Chen H. Romsos D. Egawa , M. Erickson J. Clegg K. Palmiter R. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Feldkircher K. Mistry A. Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M. Skoda R. Defective STAT signaling by the leptin receptor in diabetic mice. Glaum S. Hara M. Bindokas V. Lee C. Polonsky K. Bell G. Miller R. Leptin, the obese gene product, rapidly modulates synaptic transmission in the hypothalamus. Halaas , J. Holt S. York D. The effects of adrenalectomy, corticotropin releasing factor and vasopressin on the sympathetic firing rate of nerves to interscapular brown adipose tissue in Zucker rat.
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Once such complication is impaired bone formation and mineralization, which is associated with decelerated bone growth, increased risk of bone fracture, and delayed fracture repair [ 3 ]. Impairment of bone formation is caused, at least in part, by leptin dysregulation [ 3 , 4 , 5 ].
Leptin is an adipokine secreted primarily by adipocytes to regulate energy homeostasis, but it also plays a role in regulating bone metabolism [ 6 , 7 ]. Longitudinal growth of long bones occurs via endochondral ossification. During this process, growth plate cartilage expands and is replaced with bone tissue. Proliferation, differentiation, and metabolic activity of chondrocytes in the growth plate are inhibited in the obese, T2DM condition [ 12 ].
Leptin-deficient mice have growth plates that are reduced in height, likely due to the downregulation of genes regulating ossification [ 10 , 13 ], although the specific effects of leptin-deficiency on three-dimensional growth plate structure remain unclear. At 9—10 weeks of age, mice were sacrificed using compressed CO 2 followed immediately by bilateral pneumothorax and tibias harvested for analysis. Length of the tibia was measured using digital calipers.
Tibias were then bisected longitudinally in the sagittal plane and the medial half was prepared for imaging using confocal laser scanning microscopy [ 14 ]. Tibias were incubated in DAPI dilution and refractive index matching solution RIMS media for 48 h to reduce tissue opacity and facilitate optical imaging at greater tissue depths.
Z stacks of the proximal tibia growth plate were digitally captured at 0. Composite Z stacks in the green—blue nm laser line and yellow—green nm laser line emission spectra were formed to capture DAPI-stained nuclei and the autofluorescence of the cartilage and surrounding tissues Fig.
Cell columns were identified from a proximal superior view and followed distally through the growth plate. Measuring processes were automated using the object manager in Vaa3D and were manually checked for accuracy. The mean height of the growth plate was also measured for each tibia. Data collection was blinded and conducted by a single observer.
Brightfield microscopy images of growth plate cartilage in a lean and b obese mice. Toluidine blue stain. Descriptive statistics for body mass and tibial dimensions are shown in Table 1.
The difference in mean body mass between groups was significantly increased at the end of the 4-week study 5. Columns of proliferating and hypertrophying chondrocytes were linearly arranged in the growth plates of lean mice Figs.
In obese mice, cell columns were shorter and less organized, particularly the hypertrophying cells in the distal portion of the growth plate Figs. Mean chondrocyte surface area was Videos displaying the growth plate Z stacks of lean and obese mice being rotated along the x, y, and z axes are shown in Additional files 1 and 2.
Confocal scans of growth plate cartilage in a lean and b obese mice. DAPI nuclear stain blue. One of these consequences is reduced long bone mass. Tibial longitudinal growth requires cartilage proliferation at the epiphyseal growth plate. Our analysis of growth plate cartilage three-dimensional structure showed obese mice have reduced growth plate height and fewer chondrocyte cell columns with cells that are reduced in volume. Cellular 3D arrangement is also more disorganized in growth plates of obese mice.
These differences may be attributed to the inhibitory effects of leptin deficiency on chondrocyte metabolism Fig. Tibial growth plate chondrocytes of leptin-deficient mice exhibit disturbed 2D columnar structure with inhibited proliferation and extracellular matrix synthesis that are reversed when the cells are treated with leptin [ 21 ].
Similarly, patients with obese T2DM exhibit impaired chondrocyte proliferation and hypertrophy, as well as reduced collagen and proteoglycan expression [ 22 , 23 , 24 , 25 ]. Stiffness of the collagen network via crosslinking with advanced glycation end products also affects cartilage structure [ 26 ]. Altered cartilage extracellular composition in the diabetic state has been reported to affect mechanical properties of the tissue, thereby increasing the risk of tissue damage and impaired function [ 27 , 28 , 29 ].
This may also explain why obese patients with T2DM are at greater risk for osteoarthritis [ 30 ]. Susceptibility of chondrocytes to injury may also be a contributing factor to growth plate structural differences that inhibit growth. Schematic of the effects of leptin and leptin deficiency on growth plate cartilage of the tibia. Adipocyte-derived leptin regulates chondrocyte proliferation and arrangement in the growth plate.
First, we targeted Leprb while Huan et al. The short isoforms predominate in WAT, but Leprb is considered to be responsible for the majority of the effects of leptin Third, while we used Cre-lox methodology, Huan et al.
This knockdown approach has been suggested to induce obesity, regardless of the target RNA Finally, we can not discount the possibility that developmental effects in either model contribute to the observed differences. Circulating hyperleptinemia was associated with the obese phenotype obtained by downregulation of leptin receptors in WAT using antisense RNA Hyperleptinemia was also observed upon reduction of Leprb in multiple peripheral tissues using a tamoxifen-inducible model, despite the mice being of normal body weight The differences in circulating levels of leptin in our mice with adipose tissue-specific knockdown of Lepr compared to control mice were not statistically significant, except in females at 6 weeks of age, when it was lower in Lepr knockdown mice.
Moreover, using an ex vivo assay, leptin secretion by WAT samples, which was in the range of what has been reported 11 , 37 , was similar between Lepr knockdown and Flox control mice, in males and females.
These findings do not support a negative feedback loop of leptin secretion via Leprb at the level of adipose tissues. During the OGTT, mice with adipose tissue Lepr knockdown had lower plasma insulin concentrations than controls. Complementary results were obtained in mice with adipose tissue Lepr reconstitution, which had higher plasma insulin concentrations during the OGTT. The cause of the altered plasma insulin responses during the OGTT is unclear, but one possibility is that Lepr knockdown or reconstitution in adipose tissues alters the expression of adipokines that modulate glucose-stimulated insulin secretion.
For instance, leptin can increase the secretion of interleukin-6 IL-6 39 , which in turn stimulates insulin secretion Crosstalk between adipose tissue and the pancreas may also explain the decrease in plasma insulin concentrations in male and female Lepr knockdown mice at 16 weeks old. Despite the reduced or augmented insulin response during the OGTT for Lepr knockdown or Lepr reconstitution mice, respectively, blood glucose concentrations were similar to those of controls throughout the OGTT and the underlying mechanism is unclear.
Glucose tolerance is determined by insulin sensitivity and concentrations as well as insulin-independent stimulation of glucose disposal and inhibition of hepatic glucose production 41 , We did not detect significant differences in insulin sensitivity between Lepr knockdown and Flox controls by ITTs at different ages and insulin-stimulated hepatic Akt activation.
Moreover, we found similar hepatic glycogen content in Lepr knockdown and Flox control mice during fasting, which suggests that the ability of glucose to promote its own disposal and storage is not improved in the liver of mice with Lepr knockdown. Lastly, during an IPGTT, while Lepr knockdown mice had similar blood glucose concentrations to controls, mice with Lepr reconstitution were mildly glucose intolerant. Our results indicate that adipose tissue leptin signalling does not greatly affect the blood glucose excursion following a glucose challenge.
Our results suggest that knockdown or reconstitution of Lepr in adipose tissues does not greatly affect lipid turnover. We did not find differences in adipose tissue lipolysis in Lepr knockdown mice vs. The concentration of lipids in plasma was also comparable between Lepr knockdown and Flox control mice. However, lipid content in the liver, an organ that often accumulates lipids when adipose tissue lipolysis is elevated, was not higher in mice with Lepr reconstitution at the same age.
Lepr reconstitution in males was without effect on the expression of mediators of lipid and glucose metabolism in pgWAT or on Ucp1 expression in BAT. Although most studies indicate that the CNS is the key tissue upon which peripherally administered leptin initially acts to trigger its glucose-lowering effects in insulin-deficient diabetes, it remains unclear if the CNS pathways involved are the same as for centrally administered leptin 44 , 45 , 46 , 47 , Perry et al.
Herein, prolonged 8 days leptin therapy resulted in a faster decrease in blood glucose in diabetic mice with adipose tissue-specific Lepr knockdown vs. Since endogenous plasma leptin levels were similar between genotypes in mice with normal levels of insulin ie. The binding of circulating leptin to its leptin receptor diminishes the clearance of leptin 52 and hence, the elevated concentration of plasma leptin receptor we observed in diabetic Lepr knockdown mice may contribute to the increased circulating leptin concentration in Lepr knockdown mice following 8 days of leptin therapy.
However, the differential effect of exogenous leptin on plasma leptin concentrations in diabetic Lepr knockdown and Flox controls depends on the duration of leptin therapy. Following an acute injection of leptin, STZ-injected mice of both genotypes had similar plasma leptin profiles and leptin half-lives. After the single leptin injection, blood glucose profiles were similar between the two groups and, as expected based on our previous studies, blood glucose was not normalized Our results indicate that obesity can be modestly prevented or augmented if Lepr expression is reduced or reconstituted, respectively, in adipose tissues of males.
In our study, we also did not find an effect on body weight in females since the maximal body weight of female mice with adipose tissue-specific reconstitution of Lepr did not differ from that of female loxTB control mice. The improved blood glucose concentrations in mice with adipose tissue Lepr reconstitution at younger ages was not due to elevations in plasma insulin and at older ages, mice with Lepr reconstitution were mildly glucose intolerant.
In contrast, we found that disruption of Lepr signalling in fat reduced body weight gain, and reconstitution of leptin signalling in fat in male mice lacking leptin signaling elsewhere increased weight gain.
Although it is difficult to reconcile these results, the aP2-Cre model can promote recombination in both adipose and non-adipose tissues In conclusion, our results indicate that peripheral leptin signalling in adipose tissues affects body weight regulation and glucose metabolism. In response to an oral glucose challenge, while mice with adipose tissue Lepr knockdown had a blunted plasma insulin profile, mice with adipose tissue Lepr reconstitution had higher plasma insulin concentrations compared to controls.
In insulin-deficient diabetes, prolonged exogenous leptin therapy induced a faster reduction in blood glucose, possibly as a result of diminished leptin clearance. Additional research is warranted to further investigate the underlying mechanisms by which adipose tissue leptin signalling affects body weight and glucose homeostasis. Scott, M. Leptin receptor expression in hindbrain Glp-1 neurons regulates food intake and energy balance in mice.
Journal of Clinical Investigation , — Dodd, G. The thermogenic effect of leptin is dependent on a distinct population of prolactin-releasing peptide neurons in the dorsomedial hypothalamus. Cell Metab 20 , — Minokoshi, Y. Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats. Diabetes 48 , — Bonzon-Kulichenko, E. Central leptin regulates total ceramide content and sterol regulatory element binding protein-1C proteolytic maturation in rat white adipose tissue.
Endocrinology , — Balthasar, N. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis.
Neuron 42 , — Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion. Article Google Scholar. Shi, H. Sexually different actions of leptin in proopiomelanocortin neurons to regulate glucose homeostasis. Berglund, E. Direct leptin action on POMC neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice.
Bates, S. The role of leptin receptor signaling in feeding and neuroendocrine function. Trends Endocrinol Metab 14 , — Moon, H. Endocr Rev 34 , — Guo, K. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities.
Huynh, F. Disruption of hepatic leptin signaling protects mice from age- and diet-related glucose intolerance. Diabetes 59 , — Huan, J. Adipocyte-selective reduction of the leptin receptors induced by antisense RNA leads to increased adiposity, dyslipidemia, and insulin resistance.
J Biol Chem , — Wang, M. Lee, K. Lessons on conditional gene targeting in mouse adipose tissue. Diabetes 62 , — Eguchi, J.
Transcriptional control of adipose lipid handling by IRF4. Cell Metab 13 , — McMinn, J. Redmann, Jr. Smagin, Steven R. Smith, Erin Rodgers, Jeffrey J. These doses also increased brown adipose tissue uncoupling protein expression.
In , Zhang et al. This protein, leptin, has the structure of a long chain helical cytokine 4 and is expressed in adipose tissue in proportion to adipocyte size 5 , 6.
The suppression of food intake is mediated by a hypothalamic splice variant of the leptin receptor, OB-Rb, which has a long intracellular domain Obese humans have high circulating concentrations of leptin, but are not responsive to its effects on food intake As there is a concentration gradient between serum and cerebrospinal fluid leptin 14 , 15 , it has been hypothesized that a rate-limited transport system prevents peripheral leptin from activating the central receptor that suppresses food intake.
The limitation on transport may be due either to binding proteins in the circulation 16 or to a specific transport protein It has been proposed that one of the leptin receptor subtypes, with a short intracellular domain, may function as a transport protein and regulate leptin uptake into the brain In lean mice, leptin has minimal effects on food intake, but causes the loss of body fat, presumably due to a leptin-induced increase in energy expenditure Leptin has been shown to increase norepinephrine NE turnover in brown, but not white, adipose tissue, suggesting that the metabolic effect of leptin is attributable to activation of the sympathetic nervous system Leptin has a circulating half-life of approximately 30 min, is released in a pulsatile manner from adipose tissue, and demonstrates a circadian rhythm in circulating levels with a nocturnal elevation in concentration 13 , We have found that this method of protein administration causes excessive, intermittent elevations of the serum leptin concentration This method of administration provided constant delivery of protein 0—1.
The objectives of the study were 2-fold. This would allow separation of physiological from potentially pharmacological responses. Louis, MO and water. Body weights and food intakes were recorded daily at h, and rectal temperatures of mice were measured four times during the study at h, twice before and twice after pump placement, using a thermistor probe Thermistor thermometer model —20, Cole Palmer Instrument Co.
Leptin was a gift from ZymoGenetics Corp. Seattle, WA. After pump placement, measurements of daily food intake and body weight were continued, and rectal temperatures were measured after 2 and 4 days of infusion. All mice were decapitated in the morning of day 7 of leptin infusion. Sigma Chemical Co. The carcass, liver, pancreas, ovaries, uterus, spleen, adrenals, heart, kidney, and inguinal, perirenal, retroperitoneal, mesenteric, gonadal, and intrascapular brown fat were dissected and weighed.
The liver, hypothalamus, brown fat, and gonadal fat were snap-frozen. Daniel Riquier. The hypothalamus, brain stem, and cortex were dissected and snap-frozen in liquid nitrogen for analysis of monoamines by HPLC, as described previously Liver tissue was frozen for subsequent analysis of glycogen by the method of Lo et al.
Citrate synthase was used as a general marker for oxidative capacity and was assayed as described by Srere Hexokinase activity was used a marker for glucose utilization potential and was assayed according to the method of Uyeda and Racker For each genotype, the response variables food intake and body weight were separately modeled as a repeated measures ANOVA over the course of the experiment.
To provide overall tests of the dosage effect of leptin on each variable, a profile analysis was effected by testing the appropriate contrasts corresponding to the parallel, coincident, and level profiles hypotheses for days 4—7 of infusion after the mice had recovered from the surgery.
The dramatic reduction in food intake on the first day of leptin infusion was partially due to surgery. Lower doses of leptin had no significant effect on food intake. Daily body weights of the mice are shown in Fig. Daily food intakes of lean and obese mice. Daily body weights of lean and obese mice. Rectal temperatures of the mice are shown in Fig. Repeated measurements of temperature caused a progressive increase in temperature of all animals. Before leptin infusion, the temperatures of all obese mice were significantly lower than those of lean animals.
There was no effect of leptin on the temperatures of lean mice. Rectal temperatures of lean and obese mice measured twice before leptin infusion and twice during leptin infusion. Superscripts indicate a significant difference between treatment groups within the obese genotype on the fourth day of infusion. There were no significant differences within lean or obese genotypes on any other day.
Expression of UCP mRNA intrascapular brown fat from lean and obese mice infused for 7 days with increasing amounts of human recombinant leptin. Superscripts indicate significant differences in expression between groups of obese mice. There was no effect of leptin on UCP expression in lean mice. The weights of different adipose depots are shown in Table 1. Gonadal fat leptin mRNA expression is shown in Fig.
Expression of leptin mRNA measured in gonadal fat from lean and obese mice after 7 days of infusion with increasing amounts of leptin. Leptin expression was measured by Northern blot analysis and expressed as a ratio to 28S ribosomal RNA. Superscripts indicate significant differences in levels of expression in fat from obese mice. There was no effect of leptin on expression in tissue from lean mice. Rp, Retroperitoneal.
Total is the sum of the weights of the five dissected fat pads. Organ weights are shown in Table 2. There was no statistically significant effect of any dose of leptin on the weight of any of the organs measured in either obese or lean mice.
Liver composition is shown in Table 3. None of the doses of leptin had any effect on liver weight, liver lipid, or liver glycogen content in lean mice. A Western blot of the short form leptin receptors is shown in Fig. Liver short form leptin receptor detected by Western blot using a polyclonal antibody raised to the extracellular membrane portion of the receptor. Both the pellet P and the supernatant s were analyzed for receptor.
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