Abstract
Aim Dysfunctional adipocytes and β-cells are crucial in the advancement of obesity-induced type 2 diabetes (T2D). Calorie restriction (CR) improves insulin sensitivity and fasting blood glucose (FBG) levels, while γ-aminobutyric acid (GABA), a neurotransmitter secreted by β-cells, exerts regenerative effects.
Methods High-fat diet (HFD) and streptozotocin (STZ) induced T2D mouse model was used to assess the effect of CR and GABA combination therapy. The mice were fed with CR diet (30% reduction of HFD) and treated with GABA (2.5 mg/kg i.p) daily for 5 weeks. Assessment of FBG, body weight (BW), insulin sensitivity, glucose tolerance, and estimation of plasma insulin and lipid profile were carried out by standard methods. The mRNA expression of target genes [liver (glucoregulatory enzymes), adipose tissue (lipid metabolism markers) and skeletal muscle (mitochondrial biogenesis markers)] was assessed by SYBR Green-qPCR. Oxygen consumption rate for mitochondrial complex I, II and III was measured in skeletal muscle using Oxytherm Clark-type oxygen electrode. The effect of monotherapies and combination treatment on pancreatic β-cell regeneration and apoptosis was analysed by IHC.
Results CR+GABA group showed significant reduction in FBG levels, improved lipid profile and whole-body insulin responsiveness as indicated by increased insulin levels, insulin sensitivity and glucose tolerance compared to the monotherapies. The transcript expression profile of glucoregulatory enzymes in the liver showed significant reduction in G6Pase, PEPCK, Glycogen Phosphorylase and increased GCK expression in CR+GABA group as compared to the monotherapies The ACC and ATG expression was up-regulated in adipose tissue; and significant increase in SIRT-1, PGC-1α and TFAM expression with up-regulated mitochondrial complex I and III activities was observed in CR+GABA group as compared to the monotherapies. Percentage of BrdU/Insulin and PDX1/Ngn3/Insulin co-positive cells was significantly higher in CR+GABA treated group than the monotherapy and HFD+STZ groups. Finally, there was a significant reduction in TUNEL/Insulin co-positive cells suggesting reduced apoptosis in CR+GABA treated group.
Conclusion Our results suggest that CR in combination with GABA ameliorates T2D in HFD+STZ treated mice, by GABA induced β-cell regeneration and CR mediated insulin sensitivity.
1. Introduction
Dysfunctional adipocytes and β-cells are involved in advancing obesity-induced type 2 diabetes (T2D) [1]. Several aspects regulate the progression of T2D, such as genetic predisposition [2-9], excess calorie intake [1, 10] and sedentary lifestyle [10], all of which cause persistent hyperglycemia and eventual β-cell apoptosis. Even though there have been many recent management therapies for T2D, none can withstand the worsening condition of β-cell loss. This wide gap has triggered the search for alternative remedies that can regenerate β-cells from either stem cells or existing β-cells and bring about insulin sensitivity.
Calorie restriction (CR), an effective dietary intervention for T2D, has been associated with longevity. CR reduces caloric intake, typically by 20–40% of ad libitum consumption, while maintaining adequate intake of protein and micronutrients to avoid malnutrition [13]. CR attenuates the degree of oxidative stress [14, 15] and increases the transcript levels of genes involved in mitochondrial function and biogenesis [15]. It also improves insulin sensitivity [13, 14], FBG, other cardiometabolic risk factors, and reduces pro-inflammatory adipokines and total cholesterol [16].
γ-aminobutyric acid (GABA) has emerged as a new antidiabetic dietary supplement. GABA, a neurotransmitter, is secreted by the central nervous system (CNS) and pancreatic β-cells. It mediates protective and regenerative effects by reducing β-cell apoptosis and increasing its replication rate [17, 18]. GABA exerts its action via two primary receptors GABAA and GABAB, which regulates insulin and glucagon secretion in the pancreatic islets [19]. GABA has shown its type 1 diabetes ameliorating effect by suppressing Treg cells in long-term therapy where GABA has demonstrated induction of β-cell regeneration by proliferation and transdifferentiation [20, 21].
We have investigated the combinatorial effect of CR and GABA in the T2D mouse model induced by high-fat diet (HFD) + streptozotocin (HFD+STZ). To evaluate the effect of CR and GABA on multiple targets and regulatory pathways, we assessed i) insulin sensitivity, glucose tolerance, and metabolic profile, ii) transcript levels of key genes involved in hepatic glucoregulation and lipid metabolism in adipose tissue (AT) and mitochondrial biogenesis in skeletal muscle (SK), iii) ETC complex I, II and III activities in SK, and iv) regenerative markers in β-cells.
2. Materials and Methods
2.1 Animals and experimental strategy
2.1.1 Ethics statement
The experimental procedures were approved by the Institutional Ethical Committee for Animal Research (IECHR), Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India (FS/IECHR/2016-9).
2.1.2 Animals
Male C57BL/6 mice (∼8-10 weeks old, approximately 20-22 grams) were procured from ACTREC, Mumbai. They were housed at 23.0±1°C with a 12-hrs light/dark cycle and acclimatised for one week. These animals had free access to standard chow/HFD/CR diet (Keval Sales Corporation, Vadodara, India) and water.
2.1.3 Development of T2D mouse model
The mice were divided into two groups. The control group (n=6) was fed with a normal chow diet (NCD). Obesity can induce multiple damaging effects on peripheral tissues. Working on similar lines, mice (n=25) were fed with HFD for 20 weeks to induce obesity and insulin resistance. After 20 weeks, these mice received three consecutive doses of 40 mg/kg BW STZ i.p. (MP Biomedicals, India) to induce β-cell loss. The HFD+STZ model is characterised by a significantly increased weight (≥30 grams), hyperglycemia (FBG ≥300 mg/dL), hyperlipidemia, insulin resistance and β-cell loss [24].
2.1.4 Treatment
The HFD+STZ treated animals were divided randomly into four groups (4-5 mice/group): 1. Diabetic control (HFD+STZ) 2. CR diet-fed (30% reduction of HFD) [22, 23] 3. GABA treated (2.5 mg/kg bw i.p) (Sigma–Aldrich, United States) [21] and 4. CR+GABA treated (30% reduction of HFD + GABA, 2.5 mg/kg bw i.p). Treatment was given daily for five weeks along with 100 mg/kg bw i.p BrdU (MP Biomedicals, India) on alternate days. CR diet comprised of 39% casein, 21% lard, 19 % sucrose, 16% vitamin-mineral premix, and 5% cellulose. The timeline is depicted in figure S1.
2.2 Metabolic and biochemical assessment
2.2.1 Metabolic profiling
FBG was measured by tail snipping weekly using glucometer (TRUEresult, Nipro, India) along with body weight (BW) as well as food and water intake. At the end of the experiment, 1 ml of blood was collected from the orbital sinus for the biochemical assays after 5 hrs of fasting. Plasma was separated and used for assessing lipid profile [total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL)] by using commercial kits (Reckon Diagnostics P. Ltd, India). Friedewald’s (1972) formula was used for calculating low-density lipoprotein (LDL).
2.2.2 Intraperitoneal Glucose Tolerance Test (IPGTT) and Intraperitoneal Insulin Sensitivity Test (IPIST)
IPGTT and IPIST were performed to evaluate glucose tolerance and insulin sensitivity at the end of 5 weeks of drug treatment. Mice were fasted for 6 hrs and injected with glucose (2g/kg BW i.p) or insulin (0.5U/kg BW i.p) for IPGTT and IPIST [25] respectively. Blood glucose levels were measured at the specified time points 0, 30, 60, 90 and 120 mins after glucose or insulin injection using glucometer.
2.2.3 Assessment of insulin and C-peptide levels
The plasma levels of insulin and C-peptide in the experimental animals were measured using commercially available ELISA kits (RayBio, USA). All the plasma estimations were carried out in duplicate to ensure the coefficient of variation (CV) was below 10%.
2.3 Gene expression profiling
After sacrificing the mice, peripheral tissues such as liver, AT and SK were dissected and stored in RNAlater™ Stabilization Solution (Thermo Fisher Scientific, USA). Total RNA was extracted by the Trizol method as described previously [8]. The expression of targeted genes and GAPDH transcripts were monitored by LightCycler®480 Real-time PCR (Roche Diagnostics GmbH, Germany) using gene-specific primers (Eurofins, India) [Table S1]. Expression of GAPDH gene was used as a reference. Real-time PCR was performed as described previously [5].
2.4 Estimation of Oxygen Consumption Rate (OCR) of mitochondria
Skeletal muscle was isolated from left thigh of the mouse and mitochondria were isolated from using mitochondria isolation kit (Thermo Scientific TM, Catalog no. 89801) using manufacturer’s protocol. The isolated mitochondria were resuspended in mitochondria respiration buffer (80 mM KCl, 0.1% BSA, 50 mM HEPES, 2 mM MgCl2 and 2.5 mM KH2PO4; pH 7.2). Outer membrane integrity of the isolated mitochondria was evaluated by impermeability to exogenous cytochrome c which was constantly >95%. The activities of respiratory chain complexes I-III were recorded using 100 mM Pyruvate & 800 mM Malate (complex I), 1M Succinate (complex II) and 10 mM α-glycerophosphate (complex III) and, the protein concentration was estimated by Lowry et al method. OCR was determined by measuring the amount of oxygen (nmol) consumed, divided by the time elapsed (min) and the amount of protein present in the assay [26].
2.5 Pancreatic tissue preparation, Immunohistochemistry (IHC), assessment of β-cell regeneration and apoptosis
At the end of 5 weeks of drug treatment, animals were sacrificed, pancreatic tissues were fixed in 10% formalin and were processed for paraffin embedding. 5µm sections were cut from the paraffin-embedded blocks. To perform IHC, these sections were deparaffinised in100% xylene and washed serially in ethanol grades (100%, 95%, 80% and 70%) and proceeded with an antigen retrieval step (1N HCl at 37°C for 45 min). Subsequently, these sections were blocked with 5% donkey serum made in phosphate buffer saline (PBS) [Jackson ImmunoResearch Laboratories, Inc. USA], and antibodies were diluted in the blocking reagent. The details of the antibodies are as indicated in table S4. The sections were incubated with primary antibody for one hour at 37°C, washed with PBS, and incubated with secondary antibody for 45 min at 37°C. These sections were stained with anti-fade DAPI (Thermo Fisher Scientific, USA). The sections were covered with a coverslip. The sections were visualised under a confocal microscope (ZEISS LSM, Oberkochen, Germany) at 60X, and the images were processed by Image J (NIH, USA). Immunofluorescence staining (IFS) with anti-insulin and anti-BrdU was used to monitor β-cell proliferation. IFS with anti-insulin, anti-PAX-4 (paired box gene 4), and anti-ARX (Aristaless Related Homeobox) was carried out for α-cell to β-cell transdifferentiation while anti-insulin, anti-NGN-3 (Neurogenin 3) and anti-PDX-1 (pancreas/duodenum homeobox protein 1) were considered for β-cell neogenesis. IFS anti-insulin and TUNEL/anti-AIF (Apoptosis Inducing Factor) monitored β-cell death. Results obtained were expressed as the percentage of specified markers for regeneration and death.
2.6 Statistical analyses
The data was analysed and expressed as the mean ± SEM, and p<0.05 was considered statistically significant. The inter-group analysis was carried out by one-way ANOVA, followed by Tukey’s test for multiple group analysis. All the analyses were carried out using the Prism software 6 (GraphPad Prism, San Diego, CA, USA).
3. Results
3.1 HFD+STZ induced T2D mouse model establishment
After 20 weeks of HFD treatment, animals turned obese and insulin resistant. There was a significant increase in BW (Table S2a; Figures S1a&b). After two weeks of STZ administration, the mice turned hyperglycaemic as indicated by FBG levels ≥300 mg/dL (Table S 2b; Figures S1 c &d).
3.2 Metabolic and biochemical assessment
3.2.1 Metabolic profiling
GABA treated group showed significantly reduced FBG levels (p<0.01), but no change was observed in BW, while CR monotherapy showed improvement in BW (p<0.05) but not in FBG levels. The BW (p<0.05) and FBG (p<0.01) levels of the CR+GABA treated group were significantly reduced at the end of 5 weeks of drug treatment compared to the HFD+STZ group, as shown in Figure 1.
3.2.3 Lipid profiling
The GABA treated group showed no significant difference in the lipid profile (p>0.05), while CR fed mice (p<0.05) showed reduced TG levels as compared to HFD+STZ group. TC, TG and LDL levels were significantly reduced in CR+GABA treated group (p<0.05) as compared to HFD+STZ group (Figure 3).
3.2.2 Glucose tolerance and insulin sensitivity
To further test our hypothesis, we performed IPGTT and IPIST post-treatment. CR monotherapy did not show an improvement in glucose tolerance and insulin sensitivity. GABA (p<0.05) and CR+GABA (p<0.01) treated groups showed a significant drop in the blood glucose levels by 120 mins as compared to HFD+STZ group (Figure 2A). The AUC plot showed a significant increase in glucose tolerance in GABA and CR+GABA treated groups (p<0.05) (Figure 2B). CR+GABA (p<0.01) treated group showed improved insulin sensitivity as the blood glucose levels were reduced to normoglycemia by 120 mins as compared to HFD+STZ group (Figure 2C & D).
3.2.4 Insulin and C-peptide levels
A significant increase in insulin and c-peptide levels along with improved insulin/c-peptide ratio was observed in GABA & CR+GABA (p<0.05) treated groups as compared to HFD+STZ group (Figure 4 A-C). No significant difference was observed in CR group.
3.2 Gene expression profiling
GABA treated group showed a significant decrease (p<0.05) in the expression of phosphoenolpyruvate carboxykinase (PEPCK-gluconeogenesis), glucose-6 phosphatase (G6Pase), and glycogen phosphorylase (GP-glycogenolysis) in the liver (Figure 5). However, there was no significant difference in the expression of adipose triglyceride lipase (ATGL-lipolysis) and acetyl-CoA carboxylase-1 (ACC-1-lipogenesis) in the adipose tissue (p>0.05) (Figure 6). Also, there was no significant difference observed in sirtuin-1 (SIRT-1), PPARG coactivator 1-α (PGC-1α), and transcription factor A mitochondrial (TFAM-mitochondrial biogenesis) transcript levels in the skeletal muscle (p>0.05) (Figure 7). CR fed mice showed a significant decrease (p<0.05) in the expression of G6Pase, PEPCK (gluconeogenesis), GLUT2 and GP (glycogenolysis) in the liver (Figure 5). However, there was no significant difference observed in the expression of ATGL and ACC-1 in adipose tissue of CR fed mice (p>0.05) (Figure 6). Further, CR fed mice showed a significant increase (p<0.05) in PGC-1α transcript levels in skeletal muscle (Figure 7). CR+GABA treated group showed a significant decrease (p<0.05) in the expression of G6Pase, PEPCK (gluconeogenesis), GLUT2 and GP (glycogenolysis) genes, and a significant increase (p<0.05) in glucokinase (glycolysis) expression (Figure 5). Interestingly, lipogenesis got up-regulated as marked by the increased ACC-1 expression. Consequently, as a compensatory mechanism, lipolysis was also up-regulated, as observed by the increased ATGL expression (p<0.05) (Figure 6). CR+GABA treated group also showed a significant increase (p<0.05) in SIRT-1, PGC-1α and TFAM transcript levels in skeletal muscle (Figure 7).
3.4 Oxygen consumption rate
The rate of oxygen consumption is a significant indicator of mitochondrial activity. GABA group showed no significant difference in the ETC complex activities in skeletal muscle (p>0.05). CR fed mice showed a significant increase (p<0.05) in OCR by ETC complex I compared to the HFD+STZ group, indicating improved complex I activity in skeletal muscle. The combination therapy group showed significant OCR by ETC complexes I-III as compared to the HFD+STZ group indicating improved complex activities in skeletal muscle (p<0.05) (Figure 8).
3.5 Pancreatic β-cell regeneration and apoptosis
β-cell proliferation and apoptosis were assessed by IHC microscopy. The GABA treated group showed a significant increase in β-cell proliferation [BrdU/Insulin co-positive cells] (Figure 9) as compared to NCD (p<0.01) and HFD+STZ treated groups (p<0.001). A similar trend was observed for neogenesis [PDX-1/NGN-3/Insulin co-positive cells] (Figure 10) as compared to NCD (p<0.001) and HFD+STZ treated groups (p<0.001). Also, GABA treated group showed a significant reduction (p<0.01) in β-cell apoptosis [TUNEL positive cells] (Figure 12). However, the CR monotherapy group showed no β-cell regeneration, and there was no improvement in β-cell apoptosis (p>0.05) (Figure 9-12). The CR+GABA treated group showed a significant increase as compared to NCD and HFD+STZ in β-cell proliferation (p<0.001; p<0.001) (Figure 9) and neogenesis (p<0.001; p<0.001) (Figure 10). The CR+GABA treated group showed significantly reduced β-cell apoptosis as compared to HFD+STZ (p<0.001) (Figure 12). The GABA and CR monotherapies, along with the GABA+CR group, did not show transdifferentiation (ARX/PAX4/Insulin co-positive cells) (p>0.05) (Figure 11). Further, to validate β-cell apoptosis we used AIF as a marker. However, no AIF translocation was observed in any of the groups (Figure 13).
4. Discussion
The management of T2D involves two main approaches: i) increasing insulin secretion from the β-cells and ii) increasing insulin-mediated glucose uptake by the peripheral tissues. Patients develop tolerance against the existing line of treatment within a few years, which poses a challenge for developing new medicines. These drugs also lead to many side-effects [1]. Thus, combination therapies involving naturally existing biomolecules are the way forward to overcome the complications of T2D. Therefore, the present study was designed to determine the therapeutic potential of a combination of CR and GABA in the T2D mouse model.
CR is a potential dietary intervention emphasizing caloric management in terms of fats, carbohydrates and proteins with sufficient vitamins and minerals. CR promotes insulin sensitivity, mitochondrial biogenesis and functionality [13, 15]. Mice fed with 30-40% of the ad libitum calorie deficit diet showed increased life expectancy, robustness against toxicity and stress [27]. Further data on similar lines showed CR’s role in reducing body weight and lipid profile markers when the therapy was prolonged [28]. Several reports have explained CR’s role as an activator of the SIRT1-PGC-1α signalling pathway at the transcriptional level [30]. CR modulates lipid metabolism via the SIRT-1 signalling pathway and reduces intracellular diacylglycerol species [29]. In the liver, PGC-1α may regulate the gluconeogenic pathway; however, the molecular mechanism remains unknown [31].
Interestingly, we have also seen similar observations of action at multiple sites with CR monotherapy reducing body weight and triglyceride levels and improving insulin sensitivity besides reducing gluconeogenesis and glycogenolysis. Civitarese et al. studied the molecular mechanism of CR in mitochondrial functionality. They explained an increased mRNA expression of the genes involved in mitochondrial biogenesis (viz. SIRT1, PGC-1α and TFAM), with SIRT1 being the direct regulator of the pathway [32]. Increased mitochondrial biogenesis and oxygen consumption rate by ETC complex I in our CR monotherapy group find agreement with their findings.
GABA has a proven role in islet□cell hormone homeostasis, preserving the β□cell mass, suppressing immune reactions and consequent apoptosis [19, 33]. The food sources of GABA are potatoes, tomatoes and brown rice, while microorganisms like E. coli, Lactococcus lactis produce it [35]. In the present study, GABA monotherapy effectively reduces FBG levels, improves glucose tolerance, increases insulin and c-peptide levels and reduces gluconeogenesis and glycogenolysis. GABAnergic system functions in many tissues, including the peripheral tissues. In the liver, GABAAR regulates PI3/Akt activities that maintain hepatocyte survival and PGC-1α expression. Thus, GABA is one of the multiple factors responsible for regulating hepatic glucose metabolism, i.e. gluconeogenic and glycogenolytic pathways [36]. Fibroblast growth factor 21 (FGF-21) predominantly expressed in the liver under normal condition also induces PGC-1α expression. It leads to the regulation of lipolysis in the adipose tissue forming liver-adipose tissue crosstalk [37]. The positive effect of GABA treatment stands documented in T1D and T2D murine models. GABA therapy protects NOD animals from diabetes, and a similar effect is reported in various in-vivo models [20, 17]. Also, GABA regulates cytokine secretion from human PBMCs and suppresses β-cell□reactive CD8+ CTLs in T1D models [19, 17], proposing the role of GABA as an immunosuppressant. GABA reportedly acts as an inducer of α-to-β-like cell conversion in-vivo upon prolonged exposure in the STZ-induced mouse model [19]. Although no β-cell transdifferentiation is seen in our GABA-treated group, a significant increase in β-cell proliferation and neogenesis and reduced β-cell apoptosis were observed. The individual effects of CR and GABA on the multiple pathways and tissues diminishe the adverse actions of T2D pathophysiology when given in combination. Our results suggest that the combination treatment improves glucose homeostasis by increasing insulin sensitivity and glucose tolerance. CR enhances insulin sensitivity by improving the mitochondrial function at two levels, elevating the expression of SIRT1, PGC-1α and TFAM and increasing mitochondrial complex I-III activities. GABA therapy boosts insulin levels by inducing β-cell proliferation and neogenesis and contributes to the existing depositary. GABA also enhances the metabolic profile by reducing BW, FBG levels, triglycerides, total cholesterol, and LDL levels. As mentioned earlier, the liver-adipose tissue crosstalk regulates these pathways at the transcriptional level. CR+GABA treated group shows a significant decrease in the expression of G6Pase and PEPCK (gluconeogenesis), GLUT2 and G6Pase (glycogenolysis), and an increase in glucokinase (glycolysis). Interestingly, lipogenesis seems up-regulated as marked by the increased ACC expression, and consequently, lipolysis also gets up-regulated as a compensatory mechanism as observed by the increased ATGL expression. CR+GABA treated group showed a significant increase in β-cell proliferation, neogenesis and reduced β-cell apoptosis. We did not observe AIF translocation for β-cell apoptosis, a marker for caspase-independent cell death. Increased oxidative stress induced by STZ and low levels of antioxidant enzymes in the pancreatic cells activate caspase-3 and caspase-9 pathway to mediate β-cell apoptosis [38-40]. Hence, the HFD+STZ model mimics caspase-dependent β-cell apoptosis. However, further validation is required for caspase mediated cell death pathways.
However, a few limitations of this study are as follows. First, we used CR as one of our therapies and monitored parameters at the transcriptional level in different peripheral tissues. As CR is first-line therapy, it is ideal for assessing energy expenditure parameters. Second, our group has not performed cell lineage tracing studies to establish GABA’s role in β-cell regeneration. Thus, extensive in vitro and in vivo investigations are needed to address the above burning question and whether the effect was due to the two different signalling cascades additive stimulus or an undefined underlying mechanism. Thus, the current study suggests that combination therapy with CR and GABA leads to amelioration of T2D pathophysiology in the T2D mouse model (Figure 14).
5. Conclusion
Our study suggests that CR in combination with GABA therapy could help overcome T2D pathophysiology by their action at multiple sites. CR regulates glucose and lipid metabolism, mitochondrial biogenesis, ETC complex activities, promotes β-cell regeneration and reduces β-cell apoptosis. CR treatment seems part of a first-line therapy that can be used with other drugs like DPP-IV inhibitors or metformin. Moreover, a CR-mimetic, for instance, resveratrol, is available commercially to easily overcome the challenge of strict dietary regimen and protocols. GABA monotherapy works on β-cell regeneration, readily available from natural sources like spinach and tomatoes. An edible vegetable extract can be formulated for diabetic patients without the manifestation of significant side effects. Thus, combination therapy can be useful for effective action at multiple sites and mechanisms to combat T2D pathophysiology. This study needs further investigation at pre-clinical and clinical trial levels to evaluate the commercial viability.
Funding
RB thanks the Department of Biotechnology, New Delhi, India, for the research grant (BT/PR21242/MED/30/1750/2016).
Competing Interests
The authors declare that there are no competing interests.
Author Contributions
RB, NR, AVR and RBS conceived the idea. NR designed and performed the experiments; did data acquisition and data analysis; and wrote the original manuscript draft. NP performed a few experiments and data acquisition. SP and RP reviewed and edited the manuscript. RB and AVR contributed to the critical revision and approval of the manuscript.
Acknowledgments
We thank Dr. Ansarullah, Institute of Diabetes and Regeneration Research, Helmholtz Center, Munich, Neuherberg, Germany for the project idea. We thank Dr. Deepak Sharma, Scientist F, BARC, Mumbai, for providing the confocal facilities. NR thanks University Grants Commission-National Fellowship for higher education for ST students, New Delhi, India, for awarding SRF. RP thanks Council for Scientific and Industrial Research, New Delhi, India, for awarding SRF. Images are created with BioRender.com
Abbreviations
- T2D
- Type 2 Diabetes
- FBG
- Fasting Blood Glucose
- TC
- Total Cholesterol
- HDL
- High Density Lipoprotein
- TG
- Triglycerides
- LDL
- Low Density Lipoprotein
- BMI
- Body Mass Index
- HFD
- High Fat Diet
- CR
- Calorie Restriction
- STZ
- Streptozocin
- GABA
- γ-Aminobutyric Acid