Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug;10(2):148-59.
doi: 10.1016/j.cmet.2009.07.001.

PET imaging of leptin biodistribution and metabolism in rodents and primates

Giovanni Ceccarini  1 , Robert R Flavell, Eduardo R Butelman, Michael Synan, Thomas E Willnow, Maya Bar-Dagan, Stanley J Goldsmith, Mary J Kreek, Paresh Kothari, Shankar Vallabhajosula, Tom W Muir, Jeffrey M Friedman
Affiliations

PET imaging of leptin biodistribution and metabolism in rodents and primates

Giovanni Ceccarini et al. Cell Metab. 2009 Aug.

Abstract

We have determined the systemic biodistribution of the hormone leptin by PET imaging. PET imaging using (18)F- and (68)Ga-labeled leptin revealed that, in mouse, the hormone was rapidly taken up by megalin (gp330/LRP2), a multiligand endocytic receptor localized in renal tubules. In addition, in rhesus monkeys, 15% of labeled leptin localized to red bone marrow, which was consistent with hormone uptake in rodent tissues. These data confirm a megalin-dependent mechanism for renal uptake in vivo. The significant binding to immune cells and blood cell precursors in bone marrow is also consistent with prior evidence showing that leptin modulates immune function. These experiments set the stage for similar studies in humans to assess the extent to which alterations of leptin's biodistribution might contribute to obesity; they also provide a general chemical strategy for (18)F labeling of proteins for PET imaging of other polypeptide hormones.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Design, synthesis, and biological activity of the tracer 68Ga-DOTA-leptin
A) Two surface rendered-views of the structure of leptin (PDB ID 1AX8). Residues which are essential for Lep-R activation are rendered in yellow, while the amines (the sites of labeling in 68Ga-DOTA-leptin) are rendered in red and the C-terminus (the site of labeling in 18F-FBA-leptin) is rendered in blue. B) Schematic of 68Ga-DOTA-leptin tracer. C) ESI-MS of DOTA-leptin revealing an average of 4 copies of DOTA per leptin molecule. D) in vitro bioactivity of DOTA-leptin was compared to recombinant leptin using a cell line stably expressing leptin receptor (Lep-Rb) and a luciferase reporter under STAT3 responsive element. The EC50 for activation was 0.42 to 0.55 nM for recombinant leptin and 0.54 to 0.66 nM for DOTA-leptin (95% confidence intervals). E) DOTA-leptin induces weight loss to a similar extent as recombinant leptin in ob/ob mice (n=4, dose = 450 ng/hr). F) Radiochemical RP-HPLC analysis of purified 68Ga-DOTA-leptin (blue) and serum recovered from a mouse 30 min post injection with 68Ga-DOTA-leptin. G) Cell binding assay of 68Ga-DOTA-leptin using a 293 cell line expressing Lep-Rb, in the presence or absence of 1 μM competing recombinant leptin (n=3, mean ± SEM).
Figure 2
Figure 2. Whole body PET images of mice and rats reveal that leptin is taken up in the cortex of the kidney
The images are overexposed to reveal areas with a lower level of uptake. A) Whole body PET maximum intensity projection (MIP) image of a leptin deficient (ob/ob) mouse injected with 68Ga-DOTA-leptin. Renal uptake of leptin in ob/ob mice is 65.1% ± 3.5% while the bladder is only 2.2 ± 0.2% over 30 min. Uptake in other organs is lower than this per volume. B) Whole body PET coronal MIP image of an ob/ob mouse injected with 18F-FBA-leptin, with inset of kidneys with lowered contrast, revealing uptake of the hormone in the renal cortex. C) Whole body PET coronal MIP of a Sprague-Dawley rat injected with 68Ga-DOTA-leptin, with inset with lowered contrast, revealing uptake of the hormone in the cortex of the kidney. D) Simplified diagram of renal anatomy.
Figure 3
Figure 3. Blocking experiments of leptin uptake in the kidney inwild type mice, and analysis of uptake in Lep-R deficient animals
A) Coronal maximum intensity projection (MIP) of a C57Bl6 wild type mouse injected with 68Ga-DOTA-leptin, with inset of radiochemical RP-HPLC analysis of homogenized kidneys one hour post injection with 68Ga-DOTA-leptin. B) Coronal MIP of a C57Bl6 wild type mouse co-injected with 68Ga-DOTA-leptin and 650 μg leptin, with inset of a radiochemical RP-HPLC analysis of the urine 30 minutes post injection. C) Time activity curves of the kidney of C57Bl6 wild type mice injected with 68Ga-DOTA-leptin in the presence or absence of 650 μg leptin. All data are reported as means (n=3) ± s.e.m. Statistical significance of the difference between the two groups was assessed by Student's t-test, p< 0.01 for all time points. D) A comparison of the uptake of 68Ga-DOTA-leptin in the kidneys of wild type C57Bl6, ob/ob, and ob/ob-Lep-RΔ mice.
Figure 4
Figure 4. Role of megalin in the in vivo uptake of leptin in the kidney and in L2 cells
A) Enzyme-Linked ImmunoSorbent Assay (ELISA) of leptin in the urine of the indicated mouse strains. The dotted line represents the limit of detection of the assay (n=3 ± SEM, statistical significance of difference between Megalinlox/loxApoECre and Megalinlox/lox: p<0.001). B) Anti-leptin western blot of the urine of megalinlox/lox ApoEcre and megalinlox/lox controls. C-D) Coronal MIP of a megalinlox/lox control mouse (C) injected with 68Ga-DOTA-leptin and of a megalinlox/lox ApoEcre mouse (D) injected with 68Ga-DOTA-leptin. E) Radiochemical RP-HPLC analysis of urine recovered from a megalinlox/lox ApoEcre mouse, which was injected with 68Ga-DOTA-leptin. f, g) Time activity curves of the uptake in the kidney (F) and bladder (G) of 68Ga-DOTA-leptin in megalinlox/lox ApoEcre and megalinlox/lox mice. All data are reported as mean ± SEM (n=3). Statistical significance of megalin kidney KO versus lox controls: * p < 0.05, ** p < 0.01. H) Leptin is degraded by an L2 yolk sac tumor cell line, and the degradation can be inhibited by competing cold leptin or receptor associated protein (RAP, 100 μg/mL) I-J) Degradation of leptin by L2 cells can be inhibited by some megalin ligands but not others.
Figure 5
Figure 5. Biodistribution studies of 125I-leptin in mice
From 8-12 KBq of 125I-leptin was injected into the tail vein of the indicated mouse strains. The animals were sacrificed 15 minutes post injection, the indicated tissues removed, weighed and the radioactivity counted. Standard uptake value on the Y-axis is defined as the % injected dose/gram of tissue, divided by % injected dose per mg of blood. This analysis is used to correct for the varying levels of leptin in the blood in the different animal groups (Supplemental Table 2). Results are represented as means ± SEM (n = 3-4 per experimental group). Statistical significance between groups was assessed by Student's t-test (*: p < 0.05, **: p < 0.01). A) Uptake in organs compared between wild type animals and wild type animals with 650 μg leptin co-injection. B-D) Uptake in organs compared between ob/ob mice, ob/ob mice with 650 μg leptin co-injection, and ob/ob-Lep-RΔ mice.
Figure 6
Figure 6. PET imaging study of leptin biodistribution in rhesus macaques
Images were acquired 10 minutes post injection of 15 MBq 68Ga-DOTA-leptin or 18F-FBA-leptin. A-B) Coronal MIP acquired 10 minutes post injection of 68Ga-DOTA-leptin (A) or 18F-FBA-leptin (B). C) Coronal MIP of a rhesus macaque acquired 10 minutes post injection of 15 MBq 68Ga-DOTA-leptin, with pre-treatment with 1 mg of rhesus leptin 5 minutes before radiotracer injection. D) Coronal PET-CT fusion revealing uptake of the tracer in the cortex of the kidney. E) Sagittal PET-CT fusion revealing uptake of the tracer in the vertebral bodies, sternum, liver, and presence of the tracer in the blood pool. F-H) Axial PET-CT sections through the sphenoid bone of the base of the skull (F), the L4 vertebral body (G), and the sacrum (H). I-J) Time activity curves of 68Ga-DOTA-leptin uptake in the bone marrow (I) and heart (J). The y axis on the TACs represents the % of total injected dose localized to that tissue.
See this image and copyright information in PMC

References

    1. Aoki S, Dillon WP, Barkovich AJ, Norman D. Marrow conversion before pneumatization of the sphenoid sinus: assessment with MR imaging. Radiology. 1989;172:373–375. - PubMed
    1. Bailey DL, Townsend DW, Valk PE, Maisey MN, editors. Positron Emission Tomography. London: Springer-Verlag; 2005.
    1. Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM. Leptin enters the brain by a saturable system independent of insulin. Peptides. 1996;17:305–311. - PubMed
    1. Bennett BD, Solar GP, Yuan JQ, Mathias J, Thomas GR, Matthews W. A role for leptin and its cognate receptor in hematopoiesis. Curr Biol. 1996;6:1170–1180. - PubMed
    1. Blebea JS, Houseni M, Torigian DA, Fan C, Mavi A, Zhuge Y, Iwanaga T, Mishra S, Udupa J, Zhuang J, et al. Structural and functional imaging of normal bone marrow and evaluation of its age-related changes. Semin Nucl Med. 2007;37:185–194. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources