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. Upcoming webinars schedule: https://dknet.org/about/webinar

1. Uncovering novel mediators and
mechanisms of leptin action
Alan Rupp
University of Michigan
February 25, 2022

2. Obesity & diabetes rates continue to climb
Source: CDC

3. Obesity risk alleles are enriched in the brain
Locke et al. Nature 2015
Leptin-deficient ob/ob mice

4. The hypothalamus coordinates energy balance
Schwartz et al. Nature 2000

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?

9. snRNA-seq to identify cell type-specific transcriptomes
Macosko et al. Cell 2015

10. Standard scRNA-seq rarely captures Lepr expression
~1%
UMAP1
UMAP2
UMAP1
UMAP2

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

14. How does leptin signaling
differ by population?

15. A celltype-specific leptin transcriptome
Lepr-Sun1 mouse
Vehicle Leptin
UMAP1
UMAP2

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

18. Glp1r/Lepr cells are in the dorsomedial hypothalamus
(DMH)

19. Glp1r cells are critical for leptin’s effects on food intake

20. Leptin responsiveness is driven by Lepr expression
Lepr
DMH.Glp1r
Leptin prob.
ARH.Pomc
LHA.Nts/Crh

21. Leptin action is broadly shared across populations

22. Leptin drives synaptic remodeling
GO enrichment of leptin-regulated genes (by cluster)
TSNE1
TSNE2

23. Leptin substantially downregulates synaptic machinery
synaptic membrane GO term genes
McGregor & Harvey. Front Cell Neurosci 2018

24. SCENIC identifies cell-specific transcription factor
networks
Cells
Regulons
Aibar et al. Nat Methods 2017
Nkx2-1

25. Leptin modules dozens of transcription factor networks
All active regulons
(colored by association with leptin treatment)
UMAP1
UMAP2

26. STAT3 targets are coordinately regulated by leptin
STAT3 target genes

27. How is leptin signaling impacted by obesity?

28. A high resolution hypothalamic atlas during diet-induced
obesity
187,843 cells from 20 samples

29. Similar celltype composition in lean and obese mice

30. Extensive hypothalamic neuronal diversity
102,769 neurons

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)

32. High-fat diet transcriptional regulation is biased toward a
small set of populations

33. Lepr-Sun1 populations have clear correlates in
broader hypothalamus
Identify correlates in
Lepr-Sun1 dataset
(Seurat CCA)
Project cluster
names

34. Lepr-Sun1 populations have clear correlates in broader
hypothalamus

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

38. Broad leptin transcription factor dysregulation in obesity
Stat3
Treatment effect on each TF regulon

39. Is leptin action conserved in primates?

40. Macaque mediobasal hypothalamus in obesity
Collaboration with Paul Kievit (OHSU)
ID Age
(yr)
Sex Diet BW
(kg)
Run Sample Region
32859 7.0 F Chow 6.1 NovaA-225 794-AA-2 VMH/DMH
32859 7.0 F Chow 6.1 NovaA-225 794-AA-3 VMH/DMH
25865 14.2 M HFD à Chow 14.0 NovaA-321 2112-AA-1 ARH
25865 14.2 M HFD à Chow 14.0 NovaA-321 2112-AA-2 DMH
25865 14.2 M HFD à Chow 14.0 NovaA-321 2112-AA-4 VMH
30790 24.1 M HFD 15.8 NovaA-333 2356-AT-1 ARH
30790 24.1 M HFD 15.8 NovaA-333 2356-AT-2 DMH
30790 24.1 M HFD 15.8 NovaA-333 2356-AT-4 VMH
20275 22.2 F HFD 9.1 NovaZ-7 2529-AT-1 ARH
20275 22.2 F HFD 9.1 NovaZ-7 2529-AT-2 DMH
20275 22.2 F HFD 9.1 NovaZ-7 2529-AT-4 VMH
24693 16.6 F Chow 8.6 2740-AT 2740-AT-1 ARH
24693 16.6 F Chow 8.6 2740-AT 2740-AT-2 DMH
24693 16.6 F Chow 8.6 2740-AT 2740-AT-4 VMH

41. Macaque mediobasal hypothalamus in obesity
1. Cell / gene filtering
2. Doublet identification (scrublet)
3. Batch harmonization (Harmony)
UMAP1
UMAP2

42. Recovery of major hypothalamic cell populations in macaque
UMAP1
UMAP2

43. Macaque and mouse cell types transcriptomes are broadly
conserved

44. Celltype composition changes between mouse and macaque

45. Macaque hypothalamic neuronal diversity
Dissection region Treatment Cluster
UMAP1
UMAP2

46. Conservation of macaque neuronal populations
Integrate samples (CCA)
Project cluster label
from mouse dataset
Identifying ortholog populations

47. Broad LEPR expression in macaque hypothalamus
Cluster

48. LEPR populations are highly conserved in primates
Learn orthologs
with scANVI

49. HFD responses in macaque neuron populations

50. HFD responses in macaque neuron populations

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

52. A leptin resistance metric
PC1
PC2
UMAP1
UMAP2
Chow HFD pseudo-Leptin
Bias toward Chow neighbors

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