Lixisenatide accelerates restoration of normoglycemia and improves human beta-cell function and survival in diabetic immunodeficient NOD–scid IL-2rg^null RIP-DTR mice engrafted with human islets

Figures & data

Table 1 Demographic characteristics of human islet donors

	Donor 1	Donor 2	Donor 3	Donor 4	Donor 5
Age, years	30	53	43	51	61
Sex (M/F)	M	F	nr	M	F
Ethnicity	Hispanic/Latino	Hispanic/Latino	nr	Hispanic/Latino	nr
Body weight, kg	95	105	nr	86	nr
BMI, kg/m^2	30.9	42.7	34.7	28.9	22.2
Time	1 day,	3 days,	1 day,	2 days,	nr
in culture*	0 hour	12 hours	3 hours	10 hours
Transplanted IEQs/mouse	2,500	2,500	3,000	3,000	3,000

Note:

* Refers to the amount of time for which the human islets were cultured following isolation until shipment to our laboratory.

Abbreviations: BMI, body mass index; F, female; IEQs, islet equivalents; M, male; nr, not recorded.

Figure 1 Blood glucose and body weights of control and lixisenatide-treated mice engrafted with human islets.

Notes: Diabetic NSG RIP-DTR mice were transplanted with human islets from five individual donors and injected sc twice daily with lixisenatide or vehicle control; n=5–6 per treatment group, mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001. (A) Blood glucose levels of the mice were monitored on the days indicated. Donor 1, **medium dose vs control; Donor 2, ***high dose vs control, low, and medium groups; Donor 4, day 3, **high dose vs control, *medium dose vs control; day 5, ***medium and high doses vs control, *low dose vs control; Donor 5, *medium and high doses vs control. (B) Body weights were compared between the time of transplant and the day when the islet graft-bearing kidney was removed.
Abbreviations: NSG RIP-DTR, nonobese diabetic–severe combined immunodeficiency (NOD–scid) IL-2 receptor common gamma chain (IL-2rg^null) rat insulin promoter-diphtheria toxin receptor; SEM, standard error of mean; sc, subcutaneously.

Figure 2 Lixisenatide treatment improves recovery from hyperglycemia in human islet-engrafted mice.

Notes: Diabetic NSG RIP-DTR mice were transplanted with human islets and treated with lixisenatide or vehicle control. Mice with blood glucose values >300 mg/dL were considered hyperglycemic. Data shown are pooled from islet transplants of Donors 1, 3, 4, and 5; n=23–25 per treatment group. *P<0.05, ***P<0.001, ****P<0.0001.
Abbreviation: NSG RIP-DTR, nonobese diabetic–severe combined immunodeficiency (NOD–scid) IL-2 receptor common gamma chain (IL-2rg^null) rat insulin promoter-diphtheria toxin receptor.

Figure 3 Fasting and glucose-stimulated human insulin levels and stimulation indices in control and lixisenatide-treated mice engrafted with human islets.

Notes: Blood was collected at fasting and 15 minutes after ip glucose injection; plasma levels of (A) fasting human insulin and (B) glucose-stimulated human insulin are shown. (C) Stimulation indices (glucose-stimulated insulin/fasting insulin) from vehicle control and lixisenatide-treated mice are shown; n=4 or 5 per group, mean ± SEM.
Abbreviations: ip, intraperitoneal; SEM, standard error of mean.

Figure 4 GTTs in control and lixisenatide-treated mice engrafted with human islets.

Note: A GTT was performed in 5- to 6-hour fasted mice engrafted with islets at 4 weeks post-transplant; n=4 or 5 mice per group, mean ± SEM; *P<0.05, **P<0.01.
Abbreviations: GTT, glucose tolerance test; SEM, standard error of mean.

Figure 5 Percent TUNEL-positive human beta cells in islet grafts recovered from control and lixisenatide-treated mice.

Notes: Human islet grafts were immunostained for insulin and TUNEL, and the percent of TUNEL-positive beta cells was determined; n=3 or 4 per group for Donor 3 and 4 studies and n=2 for Donor 5 study, mean ± SEM.
Abbreviations: SEM, standard error of mean; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 6 Percent human beta- and alpha-cell proliferation in each islet graft recovered from control and lixisenatide-treated mice.

Notes: (A) Percent BrdU⁺ beta cells and (B) percent BrdU⁺ alpha cells are shown. Beta and alpha cells were identified by immunostaining for insulin and glucagon, respectively. Each dot represents data obtained from a single islet graft recovered from one mouse. The data from Donor 3 (black dots), 4 (green dots), and 5 (red dots) transplant studies were pooled. The bar represents the mean of n=10 or 11 individual islet grafts in each treatment group; >62,000 total beta cells and >24,000 total alpha cells were counted in each group.
Abbreviation: BrdU⁺, bromodeoxyuridine positive.

Figure 7 Total beta- and alpha-cell counts in human islet grafts recovered from control and lixisenatide-treated mice.

Notes: Human islet grafts of control and lixisenatide-treated mice were serially sectioned through the entire graft and (A) insulin⁺ beta cells and (B) glucagon⁺ alpha cells were counted; n=3 or 4 per group, mean ± SEM.
Abbreviation: SEM, standard error of mean.

Figure 8 Photomicrographs of human islet grafts from control and lixisenatide-treated mice.

Notes: Human islets from a single donor (Donor 5) engrafted in three vehicle control mice (top panel) and representative islet grafts from low-, medium-, and high-dose lixisenatide-treated mice (bottom panel, 1 of 3 islet graphs from each lixisenatide treatment group is shown) are shown; red, green, and blue indicate insulin, glucagon, and DAPI staining, respectively.
Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Figure S1 Pharmacokinetic analysis of control and lixisenatide treatments.

Notes: NSG mice were given a single sc injection with vehicle control or three different doses of lixisenatide; plasma levels of lixisenatide were measured at the time points indicated; n=3 mice per group at each time point (n=96 mice total + 3 untreated mice at time 0). The data from one low dose mouse at the 24-hour time point were deemed a technical failure and removed from analysis.
Abbreviations: NSG, nonobese diabetic–severe combined immunodeficiency (NOD–scid) IL-2 receptor common gamma chain (IL-2rg^null); sc, subcutaneously; conc, concentration.