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Article https://doi.org/10.1038/s41467-022-35544-3
A randomized trial of oral gamma aminobu-
tyric acid (GABA) or the combination of
GABA with glutamic acid decarboxylase
(GAD) on pancreatic islet endocrine function
in children with newly diagnosed type 1
diabetes
Alexandra Martin
1,4
,GailJ.Mick
1,4
, Heather M. Choat
1
,
Alison A. Lunsford
1
,HubertM.Tse
2
,GeraldG.McGwinJr.
3
&
Kenneth L. McCormick
1
Gamma aminobutyric acid(GABA) is synthesized by glutamate decarbox-
ylase(GAD) in β-cells. Regarding Type 1 diabetes(T1D), animal/islet-cell studies
found that GABA promotes insulin secretion, inhibits α-cell glucagon and
dampens immune inflammation, while GAD immunization may also preserve
β-cells. We evaluated the safety and efficacy of oral GABA alone, or combina-
tion GABA with GAD, on the preservation of residual insulin secretion in
recent-onset T1D. Herein we report a single-center, double-blind, one-year,
randomized trial in 97 children conducted March 2015 to June
2019(NCT02002130). Using a 2:1 treatment:placebo ratio, interventions
included oral GABA twice-daily(n=41), or oral GABA plustwo-dosesGAD-
alum(n= 25), versus placebo(n= 31). The primary outcome, preservation of
fasting/meal-stimulated c-peptide, was not attained. Of the secondary out-
comes, the combination GABA/GAD reduced fasting and meal-stimulated
serum glucagon, while the safety/tolerability of GABA was confirmed. There
were no clinically significant differences in glycemic control or diabetes anti-
body titers. Given the low GABA dose for this pediatric trial, future investiga-
tions using higher-dose or long-acting GABA formulations, either alone or with
GAD-alum, could be considered, although GABA alone or in combination with
GAD-alum did nor preserve beta-cell function in this trial.
The pathogenesis of type 1 diabetes mellitus (T1D) entailsautoimmune
destruction of pancreatic beta cells1–3. Once hyperglycemia appears,
more than 70% of islet beta cell mass has been eradicated4. Prolifera-
tion of surviving β-cells, pancreatic progenitor cells, plus transdiffer-
entiation of alpha, acinar, ductal or hepatic cells, all have the potential
to revitalize insulin production5,6.
Multiple immunological abnormalities have been reported in T1D
patients including autoantibody production against the insulin mole-
cule, the 65 kD isoform of glutamic acid decarboxylase (GAD65), var-
ious islet antigens, and the zinc transporter 8 (ZnT8) as well as
decreased regulatoryT cell (Treg) capacity to suppress T-cell mediated
destruction of the islets of Langerhans3. To date, many studies
Received: 27 October 2021
Accepted: 6 December 2022
Check for updates
A full list of affiliations appears at the end of the paper. e-mail: gjmick@uabmc.edu;klmccormick@uabmc.edu
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attempting to ward off or reverse T1D have focused on immune sup-
pression or modulation7–11, which may engender long-term side-
effects. However, the recent antiCD3 antibody trials have shown a
3-year delay in clinical diagnosis of T1D12,13.
Animal and in vitro studies maintain that gamma aminobutyric
acid (GABA) and glutamicacid decarboxylase(GAD) play fundamental
metabolic roles in the pancreas and may be potential therapeutic tar-
gets in T1D. As for GAD65 antigen (GAD-alum) treatment per se in new
onset T1D, an initial 2008 report of70 patients was auspicious insofar
as residual beta-cell function over 30 months was somewhat pre-
served. Yet a later, and more comprehensive, study with 334
patients failed to replicate this finding14. However, individual level
analysis of these two studies and another15 found that study partici-
pants positive for HLA DR3-DQ2, but negative for HLA-DR4-DQ8,
demonstrate enhanced beta cell preservation following GAD-alum
monotherapy16.
GABA, a major inhibitory neurotransmitter, is abundant within
pancreatic islets17,18 and participates in paracrine regulation of βand α
cells19,20. GAD, the enzyme that decarboxylates glutamate to form
GABA, is a major autoantigen in T1D3,21. In vitro experiments found that
isolated human islets treated with GABA receptor blockade have
decreased insulin secretion at physiologic glucose concentrations18.
Further, GABA-deficient islets did not show appropriate glucagon
inhibition in response to increasing glucose concentrations in vitro22,
suggesting that GABA is directly involved in the suppression of glu-
cagon secretion in pancreatic alpha cells. GABA activates the Ca2+ -
P13K/Akt growth and survival pathway and averts stress-induced
apoptosis in islet cell lines treated in vivo with streptozotocin (STZ)19.
In vivo, GABA delays diabetes onset in both the non-obese diabetic
(NOD) and the STZ-treated mouse if given early in life19.And,ifGABA
treatment was initiated in NOD and STZ mice after diabetes had
already commenced, normoglycemia ensued19. The mechanisms are
not fully understood, but are proposed to involve tempering of the
pancreatic autoimmune milieu and systemic inflammation.
Apart from demonstrating β-cell regeneration and glucagon
suppression with GABA in two distinct diabetic mouse models, Soltani
and colleagues described significant decreases in inflammatory cyto-
kine expression19. Functional GABA receptors are present on T-cells
and increases expression of splenic T regulatory cells, in turn poten-
tially arresting or slowing T cell mediated beta cell destruction19,23,24.In
vivo, GABA inhibits adoptive transfer of T1D following transplant of
diabetogenic splenic T cells into a NOD/SCID mouse model. Individu-
ally, GABA and GAD-alum promote survival of transplanted beta cells
in the NOD mouse, while combination therapy promoted synergistic
and dose dependent beta cell survival25. To date, neither GABA alone,
nor GABA-GAD in tandem, has been explored as therapeutic agents in
study participants with T1D. Here we show, in this human trial of low-
dose GABA, alone or as co-therapy GABA/GAD, that while the primary
outcome, β-cell function, was not statistically proven, the combination
GABA/GAD reduced fasting and meal-stimulated serum glucagon.
Glycemic control, proinsulin and diabetes autoantibodies, all second-
ary outcomes, were similar between GABA/GAD and placebo. More-
over, the safety and tolerability of the treatments was established.
Results
Recruitment and tolerability of intervention
BetweenMarch2015andJune2018,350patientswerescreenedanda
total of 97 patients enrolled (Fig. 1). There were six unrelated serious
adverse events recorded that required uneventful 1–2day
hospitalizations26.
Patient characteristics
The baseline patient characteristics for each treatment group are
summarized in Table 1. The age-stratified randomization was suc-
cessful. The ethnic distribution was as follows: 90% Caucasian, 7%
African American, 2% Hispanic and 1% Native American. All patients
were diabetes antibody positive with most retaining positivity in three.
There were no statistical differences regarding initial presentation,
including, age, diabetes ketoacidosis, the number of positive diabetes
antibodies, body mass index, HbA
1c
, fasting c-peptide or glucagon. All
patients were enrolled by 5–6 weeks post diagnosis of T1D.
Effect of GABA alone and GABA/GAD in combination on
c-peptide and glycemic control
There was no statistical effect of oral GABA alone or combination
GABA/GAD therapy on the primary outcome measure c-peptide,
including both fasting and MMTT-stimulated area under the curve
(Fig. 2a, b). The 90 min post MMTT c-peptide values for each study
group are shown in Supplementary Fig. 1. As expected, there was a
gradual diminution in c-peptide post diagnosis. A tabular summary of
the statistical comparisons for the primary and secondary outcomes
are presented in Supplementary Table 1. There was no statistical dif-
ferences in HbA
1c
outside of a small disparity in GABA versus placebo
only at the 5-month visit, and none in GABA/GAD versus placebo at all
study visits. To address this further, an analysis of area under the curve
(AUC) glucose at baseline and 12-months as well as fasting glucose at
baseline, 1 month, 5-months and 12-months showed no differences
(Supplementary Fig. 9). Insulin dose-adjusted A1c (IDAA1c)27 was 12%
increased in GABA compared to placebo at 5 and 12-months. By con-
trast, IDAA1c in GABA/GAD was not different from placebo at any time
point (Fig. 3a, b). Importantly, applying the gold-standard reference
used to establish IDAA1c, namely, a meal-stimulated c-peptide
>300 pM27, did not reveal statistical differences between the groups
(Supplementary Fig. 8a). Moreover, a sub-analysis of IDAA1c in those
participants who transitioned from basal/bolus injections to insulin
pumps –which provide far greater accuracy as to total daily insulin
dose (TDD) - between the 8–12 study visits revealed no statistical dif-
ferences in IDAA1c between the groups (Supplementary Fig. 8b).
Effect of GABA alone and GABA/GAD in combination on
glucagon
As shown in (Fig. 4a), the mean fasting glucagon in the placebo group
increasedby16.8%overthecourseforthestudy(baselineto12-
months) in contrast to the two study groups wherein this change over
time was curtailed: 0.4% in the GABA group and 0% in the GABA/GAD
group. At5 months, the mean fasting glucagon value in the GABA/GAD
group was attenuated by 10.7% compared to placebo (p= 0.086) and
11.1% compared to the GABA group (p= 0.007). By 12-months, the
mean fasting glucagon in the GABA/GAD group significantly dimin-
ished compared to the placebo patients (p= 0.035), but there were no
statistical differences relative to the GABA group.
Similar to the fasting glucagon data, the mean area under the
curve (AUC) glucagon levels increased from baseline to 12-month in all
groups (placebo group (24% increase), GABA group (13.7% increase)
and in the GABA/GAD group (13.1%). At 12-months, the AUC glucagon
in the GABA/GAD group was significantly reduced compared to pla-
cebo (p=0.041)(Fig.4b).
Based on the association between elevated glucagon and hyper-
glycemia in T1D28–32, we examined the correlation between glucos eand
glucagon. At first visit (baseline), both fasting glucose (p=0.0017)and
AUC glucose (p= 0.04) correlated with glucagon (Supplementary
Fig. 2a, b). Similar correlations for the 12-month visit were apparent
(Supplementary Fig. 2c, d).
Proinsulin levels and diabetes autoantibody titers
Fasting and 90 min post mixed meal plasma proinsulin and the
proinsulin/c-peptide ratio was examined in the three study groups at
baseline (before treatment), 5 and 12-months. No differences were
detected related to treatment (Supplementary Fig. 3). The time course
of diabetes autoantibodies (GAD65, ZnT8, ICA512) is presented in
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 2
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Supplementary Fig. 4a–c. The percent positivity for ZnT8 and ICA512 is
presented in Supplementary Fig. 4d. Overall, there were no statistical
trends or differences in the diabetes antibodies over time.
GABA levels
PlasmaGABA levels are presented in Supplementary Fig. 5.Participants
swallowed their study drug immediately before the mixed meal. There
were no differences between the study groups in the 0min (fasting)
GABA levels at either the baseline (initial) or 12-month visit. Not
unexpectedly, given the short half-life of GABA, the morning fasting
values were statistically the same in all three cohorts. The veracity of
the GABA study drug versus placebo is evidenced by the increase in
plasma GABA at 60 and 120min relative to baseline in the GABA and
GABA/GAD groups, with no change in the placebo group.
HLA haplotypes
The primary outcome, fasting and meal-stimulated c-peptide, was re-
examined after subdividing each treatment group according to high-
risk HLA haplotypes. Results did not show statistically significant dif-
ferences (Supplementary Table 2).
Discussion
This prospective, randomized, control trial of GABA and combined
GABA/GAD in children with new-onset T1D confirmed the safety and
tolerability of oral GABA, but did not attain its primary objective, the
preservation of β-cell function (Fig. 2). However, a secondary outcome
revealed a significant decrease in fasting, as well as nutrient-stimu-
lated, glucagon secretion following 12-months of oral GABA/GAD
treatment (Fig. 4). This observation corroborates favorably with ani-
mal/cell studies in which GABA (or GABA/GAD) has a paracrine inhi-
bition on α-cells.
GABA, secreted from β-cells, reportedly has both an autocrine
effect on insulin secretion as well as a paracrine inhibition of α-cell
glucagon production.Whereas a distinct GABA autocrine role remains
unsettled, the physiologically-relevant, paracrine inhibition of gluca-
gon secretion or diminution of α-cell mass has been repeatedly
documented in isolated cells or islets, perfused or biopsied pancreata,
or in vivoanimal studies. Upon binding toits cognate chloride channel,
GABA begets α-cell membrane hyperpolarization, thereby hampering
voltage-dependent calcium channels, which curtails glucagon output.
For example, in streptozotocin (STZ)-induced diabetic mice, 12 days of
daily intraperitoneal GABA (10 mg/kg) quenched the robust 7- fold
increase in α-cell mass, which occurred in controls. And, relevant to β-
cells, GABA augmented the proliferation of α-cells expressing GLP–1.
The latter, in turn, could plausibly enhance β-cell function and
growth33.
In another Type 1 diabetes model (multiple low dose STZ, MDSD),
GABA, when added to the drinking water (6 mg/ml), reduced both
Screened for eligibility (n=350)
Excluded (n=253)
iNot meeting inclusion criteria (n=240)
iScreen failed (n=13)
Analyzed (n= 39)
iExcluded from analysis (n= 2)
Baseline MMTT: all c-peptide ≤0.2 nM
GABA (n=41)
Allocated to intervention (n= 41 )
iReceived allocated (N=41)
Partial visits
Lost to follow-up (n=2)
completed two visits (n=1)
completed four visits (n=1)
Partial visits
Lost to follow-up (n=4)
completed two visits (n=2)
completed three visits (n=2)
Discontinued intervention (n=1)
Subject stopped intervention
completed th ree visits
Analyzed (n=22)
iExcluded from analysis (n= 3)
Baseline MMTT: all c-peptide ≤0.2 nM
Randomized (n=97)
Interviewed for potential interest
in participation (n= 830)
Excluded (n= 480)
ideclined for unknown reasons (n=475)
inot interested in research (n=5)
Placebo (n=31)
Allocated to intervention (n= 31)
iReceived allocated (N=31)
GABA/GAD (n=25)
Allocated to intervention (n= 25)
iReceived allocated
(
N=25
)
Partial visits
Lost to follow-up (n=3)
completed four visits (n=1)
completed two visit (n=1)
completed one visit (n=1)
Allocation
Analyzed (n= 30)
iExcluded from analysis (n= 1)
Baseline MMTT: all c-peptide ≤0.2 nM
Follow-U
p
Anal
y
sis
Fig. 1 | Consort profile. Participants, aged 4–18 years old, were screened at diagnosis with T1D and enrolled at our tertiary care university center at Children’s Hospital of
Alabama in Birmingham, Alabama. Nine study participants were from out-of-state.
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 3
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serum glucagon and α-cell mass34.Asimilartandemαand βcellular
GABA effect was also found in MDSD mice treated with 20 µmol/kg
intraperitoneal GABA prior to diabetes induction, and in a series of mice
previously rendered diabetic with severe hyperglycemia19.Isletstudies
unfailingly corroborate the inhibitory action of GABA on glucagon
secretion. In rat islets, GABA was noted to dampen glucose–stimulated
glucagon secretion35 and, in normal mice islets or perfused pancreas, an
inhibition of glucagon secretion was observed36. Finally, when a non-
ab
0.4
0.6
0.8
1.0
1.2
Time on study (months)
Fasting c-peptide (ng/ml)
01 5 12
GABA
GABA/GAD
Placebo
mean± 95% CI
GABA 39 39 3 6 33
GABA/GAD 22 21 21 20
Placebo 30 29 28 26
210
Time on study (months)
c-peptide AUC
ng/ml/min
0.5
1.0
1.5
2.0
2.5
GABA
GABA/GAD
Placebo mean±95% CI
GABA 39 33
GABA/GAD 22 20
Placebo 30 26
Fig. 2 | Fasting and AUC c-peptide in study groups over time. Fasting c-peptide
(a) was measured in the three study groups (GABA-red, GABA/GAD-blue, and pla-
cebo-black) at baseline (Time= 0,prior any treatment) and at 1, 5 and 12-months
thereafter. AUC c-peptide (b) was calculated over time in the three study groups.
Results are given as mean ±95% CI. No statistical differences were noted by two-
sided analysis of covariance (complete statistical data is summarized in Supple-
mentary Table 1). Source data are provided as a Source Data file.
Table 1 | Baseline participant characteristics
Parameter GABA n=39 GABA/GAD n=22 Placebo n= 30 GABA vrs placebo GABA/GAD vrs placebo
Age, years 11.2 ± 3.9 11.6 ± 3.2 11.1 ± 3.5 0.887 0.594
4–8 yrs (%) 31% 32% 30% 0.633 0.814
9–11 yrs(%) 44% 45% 53%
14–18 yrs (%) 26% 23% 17%
Sex Male %(n) 54%(21) 64%(14) 43%(13) 0.470 0.171
Female %(n) 46%(18) 36%(8) 57%(17)
BMI (kg/m2) 19.6 ± 3.4 19.3 ± 3.4 19.0 ± 3.2 0.435 0.770
BMI percent ile 66.9 ± 29.1 61.4 ± 26.5 60.3 ± 28.1 0.343 0.886
Ethnicity %(n)
White 92.3% (36) 90.9% (20) 86.7% (26) 0.387 0.650
African American 5.1% (2) 9.1% (2) 6.7% (2)
Hispanic 0 0 6.7% (2)
Native American 2.6% (1) 0 0
Days from diagnosis to baseline visit 25.3 ± 7.2 26.6± 6.3 25.8± 8.2 0.821 0.678
Diabetes ketoacidosis at diagnosis %(n) 23.0% (9) 22.7% (5) 36.6% (11) 0.287 0.368
Diabetes autoantibodies (% positive)a
Anti-ICA 512 81% 87% 83% 0.814 1.000
Anti-Zinc Transporter-8 94% 74% 87% 0.407 0.282
Number of autoantibodies positive (% patients)
1 3.9% 7.1% 8.3% 0.862
2 34.6% 28.6% 20.8% 0.503
3 61.5% 64.3% 70.8%
HbA1C % 11.0± 2.5 10.4 ± 2.2 11.1 ± 2.5 0.982 0.349
Total Daily Dose insulin (units/kg/day) 0.56 ± 0.21 0.47 ± 0.24 0.56 ± 0.21 0.984 0.167
C-peptide AUC at baseline (ng/ml/min) 1.85 ± 1.21 2.13 ± 1.16 1.87 ± 1.3 0.883 0.533
C-peptide fasting at baseline (ng/ml) 0.74 ± 0.55 0.78 ± 0.48 0.72± 0.60 0.884 0.630
Glucagon AUC at baseline (pg/ml/min) 78.05 ± 26.35 70.73 ± 24.51 77.90 ± 16.87 0.933 0.278
Glucagon fasting at baseline (pg/ml) 65.35 ± 16.10 61.11 ± 16.56 62.62 ± 13.62 0.906 0.770
Results arepresented as mean± SD unless otherwise specified.aAnti GAD65 was a studyinclusion criterion. Statisticalcomparisons wereby two-tail analysis of variance or Chi square as indicated
and as in Methods. GABA (gamma aminobutyric acid), GAD (GAD-alum).
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 4
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curative mass of normal human islets was transplanted into diabetic
mice (NOD –seid- ϒ), after 5 weeks of drinking water with GABA added
(6 mg/ml), serum glucagon was reduced roughly 80%37.
Antagonism of the glucagon receptor, or by genetic knockout,
especially in the face of insulin deficiency, promotes normoglycemia.
Take, for example, the following observations: (i.) Even without sup-
plemental insulin, by blocking the glucagon receptor in diabetic obese
mice, hyperglycemia was normalized38, (ii.) In the high-fat type 2 dia-
betic mouse, knockout of the glucagon receptor aborted obesity,
hyperinsulinemia and abnormal lipogenesis and, notably, prevented
hyperglycemia39, (iii.) In glucagon receptor null mice, following mas-
sive streptozotocin β−cell destruction, and despite marked hyperglu-
cagonemia (14-fold increase over wild-type), normal blood glucose
prevailed40, (iv.) Glucagon receptor antibody alone, i.e., no insulin
therapy, can normalize hyperglycemia of type 1 diabetic NOD mice41,
and finally, (v.) In humans with T1D, a single subcutaneous dose of a
glucagon receptor antibody resulted within days in a 14% reduction in
insulin dose and improved glycemic control as assessed by continuous
glucose monitoring31. Most recently, a monoclonal glucagon receptor
antagonist (Ab-4) corrected both glycemia and provoked restoration
of β−cells in type 1 diabetic rodents (NOD and PANIC-ATTAC mouse
models) as well as in mouse-implanted human islet xenografts42.
Indeed, in the NOD mouse, the Ab-4 antibody increased insulin islet
area approximately 900% versus control.
In concordance with previous reports, we found a progressive
increase in serum glucagon over the first year following T1D diagnosis
(Fig. 4), a phenomena which can persist for 3–5years
43–47.Glucagon
may worsen glycemic control28,29 by peripheral effects on hepatic,
adipose, and neural metabolism. Even in non-diabetic adults, fasting
glucagon correlates inversely with longitudinal β−cell function- infer-
ring that α-cell dysfunction is an incipient stage in disturbed glucose
metabolism48. Although suppression in serum glucagon by GABA/GAD
was found in our study, the percent lowering may not be sufficient to
impact glycemic control (Fig. 3), namely, the insulin-adjusted A1c
(IDAA1c) in this group. Of interest, using the reference standard for
IDAA1c, a meal stimulated c-peptide >300 pM, there was a trend sug-
gesting improvement in GABA/GAD group at 5-months (Supplemen-
tary Fig 8a). As evidenced in Supplementary Fig. 2, serum glucagon
correlates positively with serum glucose, which infers a role of gluca-
gon in glucose homeostasis. In our placebo cohort, the AUC glucagon
Fig. 3 | Glycemic control in study groups over time. Glycosylated Hemoglobin
(HbA
1c
)(a) and insulin adjusted A1c (IDAA1c)(b) were measured inthe three study
groups. Results are shown as mean ±95% CI and statistical comparisons were by
two-sided analysis of covariance. Regarding HbA1c (a) at 5-months GABA vrs.
Placebo **p= 0.003 and GABA vrs GABA/GAD *p=0.041. For IDAA1c (b)at5-
months,GABA vrs. Placebo**p= 0.007 and GABA vrs. GABA/GAD **p= 0.002. At 12-
months, GABA vrs. Placebo *p=0.020. Source data are provided as a Source
Data file.
ab
Time on stu dy (months)
Fasting glucagon (pg/ml)
01
50
60
215
70
80
GABA
GABA /GAD
Placebo
**
GABA 39 38 34 32
GABA/GAD 22 21 20 20
52 82 92 03 obecalP
210
60
80
100
Time on study (months)
Glucagon AUC
(pg/ml/min)
GABA
GABA/GAD
Placebo
*
GABA 39 33
GABA/GAD 22 20
Placebo 30 26
Fig. 4 | Fasting and AUC glucagon in study groups over time. Fasting glucagon
(a) was measured in the three study groups (GABA-red, GABA/GAD-blue, and pla-
cebo-black) at baseline (Time=0, prior any treatment) and at 1, 5 and 12-months
thereafter. AUC glucagon (b) was calculated over time in the three study groups.
Resultsare given as mean ±95% CI. Statistical differences were by two-sided analysis
of covariance. aAt 5 month GABA/GAD vrs. GABA, p= 0.007 and GABA/GAD vrs.
Placebo, p= 0.086. At 12- months, GABA/GAD vrs. Placebo,**p=0.035. Regarding
AUC glucagon (b), at 12-monthsGABA/GAD vrs. Placebo, *p= 0.041. Sour ce data are
provided as a Source Data file.
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 5
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at one yearwas 24% elevated versus baseline. This compares favorably
to the postprandial increases of 37 and 51% previously reported in
children with T1D (references45 and43, respectively).
The slight increase in IDAA1c in the GABA group (Fig. 3b) warrants
discussion. This calculated metric of glycemic control27 is the least
objective index insofar as it incorporates TDD, which in our study
depended on participant paper records and recall. Furthermore, TDD
is influenced by exercise, carbohydrate load, intercurrent illness and
other factors. As aforementioned, using a meal-stimulated c-peptide
>300 pmol/l27, there was no difference in glycemic control between the
three groups (Supplementary Fig. 8a). Likewise those patients who
transitioned to insulin pumps, which provide a more precise digital
assessment of TDD, showed no differences in IDAA1c (Supplementary
Fig. 8b).Finally, fasting and AUC glucose were not different amongst
the groups at 12 months (Supplementary Fig. 9).
This study has many strengths. Foremost, as an adjunct agent, it is
the first GABA study conducted in newly diagnosed humans. Further,
by studying an exclusively pediatric population, we were able to enroll
very young patients with T1D who typically have a more rapid deci-
mation of βcells than adolescents49–53. Forty percent of our study
participants were <10 years. Considering the array of confounding
factors in β-cell loss, age of onset is the major determinant in the
temporal decline in serum c-peptide. Most other potential ther-
apeutics are first investigated in the adult population, making it
impossible to reliably exclude patients with latent autoimmune dia-
betes of adulthood (LADA)54. Thirdly, our study was able to enroll all
children within the first 5 weeks after diagnosis, allowing exposure of
the pancreatic islets tothe intervention before near-total autoimmune
β-cell eradication.
Our study established that oral GABA is tolerable. The basis for
this “low-dose”designation merits consideration. The daily dose of
GABA used in animal studies, mostly mice, are sweeping, ranging
0.25mg to 1500 mg/kg. Under FDA constraints, our dose of 1 gram/M2
(about 35 mg/kg) was far below nearly all in vivo studies in which
salutary outcomes were reported (Supplementary Fig. 6).
It is speculative as to the mechanism whereby the GABA/GAD
tandem attenuated glucagon more than GABA alone. However, the
combination GABA/GAD strikingly extended, and in a synergistic
manner, the time to develop hyperglycemia in diabetic NOD mice with
transplanted β-cells25. It is conceivable that GAD-alum may have
increased ambient islet cell insulin concentrations - despite no detec-
tible change in the systemic serum levels - thereby reducing adjacent
alpha cell glucagon release. To the point, we could have included a
GAD-alum group alone, however, we did not because of the previous
single and multicenter GAD-alum studies14,15,55–57.
Proinsulin and the proinsulin/c-peptide ratio are recognized
markers of β-cell stress in T1D, likely related to aberrant proinsulin
processing58,59. We investigated whether proinsulin or the proinsulin/c-
peptide ratio was modified by treatment with GABA, or the combina-
tion GABA with GAD, due to their recognized immunosuppressive
actions in diabetes25 (Supplementary Fig. 3), no statistical differences
were identified. Likewise, there was no difference in baseline or sub-
sequent diabetes antibody titers or positivity in the treatment groups
(Table 1and Supplementary Fig. 4) which is not unexpected for a one-
year T1D trial60.
Considering the role of the DR3-DQ2 haplotypes which confer T1D
risk and disease course61, we screened our study cohorts accordingly.
Based on previous evidence demonstrating HLA haplotype specificity
to GAD-alum therapy16, we examined whether the presence or absence
of HLA DR3-DQ2 altered the primary outcome in the three treatment
groups. No differences weredetected; however, a larger cohortmay be
required to detect statistical distinctions (Supplementary Table 2).
The study has limitations, most notably the unpropitious com-
pliance (assessed by pill counts and recall,) as is commonly encoun-
tered in the real-clinic setting (Supplementary Fig. 7). Based on in vivo
animal trials, the dose of GABA (alone) may have been inadequate,
namely, beneath a threshold response (Supplementary Fig. 6). As
aforementioned, a further weakness of our study was that the GABA
preparation was relatively short acting and taken only twice daily
(Supplementary Fig. 5). Alternatively, long-acting preparations of
GABA and/or currently available GABA-ergic drugs that have longer
half-lives of action offer promise. And, based on affirmative β-cell
studies in human islets, co-treatment of GABA with an allosteric posi-
tive modulator (Ly49) of its cognate receptor is an ingenious notion62.
To sum, in this prospective, randomized controlled trial of twice-
daily GABA, or co-treatment with GABA/GAD, in humans with T1D, we
demonstrate a significant decrease in fasting and AUC glucagon in the
GABA/GAD group, with a non-significantreductionintheGABAgroup
at 12-months. There were no statistically significant changes in the
primary outcome, namely, fasting and meal-stimulated c-peptide
between the cohorts. Notwithstanding the necessarily low GABA
dose for this trial in TID children, in combination with the compliance
challenge, the reduction in serum glucagon augurs well for further
studies to conceivably preserve β-cell function or mass. Indeed, in the
sole study using co-therapy with GABA/GAD, β-cell preservation was
dependent on the dose25. Lastly, bearing in mind that GABA/GAD
attenuated glucagon production, this could in turn expand β-cell mass
and/or improve glucose homeostasis. Case in point, in diabetic mice,
blocking glucagon action begets a nearly 8-fold increase in insulin-
positive islet cell mass and mediates β-cell regeneration42,63. Insofar as
GABA tempers immune inflammation at higher doses in rodents, and
our study was constrained to relatively low-dose GABA dosing in this
pediatric trial in T1D, it is plausible that increased GABA doses, or long-
acting preparations, could offer sufficiently prolonged, above-
threshold GABA concentrations to preserve islet cells, particularly
during stage 1 diabetes.
Methods
The detailed rationale and methods for this study have been described
elsewhere with minor modification26. A succinct summary follows:
Study design and treatment
This is a prospective, one-year randomized, double blind, placebo
controlled trial to evaluate the safety and efficacy of GABA alone and
combination GABA/GAD-alum® in children with newly diagnosed T1D
(https://clinicaltrials.gov/ct2/show/NCT02002130). Patients were ran-
domized into one of three study arms (Fig. 1). The original clinical-
trial.gov posting (2013) predates the final protocol submission (2015).
We had a protracted period (2 years) prior to study launch in order to
obtain FDA approval to administer GABA in children (first human trial).
The formal study protocol approvals and funding were in place by 2015
and the first patient enrolled 3/2/2015. Suboptimally, we noticed the
documentation discordance from 2013 and updated the clinical-
trial.gov outcomes in July 2019 to align with the 2015 study protocol.
Participants and eligibility criteria
Participants were screened at the time of diagnosis with T1D, as
defined by ADA criteria. All patients were enrolled from the
clinics and in-patient wards at Children’s of Alabama (CoA), a
tertiary care university-associated referral center. The majority of
patients were residents of the state of Alabama. There were 11 out
ofstateparticipants(AZ,GA,MS,MO,NC,ND,TX,VA).Thefirst
participant was enrolled 3/2/2015 and the last study visit was 6/
24/2019. Inclusion criteria: children 4–18 years of age, positivity
for autoantibody GAD65, and enrollment within 5 weeks of
diagnosis. If the participant was female and not abstinent, two
forms of contraception were required. Exclusion criteria: preg-
nancy, systemic or inhaled steroid use, neurologic/seizure dis-
orders, adjunct oral therapies that might affect glucose or GABA
metabolism26. Six randomized patients were excluded from
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved
analysis because all c-peptide values, fasting and MMTT stimu-
lated, were <0.6 ng/ml at the initial baseline study visit64,65.Par-
ticipants received a $60 gift card as compensation for every
blood draw.
Randomization
Patients were randomized into one of three regimens (GABA, GABA/
GAD-alum, or placebo) stratified by age and in balanced blocks of three
(1:1:1) for the first 75 patients using a pre-set randomization list (gen-
erated by using a computerized procedure) known only to the un-
blinded pharmacist. This second protocol was a consequence of
unanticipated additional funding that afforded trial extension for the
GABA versus placebo groups only.
Study drugs
Oral gamma-aminobutyric acid (GABA). GABA and placebo capsules
were prepared commercially (NOW Foods, Bloomingdale, IL). GABA or
placebo was administered using premeasured capsules (1 gram/M2/
day up to maximum of 1.5 gram/day) divided into two daily doses
(morning and evening). The purity of both the GABA and placebo
products was verified by LC/MS/MS prior to study enrollment. The
control GABA that was used for mass spectroscopy analysis
was obtained from Sigma-Aldrich Chemical Company (St. Louis, MO).
Placebo and GABA capsules were, taste-wise and visually,
indistinguishable.
Glutamic acid decarboxylase (GAD-alum). GAD-alum and placebo
were prepared as a suspension with recombinant GAD enzyme and the
vaccine adjuvant Alhydrogel ® (alum) by Diamyd Medical (Stockholm,
Sweden). The subcutaneous GAD-alum injections (20 μg/dose), or
placebo, were given in clinic by the research nurse.
Mixed meal tolerance testing (MMTT)
MMTT occurred according to the visit schedule outlined in Table 2and
as described previously26.
Safety monitoring
Safety assessments included observations of reactions at the injection
site, occurrence of all adverse events (AEs)/serious adverse events
(SAEs), laboratory measurements (chemistry panel, complete blood
counts with differential, and urinalysis), neurological assessments, and
physical examination.
Adherence and retention measures
Treatment adherence of the oral capsules was assessed subjectively by
patient recall, and objectively by calculating the unused capsule count
at each visit. Study participants were asked to return any unused study
drug for safe disposal and queried whether any capsules were
destroyed or lost.
Investigative endpoints
The primary outcome measure was the effect of GABA or GABA/GAD on
fasting and meal-stimulated serum c-peptide compared to placebo at
baseline,1-month, 5-months and 12-months. Secondary endpoints
included (1) fasting and meal-stimulated glucagon and proinsulin (2)
glycemic control (HbA
1c
, IDAA1c27), (3) diabetes autoantibodies, and (4)
immune studies in peripheral blood mononuclear cells (to be pre-
sented in a separate manuscript). Exploratory endpoints included
plasma GABA levels and the proinsulin/c-peptide ratio before and after
meal-stimulation. Also, we examined the effect of diabetes-related HLA
risk haplotype on the primary outcome.
Endocrine assays
C-peptide, glucose and glucagon were measured in the University of
Alabama Core Metabolic Laboratory and as previously noted26.
C-peptide was measured bya two-site immunoenzymometric analyzer
(900 AIA-Pack, TOSOH, San Francisco, CA) and glucagon by radio-
immunassay (Millipore Sigma, Burlington, MA). Antibodies to GAD65,
IA512, and Zinc 8 Transporter were assayed commercially by Labcorp
(Burlington, NC) as standard of care.
Plasma GABA
Plasma GABA levels were obtained during mixed meal tolerance test
(MMTT) at both the baseline (initial study visit) and 12 month visits.
Patients swallowed oral study drug dose at 0 min, immediately prior to
ingesting mixed meal drink. GABA levels were determined at 0, 60 and
120 min.
GABA analysis
Materials and sample preparation. Solid stocks of GABA and GABA-
d6 were purchased from Sigma & CDN Isotopes respectively. Stan-
dards were reconstituted in methanol. The analytical range was
1–5000 ng/ml over 8 calibrators. Plasma samples were thawed on ice
and spiked with 10 µl of 500 ng/ml GABA-d6. They were transferred
quantitively to 1 cc Phree SPE cartridges (Phenomenex, Torrance, CA)
containing 600 µl of 1% formic acid acetonitrile, incubated at room
temp for 5 min and centrifuged at 1000 gfor 5 min. The flow-through
was retained and transferred to a Biotage N2 evaporator to dry.
Samples and standards were reconstituted in 100 µl of 1.0% formic
acid before analysis. LC-MS Conditions. Separation and detection
were carried out by Shimadzu Prominence 20 series HPLC in tandem
with a Sciex API 4000 triple quadrupole mass spectrometer
(MS) utilizing a modified method from Imtakt66. Chromatographic
separation occurred with a Intrada Amino Acid column 3µM
Table 2 | Study treatment visit schedule
Study visit
Study group Treatments Baseline Visit #1 Month 1 Visit #2 Month 5 Visit #3 Month 8 Visit #4 Month 12 Visit #5
GABA GABA
oral twice daily
Placebo-GAD
one injection:
visits #1 and #2
MMTTaMMTT MMTT HbA1c
Insulin- dose
MMTT
GABA/GADbGABA
oral twice daily
GAD
one injection:
visits #1 and #2
MMTT MMTT MMTT HbA1c
Insulin- dose
MMTT
Placebo Placebo-GABA
oral twice daily
Placebo-GAD
one injection:
visits #1 and #2
MMTT MMTT MMTT HbA1c
Insulin- dose
MMTT
aMMTT-Mixed meal tolerance test. bGAD = GAD-alum(Diamyd,Stockholm, Sweden)was administered on visit #1 and the seconddose was administered on visit #2.GABA(gamma aminobutyric acid),
insulin-dose (total daily insulin dose).
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
50 ×3 mm at 60 degrees C. Mobile phases were A) 0.3% formic acid in
MeCN and B) 100 mM ammonium formate. Gradient schedule was as
follows: 0 min 30% B, 4 min 35% B, 5 min 100% B, 5.1 min 30% B, and
5.5 min stop. The flowrate of 0.6 ml/min. Injection volume was 5 µl.
Analyst v1.7.2 was used for instrument control & data acquisition. The
MS was operated in positive polarity electrospray ionization. MS
source parameters were as follows: collision gas 5, curtain gas 25, GS1
40, GS2 45, IS 2000, and temperature 600. Compound mass transi-
tions were 104.1 m/z à 87 m/z & 110 m/z à 93 m/z for GABA and GABA-
d6 respectively. Compound parameters were as follows: collision
energy 15, cell exit potential 6, and declustering potential 60. Data
processing occurred in MultiQuant v3.0.3. The standard curve was
regressed linear with 1/x2 weighting
HLA genotyping in study participants
The Histocompatibility and Immunogenetics Laboratory at the Uni-
versity of Alabama at Birmingham performed HLA typing on genomic
DNA that was isolated from frozen peripheral blood mononuclear
cells (PBMC).
Statistical analysis
Baseline demographic and other clinical characteristics were com-
pared between the treatment groups using t-andchi-squaretests(or
their non-parametric equivalents) for continuous and categorical
variables, respectively. Analysis of covariance was used to compare
changes in C-peptide levels between the treatment groups. For these
analyses, the 12-month measurement served as the dependent variable
with two independent variables: (1) a categorical variable for treatment
group and (2) the baseline C-peptide measurement. A similar analytical
approach was used for the other study outcomes of interest including
glucagon, hemoglobinA1C, IDAA1C, and total daily insulin dose. Mixed
statistical models were used to conduct longitudinal analyses of
C-peptide and hemoglobin A1C measurements, and daily insulin
requirements, incorporating all three measurements. This study uti-
lized REDCap (ResearchElectronic Data Capture, version 12.3.3 https://
www.project-redcap.org), a software toolset and workflow methodol-
ogy for electronic collection and management of clinical and research
data. Data analysis of for primary, secondary and exploratory out-
comes used SAS/STAT software, version 9.4 of the SAS System.
Copyright, SAS Institute Inc. Cary, NC, USA. Graphs were prepared with
GraphPad Prism 9.0 for Windows, GraphPad Software, San Diego, CA,
USA, www.graphpad.com. Correlations and Fisher’s exact analyses
were by GraphPad.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
Following de-identification, all of the individual participant data col-
lected during this trial, as well as data dictionaries, will be available to
any researcher who provides a methodologically-sound proposal for
academic purposes. Requests should be directed to the corresponding
author and is subject to a material transfer agreement. Proposals may
be submitted up to 36-months following publication. Source data are
provided with this paper. The study protocol is available online
(https://clinicaltrials.gov/ct2/show/NCT02002130). Source data are
provided with this paper.
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Acknowledgements
This study was funded by the Juvenile Diabetes Research Foundation
(#201303440) K.L.M., Diamyd Medical (Stockholm, Sweden), Janssen
Pharmaceuticals (Raitan,NJ), and NIH R01 grant awards: DK126456(HMT)
and DK127497(H.M.T.). NOW foods (Bloomingdale, IL) supplied the
GABA and the placebo capsules. We are grateful to the children and
families for their participation in this clinical trial and Sharon May, our
research clinician specialist. Purchase of the AB Sciex 4000 mass
spectrometer in the Targeted Metabolomics and Proteomics Laboratory
came from the University of Alabama Birmingham Health Services
General Endowment Fund. Sponsors were not involved in the study
design, data collection and analysis or manuscript writing.
Author contributions
M.A., K.L.M and G.J.M designed the studies, wrote protocols and
enrolled and cared for patients, K.L.M and A.A.L obtained the Federal
IND for GABA, H.M.C. was responsible for patient care and protocol
oversight, G.G.M. provided statistical analysis. K.L.M., G.J.M. and H.M.T.
acquired data and analyzed the results, and K.L.M., G.J.M., and M.A.
wrote the manuscript.
Competing interests
The authors declare no competing interests.
Ethical standard
The protocol and consent documents were approved by the University
of Alabama at Birmingham (UAB) Institutional Review Board.
Informed consent
Written informed consent was obtained from each participant or from
the participant’s parent or legal guardian. Also, each participant
assented.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-022-35544-3.
Correspondence and requests for materials should be addressed to
Gail J. Mick or Kenneth L. McCormick.
Peer review information Nature Communications thanks the anon-
ymous reviewers for their contribution to the peer review of this work.
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© The Author(s) 2022
1
Department of Pediatrics, Division of Pediatric Endocrinology, University of Alabama at Birmingham, Birmingham, AL, USA.
2
Department of Microbiology,
Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL, USA.
3
Department of Epidemiology, School of Public Health,
University of Alabama at Birmingham, Birmingham, AL, USA.
4
These authors contributed equally: Alexandra Martin, Gail J. Mick.
e-mail: gjmick@uabmc.edu;klmccormick@uabmc.edu
Article https://doi.org/10.1038/s41467-022-35544-3
Nature Communications | (2022) 13:7928 10
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