Increased Neuronal Depolarization Evoked by Autoantibodies
in Diabetic Obstructive Sleep Apnea: Role for
Inflammatory Protease(s) in Generation of Neurotoxic
Mark B. Zimering1,2*,Zui Pan3
1Veterans Affairs New Jersey Healthcare System, East Orange, NJ
2Endocrinology, Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ
3College of Nursing and Health Innovation, University of Texas at Arlington, Arlington,
Mark B. Zimering, Medical Service (111), Veterans Affairs New Jersey
Healthcare System, Lyons, NJ 07939, USA, Tel: 908 647-0180; FAX: 908 604-5249; E-mail: @
Received:December 23, 2016; Accepted: January 08, 2017; Published:January 22, 2017
Mark B. Zimering,Zui Pan (2016) Increased Neuronal Depolarization Evoked by Autoantibodies in Diabetic Obstructive Sleep Apnea: Role forInflammatory Protease(s) in Generation of Neurotoxic Immunoglobulin Fragment. J Endocrinol Diab 4(1): 1-10 DOI: http://dx.doi.org/10.15226/2374-6890/4/1/00168
Obstructive sleep apnea increases in diabetes and morbid
obesity. We tested a hypothesis that circulating autoantibodies
in adult type 2 diabetes which increase in
association with morbid obesity are capable of causing
long-lasting neuronal depolarization and altered calcium
release in mouse atrial cardiomyocytes.
Protein-A eluates from plasma of 14 diabetic obstructive
sleep apnea patients and 17 age-matched diabetic
patients without sleep apnea were tested for effects on
depolarization and neurite out growth in N2a mouse
neuroblastoma cells. The mechanism of autoantibodymediated
neurite outgrowth inhibition was investigated
in co-incubation experiments of diabetic obstructive
sleep apnea autoantibodies with specific antagonists of
G-protein coupled receptors or the RhoA/ Rho kinase
signaling pathway. Following long-term storage of the
protein-A eluates (to allow spontaneous proteolysis and
IgG subunit dissociation), plasma autoantibodies from
diabetic obstructive sleep apnea, cancer or control patients
were compared for enhancement of inhibitory effects
on endothelial cell survival. Size exclusion chromatography
performed (in the presence or absence of a
specific membrane type 1- matrix metalloproteinase inhibitor)
was used to characterize the IgG autoantibody
subunit(s) or fragments associated with peak neurotoxicity
in diabetic obstructive sleep apnea.
Diabetic obstructive sleep apnea (n = 14) autoantibodies
caused a significant increase (P = 0.01) in membrane
depolarization in N2a mouse neuroblastoma cells
compared to control diabetic patients (n = 15) not suffering
with obstructive sleep apnea. Process extension
in N2A mouse neuroblastoma cells was significantly inhibited
(P = 0.01) by diabetic obstructive sleep apnea
(n = 9) autoantibodies compared to effects from identical
10 mg/ mL concentrations of control diabetic autoantibodies
in patients without obstructive sleep apnea.
Ten micromolar concentrations of SCH-202676,
a G-protein coupled receptor antagonist (n = 5) or ten
micromolar concentration of Y27632, a selective Rho
kinase inhibitor (n = 6), each significantly prevented
(P < 0.001) neurite outgrowth inhibition by diabetic
obstructive sleep apnea autoantibodies. Autoantibodies
in representative patients with obstructive sleep apnea
and either atrial fibrillation or left ventricular hypertrophy
evoked acute large increases in intracellular Ca2+
in HL-1 mouse atrial cardiomyocytes. The magnitude
of intracellular Ca2+ release was dose-dependently significantly
correlated to the electrocardiographic Cornell
voltage-duration product. Gel filtration of diabetic obstructive
sleep apnea autoantibodies revealed peak neurotoxicity
associated with MWs corresponding to IgG
light chain dimer(s), monomers or half-light chains as
well as a novel 5.5 kD putative light chain fragment.
These results suggest that diabetic obstructive sleep apnea
autoantibodies may induce strong depolarization in
neuronal cells and alter Ca2+ signaling in atrial cardiomyocytes
consistent with a role in pathophysiology
in subsets of diabetic obstructive sleep apnea having comorbid
atrial fibrillation or another clinically significant
cardiac rhythm disturbance.
Diabetes mellitus; Obstructive sleep apnea; Neurotoxicity;
Obstructive sleep apnea (OSA) is characterized by sleepdisordered
breathing and excessive daytime sleepiness . Obstructive
sleep apnea increases in adult type 2 diabetes (T2DM)
and may affect as many as four in ten older men with T2DM .
Visceral obesity is an important underlying risk factor in OSA ,
although the mechanism for the association is not well understood.
Airway patency is normally maintained by motor neuron
outflow to pharyngeal dilator muscles during wakefulness and
sleep . Since synaptic input to pharyngeal dilator motor neurons
normally decreases during sleep  a humoral factor which
suppresses local neuronal excitability might contribute to apnea
In prior studies, we reported circulating autoantibodies which altered
spontaneous activity in electrically-excitable cells. The autoantibodies
were increased in plasma of diabetic patients having
a cluster of complications including: painful neuropathy ,
atrial fibrillation , major depressive disorder ; and in cancer
In the present study we tested a hypothesis that plasma IgG autoantibodies
in diabetic obstructive sleep apnea cause long-lasting
neuronal depolarization compared to autoantibodies in diabetic
patients without obstructive sleep apnea. The mechanism of diabetic
OSA autoantibodies’ inhibitory effect on neurite outgrowth
in N2A neuroblastoma cells was evaluating using specific antagonists
of G-protein coupled receptors or the RhoA/ Rho kinase
Subjects and Methods
Diabetic obstructive sleep apnea and control patients
Informed consent was obtained from all study patients prior to
blood drawing. Patients were enrolled from the diabetes and endocrinology
outpatient clinics at the Veterans Affairs New Jersey
Healthcare System, East Orange, NJ. The baseline clinical characteristics
in the patients whose autoantibodies were tested for
ability to evoke neuronal depolarization are summarized in Table
1. Painful diabetic neuropathy is defined according to previously
reported criteria . Diabetic nephropathy is defined as urinary
Table 1:Baseline clinical characteristics in study participants
No OSA (n=17)
66.2 + 13.3
64.6 + 7.7
39.0 + 9.3
30.5 + 6.6
Painful neuropathy (yes/no)
Atrial fibrillation (yes/no)
OSA- obstructive sleep apnea; BMI- body mass index
^P-value from T-test (continuous variables) or c2 test
(dichotomous variables) comparing patients with or without OSA
albumin excretion 300 milligrams/gram creatinine or urinary
protein excretion 500 milligrams/ gram creatinine. Diagnostic
criteria and clinical evaluation in diabetic depression patients
were previously reported . A diagnosis of obstructive sleep
apnea was based on chart review and included patients with concurrent
use of a nasal or face-mask continuous positive airway
pressure (CPAP) device at night with or without diagnostic results
from overnight polysomnography testing. Moderate obstructive
sleep apnea is herein defined as an apnea-hypopnea index (AHI)
of 15-30 per hour; severe OSA as an AHI > 30 per hour. Morbid
obesity is defined as body mass index 35-40 kg/m2 or above with
associated medical co-morbidities and ‘super obesity’ as BMI 50
kg/m2 or higher.
Obstructive sleep apnea and atrial fibrillation or dementia
Pt 1: 59-year-old male with super obesity (BMI 60 kg/m2), type 2
diabetes and obstructive sleep apnea. He suffers from paroxysmal
atrial fibrillation, asthma, and pituitary macro-prolactinoma. Pt
2: 69-year-old male with morbid obesity, type 2 diabetes and severe
obstructive sleep apnea. He developed atrial fibrillation with
slow ventricular response requiring permanent pacemaker placement
Pt 3- 68–year- old male with super obesity (BMI 58 kg/m2),
type 2 diabetes, obstructive sleep apnea and permanent atrial fibrillation.
Results of overnight polysomnography testing included:
apnea-hypopnea index (AHI) 34 per hour, minimum O2 saturation
88%. Pt 4- A 68-year-old Caucasian male (with family history
of Alzheimer’s dementia) who suffered with obstructive sleep
apnea, moderate obesity, atrial fibrillation requiring pacemaker
placement, hypertension, diabetic nephropathy, painful neuropathy,
and depression. The patient died of unknown causes after
two-years’ follow-up observation and treatment.
Pt 5- A 68-year-old Caucasian male with morbid obesity, severe
obstructive sleep apnea, left ventricular hypertrophy, and hypertension.
He experienced several episodes of hard syncope re-
sulting in multiple rib fractures. He was diagnosed with bradycardia
secondary to sick sinus syndrome and was treated with
a dual chamber cardiac pacemaker. He underwent overnight
polysomnography: apnea/ hypopnea index (AHI) was 32.8 per
hour, his lowest oxygen saturation (O2 sat) was 72% and he experienced
more than 200 episodes of bradycardia with duration
of 392 minutes in 456 minutes total recorded time. He was doing
well until age 73 years, when he died of unknown causes.
Pt 6- A 73-year-old moderately- obese, African-American
male with atrial fibrillation, OSA and glaucoma, Overnight
polysomnography parameters included: AHI 33 per hour, and
minimum O2 saturation 79%.
Pt 7- 73-year-old obese Caucasian male with seizure disorder,
OSA, and diabetic depression who later developed dementia
Pt 8- 53-year-old thin adult-onset type 1 diabetes with seizure disorder,
schizophrenia, nephropathy, and dementia
Pt 9- 74-year-old obese type 2 diabetes male with microalbuminuria,
congestive heart failure and peripheral vascular disease
Baseline plasma samples were obtained from study participants
prior to the initiation of study procedures.
Protein A chromatography
Protein- A chromatography was carried out as previously described
. The protein-A-eluate fractions consisted of total IgG
isolated from plasma upon low pH elution from the protein-A column.
The active protein-A- eluate caused significant inhibitory
activity in endothelial cell survival.
Endothelial cell survival assay
Bovine pulmonary artery endothelial cells (Clonetics, Inc. San
Diego, CA) were grown in Medium 199 plus 10% fetal calf serum
and endothelial cell growth medium (EGM, Clonetics, Inc., San
Diego, CA). Endothelial cell number assays were carried out as
previously reported . After 48 hours’ incubation in the presence
of protein-A-eluate fractions, cells were washed with PBS
and processed for the colorimetric estimation of cell number, i.e.
cell associated acid phosphatase activity, as previously described
. Growth-promoting activity is expressed as a percentage of
the control cell number for cells grown in the absence of protein-
Mouse neuroblastoma cells
Mouse neuroblastoma N2A cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM), containing 10% fetal
bovine serum, (FBS) (Invitrogen, Carlsbad, CA) and penicillin/
streptomycin (100 U/ mL and 100 mg/ mL, respectively)
at 370C and 5% CO2. Cells were plated in 96- well plates for 3
days prior to membrane depolarization experiments.
N2A cell/ neurite out growth
Cells were plated at low density in 35 mm dishes. Next recombinant
human bFGF (10 ng/ mL) in the presence or absence of human
IgG test fractions were added to dishes in triplicate. Groups
of 50–100 cells/dish were counted 2 days after the addition of test
factors. Neurite outgrowth represents the percentage of N2A cells
expressing more than one neurite. A neurite is defined as a cell
process that is at least 2 cell diameters in length of the cell body.
Results are expressed as % N2A cells expressing neurites (which
represent the mean SD of triplicate determinations) compared
to neurite expression in cells grown with 10 ng/mL human bFGF,
but without added test protein-A-eluate fractions.
Membrane depolarization assays
After cell attachment, growth medium was removed and cells
were washed and then incubated in modified Tyrode’s solution
consisting of: 150 mM NaCl, 3 mM KCL and 30 mM HEPES,
10 mM D-glucose and 2 mM CaCL2, pH 7.4. Test fractions (human
IgG fractions) were added in the presence of 97 nM DiBAC4
(Molecular Probes, Eugene, OR) - as previously reported . Fluorescence
was measured after 5 min or longer at room temp using
a Fluoroskan Ascent FL (VWR, Inc., Franklin, MA); Ex = 485 nm,
Em = 538 nm. Results are expressed as percent of change in gross
fluorescence compared to cells to which no test protein-A-eluate
fractions were added.
HL-1 cell culture
HL-1 atrial cardiomyocytes were developed (and generously provided)
by Dr. William Claycomb (Louisiana State University Medical
Center, New Orleans, LA). They were maintained in 5%
CO2/ 95% air at 370C in Claycomb media (Sigma, St. Louis,
MO) containing 10% FBS (Biocell, Rancho Dominguez, CA), 100
U/mL:100 ug/ mL penicillin-streptomycin (Invitrogen, Carlsbad,
CA), 0.1 mM norepinephrine (Sigma, St. Louis, MO), and 2 mM
L-glutamine (Invitrogen, Carlsbad, CA).
Intracellular calcium measurement
HL-1 cells were grown in -dT3 dishes (Bioptechs, Inc., Butler, PA)
and loaded with 2 mM Fura-2 acetoxymethyl ester(Invitrogen,
Carlsbad, CA) for 30 min at 370C in a balanced salt solution (BSS)
(140 mM NaCl, 2.8 mM KCL, 2 mM CaCL2, 2 mM MgCL2, 10 mM
HEPES, pH 7.2) as previously reported . The cells were then
left for a further 10-minute period in the bathing solution at room
temperature to allow ester hydrolysis to go to completion. Using
a dual-wavelength spectrofluorometer (Photon Technology International,
Monmouth Junction, NJ) with excitation wavelengths
at 340 and 380 nm and emission at 510 nm fluorescence measurements
were performed at room temperature on the stage of
an inverted fluorescence microscope (Nikon TE200). The release
of intracellular Ca2+ was measured following exposure to 1mg/
mL concentrations of the protein-A eluate(s).
Size exclusion (gel filtration) chromatography
G75 superfine Sephacryl (Pharmacia, Piscataway, NJ) equilibrated
in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4 was
packed into a 5 x 0.7 cm column (Sigma, St. Louis, MO) having a
volume of 2.2 mL. An aliquot of the protein- A-eluate (0.05 mL)
was added to the column and eluted in 10 mM sodium phosphate,
0.15 M NaCl, pH 7.4 at a flow rate of 0.22 mL/ min, at 20C. Each
individual collected fraction (0.11 mL) was assayed for effects on
N2A neurite outgrowth. For the experiments involving inhibition
of endogenous MT1-MMP, protein- A- eluates (100 uL) were incubated
in the presence or absence of NSC 405020 (200 mM) in
50 mM HEPES, pH 6.8, containing 10 mmCaCl2, at 370C for 14
hours prior to size exclusion chromatography.
Protein- A agarose was obtained from Pierce Chemical
Co., (Rockford, IL). N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-
ylidene)methanaminehydrobromide (SCH-202676) and 3,4-
dichloro-N-(1-methylbutyl)-benzamide, (NSC 405020) were
from Sigma Chem. Co., Inc. (St Louis, MO). All other chemicals
and reagents were analytical grade.
Protein concentrations were determined by a bicinchoninic acid
protein assay kit (Pierce Chemical Co., Rockford, IL).
All data are the mean 1 SE as indicated. Comparisons were
made by Student’s t˘ test for a continuous variable, or by Chisquare
(c2) test for dichotomous variables with a significance
level, P = 0.05.
Baseline clinical characteristics in study groups
Body mass index was significantly increased in diabetes having
obstructive sleep apnea (39.0 vs 30.3 kg/ m2, P = 0.011, Table
1) compared to age-matched diabetic patients without obstructive
sleep apnea. Baseline atrial fibrillation was also significantly
increased in diabetes having OSA vs. no OSA (50% vs 11.8%, P
= 0.04, Table 1).
Depolarization induced by diabetic obstructive sleep apnea
Autoantibodies in diabetic obstructive sleep apnea (10 mg/mL)
caused a near doubling in the mean level of N2A depolarization
(129.1 vs 115.1 %, P = 0.012, Figure 1) compared to identical
concentrations of autoantibodies in age-matched diabetic patients
without OSA. These data are the first to suggest that circulating
autoantibodies in diabetes having obstructive sleep apnea cause
increased neuronal depolarization.
Neurite inhibition by diabetic obstructive sleep apnea
Autoantibodies in diabetes with obstructive sleep apnea (10
mg/mL) caused significantly greater inhibition of neurite outgrowth
in N2A cells (33% vs 50 %, P = 0.01, Figure 2A)
compared to identical concentrations of autoantibodies in agematched
diabetic patients without OSA. Co-incubation of diabetic
OSA autoantibodies (N = 5 patients) with (10mM) concentrations
Figure 1: Diabetic obstructive sleep apnea (OSA) auto
antibodies caused significantly greater mean depolarization
in N2A neuroblastoma cells compared to control diabetic
auto antibodies (No OSA). Results are mean +/- SE; P =
0.01 for the difference; N = number of participants in each
of the G protein- coupled receptor antagonist SCH-202676 significantly
prevented (P < 0.001) N2A neurite inhibition from the
autoantibodies (Figure 2B). Neurite outgrowth inhibition by diabetic
OSA autoantibodies (N = 6 patients) was also significantly
prevented (P < 0.001) by co-incubating N2A cells with 10 mM
concentrations of the selective Rho kinase inhibitor Y27632 (Figure
2C). Taken together, these results suggest involvement of one
or more G-protein coupled receptor(s) and downstream RhoA/
Rho kinase signaling in the mechanism of autoantibody-mediated
Long-term storage unmasks inhibitory endothelial cell
activity in diabetic OSA protein-A eluates
In prior studies, we reported that peak EC inhibitory activity and
neurotoxicity was unmasked following long-term storage (or furin
treatment) in diabetic protein- A- eluates from patients having
a co-morbid neurodegenerative disorder, i.e. glaucoma or dementia
. In the present study, storage (9-60 months at 0-40C) (n
= 5) unmasked significant EC inhibitory activity preferentially in
diabetic OSA protein- A- eluates compared to the stored protein-
A- eluates of diabetes without OSA (n = 17) (Table 2). Patient
subgroup having diabetes plus OSA included significantly more
glaucoma suspects (4/5 vs. 4/17; P = 0.04; Fischer’s exact test)
than the diabetes subgroup without OSA (not shown in Table 2).
Potent EC- inhibitory activity was also unmasked following storage
of a metastatic lung cancer protein- A- eluate (n = 1), or in
the freshly-isolated autoantibodies from breast cancer (n = 4),
but not in older adults without cancer or diabetes(n = 3) (Table
2). These data suggest a shared role for specific proteases
elaborated in certain cancers (or in OSA) in unmasking latent EC
inhibitory autoantibody activity.
Peak neurite-inhibitory in diabetic OSA autoantibodies:
In prior studies, peak neurotoxicity in cancer fatigue/depression
 and diabetic depression protein-A-eluates had apparent MWs
Figure 2: Diabetic obstructive sleep apnea (OSA) auto
antibodies A) caused significantly greater mean inhibition of
neurite outgrowth in N2A neuroblastoma cell compared to
control diabetic auto antibodies (No OSA). B) Inhibition of
neurite outgrowth by diabetic OSA auto antibodies (N= 5
pts) was completely blocked by co-incubation with (10 mM)
SCH-202676. C) Diabetic OSA auto antibodies’ neurite
inhibition (N=6) was also significantly antagonized by
co-incubated N2A cell with (10 mM) Y27632, a selective Rho
kinase inhibitor. Results are mean+/- SE.
corresponding to IgG light chains (23 kD) or half -light chains
(11.5 kD) . In the present study, peak neurotoxicity in diabetic
OSA/atrial fibrillation protein-A-eluates, (i.e. Pts 1, 2) had apparent
MWs of& 22 kD, and 5.5 kD (Figure 3A). An additional
neurotoxic peak in a third diabetic OSA/AF patient had apparent
MW11 kD (Pt 3 eluate; Figure 3B).
Diabetic OSA autoantibodies induce Ca2+ release in
HL-1 atrial cardiomyocytes
Protein A eluates (1 mg/ mL) from two representative diabetic
patients with OSA and either atrial fibrillation (Pt 4) or symptomatic
bradycardia requiring permanent pacemaker placement
(Pt 5) caused large increases in intracellular Ca2+ (Figure 4AB)
in atrial cardiomyocytes associated with interruption in spontaneous
Ca2+ oscillations (Figure 4A-B). Autoantibody-induced
intracellular Ca2+ release (in HL-1 cardiomyocytes) was present
in a significantly higher proportion of diabetic patients with comorbid
OSA plus atrial fibrillation or another symptomatic cardiac
dysrhythmia (6/7 vs. 1/6; P= 0.03; Fischer’s exact test)
compared to type 2 DM without OSA, AF or symptomatic cardiac
dysrhythmia (not shown in Figure 4). In an age-matched subset
of type 2 DM having AF or left ventricular hypertrophy, peak
Table 2:Unmasking of EC inhibitory activity in protein A eluates after
Diabetes & OSA (n=5)
98 + 4%
80 + 15%
Diabetes without OSA(n=17)
92 + 15%
93 + 26%
Diabetes & Lung cancer (n=1)
85 + 7%
28 + 10%
Breast cancer (n=4)
66 + 15%^^
Non-diabetic control (n=3)
102 + 3%
^T-test: comparing activity before and after storage
^^P = 0.02: compared to non-diabetic controls
amplitude of Ca2+ release (in HL-1 cells) was significantly correlated
with the Cornell voltage-duration product (r = 0.708, P =
0.007)(Figure 4C). The Cornell product is an estimate of the timevoltage
integral of the electrocardiographic QRS complex which
is useful in the ECG detection of left ventricular hypertrophy .
Possible involvement of MT1-MMP in unmasking neurotoxicity
in diabetic OSA autoantibodies
Membrane type 1 matrix metalloproteinase (MT1-MMP) is a
broad spectrum proteinase implicated in prostate, breast and lung
cancer invasion through its ability to degrade collagen and noncollagenous
proteins in the extracellular matrix [11, 12]. MT1-
MMP expression also increases in adipogenesis (e.g. morbid obesity)
, under atrial stretch conditions  and via effects of
pro-inflammatory cytokines in vascular cells . MT1-MMP has
catalytic andpexin domains which bind and unfold triple helical
collagen  prior to collagenolysis. NSC 405020 is a novel pexin
domain-specific MT1-MMP inhibitor . To test for involvement
of MT1-MMP in the generation of neurotoxic autoantibody fragments,
we incubated diabetic OSA/glaucoma (or/dementia) protein
A eluates (Pt 6, 7)in the presence or absence of half-maximal
inhibitory concentrations of NSC 405020 followed by gel permeation
chromatography. Protein-A- eluates not exposed to NSC
405020 displayed peak neurotoxicity having an apparent MW of
5.5 kD (Fig 5A). Protein- A- eluates exposed to NSC405020 displayed
a shift in peak neurotoxicity toward a higher MW species
( 43 kd) (Figure 5B).
Diurnal variation in plasma EC autoantibody growth activity
The 25-75% ammonium sulfate pellet fraction of adult microalbuminuric
diabetes plasma includes the IgG fraction and was
reported to contain FGF-like, EC growth stimulatory activity .
Plasma EC stimulatory activity displayed a diurnal variation: peak
activity (e.g. Pt 8, 9) occurred in the morning (0600 – 1000
hours) with much less, if any, stimulatory activity in the evening
Figure 3: Size exclusion (G75 Sephacryl) chromatography of
autoantibodies in three patients having diabetes, morbid
obesity, atrial fibrillation and obstructive sleep apnea.A)
Peak neurotoxicity in Patient 1, (pituitary prolactinoma)
protein-A eluate had MWs of > 100 kD and 5.5 kD; Patient 2
protein- A eluate had peak neurotoxicity associated with
MWs of 22 kD and 5.5 kD. B) Peak neurotoxicity in Patient 3
protein-A eluate had apparent MW of 11 kD.
(2000-2300 hours) (Figure 6A-B). Potent EC inhibitory activity
was present at nighttime (2300 hours) in a 53 year old, type 1 diabetic
patient suffering with co-morbid neuropsychiatric disorder
and dementia (Pt 8), but not at any other time point during a 24-
hour sampling period (Figure 6B).
Morbid obesity is strongly associated with obstructive sleep apnea
in older men  and women  although the underlying
mechanism(s) are unknown. Pro-inflammatory cytokines which
increase in visceral adiposity  cause increased expression of
inflammatory proteases, e.g. MT1-MMP  also implicated in
cancer invasion . The present data suggest involvement of an
unknown G-protein coupled receptor(s) and downstream RhoA/
Rho kinase signaling in the mechanism of autoantibody-induced
neurite outgrowth inhibition. Overlapping subfamilies of heterotrimeric
G- proteins, (i.e. Gq=11 or G12=13) not only mediate
RhoA-associated neurite retraction, but also couple to multiple
downstream signaling pathways leading to IP3-mediated intracellular
Ca2+ release  or cell depolarization . Our finding of
a dose-dependent association between 2+ release in atrial cardiomyocytes
and increased left ventricular mass (QRS voltageduration)
suggests the autoantibodies may be causally- related
to cardiac hypertrophy. Diabetic AF/OSA autoantibodies caused
quite large intracellular Ca2+ release in atrial cardiomyocytes indicative
of a role in altered Ca2+ signaling underlying clinicallysignificant
cardiac rhythm disturbances.
Hypertension and left ventricular hypertrophy both increase substantially
in obstructive sleep apnea . They cause cyclic
stretch in vascular endothelial cells  or static stretch in atrial
myocytes , respectively which activates MT1-MMP expression
Figure 4: Representative diabetic patients with obstructive
sleep apnea and either A) atrial fibrillation (Pt 4) or B) left
ventricular hypertrophy, sick sinus syndrome, and
symptomatic bradycardia (Pt 5) had auto antibodies which
evoked (at the arrows), acute large (1.5-2.5 fold) increases
in Ca2+ in HL-1 atrial cardiomyocytes. Similar results were
obtained in three or more experiments. C) Plot of Cornell
voltage- duration product as a function of intracellular Ca2+
release in HL-1 cells in a subset of thirteen diabetic patients
required for extracellular matrix remodeling. Integrin (b1) responds
to mechanical force, e.g. stretch, by activating G12/13
family heterotrimeric G- proteins capable of activating downstream
RhoA/ ROCK signaling . In a prior report, diabetic EC
inhibitory autoantibodies caused loss of endothelial cell adhesion
in association with stress fiber activation and RhoA/ Rho kinase
signaling . Although the receptor is unknown, the autoantibodies
may bind to site(s) in the extracellular matrix affecting
HSPG-integrin interaction(s) perhaps triggering intracellular signaling
Glaucomatous optic atrophy is characterized by remodeling of the
optic nerve head associated with increased local expression of
MT1-MMP . Our finding of a significant association between
latent neurite inhibitory activity in diabetic OSA autoantibodies
and glaucomatous cupping (i.e. glaucoma suspect) is novel and
perhaps consistent with a reported association between glaucoma
and OSA . It suggests that tissue-specific proteolytic remodeling
may contribute to optic disc neurite loss in part by unmasking
(latent) neurotoxicity in a subset of circulating autoantibodies.
Endothelial cell inhibitory activity in the stored or freshly-isolated
protein-A eluates of plasma in metastatic lung and breast cancer
(Table 2) may be consistent with elaboration of proteolytic activity,
(e.g. MT1-MMP or closely-related MMPs)reported in these
Peak neurotoxicity having apparent MWs of 11 kD, 23 kD
and 43 kD are characteristic of half-light chains, light chains
and LC dimers, respectively. Yet more study is needed to determine
whether a novel 5.5kD diabetic OSA autoantibody fragment
is derived from specific light chains. A preliminary search
Figure 5: Size exclusion (G75 Sephacryl) chromatography of
autoantibodies in two patients having diabetes, obstructive
sleep apnea, and a neurodegenerative disorder, i.e.
glaucoma (Pt 6), dementia and depression (Pt 7) following
incubation in the absence(A) or presence (B) of a specific
MT1-MMP inhibitor. A) Peak neurotoxicity in the
autoantibodies incubated without MT1-MMP inhibitor had
apparent MW of 5.5 kD. B) MT1-MMP inhibition was
associated with a shift in peak neurotoxicity toward higher
MW species, i.e. 43 kD perhaps corresponding to light chain
of database(s) of light chain variable genes revealed that lambda
gene family (II or III) members (i.e. 2e2.2 or DPL 23)contain
an unusual dibasic K-R amino acid pair at positions 52,53 in
complementarity- determining region 2 (CDR2) . DPL23 is a
l3 light chain variable gene which also contains a potential MT1-
MMP consensus recognition sequence, P-X-G-I , at amino acid
positions 55-58 immediately adjacent to CDR2. Cleavage near
dibasic residues within CDR2 might generate a light chain fragment
having the observed MW of 5-6 kD.
DPL23gene usage was reported in anti-neurotrophic viral glycoprotein
antibodies such as anti-rabies  and anti- HIV-1 antibody
light chains. The crystallographic structure of a DPL 23-
derived light chain component in anti-HIV-1 envelope antibodies
was solved by W.D. Tolbert, X. Wu, M. Pazgierand is reported
("4fze-id,") in the Protein Database, European Pazgier and Molecular
Biology Laboratory-EBI, (Welcome Genome Campus, Hinxton,
Cambridge shire, UK). The light chain has a secondary structure
which is rich in hydrogen-bonded, b turns spanning the
CDR2 region, i.e. amino acid residues 48-58 (Protein Database,
EMBL-EBI). A postulated function of the pexin domain in MT1-
MMP is to disrupt hydrogen- bonding in triple helical collagen
causing unfolding which permits access to the MT1-MMP catalytic
domain . DPL23 is among a restricted group of k or l LC
genes reported to encode anti-SS-A/Ro autoantibodies, such as
were isolated from salivary gland- infiltrating B cells in primary
Sjogren’s syndrome . Evidence for antigenic selection in anti-
SS-A/Ro autoantibody LCs includes a somatic mutation in the Vk
gene L6 which replaces asparagine (N) at position 51 with lysine
Figure 6: Diurnal variation in plasma endothelial cell
growth-promoting activity in the 25-75% ammonium sulfate
pellet fraction of adult type 1 or type 2 diabetes with (Pt 8)
or without (Pt 9) severe co-morbid neuropsychiatric and
neurodegenerative disorders. Nocturnal peak (@ 2300
hours) in endothelial cell inhibitory activity was specific to
autoantibody-containing fraction of plasma in type 1
diabetes having neuropsychiatric/neurodegenerative
co-morbidities. Protein- A eluate in Pt 8 previously caused
significant inhibition of neurite outgrowth in N2A cells .
(K) resulting in a dibasic KR amino acid pair in CDR2, analogous
to the dibasic KR pair in ‘unmutated’ DPL23 gene-encoded
LCs. It is of interest that a subset of anti-SS-A/ Ro auto antibodies
in lupus are disease-causative of neonatal congenital heart
blocks . Evidence for antigenic selection as a driving force in
certain pathogenic autoantibody LC fragments comes from "hypermutation"
in theDPL 23-derived, anti-HIV-1 envelope antibody
LC ("4fze") Among other amino acid replacements, the 4fze LC
CDR2 region contains an unusual aspartic acid tandem repeat
(DD) aa (positions 50-51) adjacent to the conserved KR dibasic
pair. Another l3 light chain variable gene product harboring
an unusually large number of acidic and charged residues within
CDR2 was reported as pathogenic in human membranoproliferative
glomerulonephritis through its ability to bind and prevent
complement binding to factor H . In lupus anti-DNA antibodies,
negatively-charged residues, i.e. aspartic acid, present in the
k LC CDR1 or CDR2 regions cause reduced binding to DNA 
which promotes self-tolerance and escape from clonal deletion.
Taken together, these findings suggest that high level of inflammation
(in HIV disease, cancer, systemic autoimmunity, morbid
obesity)may drive immunity to self-antigens leading to the selection
of a restricted subset of l light chain gene products prone to
cleavage(by inflammatory proteases) resulting in formation of a
neurotoxic fragment or fragments.
Two-fold increased neuronal depolarization evoked by diabetic
OSA autoantibodies is significant. It suggests a novel humoral
immune-mediated mechanism of disordered neural regulation of
breathing in obstructive sleep apnea affecting older morbidlyobese
type 2 diabetes. Our preliminary data showing peak plasma
neurotoxicity at nighttime is of interest. One possibility is enhanced
release of a protease which has a normal nocturnal pattern
of expression. One such candidate is MMP2, activated by
MT1-MMP, which showed increased expression at nighttime in
orchestrating physiologic desquamation of surface corneal epithe-
lial cells . Continuous positive airway pressure is an established
and proven effective treatment in reducing life-threatening
OSA-related complications , however, long-term compliance
with nasal or face mask CPAP may be possible in only approximately
one-half of affected patients . Pharmacologic modulation
of the neural- mediated pathway(s) involved in nocturnal
disordered breathing  could provide an alternative treatment
approach in non-anatomic forms of obstructive sleep apnea,
which requires more study.
A limitation of our study is that it may only pertain to older men
with type 2 diabetes. More study is needed in women in whom
morbid obesity (as opposed to anatomical upper airway narrowing)
is an important contributing factor in OSA causation .
In summary, diabetic obstructive sleep apnea was associated with
autoantibodies which caused strong depolarization in N2A cells as
well as mediating neurite retraction via a G-protein coupled receptor,
and RhoA/ ROCK downstream signaling pathway. A novel,
low MW (5.5 kD) putative light chain fragment was found in
diabetic OSA plasma in association with morbid obesity and complications
affecting electrically-excitable tissues.
In conclusion, the current data suggest a novel mechanism in
which circulating strongly depolarizing autoantibodies may contribute
to pathophysiology in a subset of diabetic obstructive sleep
Supported by grants from the Veterans Biomedical Research Institute
(East Orange, NJ) to MBZ and from the American Diabetes
Association (1-13-IN-40-BD) to ZP & MBZ, We thank Kalashree
Gopal for technical assistance.
- Punjabi NM, Sorkin JD, Katzel LI, Goldberg AP, Schwartx AR, Smith PL. Sleep-disordered breathing and insulin resistance in middle-aged and overweight men. Am J RespirCrit Care Med 2002;165(5):677–682. DOI:10.1164/ajrccm.165.5.2104087.
- Elmasry A, Lindberg E, Berne C, Janson C, Gislason T, Tageldin MA, et al. Sleep-disordered breathing and glucose metabolism in hypertensive men: a population-based study. J Intern Med. 2001;249(2):153–161.
- Strobel RJ, Rosen RC. Obesity and weight loss in obstructive sleep apnea: a critical review. Sleep. 1996;19(2):104–115.
- Kuna ST, Remmers JE. Neural and anatomic factors related to upper airway occlusion during sleep. Med Clin North Am. 1985;69(6):1221-1242.
- Zimering MB, Alder J, Pan Z, Donnelly RJ. Anti-endothelial and anti-neuronal effects from auto-antibodies in subsets of adult diabetes having a cluster of microvascular complications.Diabetes Res ClinPract. 2011;93(1):95-105. doi: 10.1016/j.diabres.2011.03.029.
- Zimering MB, Mirkovic N, Pandya M, Zimering JH, Behnke JA, et al. Toxic Immunoglobulin Light Chain Auto antibodies are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes. J EndocrinolDiab. 2016;3(1):1-14. DOI: dx.doi. org/10.15226/2374-6890/3/1/00141.
- Zimering MB, Behnke JA, Thakker-Varia S, Alder J. Auto antibodies in Human Diabetic Depression Inhibit Adult Neural Progenitor Cells In vitro and Induce Depressive-Like Behavior in Rodents. J EndocrinolDiab 2015;2(2):1-11.DOI:dx.doi.org/10.15226/2374-6890/2/2/00119.
- Zimering MB, Thakker-Varia S. Increased fibroblast growth factor-like auto antibodies in serum from a subset of patients with cancer-associated hypercalcemia. Life Sci. 2002; 71(25):2939-2959. DOI:10.1016/S0024-3205(02)02160-4.
- Zimering MB, Moritz TE, Donnelly RJ. Anti-neurotrophic effects from autoantibodies in adult diabetes having primary open angle glaucoma or dementia. Front Endocrinol (Lausanne). 2013;4:58. Doi:10.3389/fendo.2013.00058.
- Molloy TJ, Okin PM, Devereux RB, Kligfield P. Electrocardiographic detection of left ventricular hypertrophy by the simple QRS voltage-duration product. J Am CollCardiol. 1992;20(5):1180-1186.
- Sato H, Seiki M. Membrane-type matrix metalloproteinases (MT-MMPs) in tumor metastasis. J Biochem. 1996;119(2):209-215.
- Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, Basset P. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc Natl AcadSci U S A. 1995;92(7):2730-2734.
- Chun TH, Hotary KB, Sabeh F, Saltiel AR, Allen ED, Weiss SJ. A pericellular collagenase directs the 3-dimensional development of white adipose tissue. Cell. 2006;125(3):577-591.
- Saygili E, Rana OR, Meyer C, Gemein C, Andrzejewski MG, Ludwig A, Weber C, et al.The angiotensin-calcineurin-NFAT pathway mediates stretch-induced up-regulation of matrix metalloproteinases-2/-9 in atrial myocytes Basic Res Cardiol. 2009;104(4):435-448. doi: 10.1007/s00395-008-0772-6.
- Rajavashisth TB, Liao JK, Galis ZS, Tripathi S, Laufs U, Tripathi J, et al. Inflammatory cytokines and oxidized low density lipoproteins increase endothelial cell expression of membrane type 1-matrix metalloproteinase. J Biol Chem. 1999;274(17):11924-11929.
- d'Ortho MP, Will H, Atkinson S, Butler G, Messent A, Gavrilovic J,et al. Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem. 1997; 250(3):751-757.
- Remacle AG, Golubkov VS, Shiryaev SA, Dahl R, Stebbins JL, Chernov AV, Cheltsov AV,et al. Novel MT1-MMP small-molecule inhibitors based on insights into hemopexin domain function in tumor growth. Cancer Res. 2012;72(9):2339-2349. Doi:10.1158/0008-5472.CAN-11-4149.
- Zimering MB, Eng J. Increased basic fibroblast growth factor-like substance in plasma from a subset of middle-aged or elderly male diabetic patients with micro albuminuria or proteinuria. J ClinEndocrinolMetab. 1996;81(12):4446-4452.
- Guilleminault C, Quera-Salva MA, Partinen M, Jamieson A. Women and the obstructive sleep apnea syndrome. Chest. 1988;93(1):104-109.
- Lurie A. Inflammation, oxidative stress, and procoagulant and thrombotic activity in adults with obstructive sleep apnea. AdvCardiol. 2011;46:43-66. DOI:10.1159/000325105.
- Tinker A, Finlay M, Nobles M, Opel A. The contribution of pathways initiated via the Gq\11 G-protein family to atrial fibrillation. Pharmacol Res. 2016;105:54-61. Doi:10.1016/j.phrs.2015.11.008.
- Postma FR, Jalink K, Hengeveld T, Bot AG, Alblas J, de Jonge HR, et al. Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor. EMBO J. 1996;15(1):63-72.
- Cloward TV, Walker JM, Farney RJ, Anderson JL. Left ventricular hypertrophy is a common echocardiographic abnormality in severe obstructive sleep apnea and reverses with nasal continuous positive airway pressure. Chest. 2003;124(2):594-601.
- Cummins PM, von Offenberg Sweeney N, Killeen MT, Birney YA, Redmond EM, Cahill PA. Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with. Am J Physiol Heart Circ Physiol. 2007;292(1):H28-42. DOI:10.1152/ajpheart.00304.200.
- Zile MR, Baicu CF, Stroud RE, Van Laer A, Arroyo J, Mukherjee R, et al. Pressure overload-dependent membrane type 1-matrix metalloproteinase induction: relationship to LV remodeling and fibrosis. Am J Physiol Heart Circ Physiol. 2012;302(7):H1429-1437. DOI:10.1152/ajpheart.00580.2011.
- Takefuji M, Krüger M, Sivaraj KK, Kaibuchi K, Offermanns S, Wettschureck N. RhoGEF12 controls cardiac remodeling by integrating G protein- and integrin-dependent signaling cascades. J Exp Med. 2013;210(4):665-673.
- Zimering MB, Pan Z. Autoantibodies in type 2 diabetes induce stress fiber formation and apoptosis in endothelial cells. J ClinEndocrinolMetab. 2009;94(6):2171-2177.
- Agapova OA, Kaufman PL, Lucarelli MJ, Gabelt BT, Hernandez MR.Differential expression of matrix metalloproteinases in monkey eyes with experimental glaucoma or optic nerve transection.Brain Res. 2003;967(1-2):132-43.
- Shi Y, Liu P, Guan J, Lu Y, Su K. Association between glaucoma and obstructive sleep apnea syndrome: A meta-analysis and systematic review. PLoS ONE . 2015;10(2):e0115625. doi:10.1371/journal.pone.0115625.
- Williams SC, Frippiat JP, Tomlinson IM, Ignatovich O, Lefranc MP, Winter G. Sequence and evolution of the human germline V lambda repertoire. J Mol Biol. 1996;264(2):220-232.
- Ikematsu W, Kobarg J, Ikematsu H, Ichiyoshi Y, Casali P. Clonal analysis of a human antibody response. III. Nucleotide sequences of monoclonal IgM, IgG, and IgA to rabies virus reveal restricted V kappa gene utilization, junctional V kappa J kappa and V lambda J lambda diversity, and somatic hypermutation. J Immunol. 1998;161(6):2895-2905.
- Pelman GR, Morrison CJ, Overall CM. Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond J BiolChem. 2004;280(3):2370-2377. DOI:10.1074/jbc.M409603200.
- Suzuki H, Takemura H, Suzuki M, Sekine Y, Kashiwagi H. Molecular cloning of anti-SS-A/Ro 60-kDa peptide Fab fragments from infiltrating salivary gland lymphocytes of a patient with Sjögren's syndrome. BiochemBiophys Res Commun. 1997;232(1):101-106. DOI:10.1006/bbrc.1997.6233.
- Jokiranta TS, Solomon A, Pangburn MK, Zipfel PF, Meri S. Nephritogenic lambda light chain dimer: a unique human miniautoantibody against complement factor H.J Immunol. 1999; 163(8):4590-4596.
- Ibrahim SM, Weigert M, Basu C, Erikson J, Radic MZ. Light chain contribution to specificity in anti-DNA antibodies. J Immunol. 1995;155(6):3223-3233.
- Wiechmann AF, Ceresa BP, Howard EW. Diurnal variation of tight junction integrity associates inversely with matrix metalloproteinase expression in Xenopuslaevis corneal epithelium: implications for circadian regulation of homeostatic surface cell desquamation. PLoS One. 2014;9(11):e113810. DOI: 10.1371/journal.pone.0113810.
- Harbison J, O’Reilly P, McNicholas WT. Cardiac rhythm disturbances in the obstructive sleep apnea syndrome: effects of nasal continuous positive airway pressure therapy. Chest 2000;118:591–595.
- Martínez-García MA, Campos-Rodríguez F, Catalán-Serra P, Soler-Cataluña JJ, Almeida-Gonzalez C, De la Cruz Morón I, et al. Cardiovascular mortality in obstructive sleep apnea in the elderly: role of long-term continuous positive airway pressure treatment: a prospective observational study. Am J RespirCrit Care Med. 2012;186(9):909-916. DOI:10.1164/rccm.201203-0448OC.
- Taranto-Montemurro L, Edwards BA, Sands SA, Marques M, Eckert DJ, White DP, et al. Desipramine increases genioglossus activity and reduces upper airway collapsibility during non-REM sleep in healthy subjects. Am J RespirCrit Care Med. 2016; 194(7):878-885.