behavior. MDA-MB-231 cells were grown for 48 h in medium with either 5% FBS or 5% lipoprotein-depleted (LD) FBS. Growth in LD medium resulted in visibly reduced lipid droplets and an 85% decrease in cell migration. Addition of LDL to the LD medium dose-dependently restored the ability to migrate in an ACAT-sensitive manner. LDL receptor (LDLR) mRNA was 12-fold higher in MDA-MB-231 cells compared to nontumorigenic ER-MCF-10A breast epithelial cells grown in LD medium. Addition of LDL to the LD medium reduced LDLR mRNA levels in MCF-10A cells only. We asked if ACAT1 activity was associated with the expression of the LDLR in MDA-MB-231 cells. LDLR mRNA in MDA-MB-231 cells was substantially reduced by inhibition of ACAT, demonstrating that high ACAT1 activity permitted higher LDLR expression. This data substantiates the association of lipid accumulation with aggressive behavior in an ER-breast cancer cell line.

Fenofibrate, a peroxisome proliferator-activated receptor α agonist, alters triglyceride metabolism in enterocytes of mice.

Fenofibrate, a drug in the fibrate class of amphiphathic carboxylic acids, has multiple blood lipid modifying actions, which are beneficial to the prevention of atherosclerosis. One of its benefits is in lowering fasting and postprandial blood triglyceride (TG) concentrations. The goal of this study was to determine whether the hypotriglyceridemic actions of fenofibrate in the postprandial state include alterations in TG and fatty acid metabolism in the small intestine. We found that the hypotriglyceridemic actions of fenofibrate in the postprandial state of high-fat (HF) fed mice include a decrease in supply of TG for secretion by the small intestine. A decreased supply of TG for secretion was due in part to the decreased dietary fat absorption and increased intestinal fatty acid oxidation in fenofibrate compared to vehicle treated HF fed mice. These results suggest that the effects of fenofibrate on the small intestine play a critical role in the hypotriglyceridemic effects of fenofibrate.