Study on the Molecular Mechanism of Berberine Regulating the Gut Microbiota-derived Metabolites 5-AVAB to Improve Type 2 Diabetes Mellitus Complicated With Depression.
Conditions: Diabetes-Related Complications
Sponsor: Affiliated Hospital of Nantong University

                                    
 
 
 
 
 
Study on the Molecular Mechanism of Berberine to 
Improve Type 2 Diabetes Mellitus Complicated With 
Depression (SBDD) 
 
Study Protocol with Statistical Analysis Plan 
 
NCT06979440 
 
18/5/2025 
 
 

                                    
 
 
Study Protocol with Statistical Analysis Plan 
 
1. Introduction 
1.1 Lack of Specific Therapeutic Interventions for Type 2 Diabetes Complicated with 
Depression 
According to the International Diabetes Federation, approximately 536.6 million 
individuals aged 20–79 worldwide were afflicted with diabetes in 2021, of whom 90% had type 
2 diabetes (T2D)[1]. Depression has emerged as a significant comorbidity of T2D, with 
epidemiological studies revealing that approximately 28% of T2D patients suffer from comorbid 
depression—a prevalence twice that of the general population[2]. The coexistence of T2D with 
depression exacerbates disease progression, impairs quality of life, diminishes treatment 
adherence, elevates mortality risk, and imposes substantial burdens on families and society. 
The underlying mechanisms linking T2D and depression remain incompletely elucidated. 
Notably, while depression incidence is notably higher in diabetic patients, no significant 
difference exists between individuals with impaired glucose metabolism and healthy controls, 
suggesting that hyperglycemia and related metabolic disturbances may not be the primary 
triggers of depression[3]. Moreover, antidepressant therapy alone fails to sustainably ameliorate 
hyperglycemia in T2D patients. Furthermore, several classes of antidepressants (e.g., selective 
serotonin reuptake inhibitors, SSRIs) are associated with weight gain, which may exacerbate 
T2D risk[4]. Given the absence of targeted pharmacotherapies for this comorbidity, deciphering 
its pathogenic mechanisms and identifying novel therapeutic targets hold urgent clinical 
significance. 
1.2 Gut Microbiota-Derived Metabolite 5-AVAB Exacerbates Hippocampal Neuronal 
Damage and Depression-like Behaviors in T2D with Depression 
The hippocampus serves as a critical neuropathological substrate in T2D with comorbid 
depression; hippocampal atrophy is a distinctive neuroimaging hallmark in affected patients[2]. 
Preclinical evidence demonstrates that diabetes diminishes hippocampal neurogenesis and 
neuroplasticity, thereby fostering the development of depressive symptoms[5]. The gut 
microbiota, a shared etiological factor in both T2D and depression[6], modulates hippocampal 

                                    
 
 
function through metabolic and multifactorial pathways[7]. Dysbiosis and increased gut 
microbial metabolites contribute to hippocampal dysfunction and behavioral impairments[8]. 
Nevertheless, the roles and mechanisms of gut microbiota and their metabolites in hippocampal 
neuronal injury in T2D with depression remain obscure. Thus, profiling characteristic gut 
microbiota and microbial metabolites in this comorbidity and exploring microbiota-targeted 
therapeutic strategies for hippocampal injury bear profound scientific and clinical implications. 
Among microbial metabolites, 5-aminovaleric acid betaine (5-AVAB) has garnered 
substantial attention as a novel bioactive molecule. 5-AVAB is a gut microbiota-derived 
metabolite increasingly implicated in metabolic diseases. A recent randomized controlled trial 
demonstrated that elevated plasma 5-AVAB levels correlate strongly with diabetes[9]. Notably, 
5-AVAB crosses the blood-brain barrier and induces neuronal dysfunction[10]. Furthermore, it 
promotes lipidotoxicity by attenuating mitochondrial β-oxidation, which may drive obesity-
related metabolic disorders[11]. These findings suggest that 5-AVAB may serve as a crucial 
mediator in diabetes-associated neuronal pathology. 
Endogenous 5-AVAB synthesis involves enzymes such as 5-aminovaleramidase (DavA) 
and L-lysine monooxygenase (DavB) and is positively associated with Pseudomonas, Bilophila, 
Coriobacteriaceae, and Enterococcus faecalis[10,12]. Reported microbial pathways for 5-
AVAB production include the trimethyllysine (substrate)-DavB-DavA cascade in 
Pseudomonas (Fig. 1-1)[13]. Our preliminary metabolomic analyses revealed significantly 
elevated fecal 5-AVAB levels in T2D with depression mice compared to controls. Moreover, 
oral gavage of 5-AVAB in C57BL/6J mice led to rapid cerebral accumulation, peaking 
within 5 minutes. In a high-fat/high-sugar diet + streptozotocin + chronic unpredictable 
mild stress (CUMS)-induced T2D with depression model, 4-week 5-AVAB administration 
(100 mg/kg) markedly exacerbated depression-like behaviors (see Preliminary Data 1). Thus, 
5-AVAB represents a potential novel biomarker promoting T2D with depression 
progression. Given the absence of prior investigations in this domain, further mechanistic 
exploration is imperative. 
Building upon our previous findings, we propose to collect serum and fecal samples from 
healthy controls and T2D with depression patients to establish a clinical microbiota model—
validating elevated 5-AVAB levels and elucidating its molecular mechanisms. 

                                    
 
 
References 
[1] Ong K L, Stafford L K, McLaughlin S A, et al. Global, regional, and national burden of 
diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis 
for the Global Burden of Disease Study 2021[J]. The Lancet, 2023, 402(10397): 203-234. 
[2] Tomic D, Shaw J E, Magliano D J. The burden and risks of emerging complications of 
diabetes mellitus[J]. Nature Reviews Endocrinology, 2022, 18(9): 525-539. 
[3] Nouwen A, Nefs G, Caramlau I, et al. Prevalence of Depression in Individuals With 
Impaired Glucose Metabolism or Undiagnosed Diabetes[J]. Diabetes Care, 2011, 34(3): 
752-762. 
[4] Gold S M, Köhler-Forsberg O, Moss-Morris R, et al. Comorbid depression in medical 
diseases[J]. Nature Reviews Disease Primers, 2020, 6(1): 69. 
[5] Ho N, Sommers M S, Lucki I. Effects of diabetes on hippocampal neurogenesis: Links to 
cognition and depression[J]. Neuroscience & Biobehavioral Reviews, 2013, 37(8): 1346-
1362. 
[6] Warma S, Lee Y, Brietzke E, et al. Microbiome abnormalities as a possible link between 
diabetes mellitus and mood disorders: Pathophysiology and implications for treatment[J]. 
Neuroscience & Biobehavioral Reviews, 2022, 137: 104640. 
[7] Kuijer E J, Steenbergen L. The microbiota-gut-brain axis in hippocampus-dependent 
learning and memory: current state and future challenges[J]. Neuroscience & 
Biobehavioral Reviews, 2023, 152: 105296. 
[8] Liu G, Yu Q, Tan B, et al. Gut dysbiosis impairs hippocampal plasticity and behaviors by 
remodeling serum metabolome[J]. Gut Microbes, 2022, 14(1): 2104089. 
[9] Haberbosch L, Kierszniowska S, Willmitzer L, et al. 5-Aminovaleric acid betaine predicts 
impaired glucose metabolism and diabetes[J]. Nutrition & Diabetes, 2023, 13(1): 17. 
[10] Mossad O, Nent E, Woltemate S, et al. Microbiota-dependent increase in δ-valerobetaine 
alters neuronal function and is responsible for age-related cognitive decline[J]. Nature 
Aging, 2021, 1(12): 1127-1136. 
[11] Liu K H, Owens J A, Saeedi B, et al. Microbial metabolite delta-valerobetaine is a diet-
dependent obesogen[J]. Nature Metabolism, 2021, 3(12): 1694-1705. 
[12] Haikonen R, Kärkkäinen O, Koistinen V, et al. Diet- and microbiota-related metabolite, 5-
aminovaleric acid betaine (5-AVAB), in health and disease[J]. Trends in Endocrinology & 
Metabolism, 2022, 33(7): 463-480. 
[13] Zhao M, Zhao L, Xiong X, et al. TMAVA, a Metabolite of Intestinal Microbes, Is Increased 
in Plasma From Patients With Liver Steatosis, Inhibits γ-Butyrobetaine Hydroxylase, and 
Exacerbates Fatty Liver in Mice[J]. Gastroenterology, 2020, 158(8): 2266-2281.e27. 
2. Study Design 
2.1 Research purpose and significance 
(1) Establish clinical microbiota and animal models to assess the efficacy of berberine-mediated 
inhibition of gut microbial 5-AVAB in ameliorating T2D with depression. 
(2) Validate 5-AVAB elevation in patients and delineate its mechanistic contributions. 

                                    
 
 
2.2 Study Framework 
(1) Investigate berberine's inhibitory effect on 5-AVAB production in in vitro gut microbiota 
from T2DM with depression patients 
① Enroll T2DM with depression (T2DD) and T2DM-only (Control) subjects. Collect fecal and 
serum samples to compare 5-AVAB levels. 
② Evaluate the ability of different concentrations of berberine (10/50/100 μg/mL) to inhibit 5-
AVAB production in fecal microbiota from T2DD patients. 
(2) Mechanistic Study of Berberine's Suppression of Key 5-AVAB-Producing Bacteria 
① Compare gut microbiota structure in fecal samples from clinical T2DD patients versus 
Control subjects. Perform correlation analysis to identify 5-AVAB-associated characteristic 
microbial taxa. 
② Identify and isolate 5-AVAB-producing bacterial strains. Screen for the key 5-AVAB-
producing bacteria. 
2.3 Study Subjects 
Control group: Patients with type 2 diabetes mellitus (T2DM) only 
T2DD group: Patients with type 2 diabetes mellitus complicated with depression (T2DD) 
2.4 Research Procedures  
(1) Study on berberine's inhibitory effect on 5-AVAB production by gut microbiota in T2DD 
patients  
This study will enroll two groups of participants, divided into control group and T2DD 
patient group, with 20 individuals in each group. Whole blood (5 mL) and fresh feces (5 g) will 
be collected from each participant, with whole blood used for subsequent serum extraction. LC-
MS/MS will be employed to measure and compare 5-AVAB content in samples from each group. 
The fecal samples will be homogenized with anaerobic culture medium, then incubated with 
berberine (10 μg/mL, 50 μg/mL, 100 μg/mL) for 2, 6, 12, and 24 hours respectively, followed 
by 5-AVAB measurement using LC-MS/MS. 
(2) Mechanism study of berberine's inhibitory effect on key 5-AVAB-producing bacteria  

                                    
 
 
Following metagenomic sequencing, bioinformatics analysis will compare gut microbiota 
in clinical T2DD patients and control subjects, analyzing differences in microbial species 
composition between groups. Highly abundant gut microbial taxa will be screened and correlated 
with fecal 5-AVAB content to identify the most significantly associated genera. Using 
culturomics, 5-AVAB-producing bacterial strains will be identified and obtained. Single colonies 
will be picked into LB liquid medium for growth at 37°C, supplemented with TML-d9 substrate 
(50 mg/mL) for continued culture. Concentration-time curves of 5-AVAB-d9 will be plotted for 
different bacterial strains to screen for key producers showing highest AUC values. Using sterile 
pure medium as control, the selected bacterial strains will be co-cultured with berberine (0, 50 
mg/mL) for 12 hours in vitro. UV-Vis spectrophotometer UV-2600 (Shimadzu, China) will 
compare OD620nm values between berberine-treated and control groups, while LC-MS/MS will 
measure 5-AVAB-d9 production. 
2.5 Evaluation Criteria 
No additional interventions are implemented in this study. Fecal samples are collected post-
defecation, and blood collection volume (5 mL) complies with ISO international standards. 
2.6 Data Management, Statistical Analysis Plan & Confidentiality Measures 
All study data will be stored on password-protected encrypted hard drives within dedicated 
research computers and retained for three years after study completion, accessible exclusively 
to authorized research personnel. Upon request, government regulatory bodies, institutional 
review boards, or ethics committees may access the data in accordance with regulations. De-
identified participant information and questionnaire responses may be utilized for future 
scientific research.III. Participant Recruitment (n=40–60). 
3. Participant Recruitment 
3.1 Inclusion Criteria 
(1) Aged 18–65 years (inclusive), regardless of gender; 
(2) Control group: Diagnosed with type 2 diabetes (T2D), with no history of depression or other 
psychiatric disorders; 
(3) T2DD patient group: Diagnosed with type 2 diabetes comorbid with depressive disorder 

                                    
 
 
(T2DD), where depression meets the DSM-5 diagnostic criteria for recurrent depressive disorder 
without psychotic symptoms or single-episode major depressive disorder (MDD), with a total 
score of ≥22 on the HAMD-17 scale; 
(4) Participants must read, fully understand the subject information, and sign the informed 
consent form. 
3.2 Exclusion Criteria 
(1) Those who do not meet the inclusion criteria; 
(2) No use of antibiotics, prebiotics, probiotics, enteral nutrition drugs, etc. within 3 months prior 
to diagnosis, and no such treatments during the study period; 
(3) Individuals with progressive severe diseases (e.g., cancer); 
(4) Those with severe aphasia, agnosia, or apraxia; 
(5) Long-term use of psychotropic medications in the previous month or participation in new 
drug trials in the past 3 months; 
(6) Pregnant or lactating women; 
(7) Individuals with alcoholism or drug abuse; 
(8) Poor mental condition, unable to cooperate; 
(9) Any other conditions deemed unsuitable for participation in the study. 
3.3 Withdrawal and Termination Criteria 
If a participant discontinues the study prior to sample collection, they will be considered 
withdrawn/terminated from the study. 
3.4 Study Participation Duration (Per Session and Total Time) 
Participation in the study is defined as the period from enrollment to the completion of 
sample collection. 
3.5 Recruitment Process 
Recruitment for this study will commence only after obtaining approval from the Ethics 

                                    
 
 
Committee of Affiliated Hospital of Nantong University. Participants will be recruited from the 
Endocrinology and Neurology Departments of the hospital, with an anticipated enrollment of 
40–60 individuals. No financial compensation will be provided. 
4. Risk/Benefit Assessment 
4.1 Benefits 
This study aims to establish the gut microbial metabolite 5-AVAB as a novel biomarker for 
diagnosing type 2 diabetes with comorbid depression (T2DD). The findings may advance 
diagnostic techniques and the development of related testing kits, as well as facilitate the 
development of new therapeutics for T2DD. Additionally, this research could provide evidence 
for identifying therapeutic targets in the comorbidity of chronic diseases such as diabetes and 
depression. 
4.2 Risks 
Since this study does not involve additional interventions beyond standard care, 
participation is unlikely to cause physical, psychological, or social harm beyond routine clinical 
management. Furthermore, it will not negatively impact disease diagnosis or treatment. 
4.3 Protection of Special Populations 
No additional interventions are applied in this study. Special populations (e.g., vulnerable 
individuals) must review and fully comprehend the subject information with the assistance of a 
family member before providing written informed consent. 
5. Ethical Considerations in the Study 
(1) Patient Privacy Protection: 
The study may involve sensitive information from participants, necessitating strict 
confidentiality measures. 
Solution: Implement rigorous privacy protection protocols, including data anonymization and 
restricted data access. 
(2) Informed Consent: 
Participants must fully comprehend the study's purpose, methodology, potential risks, and 

                                    
 
 
benefits before voluntary enrollment. 
Solution: Obtain explicit informed consent prior to study commencement, accompanied by 
detailed participant information materials. 
6. Annual Research Plan 
This project is scheduled for completion over three years. 
Year 1 (January 2025 – December 2025): 
Recruit T2DD patients and healthy controls; prepare for clinical sample collection. 
Complete annual progress report and draft relevant manuscript sections. 
Year 2 (January 2026 – December 2026): 
Conduct clinical sample collection and laboratory testing. 
Complete annual progress report, finalize related manuscripts, and initiate journal submissions. 
Year 3 (January 2027 – December 2027): 
Finalize all clinical sample collection and testing. 
Submit annual progress report and publish research findings. 
7. Additional Notes 
7.1 Collaborations 
This study is funded by the National Natural Science Foundation of China (NSFC, Grant 
no. 82404740), Jiangsu Pharmaceutical Association ("Yao" Yan Xin Sheng Project, Grant no. 
202495102). 
7.2 Publication Plans & Authorship 
Research findings will be disseminated through peer-reviewed scientific papers. Authorship 
will be determined based on intellectual contribution.