6-Month Project Update
To examine how LDs specifically
affect human islet α cells, I applied an
immunomagnetic positive selection
strategy to enrich α cells from
dissociated non-diabetic adult human
islets. Using an anti-CD26 antibody
as the selection marker, I was able to
produce human pseudo islets greatly enriched for α cells (Fig. 1A, termed α
+HpI or α plus Human pseudo islets). Lentiviruses carrying a universal
promoter driving shPLIN2 or PLIN2 were used to knockdown (KD) or over-express (OE) the protein. In
addition, a GFP or Cherry reporter was used to mark cells infected in α
+HpI. Around 70% of α cells (i.e.,
GCG+) expressed GFP or Cherry (Fig. 1B-C), and both PLIN2 mRNA and protein expression was significantly
impacted in KD and OE pseudo islets (Fig. 1D-E). Consistently, LD status evaluated by BODIPY staining
revealed that PLIN2 KD compromised, while PLIN2 OE elevated LD levels, as expected (Fig. 1E). No overt cell
death was observed in any of the experimental groups.
To evaluate how PLIN2 levels regulate glucagon secretion, I performed static incubation with a low stimulating
(2.5mM) and high inhibiting (16.7mM) [glucose]. Interestingly, PLIN2 KD α+HpI elevated glucagon secretion at
2.5mM glucose and a trend towards higher secretion at 16.7mM. The secretion profile of PLIN2 OE α
resembled Sham pseudo islets (Fig. 1F), and no overt changes in glucagon content were observed in
PLIN2KD or PLIN2OE (Fig. 1G). This same pattern was observed in the two donors analyzed. In summary, my
preliminary data highlight that limiting LD formation directly impacts human α cell glucagon secretion.
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There is increasing evidence that Type 1 diabetes (T1D) is caused by the dysfunction of both insulin-producing β cells and glucagon-producing α cells in the islet of the pancreas. While efforts have focused on understanding changes occurring in β cells, little is known about the genetic or molecular factors that regulate α cell activity in T1D, or even under healthy conditions. Notably, in addition to the activation of proteins involved in inflammation, abnormally high lipid levels are also commonly seen in the T1D population. This is called lipotoxicity, and it contributes to β cell dysfunction and likely α cell dysfunction. However, it’s presently unclear how these lipids are normally regulated by human islet α cells, and whether their misregulation contributes to dysfunction in T1D.
Intracellular lipid droplets (LDs) typically serve as an important storage depot for excess lipids. However, no one has studied the impact of LDs in α cells. My research discovered that LDs accumulate in an age- and the diabetes-dependent manner in human islet α and β cells. Interestingly, LD levels were significantly higher in the α cells of long-standing T1D islets. To examine the relationship between LD accumulation and islet cell function, I have used human β cells to obtain experimental and mechanistic insight. I demonstrated that LDs play a critical role in maintaining β cell function and health by protecting against lipotoxicity.
In this study, I will directly examine how LDs influence islet α cell function under both healthy and diabetic lipotoxic conditions. I hypothesize that the genetic/nutritional factors involved in LD formation and/or lipid handling influence α cell activity. These studies will include examining if cellular LD levels impact the inflammation and lipotoxicity associated with T1D islets.