dkNET New Investigator Pilot Program in Bioinformatics Awardee Seminar Series
Presenter: Alan Rupp, Ph.D. Research Investigator in Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School.
Abstract
Rates of obesity and diabetes continue to rise, impacting the health and wellbeing of millions. Treatment options are limited, in part because of our incomplete understanding of the biology of hunger and energy expenditure. The hormone leptin is produced by adipose tissue and signals the repletion of adipose energy stores to leptin receptor (Lepr)-expressing neurons in the hypothalamus. Leptin- and Lepr-deficient humans and rodents display marked hyperphagia, reduced energy expenditure, and extreme obesity. The crucial cellular targets (i.e., Lepr neurons) and transcriptional mechanisms that mediate these responses remain largely unknown, however. To reveal the cellular architecture of Lepr cells, we performed single nucleus RNA-seq of the hypothalamus in lean and obese rodents and macaques and in enriched mouse Lepr neurons. We identified over a dozen distinct Lepr neuron populations distributed across multiple hypothalamic nuclei, including a novel conserved population of Lepr neurons that is marked by Glp1r expression and which displays strong transcriptional responses to diet-induced obesity. Deleting Lepr from these Lepr/Glp1r cells resulted in excessive food intake and weight gain, revealing the importance of these for the control of energy balance by leptin. In contrast, we found that ventromedial hypothalamic (VMH) Lepr neurons represent a distinct class of VMH neurons that promote energy expenditure. Finally, we showed that leptin signaling during obesity remains intact in a subset of hypothalamic Lepr populations, while other Lepr neurons that play key roles in energy balance exhibited blunted responses. Overall, these studies reveal the neuronal structure of leptin action and highlight cell populations and molecular pathways that represent potential targets for obesity therapy.
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5. Leptin receptor (Lepr) expression
Leptin responsiveness is broadly distributed
LHA.Nts
(Leinninger et al. 2011)
DMH.Prlh
(Dodd et al. 2014)
ARH.Ghrh
(Rupp et al. 2019)
VMH.Nr5a1
(Dhillon et al. 2006)
6. Leptin action is driven primarily through STAT3 signaling
Pan et al. Mol Metab 2019
Pan and Myers, Nat Rev Neurosci 2018
7. Excessive leptin in obesity leads to leptin resistance
Munzberg et al. Endocrinol 2007
phospho-STAT3 immunohistochemistry
Leptin
8. Major questions
• What hypothalamic cell populations express Lepr?
• Does leptin signaling differ by population?
• How does metabolic dysfunction (such as obesity) impact leptin signaling?
• Are Lepr populations and responses conserved in primates?
11. A mouse model for genetic celltype nucleus enrichment
Mo et al. Neuron 2015
Tag Lepr-
expressing
nuclei with GFP
PMv
PVp
VMH
ARH
DMH
LHA
Cross with Lepr-
Cre mouse line
LeprCre
; R26-LSL-Sun1:sfGFP
12. Remove low
quality cells and
low detection
genes
Robust enrichment of Lepr neurons in Lepr-Sun1 model
UMAP1
UMAP2
Identify celltypes
UMAP1
UMAP2
Expression
matrices
Remove
doublets
Find variable
genes
Embed in lower
dimension
(UMAP)
13. A catalog of Lepr hypothalamic populations
UMAP1
UMAP2
Hypothalamic neurons
16. Leptin response is biased toward a small set of populations
Burkhardt et al. Nat Biotechnol 2021
DMH.Glp1r
ARH.Pomc
GLUT1
LHA.Nts/Crh
PMv.Tac1
DE genes from 100 random
downsamples (fixed size)
17. Leptin response is biased toward a small set of
populations
DMH.Glp1r
ARH.Pomc
GLUT1
LHA.Nts/Crh
PMv.Tac1
31. Identifying consensus clusters from published datsets
Consensus names
arc: arcuate nucleus (Campbell et al 2017)
poa: preoptic area (Moffitt et al. 2017)
lha: lateral hypothalamic area (Mickelsen et al. 2019)
vmh: ventromedial hypothalamus (Kim et al. 2019)
vph: ventral posterior hypothalamus (Mickelsen et al. 2020)
pvh: paraventricular hypothalamus (Xu et al. 2020)
35. Leptin responses are present in Lepr populations in obesity
Hypothalamic populations
Lepr-Sun1
populations
Correlation of fold-change values in obesity or leptin treatment
High Lepr
populations
36. Variable dampening of leptin response in obesity
Integrate samples (CCA)
Predict treatment for hypothalamic cells
(Vehicle or Leptin)
Is obese transcriptome similar to leptin?
37. Loss of Stat3 target regulation in Agrp cells during obesity
Munzberg et al. Endocrinol 2007
STAT3 target genes phospho-STAT3 IHC
51. Generating leptin responses in naïve cells
Lotfollahi et al. Nat Methods 2019
Embed mouse vehicle,
chow, and leptin cells in
a common latent space
Estimate leptin
effects by population
Infer leptin responses
for chow cells
53. Macaque AGRP & POMC populations are predicted to
undergo leptin resistance
Infer leptin responses
for chow cells
Identify macaque
ortholog cells from Lepr-
Sun1 dataset
Embed mouse vehicle
and leptin and
macaque chow in a
common latent space
Estimate leptin
effects by population
54. Conclusions
• The Lepr population structure is complex and shared in macaque
• Leptin signaling is similar across major Lepr cell types
• Major leptin transcriptional targets are blunted in chronic obesity
• Primate hypothalamic cell types are broadly conserved in mouse
• Macaque arcuate cell types show signs of leptin resistance in obesity
• What aspects of acute leptin signaling remain and are blunted in obesity?
• Can aspects of leptin signaling be rescued with ER stress relievers (e.g. Celastrol)?
Future directions
55. Acknowledgements
Martin Myers lab
Abbey Tomlinson
Alison Affinati
Paul Sabatini
Jon Flak
Wenwen Cheng
Jim Dell’Orco
Alex MacKenzie
David Olson lab
Aristides Diamant
Randy Seeley lab
Nadejda Bozadjieva
Stace Kernoodle
Novo Nordisk
Chien Li
Lotte Bjerre Knudsen
Center for
Gastrointestinal
Research
Funding
Paul Kievit lab (OHSU)
Cadence True
Sarah Lindsley
Melissa Kirigiti
Tune Pers (U Copenhagen)
dkNET New Investigator in
Bioinformatics Pilot Program