Abstract

   Increased methylglyoxal (MG) formation is associated with diabetes and
   its complications. In zebrafish, knockout of the main MG detoxifying
   system Glyoxalase 1, led to limited MG elevation but significantly
   elevated aldehyde dehydrogenases (ALDH) activity and aldh3a1
   expression, suggesting the compensatory role of Aldh3a1 in diabetes. To
   evaluate the function of Aldh3a1 in glucose homeostasis and diabetes,
   aldh3a1^−/− zebrafish mutants were generated using CRISPR-Cas9.
   Vasculature and pancreas morphology were analysed by zebrafish
   transgenic reporter lines. Corresponding reactive carbonyl species
   (RCS), glucose, transcriptome and metabolomics screenings were
   performed and ALDH activity was measured for further verification.
   Aldh3a1^−/− zebrafish larvae displayed retinal vasodilatory
   alterations, impaired glucose homeostasis, which can be aggravated via
   pdx1 silencing induced hyperglycaemia. Unexpectedly, MG was not
   altered, but 4-hydroxynonenal (4-HNE), another prominent lipid
   peroxidation RCS exhibited high affinity with Aldh3a1, was increased in
   aldh3a1 mutants. 4-HNE was responsible for the retinal phenotype via
   pancreas disruption induced hyperglycaemia and can be rescued via
   l-Carnosine treatment. Furthermore, in type 2 diabetic patients, serum
   4-HNE was increased and correlated with disease progression. Thus, our
   data suggest impaired 4-HNE detoxification and elevated 4-HNE
   concentration as biomarkers but also the possible inducers for
   diabetes, from genetic susceptibility to the pathological progression.

   Keywords: Aldh3a1, Reactive carbonyl species, 4-hydroxynonenal, Glucose
   homeostasis, Diabetes

Graphical abstract

   Image 1
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Highlights

     * •
       Aldh3a1 mutant was generated using CRISPR/Cas9 and displayed
       impaired glucose homeostasis.
     * •
       Elevated 4-Hydroxynonenal (4-HNE) was responsible for
       hyperglycaemia in aldh3a1 mutants and was rescued by Carnosine.
     * •
       Patient serum 4-HNE level was correlated with HbA1c and fasting
       glucose.
     * •
       Impaired 4-HNE detoxification acts as possible inducers for
       diabetes, from genetic susceptibility to pathological progress.

Abbreviations

   4-HNE
          4-hydroxynonenal

   AGEs
          advanced glycation end products

   ALDH
          aldehyde dehydrogenase

   Aldh3a1
          aldehyde dehydrogenase 3 families, member A1

   DR
          diabetic retinopathy

   IR
          insulin resistance

   MG
          methylglyoxal

   Pdx1
          pancreatic and duodenal homeobox 1

   RCS
          reactive carbonyl species

   ROS
          reactive oxygen species

   T2DM
          type 2 diabetes mellitus

1. Introduction

   The prevalence of diabetes in people aged 20–79 years is rising
   rapidly, from 151 million (4.6% of the global population) in 2000 to
   463 million (9.3%) in 2019 and is expected to 700 million (10.9%) in
   2045 [[44]1]. Diabetic patients have an increased risk of developing
   microvascular and macrovascular complications, which can cause
   blindness, kidney failure, and lower limb amputation [[45]2]. For
   instance, diabetic retinopathy (DR) is a common microvascular
   complication of diabetes and the leading cause of vision loss in
   working-age people [[46]3]. Since patients with severe degrees of DR
   suffer weakened life quality by decreased physical, emotional and
   social well-being and increased health-care resources [[47]4], early
   diagnose and timely treatment of DR is essential for preventing sight
   impairment and blindness.

   As a reactive carbonyl species (RCS), methylglyoxal (MG) is the main
   precursor of advanced glycation end products (AGEs) [[48]5]. MG is
   elevated in the plasma and tissue of diabetic patients [[49][6],
   [50][7]] and AGEs are strongly linked to the development of
   microvascular complications [[51]8,[52]9]. Apart from glyoxalase, the
   central MG detoxification system [[53]6], MG can also be detoxified by
   aldo-keto-reductase and aldehyde dehydrogenase (ALDH) [[54]10,[55]11].
   A recent study showed knockout of glyoxalase 1 (Glo1) only led to a
   1.5-fold MG elevation in zebrafish [[56]12]. Whereas a two-fold
   increase in ALDH activity and significantly raised aldh3a1 expression
   were observed in the glo1 mutants, suggesting Aldh3a1 as an alternative
   protein for the detoxification of corresponding RCS [[57]12].

   In addition to MG, Aldehyde dehydrogenase has a board spectrum of
   substrates of RCS such as acetaldehyde and 4-hydroxynonenal (4-HNE)
   [[58]13]. As the most prominent lipid peroxidation specific aldehydes,
   4-HNE has drawn significant attention during the last 40 years
   [[59]14], leading to several damaging effects such as apoptosis,
   mitochondrial dysfunction, inflammation and proteasome dysfunction in
   pathological condition [[60]15]. Due to the high reactive property,
   4-HNE is associated with the progression of several diseases, including
   but not limited to, Alzheimer's disease (AD), Parkinson's disease (PD),
   heart disease, atherosclerosis, cancer and diabetes [[61][16],
   [62][17], [63][18], [64][19], [65][20]].

   The role of 4-HNE in diabetes is not well understood and preliminary
   data also suggest concentration-dependent effects. Below the cytotoxic
   amount, 4-HNE reacts to high glucose by active peroxisome
   proliferator-activated receptor δ (PPARδ) complexes and ultimately
   increase the secretion of insulin in INS-1E beta cells [[66]21]. When
   the neutralization capacity of 4-HNE is exceeded, it modifies
   macromolecules structure, conformation and function, accompanied by
   AGEs and advanced lipoxidation end products (ALEs)’ elevation,
   resulting in β cell dysfunction [[67]22]. 4-HNE exhibits strongly
   correlation with diabetic nephropathy [[68]23], neuropathy [[69]24] and
   retinopathy [[70]25]. Yet, appropriate animal models for an endogenous
   4-HNE increase are missing to analyse its damaging mechanisms in vivo.

   Aldehyde dehydrogenase 3 families, member A1 (Aldh3a1) is a metabolic
   enzyme that oxidizes mainly toxic lipid peroxidation aldehydes to their
   corresponding carboxylic acids [[71]26]. Several studies have proven a
   high ALDH3A1 affinity for 4-HNE but low ability to detoxify
   malondialdehyde (MDA), in supporting its multifaceted function
   [[72][27], [73][28], [74][29]]. However, it remained unclear if a
   permanent knockout of Aldh3a1 causes increased 4-HNE concentration and
   results in diabetes and organ damage in the end.

   Therefore, the study aimed to evaluate the internal level of RCS and
   consequent effects of Aldh3a1 knockout on glucose metabolism and the
   retinal vascular system in zebrafish, while also to investigate the
   clinical relevance of 4-HNE in the sera of diabetic patients. Our data
   suggest impaired 4-HNE detoxification and elevated 4-HNE concentration
   as biomarkers but also the possible inducers for diabetes, from genetic
   susceptibility to the pathological progression.

2. Results

2.1. Sequence alignment of ALDH3A1 among different species and analysis of
the aldh3a1 mRNA expression in zebrafish

   ALDH3A1 enzyme system exists in human, mouse and zebrafish
   [[75]30,[76]31], but the similarity of the enzyme protein in the
   different species has not been described yet. At first, we aligned the
   amino acid sequence and the alignment showed zebrafish Aldh3a1 shares
   61.6% similarity of protein with human ALDH3A1 and 57.6% with mouse; as
   an enzyme-encoding gene, the active sites: cysteine and glutamic acid
   are the same in these three species ([77]Fig. 1A). Then, RT-qPCR was
   performed for a better understanding of the potential expression
   differences of aldh3a1 mRNA level in zebrafish regarding the
   development and organ distribution. Results showed an increased trend
   of aldh3a1 mRNA as the larva developed by around 3-fold elevation at
   96 hours after post fertilization (hpf) than 24 hpf. While in adult
   fish, brain and eye were the most expressed organs, with 11- and 7-fold
   significant elevation compared to heart ([78]Fig. 1B). Altogether, the
   data have identified the existence of aldh3a1 in zebrafish larvae and
   different adult organs, and the possibility for studying ALDH3A1 enzyme
   system by using zebrafish as a model.

Fig. 1.

   [79]Fig. 1
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   Sequence alignment of Aldh3a1 across different species and expression
   of aldh3a1 mRNA in zebrafish.

   (A). Amino acid alignment showed a high similarity and same active
   sites (red frame) between different species: First line, zebrafish
   Aldh3a1; Second line, human ALDH3A1; Third line, mouse ALDH3A1. (B).
   Heatmap of aldh3a1 mRNA expression in wild type zebrafish showed an
   increasing trend from 24 hpf to 96 hpf in larvae, and brain and eye
   were the most expressed adult organs. The higher and lower expression
   is displayed in pink and blue, respectively. Expression of genes was
   determined by RT-qPCR and normalized to b2m. The average values of 24
   hpf wild type larvae and heart (adult organs) were standardized to 1;
   Larvae: n = 3 clutches with 30 larvae, adult organs: n = 4 with one
   organ per sample. For statistical analysis one-way ANOVA followed by
   Sidak's multiple comparison test was applied, **p < 0.01, ***p < 0.001.
   RT-qPCR, real-time quantitative polymerase chain reaction; hpf, hours
   after post fertilization; b2m, β2 microglobulin. (For interpretation of
   the references to colour in this figure legend, the reader is referred