Relationship between cardiovascular risk factors and adipocytokines gene expression in local fat depots

In this study we studied the relationships between adipokines (leptin, soluble leptin receptor) in adipose tissue (AT) and cardiovascular disease (CVD) risk factors. Methods Fat tissue biopsies were obtained from 134 patients median aged 64.0 (48.0; 68.0) with stable CAD undergoing coronary artery bypass grafting. Traditional cardiovascular risk factors and patient treatment have been recorded. AH was defined as systolic blood pressure >140 mm Hg Art., diastolic blood pressure >90 mm Hg. Dyslipidemia was defined as a previously detected increase in total serum cholesterol (>200 mg/dl), triglycerides (>200 mg/dl), or low-density lipoprotein (LDL) cholesterol (>150 mg/dl) for at least 1 year, or use of lipid-lowering drugs. Smoking was classified as current or former smokers; current smoking status was defined as at least one cigarette daily over the last year. Adipocytes were isolated from subcutaneous (SAT), epicardial (EAT), and perivascular AT (PVAT) samples. Isolated adipocytes were cultured for 24-h after which, Then adipocytes were immediately processed to RNA extraction and determination of adipokines gene expression. Results In this study, we showed that men with CAD LEP and LEPR expression were lower in PVAT, whereas only the latter was decreased in SAT. Thus, LEPR expression in local fat depots appeared to differ in men and women, which is consistent with previous reports. Men with CAD showed lower LEP expression than women in EAT, whereas no significant differences were observed in SAT. Patients 51–59 years old were characterized by the lowest LEP expression in SAT and the highest LEP and LEPR expression in EAT and PVAT. Those >60 years presented the highest levels of LEPR in SAT. The presence of dyslipidemia in patients with CAD correlated with decreased LEPR expression in EAT, which is an unfavorable sign and may potentially lead to the development of leptin resistance. In patients with AH, LEP expression in EAT and LEPR expression in SAT and PVAT is increased. AH >20 years was associated with increased LEP in ATs. Smokers with CAD exhibited higher levels of LEP expression in SAT and EAT, and LEPR expression in SAT. Conclusions The results of the study indicate a close association of traditional CVD risk factors with adipocytokine imbalance in patients with CAD. The results indicate the potential of the fat depot and leptin and its receptor data as a drug application point. The study of the molecular basis of PVAT and EAT function can provide a more complete understanding of the etiopathogenetic mechanisms of CVD and develop an effective strategy for their prevention and control. FUNDunding Acknowledgement Type of funding sources: None.

Leptin Receptor Expression in GABAergic Cells is Not Sufficient to Normalize Metabolism and Reproduction in Mice

Previous studies indicate that leptin receptor (LepR) expression in GABAergic neurons is necessary for the biological effects of leptin. However, it is not clear whether LepR expression only in GABAergic neurons is sufficient to prevent the metabolic and neuroendocrine imbalances caused by LepR deficiency. In the present study, we produced mice that express the LepR exclusively in GABAergic cells (LepRVGAT mice) and compared them with wild-type (LepR+/+) and LepR-deficient (LepRNull/Null) mice. Although LepRVGAT mice showed a pronounced reduction in body weight and fat mass, as compared with LepRNull/Null mice, male and female LepRVGAT mice exhibited an obese phenotype relative to LepR+/+ mice. Food intake was normalized in LepRVGAT mice; however, LepRVGAT mice still exhibited lower energy expenditure in both sexes and reduced ambulatory activity in the females, compared with LepR+/+ mice. The acute anorexigenic effect of leptin and hedonic feeding were normalized in LepRVGAT mice despite the hyperleptinemia they present. Although LepRVGAT mice showed improved glucose homeostasis compared with LepRNull/Null mice, both male and female LepRVGAT mice exhibited insulin resistance. In contrast, LepR expression only in GABAergic cells was sufficient to normalize the density of agouti-related peptide (AgRP) and α-MSH immunoreactive fibers in the paraventricular nucleus of the hypothalamus. However, LepRVGAT mice exhibited reproductive dysfunctions, including subfertility in males and alterations in the estrous cycle of females. Taken together, our findings indicate that LepR expression in GABAergic cells, although critical to the physiology of leptin, is insufficient to normalize several metabolic aspects and the reproductive function in mice.

Leptin Receptor Expressing Neurons in the Substantia Nigra Regulate Locomotion, and in The Ventral Tegmental Area Motivation and Feeding

Leptin is an anorexigenic hormone, important in the regulation of body weight. Leptin plays a role in food reward, feeding, locomotion and anxiety. Leptin receptors (LepR) are expressed in many brain areas, including the midbrain. In most studies that target the midbrain, either all LepR neurons of the midbrain or those of the ventral tegmental area (VTA) were targeted, but the role of substantia nigra (SN) LepR neurons has not been investigated. These studies have reported contradicting results regarding motivational behavior for food reward, feeding and locomotion. Since not all midbrain LepR mediated behaviors can be explained by LepR neurons in the VTA alone, we hypothesized that SN LepR neurons may provide further insight. We first characterized SN LepR and VTA LepR expression, which revealed LepR expression mainly on DA neurons. To further understand the role of midbrain LepR neurons in body weight regulation, we chemogenetically activated VTA LepR or SN LepR neurons in LepR-cre mice and tested for motivational behavior, feeding and locomotion. Activation of VTA LepR neurons in food restricted mice decreased motivation for food reward (p=0.032) and food intake (p=0.020), but not locomotion. In contrast, activation of SN LepR neurons in food restricted mice decreased locomotion (p=0.025), but not motivation for food reward or food intake. Our results provide evidence that VTA LepR and SN LepR neurons serve different functions, i.e. activation of VTA LepR neurons modulated motivation for food reward and feeding, while SN LepR neurons modulated locomotor activity.

Cross-Species Analysis Defines the Conservation of Anatomically-Segregated VMH Neuron Populations

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing Translating Ribosome Affinity Purification with RNA sequencing (TRAP-seq) data with snRNA-seq data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of 6 major genetically- and anatomically-differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously-suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

Altered Brain Leptin and Leptin Receptor Expression in the 5XFAD Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles. Interestingly, individuals with metabolic syndromes share some pathologies with those diagnosed with AD including neuroinflammation, insulin resistance and cognitive deficits. Leptin, an adipocyte-derived hormone, regulates metabolism, energy expenditure and satiety via its receptor, LepR. To investigate the possible involvement of leptin in AD, we examined the distribution of leptin and LepR in the brains of the 5XFAD mouse model of AD, utilizing immunofluorescent staining in young (10–12-weeks; n = 6) and old (48–52-weeks; n = 6) transgenic (Tg) mice, together with age-matched wild-type (WT) controls for both age groups (young-WT, n = 6; old-WT, n = 6). We also used double immunofluorescent staining to examine the distribution of leptin and leptin receptor expression in astrocytes. In young 5XFAD, young-WT and old-WT mice, we observed neuronal and endothelial expression of leptin and LepR throughout the brain. However, neuronal leptin and LepR expression in the old 5XFAD brain was significantly diminished. Reduced neuronal leptin and LepR expression was accompanied by plaque loading and neuroinflammation in the AD brain. A marked increase in astrocytic leptin and LepR was also observed in old 5XFAD mice compared to younger 5XFAD mice. We postulate that astrocytes may utilize LepR signalling to mediate and drive their metabolically active state when degrading amyloid in the AD brain. Overall, these findings provide evidence of impaired leptin and LepR signalling in the AD brain, supporting clinical and epidemiological studies performed in AD patients.

Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.

Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression

Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.

Insulin regulates hepatic leptin receptor expression in early lactating dairy cows

Energy balance controls the expression of the leptin receptor (Lepr) in the ruminant hypothalamus but whether similar regulation occurs in peripheral tissues is unknown. To address this issue, we measured Lepr expression in the liver and adipose tissue of dairy cows during the transition from late pregnancy (LP) to early lactation (EL). This period is characterized by the development of a profound state of energy insufficiency and is associated with reduced plasma insulin and leptin and with increased plasma growth hormone. Hepatic expression of the short (Lepr-a) and long (Lepr-b) isoforms was 40% higher during EL (8 days postpartum) than LP (30 days prepartum). A similar effect was observed when negative energy balance was induced in nonpregnant, late-lactation dairy cows by food restriction, implicating energy insufficiency as a specific cause in EL. The stimulation of hepatic Lepr expression was reversed after a 48-h period of hyperinsulinemic euglycemia in EL. Changes in hepatic Lepr expression during chronic elevation of plasma leptin in EL or plasma growth hormone in nonpregnant, late-lactation cows did not support a role for these hormones in mediating the effects of energy insufficiency on hepatic Lepr expression. In adipose tissue, Lepr expression was increased 10-fold during the transition from LP to EL. Overall, these data indicate that hypoinsulinemia is partly responsible for the induction of Lepr expression in the liver, and perhaps adipose tissue, of energy-deficient dairy cows.