Pathophysiology
Pancreatic β-Cell Rest Replenishes Insulin Secretory Capacity and Attenuates Diabetes in an Extreme Model of Obese Type 2 Diabetes

Brandon B. Boland1,2⇑, Charles Brown Jr.1, Michelle L. Boland1,2, Jennifer Cann1, Michal Sulikowski1, Gitte Hansen2, Rikke V. Grønlund2, Wanda King1, Cristina Rondinone1, James Trevaskis1, Christopher J. Rhodes1 and Joseph S. Grimsby1

1Division of Cardiovascular and Metabolic Disease, MedImmune LLC, Gaithersburg, MD
2Gubra ApS, Hørsholm, Denmark

Abstract
The onset of common obesity-linked type 2 diabetes (T2D) is marked by exhaustive failure of pancreatic β-cell functional mass to compensate for insulin resistance and increased metabolic demand, leading to uncontrolled hyperglycemia. Here, the β-cell–deficient obese hyperglycemic/hyperinsulinemic KS db/db mouse model was used to assess consequential effects on β-cell functional recovery by lowering glucose homeostasis and/or improving insulin sensitivity after treatment with thiazolidinedione therapy or glucagon-like peptide 1 receptor agonism alone or in combination with sodium/glucose cotransporter 2 inhibition (SGLT-2i). SGLT-2i combination therapies improved glucose homeostasis, independent of changes in body weight, resulting in a synergistic increase in pancreatic insulin content marked by significant recovery of the β-cell mature insulin secretory population but with limited changes in β-cell mass and no indication of β-cell dedifferentiation. Restoration of β-cell insulin secretory capacity also restored biphasic insulin secretion. These data emphasize that by therapeutically alleviating the demand for insulin in vivo, irrespective of weight loss, endogenous β-cells recover significant function that can contribute to attenuating diabetes. Thus, this study provides evidence that alleviation of metabolic demand on the β-cell, rather than targeting the β-cell itself, could be effective in delaying the progression of T2D.

Introduction
Chronic hyperglycemia resulting from the failure of pancreatic β-cell function and mass to compensate for insulin resistance marks the onset of obesity-linked type 2 diabetes (T2D). Early in the pathogenesis of obese T2D, a compensatory increase in β-cell function and/or mass can cater to increased metabolic load and insulin resistance. With time, however, the chronic demand on the β-cell becomes exhaustive, leading to inadequate functional β-cell mass through apoptosis or loss of identity and ultimately to frank T2D (1–4). This progressive β-cell exhaustion in the pathogenesis of T2D has led to the concept that transient β-cell rest might be beneficial to the treatment of T2D by decreasing β-cell demand and subsequently improving function and/or promoting the survival of remaining endogenous β-cells (5). There is some precedence for this by improving insulin sensitivity in specific situations, such as gestational diabetes (6), marked calorie restriction (7), or short-term direct inhibition of endogenous insulin secretion in obese T2D (8). However, attenuating metabolic demand to improve β-cell function has not been examined in detail at the level of the β-cell using current T2D therapeutic approaches.

Whether failure of β-cell function or reduced β-cell mass is key to the onset of T2D often has been debated, but in humans, disease pathogenesis is likely to be variable and contributed by both (9). For β-cell dysfunction in T2D, there is a characteristic loss of glucose sensing, blunted first-phase glucose-induced insulin secretion, and presumed inadequate insulin production (10). In contrast, obese T2D mouse models have indicated that β-cell insulin production is markedly elevated despite severely depleted intracellular insulin stores (11). Of note, when pancreatic islets isolated from these obese T2D mice were incubated at normal glycemia (5.6 mmol/L) overnight, the rate of insulin production normalized, intracellular insulin secretory granule stores replenished, and glucose-induced biphasic insulin secretion was restored (11).

We examined whether a glucose-lowering therapeutic strategy (by the glucagon-like peptide 1 receptor [GLP-1R] agonist liraglutide and/or sodium/glucose cotransporter 2 inhibition [SGLT-2i] with dapagliflozin) or an insulin sensitizing approach (using the thiazolidinedione [TZD] rosiglitazone alone or in combination with dapagliflozin) alleviates the demand on the β-cell in vivo in BKS.Cg-Dock7m +/+ Lebrdb/J (hereafter referred to as KS db/db) mice (an extreme model of obese T2D because of failed β-cell compensation) to restore endogenous β-cell function. We chose this model (rather than polygenic or diet-induced models of diabetes) because it presents with a catastrophic and early loss of β-cell function. Thus, if therapeutic efficacy can be observed in such an extreme model, we anticipate that it would extend to less deleterious pathophysiologies of the same disease. In addition, we used lean C57BLKS/J mice as comparative controls. Our findings highlight that glycemic lowering with established T2D therapies, especially in combination, also can instigate β-cell rest in vivo by using the inherent adaptive flexibility of β-cells to improve their secretory capacity and function, thereby slowing progression of the disease.

Research Design and Methods
Experimental Design
Four cohorts of male KS db/db mice were used. C57BLKS/J animals were used as lean controls. Animals were group housed with ad libitum access to water and rodent chow in a controlled environment. Cohorts 1 and 2 were used to study the combination of dapagliflozin/liraglutide, whereas cohorts 3 and 4 were used to study the combination of rosiglitazone/dapagliflozin. Cohorts 1 and 3 were used to demonstrate the efficacy of combination therapies, pancreatic insulin content, and immunohistochemistry, whereas cohorts 2 and 4 were used for islet experiments. Cohorts 2, 3, and 4 were purchased from The Jackson Laboratory (Farmington, CT) and studied at MedImmune LLC, whereas cohort 1 was purchased from Charles River Laboratories Italia (Calco, Italy) and studied at Gubra ApS. All cohorts were purchased at 7 weeks of age, acclimated for 1 week, randomized according to body weight and percent HbA1c (%HbA1c), and studied for 4 weeks. Animals were dosed daily on the basis of body weight. Rosiglitazone (U.S. Pharmacopeia, Rockville, MD) and dapagliflozin (AstraZeneca UK) were dosed orally at 3 mg/kg and 1 mg/kg, respectively using hydroxypropyl methylcellulose (HPMC) vehicle. Liraglutide (Novo Nordisk, Bagsværd, Denmark) was dosed subcutaneously (0.2 mg/kg) using PBS vehicle. Control animals in cohorts 3 and 4 received oral HPMC daily; control animals in cohorts 1 and 2 received both oral HPMC and subcutaneous PBS daily. At the end of the 3rd study week, intraperitoneal glucose tolerance test (ipGTT), fasting plasma glucose (FPG), fasting plasma insulin, and %HbA1c were determined. At the end of the 4th study week, body composition was determined, and animals were sacrificed for the collection of pancreas, islets, plasma, or liver as appropriate. Animal care, use, and experimental protocols were approved by the institutional animal care and use committees of MedImmune and Gubra.

ipGTT
Six-hour fasted mice were injected intraperitoneally with 1.5 g/kg glucose in saline. Blood glucose was determined at 0, 5, 10, 15, 60, and 180 min for cohort 1 and 0, 15, 30, 60, 120, and 240 min for cohort 2. The circulating plasma glucose levels of the mice were determined colorimetrically, whereas the plasma insulin level was determined through ELISA at 15 min.

Analysis of Pancreatic Insulin Content, Circulating Factors, and Nuclear Magnetic Resonance
Pancreatic insulin content was determined from whole pancreas through acid-ethanol extraction and insulin ELISA. Plasma glucose was determined through colorimetric glucose oxidase kit (Cayman Chemical, Ann Arbor, MI). Plasma insulin was determined through ELISA (Meso Scale Diagnostics, Rockville, MD). The %HbA1c was determined colorimetrically from whole blood (Crystal Chem, Elk Grove Village, IL). Plasma cholesterol, triglycerides, AST, and ALT were determined using the cobas c111 autoanalyzer (Roche, Basel, Switzerland). Liver fat was determined using the Bruker minispec mq (Bruker, Billerica, MA). Body composition was determined using the Bruker LF90II analyzer.

Immunohistochemistry and β-Cell Mass Analysis
For immunohistochemistry, pancreata were fixed, embedded, and cut into 5-μm sections. Sections were stained on the BOND RX system (Leica Microsystems, Buffalo Grove, IL) with rabbit anti-MafA (Bethyl Laboratories, Montgomery, TX) or rabbit anti-Ki67 (Abcam, Cambridge, MA) followed by antigen retrieval, 3,3′-diaminobenzidine staining, and glucagon/insulin detection as previously described (12). β-Cell and α-cell mass were quantified from insulin/glucagon (brown/pink) stained sections (n ≥ 3) using HALO software (Indica Laboratories, Corrales, NM). Here, insulin/glucagon/hematoxylin chromogenically stained slides were scanned and uploaded to the HALO software suite. HALO discriminates between stained/unstained area and thus can quantitate the area of the pancreas occupied by either insulin or glucagon. From these values, we can determine the area ratios of pancreas to β-cell or α-cell (i.e., β-cell mass, α-cell mass, respectively).

Quantitative Electron Microscopy
Freshly isolated islets were fixed in 0.1 mol/L cacodylate buffer containing 4% paraformaldehyde/2% glutaraldehyde. Samples were resin embedded, sectioned, and stained as previously described (11). Micrographs were imaged and quantified as previously described (12). Mature insulin granules and immature insulin granules were quantified per total cytoplasmic area from ≥20 electron micrographs per group (n ≥ 3 biological replicates, ≥1.0 mm2 total cytoplasmic area).

Islet Isolation, Glucose-Stimulated Insulin Secretion, Perifusion, and Quantitative RT-PCR
Pancreatic islets from C57BL/6J, KS db/db, or C57BLKS/J mice were isolated by collagenase digestion, as previously described (13). Human islets were obtained from Prodo Laboratories (Aliso Viejo, CA). Glucose-stimulated insulin secretion experiments were in C57BL/6J islets cultured overnight in RPMI media (10% FBS, 5.6 mmol/L glucose) (Thermo Fisher Scientific, Waltham, MA). Insulin secretion was assessed by 1-h static incubation in the presence of rosiglitazone (10 μmol/L), dapagliflozin (10 μmol/L), or both dissolved in 0.2% DMSO, as previously described (11), after preincubation at 37°C for 4 h with the same study compounds. For quantitative RT-PCR (qRT-PCR) and perifusion, freshly isolated islets from KS db/db and C57BLKS/J mice were used. Islet RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized using the Superscript III First-Strand Synthesis System (Thermo Fisher Scientific). qRT-PCR analysis was performed using Taqman Gene Expression Assay probe/primer sets (Thermo Fisher Scientific) for Ins1, Ins2, Gck, Slca2a, Pdx1, and Rna18s; PrimePCR Probe Assay (Bio-Rad, Hercules, CA) was used for Mafa. Results are shown as the target gene expression relative to Rna18s expression and normalized to vehicle expression using the 2(−ΔΔCt) method. For perifusion, an equivalent number of medium-sized islets were placed into individual perifusion columns connected to a 12-channel perifusion apparatus (Biorep Technologies, Miami Lakes, FL). After 90 min of preperifusion with 2.8 mmol/L Krebs-Ringer bicarbonate HEPES buffer (KRBH), biphasic islet insulin secretion was assessed. KRBH (2.8 mmol/L glucose) was pumped through the chambers followed by KRBH (16.7 mmol/L glucose), as previously described (14). The perfusate was collected at 2-min intervals in 96-well plates and maintained at 4°C. Islets were lysed for protein content, and perfusate insulin concentrations were determined through ELISA.

Statistical Analysis
Data normality was determined by the D’Agostino and Pearson test. Parametric data were analyzed by one-way or two-way ANOVA followed by Tukey test. Nonparametric data were analyzed by Kruskal-Wallis test followed by Dunn multiple comparisons test. For islet experiments (Figs. 3 and 4), data for individual cohorts were analyzed separately to confirm that no statistical differences existed between combined cohort data and individual cohort data (data not shown). Data are presented as mean ± SD or box and whisker plots ± minimum/maximum, as appropriate. All data were analyzed using GraphPad Prism 7.02 (GraphPad Software, San Diego, CA). Statistical significance was set at P ≤ 0.05.

Results
Antidiabetic Efficacy of SGLT-2i in Combination With GLP-1 or TZD Therapies
Eight-week-old male KS db/db mice were treated with select diabetes therapies: peroxisome proliferator–activated receptor-γ (PPARγ agonism (rosiglitazone), GLP-1R agonism (liraglutide), SGLT-2i (dapagliflozin), or the combination of SGLT-2i plus GLP-1R or PPARγ agonism over 4 weeks. Study animals displayed markedly different weight profiles (Figs. 1A–D and 2A–D), depending on GLP-1 or PPARγ therapy. Vehicle-treated animals had increased body weight by ∼10% (Figs. 1E and 2E), whereas liraglutide monotherapy led to a 7.5% increase, similar to lean animals (Fig. 1E); dapagliflozin monotherapy led to an ∼15% increase (Figs. 1E and 2E); and rosiglitazone monotherapy led to a 29% increase (Fig. 2E). The combination of dapagliflozin/rosiglitazone led to a 34% increase in body weight in contrast to the only 8% increase observed for dapagliflozin/liraglutide, a fourfold difference (Figs. 1E and 2E). Body composition analysis revealed reductions in fat mass after treatment with dapagliflozin (Fig. 2F), liraglutide, and dapagliflozin/liraglutide (Fig. 1F). Lean mass correspondingly was increased by liraglutide and the combination of dapagliflozin/liraglutide but unaffected by dapagliflozin or rosiglitazone (Figs. 1G and 2G). Rosiglitazone administration increased liver fat, liver weight, and plasma ALT, AST, and cholesterol but sharply reduced plasma triglycerides (Supplementary Table 1). Despite not influencing liver fat, dapagliflozin monotherapy increased liver weight (Supplementary Table 1).

Figure 1
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Figure 1
Physiological parameters of dapagliflozin/liraglutide cohorts. In cohorts 1 and 2, 8-week-old male KS db/db mice were treated with PBS vehicle, dapagliflozin, liraglutide, or dapagliflozin/liraglutide for 4 weeks. C57BLKS/J mice were used as lean controls but were not included in the statistical analysis. A and B: Percent weight change and absolute weight change in cohort 1 during the experimental period. C and D: Percent weight change and absolute weight change in cohort 2 during the experimental period. E: Percent weight change of cohort 1. F and G: Body composition analysis of cohort 1 as percent body fat (fat mass per body weight) and percent lean mass (lean mass per body weight) at the end of the experimental period. H: Change in cohort 1 %HbA1c over the 4-week experimental period. I and J: ipGTT (2 g/kg glucose) after a 6-h fast in cohort 1 at day 21 of the experimental period and the corresponding total glucose excursions for the ipGTT as AUC for cohort 1. K: Plasma insulin levels at 15 min during the ipGTT. L and M: Six-hour FPG and insulin of cohort 1 at day 21 of the experimental period. N: HOMA-IR as calculated from the 6-h FPG and insulin levels. n ≥ 9 for cohort 1, n ≥ 8 for cohort 2, and n ≥ 4 for panels F and G. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs. vehicle. Asterisks above a line indicate significance vs. the indicated group. &P ≤ 0.05 liraglutide, $P ≤ 0.05 dapagliflozin/liraglutide vs. vehicle. D, dapagliflozin; L, liraglutide; NS, not significant; V, vehicle.

Figure 2
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Figure 2
Physiological parameters of rosiglitazone/dapagliflozin cohorts. In cohorts 3 and 4, 8-week-old male KS db/db mice were treated with PBS vehicle, rosiglitazone, dapagliflozin, or rosiglitazone/dapagliflozin for 4 weeks. A and B: Percent weight change and absolute weight change in cohort 3 during the experimental period. C and D: Percent weight change and absolute weight change in cohort 4 during the experimental period. E: Percent weight change of cohort 3. F and G: Body composition analysis of cohort 3 as percent body fat (fat mass per body weight) and percent lean mass (lean mass per body weight) at the end of the experimental period. H: Change in cohort 3 %HbA1c over the 4-week experimental period. I and J: ipGTT (2 g/kg glucose) after a 6-h fast in cohort 3 at day 21 of the experimental period and the corresponding total glucose excursions for the ipGTT as AUC for cohort 3. K: Plasma insulin levels during the ipGTT. L and M: Six-hour FPG and insulin of cohort 3 at day 21 of the experimental period. N: HOMA-IR as calculated from the 6-h FPG and insulin levels. n ≥ 9 for cohort 3, n ≥ 12 for cohort 4, and n ≥ 4 for panels F and G. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs. vehicle. Asterisks above a line indicate significance vs. the indicated group. ^P ≤ 0.05 dapagliflozin; #P ≤ 0.05 rosiglitazone. D, dapagliflozin; NS, not significant; R, rosiglitazone; V, vehicle.

Vehicle-treated animals displayed a 2-point increase in %HbA1c, which was significantly mitigated by monotherapy treatments (Figs. 1H and 2H). However, both combination strategies were more effective than the respective monotherapies; dapagliflozin/rosiglitazone led to a 1-point reduction in %HbA1c (Fig. 2H), whereas dapagliflozin/liraglutide maintained %HbA1c, similar to lean animals (Fig. 1H). The observed improvements in %HbA1c were paralleled by improved glucose tolerance. Combination-treated animals displayed improved glucose excursions compared with vehicle controls (Figs. 1I and 2I). The total glucose area under the curve (AUC) values for both combination-treated groups were significantly reduced, nearly to the level of the lean animals (Figs. 1J and 2J). In vivo insulin secretion during the ipGTT was significantly improved by both liraglutide and rosiglitazone, although this effect was far more pronounced by liraglutide in line with its insulinotropic action (Figs. 1K and 2K). All monotherapies reduced FPG levels versus vehicle-treated animals, but combination strategies elicited a more robust decrease comparable to lean animals (Figs. 1L and 2L). The restoration of plasma glucose to near-normal levels by both combination strategies was facilitated by correspondingly greater plasma insulin levels capable of overcoming the inherent insulin resistance in KS db/db animals (Figs. 1M and 2M). Although clear differences exist in the absolute levels of both fasting glucose and insulin between cohorts, the relative changes in glucose confirm the largely similar therapeutic efficacies of the glucose-lowering strategy (liraglutide ± dapagliflozin) or by improving insulin sensitization (rosiglitazone ± dapagliflozin) (Supplementary Fig. 1A and B, respectively). Finally, HOMA of insulin resistance (HOMA-IR), a relative measure of insulin sensitivity, was unaffected by liraglutide therapies but had a tendency to improve with rosiglitazone therapies, although not statistically significantly (Figs. 1N and 2N).

Restoration of Pancreatic MafA Expression and β-Cell Insulin Content
Despite stark differences in body weight, combinations of dapagliflozin and liraglutide or rosiglitazone increased pancreatic insulin content more than threefold (Fig. 3A). Of note, no significant change in β-cell mass was observed in combination-treated animals (Fig. 3B and Supplementary Fig. 2A). Correspondingly, no difference in islet β-cell replication was observed (Supplementary Fig. 3 and Supplementary Table 2). However, combination-treated mice displayed a greater number of mature insulin granules (Fig. 3C), although the content of immature granules was unchanged (Fig. 3D). Representative electron micrographs of β-cells from untreated KS db/db mice (Fig. 3E) paralleled the morphology observed in previous studies of obesity-linked T2D (11): marked mature insulin secretory granule degranulation, a greater proportion of immature secretory granules relative to mature secretory granules, and an expansion of the Golgi apparatus and endoplasmic reticulum. Rosiglitazone (Fig. 3F), dapagliflozin (Fig. 3G), and liraglutide (Fig. 3H) all demonstrated some ability to increase insulin granule number. However, the combination of dapagliflozin/rosiglitazone (Fig. 3I) or dapagliflozin/liraglutide (Fig. 3J) induced marked insulin regranulation and reduced endoplasmic reticulum area. Staining for MafA, a key pancreatic transcription factor exclusive to β-cells (15), increased proportionally to the intensity of insulin in lean control and combination-treated islets (Fig. 3P–R) versus monotherapy-treated (Fig. 3M–O) and vehicle control islets (Fig. 3L), suggesting that β-cell recovery of endogenous insulin secretory capacity underlies significant antidiabetic efficacy (Supplementary Table 3).

Figure 3
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Figure 3
Pancreatic insulin content, immunohistochemistry of fixed pancreata, and electron microscopy of freshly isolated pancreatic islets. A: Pancreatic insulin content (n ≥ 6). B: β-Cell mass from insulin/glucagon dual-stained immunohistochemistry sections (n ≥ 4). C and D: Mature and immature insulin granule quantification from electron micrographs (n ≥ 3 from ≥10 electron micrographs). E–K: Representative electron micrographs of freshly isolated islets from KS db/db mice treated with vehicle, rosiglitazone, dapagliflozin, liraglutide, rosiglitazone/dapagliflozin, dapagliflozin/liraglutide, and untreated lean C57BLKS/J mice (n ≥ 3). Scale bars = 1 μm. L–R: Immunohistochemical staining of insulin (yellow), MafA (brown), and glucagon (32) of fixed pancreata from KS db/db mice treated with vehicle, rosiglitazone, dapagliflozin, liraglutide, rosiglitazone/dapagliflozin, dapagliflozin/liraglutide, and untreated lean C57BLKS/J mice (n ≥ 4). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs. vehicle. Asterisks above a line indicate significance vs. the indicated group. D, dapagliflozin; L, liraglutide; R, rosiglitazone; V, vehicle.

Synergistic Improvement in β-Cell Function
Expression of key genes related to specific β-cell function were examined from isolated islets after 4-week treatment. Ins1 transcript levels were significantly increased from both combination-treated animals by approximately eightfold (Fig. 4A), whereas Ins2 transcript levels were significantly increased in islets from liraglutide monotherapy (fivefold) and liraglutide/dapagliflozin combination-treated animals (sixfold) (Fig. 4B). The mRNA levels of Slc2a2 and Gck were increased in combination-treated animals versus vehicle controls (Fig. 4C and F) as were Mafa transcript levels, particularly rosiglitazone/dapagliflozin (12-fold) (Fig. 4D), complementary to MafA immunostaining (Fig. 3). Pdx1 expression also was significantly increased in islets from animals treated with liraglutide monotherapy and combination therapies (Fig. 4E).

Figure 4
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Figure 4
β-Cell functional gene analysis and perifusion of freshly isolated islets. A–F: Freshly isolated islet qRT-PCR for Ins1, Ins2, Slca2a, Mafa, Pdx1, and Gck (n ≥ 4). Gene expression was normalized to the housekeeping gene Rna18s and presented as relative expression compared with vehicle. G: Perifusion of freshly isolated islets. From 0 to 42 min, islets were perifused with 2.8 mmol/L glucose; from 44 to 88 min, islets were perifused with 16.7 mmol/L glucose. At the end of perifusion, islets were lysed and analyzed for protein content. H and I: AUC of first-phase (44–56 min) and second-phase (58–88 min) insulin secretion (n ≥ 4). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs. vehicle. Asterisks above a line indicate significance vs. the indicated group. D, dapagliflozin; L, liraglutide; R, rosiglitazone; RQ, relative quantification; V, vehicle.

Because loss of first-phase insulin secretion is a hallmark of β-cell secretory dysfunction (16), we assessed the insulin secretory response in islets from vehicle controls, treated mice, and lean controls. Immediately after isolation, islets were preperifused with basal 2.8 mmol/L glucose KRBH for 60 min followed by perifusion with 2.8 mmol/L glucose KRBH and then stimulatory 16.7 mmol/L glucose KRBH to assess biphasic insulin secretion. Islets from monotherapy-treated animals demonstrated some recovery of first-phase insulin secretion and improved second-phase insulin secretion versus vehicle control islets (Fig. 4G). However, islets from combination-treated animals showed robust first-phase insulin secretion and sustained second-phase insulin secretion, even compared with islets from monotherapy-treated animals. Islets from combination-treated animals elicited significantly greater first-phase (Fig. 4H) and second-phase (Fig. 4I) insulin secretion relative to vehicle controls, indicating a synergistic improvement in recovery of functional insulin secretory capacity over monotherapy treatments.
　

讨论
常见的与肥胖相关的T2D的发作以功能性β细胞质量的丧失为特征，后者不再维持正常的葡萄糖稳态。然而，β细胞功能和/或质量是否驱动T2D疾病的发病机理仍不清楚（3）。

在这里，使用KS db / db小鼠（一种由高血糖/高胰岛素血症引起的肥胖症引起的胰岛素抵抗的极端模型）[17]，已揭示出β细胞功能障碍是T2D的主要驱动因素。

在同一只小鼠模型中，研究表明，剩余的β细胞中的内源性胰岛素储存显着减少，这些动物具有高胰岛素血症。但是，胰岛素的产生明显上调，新合成的（原）胰岛素迅速分泌出来，以补偿新陈代谢的需求（11）。在这种情况下，整个β细胞超微结构适应于一种胰岛素生产而不是一种储存，这表明明显的β细胞功能障碍是β细胞辛勤工作的结果，而不是固有的功能缺陷（11 ）。

此外，由于在这种肥胖的糖尿病胰岛中β细胞脱粒，使用胰岛素作为免疫组织化学标记可能低估了β细胞量的测量。值得注意的是，

在正常血糖下孵育过夜的小鼠分离的胰岛β细胞中进行的并行离体研究导致β细胞超微结构正常化，β颗粒胰岛素存储和分泌能力的恢复，以及葡萄糖调节的胰岛素分泌的正常化（11 ）。

这项研究支持以下假设：使用常见的T2D治疗方法（特别是联合使用）可以在体内降低β细胞的代谢需求，从而通过间接恢复内源性β细胞功能和独立于胰岛素的分泌能力，为T2D的治疗带来有益的后果，而与体重变化无关。然而，值得一提的是KS db / db小鼠与人类肥胖/ T2D的疾病病理生理学。选择该动物模型作为极端肥胖/胰岛素抵抗之一，其功能性β细胞质量明显受损，以更好地揭示体内刺激β细胞静息的方法可以改善内源性β细胞分泌能力和功能。但应该保留的是，瘦素信号传导的丧失不是常见的人类肥胖/ T2D的主要因素，并且这些小鼠中存在的严重高胰岛素血症与在人类T2D进程中观察到的相对中等的高胰岛素血症形成鲜明对比（18）。然而，尽管有这些担忧，但这项研究通过使用不太传统的二氮嗪治疗策略抑制人的内源性胰岛素分泌（8）或小鼠的胰岛素治疗（19），补充了先前促进β细胞静息的临床研究。使用热量摄入急剧减少的饮食限制可以改善β细胞功能（7）。在这种情况下，减肥引起的胰岛素敏感性改善可能是这种功能逆转的主要驱动力。医师辅助热量限制的最新研究还发现，糖尿病缓解率与体重减轻程度相关（20），减肥手术也是如此（21）。目前，一项正在进行的恢复胰岛素分泌（RISE）的临床试验正在研究在成年和小儿糖尿病前期和早期T2D患者中联合药物干预（二甲双胍±利拉鲁肽）后β细胞功能的保存（22）。在这里，选择了更常规的治疗策略，以通过对胰腺β细胞不一定具有直接作用的独特作用机制来降低体内葡萄糖稳态。 SGLT-2i通过防止近端肾脏重新摄取葡萄糖来降低循环葡萄糖（23），而不会影响β细胞胰岛素的分泌（补充图4）。TZD是PPARγ的部分激动剂，主要起胰岛素增敏剂的作用，对胰岛素的分泌几乎没有影响（补充图4），并且在延缓啮齿类动物T2D发病过程中延迟β细胞的净损失方面显示出希望（24）。尽管GLP-1R激动剂与利拉鲁肽等GLP-1类似物可作为葡萄糖依赖性胰岛素促分泌剂（25），但在肥胖的T2D中，它在诱导饱腹感和体重减轻，以及延长排空和改善葡萄糖稳态方面非常有效（26，27）。作为单一疗法，这三种不同的治疗策略在严重的肥胖糖尿病糖尿病KS db / db小鼠模型上显示出一定程度的改善葡萄糖稳态，但是在联合使用时它们的协同作用更为有效。确实，新兴的临床数据支持了这些结果。例如，发现SGLT-2i可改善患有不受控制的T2D的个体的肠降血糖素敏感性（28）。尽管两个联合治疗组的体重有显着差异，但SGLT-2i联合GLP-1R激动剂或TZD治疗可导致HbA1c，FPG和葡萄糖耐量的加和改善（图1和2）。 SGLT-2i / GLP-1R激动剂或SGLT-2i / TZD组合策略均显着改善了内源性β细胞功能。随着FPG降低，空腹胰岛素水平改善；胰腺胰岛素含量显着增加，与此同时，内源性β-颗粒胰岛素储库大量重新调节，这反过来又补充了胰岛素分泌能力并恢复了正常的葡萄糖诱导的双相胰岛素分泌。此外，两种联合疗法均增加了关键β细胞转录因子Mafa和Pdx1的表达，这些因子控制了其他β细胞功能基因的表达（29），这些基因也因这些治疗而增加（图4）。值得注意的是，在使用任一联合疗法的这些KS db / db小鼠中，β细胞的质量均未发生明显变化，强调对胰岛素抵抗的补偿主要是通过β细胞功能的改变而发生的（30）。

　

胰腺β细胞对代谢状态具有显着适应性。例如，在长期禁食期间，β细胞的胰岛素分泌能力基本被关闭，作为针对低血糖的一种保护措施，而禁食的β细胞仍然保持着准备，可以在重新喂养后迅速恢复有效的胰岛素生产时间（12）。确实，在接受胃旁路手术的患者中，甚至在手术后许多年，观察到的β细胞对葡萄糖的敏感性都降低了，这意味着要采取适当的预防措施来防止餐后促分泌素诱导的胰岛素分泌过多引起的低血糖（31）。当前的发现表明，胰岛素抵抗的治疗性减轻或葡萄糖稳态的改善降低了对内源性β细胞增加T2D中胰岛素产生的需求，从而使β细胞适应性迅速恢复了胰岛素分泌能力并使胰岛素分泌正常化。降低血糖负担的适应性结果是改善了β细胞功能，这最终有助于减弱T2D。这些发现可能对人类普通肥胖T2D的治疗具有启发意义。首先，对T2D的早期诊断和随后的有效联合治疗应保留内源性β细胞功能，从而延缓疾病进展。其次，β细胞本身不必一定是直接的治疗靶标；相反，应主要针对增加对β细胞需求的T2D病理生理学，以使β细胞休息并因此恢复正常的β细胞功能和葡萄糖稳态。实际上，在缺乏增强的分泌功能的情况下，针对T2D中已经过度劳累的β细胞的直接促胰岛素疗法可能有害，并加速了疾病的发病机理。

Discussion
The onset of common obesity-linked T2D is marked by loss of functional β-cell mass that no longer maintains normal glucose homeostasis. However, whether β-cell function and/or mass drive T2D disease pathogenesis remains unclear (3). Here, use of the KS db/db mouse, an extreme model of hyperglycemic/hyperinsulinemic obesity-induced insulin resistance (17), has revealed a bias toward β-cell dysfunction as a major driver in T2D. In the same mouse model, it was shown that endogenous insulin stores in remaining β-cells are markedly depleted, and these animals were profoundly hyperinsulinemic. However, insulin production was markedly upregulated, and the newly synthesized (pro)insulin rapidly secreted in efforts to compensate for increased metabolic demand (11). In this instance, the whole β-cell ultrastructure adapted to one of insulin production rather than to one of storage, implicating that apparent β-cell dysfunction is a consequence of a hard-working β-cell rather than of an inherent functional defect (11). Moreover, because of the degranulation of β-cells in such obese diabetic pancreatic islets, the use of insulin as an immunohistochemical marker likely underestimated measures of β-cell mass. Of note, parallel ex vivo studies in isolated islets from KS db/db mice incubated at euglycemia overnight led to normalized β-cell ultrastructure, restoration of β-granule insulin stores and secretory capacity, and consequent normalization of glucose-regulated insulin secretion (11).

This study supports the hypothesis that metabolic demand on the β-cell can be reduced in vivo using common T2D therapeutics (especially in combination) to yield beneficial consequences for the treatment of obese T2D by indirectly restoring endogenous β-cell function and insulin secretory capacity independent of changes in body weight. However, the disease pathophysiology in the KS db/db mice versus human obese/T2D warrants mention. This animal model was chosen as one of extreme obesity/insulin resistance with significantly compromised functional β-cell mass to better reveal that approaches to instigate β-cell rest in vivo could improve endogenous β-cell secretory capacity and function. But there should be reservation in that loss of leptin signaling is not a primary contributor to common human obese/T2D, and the severe hyperinsulinemia present in these mice contrasts with a relatively moderate hyperinsulinemia observed in the progression of human T2D (18). However, despite these concerns, this study complements previous clinical work of instigating β-cell rest by using the less orthodox therapeutic strategies of diazoxide to inhibit endogenous insulin secretion in humans (8) or insulin therapy in mice (19). Dietary restriction using sharp reductions in caloric intake can result in improved β-cell function (7). In this case, it is likely that improvement in insulin sensitivity as a consequence of weight loss was the primary driver of this functional reversal. A recent study of physician-assisted caloric restriction also found that the rate of diabetic remission correlated with the degree of weight lost (20), as does bariatric surgery (21). Currently, the ongoing Restoring Insulin Secretion (RISE) clinical trial is investigating the preservation of β-cell function after combinatorial pharmaceutical intervention (metformin ± liraglutide) in adult and pediatric patients with prediabetes and early T2D (22). Here, the more conventional therapeutic strategies were chosen to lower glucose homeostasis in vivo by distinct mechanisms of action that did not necessarily have direct effects on pancreatic β-cells. SGLT-2i lowers circulating glucose by preventing glucose reuptake in the proximal kidney (23), without effects on β-cell insulin secretion (Supplementary Fig. 4). TZDs are partial PPARγ agonists and primarily act as insulin sensitizers, with little effect on insulin secretion (Supplementary Fig. 4), and have shown promise in delaying the net loss of β-cell mass during the pathogenesis of rodent T2D (24). Although GLP-1R agonism, with GLP-1 analogs such as liraglutide, acts as a glucose-dependent insulin secretagogue (25), in obese T2D, it is also quite effective at inducing satiety and weight loss that, together with effects on delaying gastric emptying, improve glucose homeostasis (26,27). As monotherapies, these three distinct therapeutic strategies demonstrated some degree of improving glucose homeostasis on the severe obese-diabetic KS db/db mouse model, but they were synergistically more effective in combination. Indeed, emerging clinical data support these results; for instance, SGLT-2i was found to improve incretin sensitivity in individuals with uncontrolled T2D (28). Here, SGLT-2i in combination with either GLP-1R agonism or TZD therapy led to additive improvements in HbA1c, FPG, and glucose tolerance, despite a dramatic difference in body weight between the two combination-treated groups (Figs. 1 and 2). Both SGLT-2i/GLP-1R agonism or SGLT-2i/TZD combination strategies markedly improved endogenous β-cell function. Fasting insulin levels improved as FPG decreased; there was a marked increase in pancreatic insulin content that was paralleled by a substantial regranulation of endogenous β-granule insulin stores, which in turn replenished insulin secretory capacity and restored normal glucose-induced biphasic insulin secretion. Furthermore, both combination therapies increased the expression of key β-cell transcription factors Mafa and Pdx1, which control the expression of other β-cell functional genes (29) that also were increased by these treatments (Fig. 4). Of note, β-cell mass did not appreciably change in these KS db/db mice with either combination therapy, emphasizing that compensation to insulin resistance occurs predominantly through alterations in β-cell function (30).

Pancreatic β-cells are remarkably adaptable in response to metabolic status. For example, β-cell insulin secretory capacity essentially is shut down during prolonged fasting as a protective measure against hypoglycemia, yet the fasted β-cells remain poised to rapidly restore effective insulin production hours after refeeding (12). Indeed, in patients who underwent gastric bypass, β-cell sensitivity to glucose was observed to be reduced, even many years after the procedure, implicating an adaptation to prevent hypoglycemia from excess postprandial secretagogue-induced insulin secretion (31). The current findings suggest that therapeutic alleviation of insulin resistance or improvement in glucose homeostasis reduces the demand on endogenous β-cells for increased insulin production in T2D, allowing β-cell adaptation to rapidly restore insulin secretory capacity and normalize insulin secretion. The adaptive consequence of reduced glycemic burden is improved β-cell function, which ultimately contributes to attenuating T2D. These findings likely have translational implications for the treatment of common obese T2D in humans. First, an early diagnosis of T2D and subsequent effective combination treatment should preserve endogenous β-cell function and, as such, delay disease progression. Second, the β-cell itself should not necessarily be a direct therapeutic target; rather, pathophysiologies of T2D that increase demand on the β-cell should be primarily targeted to then allow the β-cell to rest and consequently restore normal β-cell function and glucose homeostasis. Indeed, direct insulinotropic therapies targeting an already overworked β-cell in T2D, in the absence of enhanced secretory function, could be detrimental and accelerate disease pathogenesis.

https://diabetes.diabetesjournals.org/content/68/1/131