Leptin-deficient Mice Research Articles

Glial Endozepines Reverse High-Fat Diet-Induced Obesity by Enhancing Hypothalamic Response to Peripheral Leptin.

Research on energy homeostasis has focused on neuronal signaling; however, the role of glial cells has remained little explored. Glial endozepines exert anorexigenic actions by mechanisms which remain poorly understood. In this context, the present study was designed to decipher the mechanisms underlying the anorexigenic action of endozepines and to investigate their potential curative effect on high-fat diet-induced obesity. We carried out a combination of physiological, pharmacological, and molecular analyses together to dissect the underlying mechanisms of endozepine-induced hypophagia. To evaluate the potential anti-obesity effect of endozepines, different model of obesity were used, i.e., ob/ob and diet-induced obese mice. We show that the intracerebral administration of endozepines enhances satiety by targeting anorexigenic brain circuitry and induces STAT3 phosphorylation, a hallmark of leptin signaling. Strikingly, endozepines are entirely ineffective at reducing food intake in the presence of a circulating leptin antagonist and in leptin-deficient mice (ob/ob) but potentiate the reduced food intake and weight loss induced by exogenous leptin administration in these animals. Endozepines reversed high fat diet-induced obesity by reducing food intake and restored leptin-induced STAT3 phosphorylation in the hypothalamus. Interestingly, we observed that glucose and insulin synergistically enhance tanycytic endozepine expression and release. Finally, endozepines, which induce ERK activation necessary for leptin transport into the brain in cultured tanycytes, require tanycytic leptin receptor expression to promote STAT3 phosphorylation in the hypothalamus. Our data identify endozepines as potential anti-obesity compounds in part through the modulation of the LepR-ERK-dependent tanycytic leptin shuttle.

215-OR: Silencing CCKBR Containing Neurons in the VMN Restrains Hepatic Glucose Production to Ameliorate Diabetes in Mice

Optimal glucose control remains elusive for many patients with diabetes despite current therapies, necessitating the development of novel therapeutic modalities. While the central nervous system (CNS) plays a crucial role in glucose homeostasis, specific CNS targets for therapeutic intervention in diabetes have remained elusive. We recently demonstrated that the cholecystokinin b receptor (CCKbR)-expressing subpopulation of ventromedial hypothalamic nucleus (VMN; VMNCCKBR) neurons not only mediate the counter-regulatory response, but also elevate blood glucose by insulin-independently modulating hepatic glucose production under normal physiologic conditions. To test the potential therapeutic utility of these neurons for the treatment of diabetes, we cre-dependently expressed tetanus toxin in the VMN of Cckbrcre mice to silence VMNCCKBR neurons in streptozotocin (STZ)-induced (insulinopenic; Type 1 model) and leptin-deficient (ob/ob; obese Type 2 model) mice. While STZ-treated control mice became hyperglycemic and lost approximately 20% of their body weight over two weeks, VMNCCKBR-silenced mice were protected from these effects of STZ, despite similar hormone levels in STZ-treated control and CCKBRTTmice. Similarly, silencing of VMNCCKBR neurons in ob/ob mice normalized fasting and ad libitum-fed blood glucose despite identical food intake, body weight and body composition in control and VMNCCKBR-silenced mice. While VMNCCKBR-silenced ob/ob mice exhibited no alterations in glucose or insulin tolerance compared to controls, glucose clamps demonstrated decreased hepatic glucose output in the VMNCCKBR-silenced animals. These data suggest that VMNCCKBR neurons represent potential targets to reduce hepatic glucose production and ameliorate hyperglycemia in diabetes. Disclosure A. Affinati: Research Support; Self; Novo Nordisk Inc. J. Flak: None. N. Bozadjieva: None. N. Kodur: None. C.M. Cras-Méneur: None. D.A. Sandoval: Research Support; Self; MedImmune, Novo Nordisk A/S. M.G. Myers: Research Support; Self; AstraZeneca, Novo Nordisk Inc. Funding National Institutes of Health (5T32DK7245-42, 1F32DK122660-01)

The Peripubertal Decline in Makorin Ring Finger Protein 3 Expression is Independent of Leptin Action.

A critical body weight is necessary for pubertal development, an effect mediated in part by leptin. The potential regulation by leptin of Makorin Ring Finger Protein 3 (MKRN3), in which loss-of-function mutations are the most common genetic cause of central precocious puberty, has not been previously explored. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if leptin contributes to the decrease in hypothalamic Mkrn3 mRNA levels observed in mice during pubertal development. We first used a leptin-deficient (ob/ob) mouse model. Mkrn3 mRNA levels in the mediobasal hypothalamus (MBH), which includes the arcuate nucleus, and in the preoptic area (POA), both showed a significant decrease with age from postnatal day (PND) 12 to PND30 in ob/ob mice in both males and females, similar to that observed in wild-type mice. To further explore the effects of leptin on Mkrn3 expression, we exposed prepubertal wild-type mice to high levels of leptin from age PND9-12, which did not result in any significant difference in Mkrn3 expression levels in either the MBH or POA. In summary, regulation of Mkrn3 expression by leptin was not observed in either the MBH or the POA, 2 hypothalamic sites important for pubertal maturation. These data suggest that the decline in Mkrn3 at the onset of puberty may occur independently of leptin and support our hypothesis that MKRN3 is a bona fide controller of puberty initiation.

SAT-588 Role of Adipose Tissue in Reproduction, Mammary Gland Development, Function and Cancer

To investigate the role of adipose tissue in the function of the mammary gland (MG) and reproductive system, we have examined lipodystrophic (LD) mice. LD mice of both sexes are sterile, but fertility was restored in both sexes with leptin injections. In addition, leptin was only needed for initial stages of pregnancy and not for parturition. A transplant of mouse embryonic fibroblasts (MEFs) led to the formation of an ectopic fat pad which also rescued the fertility in both sexes. However, pups born to rescued LD mothers died shortly after birth. We therefore examined the mammary glands of these mothers. MGs from LD mice were rudimentary and lacked terminal end buds. Leptin-injected and MEF rescued LD mice were able to become pregnant, showed normal pregnancy-associated glandular proliferation despite a smaller glandular area, were able to produce a small amount of milk that had grossly normal content of milk proteins and neutral lipids, but could not sustain pups to weaning. In order to separate the individual requirements for 1) adipokines such as leptin, 2) estradiol, and 3) epithelial-adipocyte interactions, we performed a series of experiments with both LD and ob (leptin-deficient) mice that received either estradiol or preadipocyte transplant. The resulting fat pad did not rescue the defect in MG development in LD mice. The defect also was not rescued with estradiol pellets. Ob/ob mice, like LD mice, lack leptin and estradiol, but retain adipose tissue. Ob mice have defective MG development. However, in striking contrast to LD mice, reconstitution of a WT fat pad in ob mice rescued the defect in MG development. Estradiol treatment did not rescue MG development in ob mice. Therefore direct interaction between mammary gland epithelia and adipocytes is a requirement for full invasion and expansion of the gland during puberty, but is not required for glandular proliferation during pregnancy and milk production.Given that excess adipose tissue is a risk factor for breast cancer we wanted to determine if breast cancer was affected by the absence of adipose tissue. LD mice were bred to MMTV-PyMT mice that develop spontaneous breast cancer. Remarkably, LD PyMT+ mice had accelerated growth of primary tumors compared to WT PyMT+ mice. Using our MEF transplant model future studies will be directed to understanding whether the accelerated breast cancer growth is due to loss of adipokines or altered epithelial-stromal interactions.

Not the usual suspect.

Chung et al. studied drivers of obesity-associated pancreatic ductal adenocarcinoma in a leptin-deficient mouse model and identified the hormone cholecystokinin, upregulated in islet beta cells in the context of obesity, to be promoting pancreatic tumorigenesis.

PPARγ-mediated microglial activation phenotype is involved in depressive-like behaviors and neuroinflammation in stressed C57BL/6J and ob/ob mice

BackgroundThere is an increased risk for obese patients with chronic low-grade inflammation to develop depression. Stress induces microglial activation and neuroinflammation that play crucial roles in the pathogenesis of depression. Peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear transcription factor, regulates microglial polarization and neuroinflammation. Our study aimed to investigate the role of PPARγ in the development of depressive symptoms and neuroinflammation induced by chronic unpredictable mild stress (CUMS) in wild-type/C57BL/6J (wt) and leptin-deficient (ob/ob) mice. MethodsCUMS was used to build a depression model with wt and ob/ob mice. Depressive-like behaviors were evaluated by sucrose preference test, open field test, tail suspension test, and Morris water maze test. Cytokines, the activated microglial state, and nuclear factor-κB (NF-κB) and PPARγ expression in the prefrontal cortex (PFC) and hippocampus (HIP) were examined by enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and western blotting. Additionally, pioglitazone, an agonist of PPARγ, was used as a treatment intervention. ResultsAfter CUMS, ob/ob mice exhibited severe behavioral disorders and spatial memory impairment, and higher levels of pro-inflammatory cytokines, M1/M2 ratios, and NF-κB activation, as well as lower levels of anti-inflammatory cytokines and PPARγ expression in the PFC and HIP compared to wt mice. Administration of pioglitazone relieved these alterations in wt and ob/ob mice. ConclusionsCUMS was able to induce severe depressive-like behaviors, neuroinflammation, and reduced expression of PPARγ in ob/ob mice as compared to wt mice. This suggests that PPARγ mediates the microglial activation phenotype, which might be related to the susceptibility of stressed ob/ob mice to develop depressive disorder.

Caloric restriction reverses left ventricular hypertrophy through the regulation of cardiac iron homeostasis in impaired leptin signaling mice

Leptin-deficient and leptin-resistant mice manifest obesity, insulin resistance, and left ventricular hypertrophy (LVH); however, LVH’s mechanisms are not fully understood. Cardiac iron dysregulation has been recently implicated in cardiomyopathy. Here we investigated the protective effects of caloric restriction on cardiac remodeling in impaired leptin signaling obese mice. RNA-seq analysis was performed to assess the differential gene expressions in the heart of wild-type and ob/ob mice. In particular, to investigate the roles of caloric restriction on iron homeostasis-related gene expressions, 10-week-old ob/ob and db/db mice were assigned to ad libitum or calorie-restricted diets for 12 weeks. Male ob/ob mice exhibited LVH, cardiac inflammation, and oxidative stress. Using RNA-seq analysis, we identified that an iron uptake-associated gene, transferrin receptor, was upregulated in obese ob/ob mice with LVH. Caloric restriction attenuated myocyte hypertrophy, cardiac inflammation, fibrosis, and oxidative stress in ob/ob and db/db mice. Furthermore, we found that caloric restriction reversed iron homeostasis-related lipocalin 2, divalent metal transporter 1, transferrin receptor, ferritin, ferroportin, and hepcidin expressions in the heart of ob/ob and db/db mice. These findings demonstrate that the cardioprotective effects of caloric restriction result from the cellular regulation of iron homeostasis, thereby decreasing oxidative stress, inflammation, and cardiac remodeling. We suggest that decreasing iron-mediated oxidative stress and inflammation offers new therapeutic approaches for obesity-induced cardiomyopathy.

Partial leptin deficiency confers resistance to diet-induced obesity in mice

ObjectiveHyperleptinemia per se is sufficient to promote leptin resistance in the obese state. Leptin sensitivity can be restored by reducing circulating leptin levels within a physiologically healthy range and is a viable antiobesity and antidiabetic strategy. However, a previous study suggests that partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice, arguing that a lower leptin level may indeed promote diet-induced obesity and its associated metabolic disorders. Here, we aim to elucidate what the impact of partial leptin deficiency is on fat mass and insulin sensitivity. MethodsWe used two different mouse models of partial leptin deficiency: an adipocyte-specific congenital heterozygous leptin knockout mouse line (LepHZ) and the well-established whole body heterozygous leptin knockout mouse (OBHZ). The metabolic studies of OBHZ and LepHZ mice were performed both on normal carbohydrate-rich chow diet and on a high-fat diet (HFD). Male and female mice were included in the study to account for sex-specific differences. Body weight, food intake, glucose tolerance, and insulin tolerance were tested. Histology of adipose tissue and liver tissue allowed insights into adipose tissue inflammation and hepatic triglyceride content. Immunohistochemistry was paired with RT-PCR analysis for expression levels of inflammatory markers. ResultsBoth OBHZ and LepHZ mice displayed reduced circulating leptin levels on the chow diet and HFD. On chow diet, male OBHZ and LepHZ mice showed elevated fat mass and body weight, while their glucose tolerance and insulin sensitivity remained unchanged. However, the inability in partially leptin-deficient mice to fully induce circulating leptin during the development of diet-induced obesity results in reduced food intake and leaner mice with lower body weight compared to their littermate controls. Importantly, a strong reduction of adipose tissue inflammation is observed along with improvements in insulin sensitivity and enhanced glucose tolerance. Additionally, partial leptin deficiency protects the mice from fatty liver and liver fibrosis. Chronically HFD-fed OBHZ and LepHZ mice remain more sensitive to exogenous leptin injection, as reflected by their reduced food intake upon an acute leptin treatment. ConclusionIn response to HFD feeding, the inability to upregulate leptin levels due to partial leptin deficiency protects mice from diet-induced obesity and metabolic dysregulation. Thus, in an obesogenic environment, maintaining lower leptin levels is highly beneficial for both obesity and diabetes management. Chronic leptin reduction represents a viable preventive strategy whose efficacy awaits clinical testing.

Beneficial effect on podocyte number in experimental diabetic nephropathy resulting from combined atrasentan and RAAS inhibition therapy.

Podocyte loss and proteinuria are both key features of human diabetic nephropathy (DN). The leptin-deficient BTBR mouse strain with the ob/ob mutation develops progressive weight gain, type 2 diabetes, and diabetic nephropathy that has many features of advanced human DN, including increased mesangial matrix, mesangiolysis, podocyte loss, and proteinuria. Selective antagonism of the endothelin-1 type A receptor (ETAR) by atrasentan treatment in combination with renin-angiotensin-aldosterone system inhibition with losartan has been shown to have the therapeutic benefit of lowering proteinuria in patients with DN, but the underlying mechanism for this benefit is not well understood. Using a similar therapeutic approach in diabetic BTBR ob/ob mice, this treatment regimen significantly increased glomerular podocyte number compared with diabetic BTBR ob/ob controls and suggested that parietal epithelial cells were a source for podocyte restoration. Atrasentan treatment alone also increased podocyte number but to a lesser degree. Mice treated with atrasentan demonstrated a reduction in proteinuria, matching the functional improvement reported in humans. This is a first demonstration that treatment with the highly selective ETAR antagonist atrasentan can lead to restoration of the diminished podocyte number characteristic of DN in humans and thereby underlies the reduction in proteinuria in patients with diabetes undergoing similar treatment. The benefit of ETAR antagonism in DN extended to a decrease in mesangial matrix as measured by a reduction in accumulations of collagen type IV in both the atrasentan and atrasentan + losartan-treated groups compared with untreated controls.

Sirtuin1 Role in the Melatonin Protective Effects Against Obesity-Related Heart Injury.

Obesity is a worldwide epidemic disease that induces important structural and functional changes to the heart and predisposes a patient to devastating cardiac complications. Sirtuin1 (SIRT1) has been found to have roles in regulating cardiac function, but whether it can help in cardioprotection is not clear. The aim of the present study was to determine whether melatonin, by modulating SIRT1 and in turn mitochondria signaling, may alleviate obesity-induced cardiac injuries. We investigated 10 lean control mice and 10 leptin-deficient obese mice (ob/ob) orally supplemented with melatonin for 8 weeks, as well as equal numbers of age-matched lean and ob/ob mice that did not receive melatonin. Hearts were evaluated using multiple parameters, including biometric values, morphology, SIRT1 activity and expression of markers of mitochondria biogenesis, oxidative stress, and inflammation. We observed that ob/ob mice experienced significant heart hypertrophy, infiltration by inflammatory cells, reduced SIRT1 activity, altered mitochondrial signaling and oxidative balance, and overexpression of inflammatory markers. Notably, melatonin supplementation in ob/ob mice reverted these obesogenic heart alterations. Melatonin prevented heart remodeling caused by obesity through SIRT1 activation, which, together with mitochondrial pathways, reduced oxidative stress and inflammation.

Antrodia cinnamomea Confers Obesity Resistance and Restores Intestinal Barrier Integrity in Leptin-deficient Obese Mice.

Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. Antrodia cinnamomea (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient (ob/ob) mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, ob+/+) and ob/ob mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in ob/ob mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in ob/ob mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.

Leptin promotes methionine adenosyltransferase 2A expression in hepatic stellate cells by the downregulation of E2F-4 via the β-catenin pathway.

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the β-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced β-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.

Amino acid-based compound activates atypical PKC and leptin receptor pathways to improve glycemia and anxiety like behavior in diabetic mice

Differences in glucose uptake in peripheral and neural tissues account for the reduced efficacy of insulin in nervous tissues. Herein, we report the design of short peptides, referred as amino acid compounds (AAC) with and without a modified side chain moiety. At nanomolar concentrations, a candidate therapeutic molecule, AAC2, containing a 7-(diethylamino) coumarin-3-carboxamide side-chain improved glucose control in human peripheral adipocytes and the endothelial brain barrier cells by activation of insulin-insensitive glucose transporter 1 (GLUT1). AAC2 interacted specifically with the leptin receptor (LepR) and activated atypical protein kinase C zeta (PKCς) to increase glucose uptake. The effects induced by AAC2 were absent in leptin receptor-deficient predipocytes and in Leprdb mice. In contrast, AAC2 established glycemic control altering food intake in leptin-deficient Lepob mice. Therefore, AAC2 activated the LepR and acted in a cytokine-like manner distinct from leptin. In a monogenic Ins2Akita mouse model for the phenotypes associated with type 1 diabetes, AAC2 rescued systemic glucose uptake in these mice without an increase in insulin levels and adiposity, as seen in insulin-treated Ins2Akita mice. In contrast to insulin, AAC2 treatment increased brain mass and reduced anxiety-related behavior in Ins2Akita mice. Our data suggests that the unique mechanism of action for AAC2, activating LepR/PKCς/GLUT1 axis, offers an effective strategy to broaden glycemic control for the prevention of diabetic complications of the nervous system and, possibly, other insulin insensitive or resistant tissues.

In vivo liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism.Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells.Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery.Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

Estradiol and high fat diet associate with changes in gut microbiota in female ob/ob mice

Estrogens protect against diet-induced obesity in women and female rodents. For example, a lack of estrogens in postmenopausal women is associated with an increased risk of weight gain, cardiovascular diseases, low-grade inflammation, and cancer. Estrogens act with leptin to regulate energy homeostasis in females. Leptin-deficient mice (ob/ob) exhibit morbid obesity and insulin resistance. The gut microbiome is also critical in regulating metabolism. The present study investigates whether estrogens and leptin modulate gut microbiota in ovariectomized ob/ob (obese) or heterozygote (lean) mice fed high-fat diet (HFD) that received either 17β-Estradiol (E2) or vehicle implants. E2 attenuated weight gain in both genotypes. Moreover, both obesity (ob/ob mice) and E2 were associated with reduced gut microbial diversity. ob/ob mice exhibited lower species richness than control mice, while E2-treated mice had reduced evenness compared with vehicle mice. Regarding taxa, E2 was associated with an increased abundance of the S24-7 family, while leptin was associated with increases in Coriobacteriaceae, Clostridium and Lactobacillus. Some taxa were affected by both E2 and leptin, suggesting these hormones alter gut microbiota of HFD-fed female mice. Understanding the role of E2 and leptin in regulating gut microbiota will provide important insights into hormone-dependent metabolic disorders in women.

Attenuation of Inflammation and Leptin Resistance by Pyrogallol-Phloroglucinol-6,6-Bieckol on in the Brain of Obese Animal Models.

Obesity induces inflammation both in the adipose tissue and the brain. Activated macrophage infiltration, polarization of macrophages to a more inflammatory type (M1), and increased levels of pro-inflammatory cytokines are related to brain inflammation, which induces leptin resistance in the brain. Pyrogallol-phloroglucinol-6,6-bieckol (PPB), a compound from Ecklonia cava, has anti-inflammatory effects. In this study, we evaluated the effects of PPB effect M1 polarization and inflammation and its ability to restore the effects of leptin, such as a decrease in appetite and body weight. We administered PPB to diet-induced obesity (DIO) and leptin-deficient (ob/ob) mice, evaluated macrophage activation, polarization, and changes of inflammatory cytokine level in adipose tissue and brain, and determined the effect of PPB on leptin resistance or leptin sensitivity in the brain. The levels of activated macrophage marker, M1/M2, and pro-inflammatory cytokines were increased in the adipose tissue and brain of DIO and ob/ob mice than control. TLR4 expression, endoplasmic reticulum (ER) stress, and NF-κB expression in the brain of DIO and ob/ob mice were also increased; this increase was related to the upregulation of SOCS3 and decreased phosphorylated STAT3, which decreased leptin sensitivity in the brain. PPB decreased inflammation in the brain, restored leptin sensitivity, and decreased food intake and weight gain in both DIO and ob/ob mice.

High fat diet modulates the protein content of nutrient transporters in the small intestine of mice: possible involvement of PKA and PKC activity

AimsChronic high fat consumption has been shown to modulate nutrient transporter content in the intestine of obese mice; however it is unclear if this regulation occurs before or after the establishment of obesity, and the underlying molecular mechanism requires elucidation. Main methodsTowards this goal C57BL/6 mice were fed a low fat diet (LFD) or high fat diet (HFD), and specific protein and gene expression levels were assessed for up to 12 weeks. Similar experiments were also performed with leptin-deficient (Ob/Ob) mice. Key findingsThe results showed that the HFD group presented decreased GLUT2, PEPT1, FAT/CD36 and NPC1L1, and increased NHE3, MTTP and L-FABP content. Animals fed an HFD also presented enhanced lipid transporter gene expression of Slc27a4, Npc1l1, Cd36, Mttp and L-Fabp. Additionally, FAT/CD36 and NPC1L1 protein levels were reduced in both HFD-induced obese and Ob/Ob mice. Ob/Ob mice also exhibited increased Slc2a2 and Slc15a1 mRNAs expression, but the protein expression levels remained unchanged. The HFD also attenuated PKA and PKC activities. The inhibition of PKA was associated with decreased FAT/CD36 content, whereas increased L-FABP levels likely depend on CREB activation, independent of PKA. It is plausible that the HFD-induced changes in NPC1L1, MTTP and L-FABP protein content involve regulation at the level of transcription. Moreover, the changes in GLUT2 and PEPT1 content might be associated with low PKC activity. SignificanceThe results indicated that an HFD is capable of reducing nutrient transporter content, possibly attenuating nutrient uptake into the intestine, and may represent a feedback mechanism for regulating body weight. Furthermore, the elevated levels of NHE3, L-FABP and MTTP may account for the increased prevalence of hypertension and dyslipidemia in obese individuals. All of these changes are potentially linked to reduced PKA or PKC activities.

Gut microbiota regulates cardiac ischemic tolerance and aortic stiffness in obesity.

The gut microbiota has emerged as an important regulator of host physiology, with recent data suggesting a role in modulating cardiovascular health. The present study determined if gut microbial signatures could transfer cardiovascular risk phenotypes between lean and obese mice using cecal microbiota transplantation (CMT). Pooled cecal contents collected from obese leptin-deficient (Ob) mice or C57Bl/6j control (Con) mice were transplanted by oral gavage into cohorts of recipient Ob and Con mice maintained on identical low-fat diets for 8 wk (n = 9-11/group). Cardiovascular pathology was assessed as the degree of arterial stiffness (aortic pulse wave velocity) and myocardial infarct size following a 45/120 min ex vivo global cardiac ischemia-reperfusion protocol. Gut microbiota was characterized by 16S rDNA sequencing, along with measures of intestinal barrier function and cecal short-chain fatty acid (SCFA) composition. Following CMT, the gut microbiota of recipient mice was altered to resemble that of the donors. Ob CMT to Con mice increased arterial stiffness, left ventricular (LV) mass, and myocardial infarct size, which were associated with greater gut permeability and reduced cecal SCFA concentrations. Conversely, Con CMT to Ob mice increased cecal SCFA, reduced LV mass, and attenuated myocardial infarct size, with no effects on gut permeability or arterial stiffness. Collectively, these data demonstrate that obesity-related changes in the gut microbiota, independent of dietary manipulation, regulate hallmark measures of cardiovascular pathology in mice and highlight the potential of microbiota-targeted therapeutics for reducing cardiovascular pathology and risk in obesity.NEW & NOTEWORTHY These data are the first to demonstrate that cecal microbiota transplantation (CMT) can alter cardiovascular pathology in lean and obese mice independent from alterations in dietary intake. Myocardial infarct size was reduced in obese mice receiving lean CMT and worsened in lean mice receiving obese CMT. Lean mice receiving obese CMT also displayed increased aortic stiffness. These changes were accompanied by alterations in short-chain fatty acids and gut permeability.

Abstract 137: Impact of Leptin Deficiency Compared to Neuronal Specific Leptin Receptor Deletion on Cardiometabolic Regulation

The main goal of this study was to compare the impact of whole-body leptin deficiency with neuronal specific leptin receptor (LR) deletion on metabolic and cardiovascular regulation. Liver fat, diacylglycerol acyltransferase-2 (DGTA2) and CD36 protein content were measured in male and female wild-type (WT, n=5/sex), nervous system LR deficient (LR/Nestin-Cre, n=6/sex), and leptin deficient (ob/ob, n=5) mice. Blood pressure (BP) and heart rate (HR) were recorded by telemetry, and motor activity (MA) and oxygen consumption (VO 2 ) were monitored at 24 weeks of age. Female and male LR/Nestin-Cre and ob/ob mice were heavier than WT mice (62±5 and 61±3 vs 31±1 g), hyperphagic (6.2±0.5 and 6.1±0.7 vs 3.5±1.0 g/day), with reduced VO 2 (27±1 and 33±1 vs 49±3 ml/kg/min) and decreased MA (3±1 and 7±2 vs 676±105 cm/hr). LR/Nestin-Cre and ob/ob mice were also hyperinsulinemic and hyperglycemic compared to WT mice. LR/Nestin-Cre mice had high plasma leptin levels while ob/ob mice had undetectable leptin levels. Despite comparable obesity, LR/Nestin-Cre mice had lower liver fat content (83% reduced) and DGTA2 and CD36 protein levels than ob/ob mice. Male WT, LR/Nestin-Cre, and ob/ob mice exhibited similar BP (111±3, 110±1 and109±2 mmHg), whereas female LR/Nestin-Cre mice had higher BP compared to WT females despite similar metabolic phenotypes compared to male LR/Nestin-Cre mice. No significant increases in plasma aldosterone concentration was observed in male LR/Nestin-Cre compared to WT mice (495±48 vs. 440±62 pg/ml); however, female LR/Nestin-Cre mice had ~14 fold higher plasma aldosterone concentration compared to female WT mice (707±97 vs. 50±3 pg/ml). These results indicate that although nervous system LRs play a crucial role in regulating body weight and glucose homeostasis, peripheral LRs also regulate liver fat deposition and modulate BP in a sex dependent manner. (NHLBI-PO1HL51971, NIGMS P20GM104357 and U54GM115428)

Effects of Type 2 Diabetes Mellitus on Gene Expressions of Mouse Meibomian Glands

ABSTRACT Purpose Type 2 Diabetes mellitus (DM) is a major health problem and its ocular complications like orbital infections, cataract and diabetic retinopathy cause blindness. Meibomian gland (MG) dysfunction and dry eye disease are also important ocular complications of type 2 DM but not enough research has been conducted on these complications. Our hypothesis suggests type 2 DM can alter significant gene expressions of MG. In our study, MGs of leptin-deficient spontaneous diabetic and non-diabetic mice were extracted, and gene expression profiles were analyzed with microarray technology. Methods Mice were divided into two groups; nine Lep b/ob spontaneous diabetic mice as type 2 DM group and nine non-diabetic Balb/c mice as controls. Blood glucose levels, tearfilm break-up time and fluorescein scores were measured in both two groups for 12 weeks. MGs were dissected and RNAs were isolated for microarray gene expression analysis. We filtered probes with standard deviation of more than 0.1 and we used 40452 of 45281 probes for processing. We performed fold change analysis and identified which genes are affected, and we analyzed the impact of genes on proteins, pathways and gene ontologies by using various databases. Results We observed 172 up-regulated and 118 down-regulated genes in type 2 diabetic mice when compared to non-diabetic mice. Interestingly, expression of collagen type I, integrin beta-I binding protein-I, pyruvate dehydrogenase kinase, TNF receptor genes up-regulated with DM; on the other hand, IL-33, cholecystokinin, plasminogen activator, IL-1 and serine peptidase inhibitor genes down-regulated significantly. Also, we have seen a significant decrease in WNT signaling and pentose phosphate pathways-related genes. Conclusion Our data show these changes in gene expression caused by endocrine and immune mechanisms of type 2 DM which result disrupted homeostasis of epithelial cells of MG. Increased expressions of apoptosis and inflammation-related genes and their effects on related pathways have proven that MGs were negatively affected by type-2 DM.