Lipid Droplet Accumulation in Human Pancreatic Islets Is Dependent On Both Donor Age and Health

Abstract

Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet β-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and β-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet β-like cells produced from human embryonic cell-derived β-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.

Figure 1

LD accumulation in human islet β-cells increases with age. A: Representative EM images of healthy human islet β-cells from a 2-, 8-, 19-, and 67-year-old donor, which illustrates that very few LDs (red arrows) are found in juvenile donors compared with adult. The yellow arrows depict lipofuscins. Scale bar = 2 μm. yr, years. B: The number of lipid granules (including lipofuscins) per donor β-cell, with the quantity of β-cells counted in parenthesis. C: Percentage of β-cells containing lipid granules in human donors of different ages. D: Representative immunofluorescent image showing LD accumulation in healthy adult islet α- and β-cells from a 55-year-old (55yr) donor. Staining was performed to detect insulin (Ins) (white), glucagon (Gcg) (red), LDs (BODIPY [green]), and nuclei (DAPI [blue]). Scale bar = 50 μm. Right: magnified view of LD build-up in α- and β-cells within the marked area in the left panel. Error bars indicate SEM.

Figure 2

Relatively few LDs are present in the juvenile human pancreas. A: Pancreatic LDs are difficult to detect by Nile Red neutral lipid staining in juvenile (11 months old [11m] to 7 years old [7yr]) in comparison with teenage (19 years old) and adult (50 years old) human donors. The islet hormone area is depicted in green and LDs (Nile red) are depicted in red and nuclei in blue (DAPI). Scale bar = 50 μm. B: Percent of the islet LD area in the various age-groups (n = 6–8 donors/group). yr, years. C: The percent of LD containing islets in the donor age-groups. All error bars indicate SEM. **P < 0.01 and ***P < 0.001 vs. 0- to 2-year-old group; $$P < 0.01 and $$$P < 0.001 vs. 3- to 11-year-old group; +++P < 0.001 vs. 16- to 20-year-old group.

Figure 3

Intrinsic human islet factors determine the LD levels. A: Workflow schematic of the healthy juvenile and adult islet transplantation studies. Islets were transplanted under the kidney capsule for 6 weeks. B: Representative images of juvenile and adult islets showing that LD number is much higher in adult transplanted islets. The islet hormone area is depicted in green and LDs are depicted (Nile red) in red and nuclei (DAPI) in blue. Scale bar = 50 μm. C: Quantification of the percent of LD⁺ cells in the transplanted hormone⁺ cell area ± SEM. m, months old; Tx, transplant; yr, years old.

Figure 4

LDs detected in human ES cell–derived eBCs. A: A simplified schematic representing human ES cell differentiation into eBCs and the transplantation of eBCs into NSG mice. The eBCs are produced by FACS isolating the insulin⁺ synaptophysin⁺ cells from day 20 (D20), cell reaggregation, and then culturing for 4–8 days. eBCs improve their secretory properties upon transplantation (24). 3 & 9mon, 3 and 9 months. B: Representative images taken of day 20 spheres, day 27 eBCs, and a 9-month eBC graft immunostained with BODIPY (green), insulin (red), SYP (white), and DAPI (blue). LDs are enriched in insulin⁺ eBC cells but are also detectible in some insulin⁻ cells. The white square illustrates the zoomed-in area. Scale bar = 50 μm. C: LD distribution within the eBC transplant insulin⁺, synaptophysin⁺, or synaptophysin⁻ cell populations. n = 6. Error bars indicate SEM. **P < 0.01. INS, insulin; SYP, synaptophysin; Tx, transplant.

Figure 5

Pancreas LDs are rarely found in the aging mouse or in rodent models of hyperlipidemia and hyperglycemia. BODIPY⁺ (green) LDs are rarely detected in the pancreas of 1.5-year-old (1.5yr) C57BL6 mice, C57BL6 mice fed a low-fat diet (LFD) or HFD, and LDLR^−/− mice (top) or SD rats, ZDF rats, control db/+ mice, and diabetic db/db mice (bottom). BODIPY staining of the 35-year-old human donor is shown for comparison (top left). Scale bar = 50 μm. The physiological conditions of the rodents prior to islet isolation are provided in Supplementary Fig. 1. 10wk, 10 weeks old.

Figure 6

Little change in human islet PLIN2 and PLIN3 mRNA levels with respect to age or T2D. Relative PLIN1, 2, 3, 4, and 5 mRNA expression for islets in juvenile humans (age range 14 months–9 years) (A), adult humans (age range 39–68 years) (B), adult male C57BL6 mice (C), juvenile and adult humans (D), adults with T2D (age range 47–61 years) (E), age-matched adults without diabetes (ND) or with T2D (F), and adult humans and C57BL6 mice (G). There are no significant changes in PLIN mRNA expression between either healthy juvenile and adult islets (D) or adult healthy and T2D islets (F). The number of independent human and mouse islet samples analyzed is shown, with the adult ND number reduced for an age-matched comparison with T2D donor islets in F. Data presented relative to PLIN2 in A–C and E and relative to juveniles in D and to adult humans without diabetes in F and G. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. PLIN1 in A–C and E or vs. adult humans in G; $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. PLIN2; +++P < 0.001 vs. PLIN3 in A–C and E.

Figure 7

LDs are enriched in T2D islets. A: Representative images contrasting the uniform BODIPY pattern in pancreas islet and acinar cells of a healthy donor to the enriched pattern of a T2D donor islet. Yellow dotted lines outline the islet area and DAPI (blue) shows the pancreas cell nuclei. B: Quantification of the percent LD area in islets. C: Quantification of the percent LD area in acinar cells. D: Quantification of the ratio of percent LD islet to acinar cell area. E: Quantitation of the islet PLIN2 or PLIN3. F: Islet PLIN2 or PLIN3 (red) colocalization with BODIPY⁺ (LD [green]) cells in the T2D pancreas. The yellow dotted lines outline the islet insulin⁺ (Ins) (white) area. Magnified insets are provided to more clearly show PLIN2/3 and LD colocalization. Scale bar = 50 μm. Error bars indicate SEM. **P < 0.01, ***P < 0.001. yr, years old.