Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in obesity.

Overnutrition engenders the expansion of adipose tissue and the accumulation of immune cells, in particular, macrophages, in the adipose tissue, leading to chronic low-grade inflammation and insulin resistance. In obesity, several proinflammatory subpopulations of adipose tissue macrophages (ATMs) identified hitherto include the conventional "M1-like" CD11C-expressing ATM and the newly discovered metabolically activated CD9-expressing ATM; however, the relationship among ATM subpopulations is unclear. The ER stress sensor inositol-requiring enzyme 1α (IRE1α) is activated in the adipocytes and immune cells under obesity. It is unknown whether targeting IRE1α is capable of reversing insulin resistance and obesity and modulating the metabolically activated ATMs. We report that pharmacological inhibition of IRE1α RNase significantly ameliorates insulin resistance and glucose intolerance in diet-induced obesity mice. IRE1α inhibition also increases thermogenesis and energy expenditure, and hence protects against high fat diet-induced obesity. Our study shows that the "M1-like" CD11c + ATMs are largely overlapping with but yet non-identical to CD9 + ATMs in obese white adipose tissue. Notably, IRE1α inhibition diminishes the accumulation of obesity-induced metabolically activated ATMs and "M1-like" ATMs, resulting in the curtailment of adipose inflammation and ensuing reactivation of thermogenesis, without augmentation of the alternatively activated M2 macrophage population. Our findings suggest the potential of targeting IRE1α for the therapeutic treatment of insulin resistance and obesity.

Drosophila transmembrane protein 214 (dTMEM214) regulates midgut glucose uptake and systemic glucose homeostasis.

The availability of glucose transporter in the small intestine critically determines the capacity for glucose uptake and consequently systemic glucose homeostasis. Hence a better understanding of the physiological regulation of intestinal glucose transporter is pertinent. However, the molecular mechanisms that regulate sodium-glucose linked transporter 1 (SGLT1), the primary glucose transporter in the small intestine, remain incompletely understood. Recently, the Drosophila SLC5A5 (dSLC5A5) has been found to exhibit properties consistent with a dietary glucose transporter in the Drosophila midgut, the equivalence of the mammalian small intestine. Hence, the fly midgut could serve as a suitable model system for the study of the in vivo molecular underpinnings of SGLT1 function. Here, we report the identification, through a genetic screen, of Drosophila transmembrane protein 214 (dTMEM214) that acts in the midgut enterocytes to regulate systemic glucose homeostasis and glucose uptake. We show that dTMEM214 resides in the apical membrane and cytoplasm of the midgut enterocytes, and that the proper subcellular distribution of dTMEM214 in the enterocytes is regulated by the Rab4 GTPase. As a corollary, Rab4 loss-of-function phenocopies dTMEM214 loss-of-function in the midgut as shown by a decrease in enterocyte glucose uptake and an alteration in systemic glucose homeostasis. We further show that dTMEM214 regulates the apical membrane localization of dSLC5A5 in the enterocytes, thereby revealing dTMEM214 as a molecular regulator of glucose transporter in the midgut.

Hydroxybenzamide derivatives protect pancreatic ß cell by suppressing unfolded protein response activation.

Endoplasmic reticulum (ER) stress-induced Pancreatic ß-cell dysfunction and death plays important roles in the development of diabetes. The 1,2,3-triazole derivative 1 is one of only a few structures that have thus far been identified that protect ß cells against ER stress, but it is limited for its narrow activity range. In this study, we designed and synthesized a series of hydroxybenzamide (HBA) derivatives in which the triazole pharmacophore was substituted with an amide linker. Structure-activity relationship studies identified WO3i (3-hydroxy-N-(4-[trifluoromethyl]benzyl)benzamide) that possesses ß-cell protective activity against ER stress at a 100% maximal activity with EC50 at 0.19 µM). We showed that WO3i suppresses the expression of CHOP, a key mediator of ER stress-induced apoptosis, and the activation of apoptotic genes. Mechanistically, we further showed that WO3i suppresses the ER stress-induced activation of all three pathways of unfolded protein response-ATF6, IRE1α, and PERK. Identification of this novel ß-cell-protective scaffold thus provides a new promising modality for the potential for drug development for the treatment of diabetes.

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut.

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. Using molecular genetic approaches, we investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, we found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. We showed that dSLC5A5 undergoes apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, we showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, our studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.

Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes.

ß-cell ER stress plays an important role in ß-cell dysfunction and death during the pathogenesis of diabetes. Proinsulin misfolding is regarded as one of the primary initiating factors of ER stress and unfolded protein response (UPR) activation in ß-cells. Here, we found that the ER stress sensor inositol-requiring enzyme 1α (IRE1α) was activated in the Akita mice, a mouse model of mutant insulin gene-induced diabetes of youth (MIDY), a monogenic diabetes. Normalization of IRE1α RNase hyperactivity by pharmacological inhibitors significantly ameliorated the hyperglycemic conditions and increased serum insulin levels in Akita mice. These benefits were accompanied by a concomitant protection of functional ß-cell mass, as shown by the suppression of ß-cell apoptosis, increase in mature insulin production and reduction of proinsulin level. At the molecular level, we observed that the expression of genes associated with ß-cell identity and function was significantly up-regulated and ER stress and its associated inflammation and oxidative stress were suppressed in islets from Akita mice treated with IRE1α RNase inhibitors. This study provides the evidence of the in vivo efficacy of IRE1α RNase inhibitors in Akita mice, pointing to the possibility of targeting IRE1α RNase as a therapeutic direction for the treatment of diabetes.

A novel peroxisome proliferator-activated receptor gamma ligand improves insulin sensitivity and promotes browning of white adipose tissue in obese mice.

OBJECTIVE: Nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a promising target for the treatment of type 2 diabetes. The antidiabetic drug thiazolidinediones (TZDs) are potent insulin sensitizers as full agonists of PPARγ, but cause unwanted side effects. Recent discoveries have shown that TZDs improve insulin sensitivity by blocking PPARγ phosphorylation at S273, which decouples the full agonism-associated side effects. PPARγ ligands that act through the blockage of PPARγ phosphorylation but lack the full agonist activity would be expected to improve insulin sensitivity without TZD-associated side effects, however, chemicals that carry such traits and bind to PPARγ with high-affinity are lacking. Moreover, TZDs are known to promote white-to-brown adipocyte conversion and energy expenditure and appear to require their full agonism on PPARγ for this activity. It is unknown whether a partial or non-TZD agonist of PPARγ is capable of promoting browning effect. In this study, we developed a novel non-TZD partial agonist of PPARγ and investigated its function on insulin sensitivity and white-to-brown conversion and energy expenditure in diet-induced obese mice. METHODS: A novel indole-based chemical WO95E was designed via medicinal chemistry and tested for PPARγ binding and activity and for the effect on PPARγ phosphorylation. Diet-induced obese mice were administered with WO95E for 4 weeks. Insulin sensitivity, glucose tolerance, body weight, fat tissue weight, adipocyte size, morphology, energy expenditure, and expression levels of genes involved in PPARγ activity, thermogenesis/browning, and TZD-related side effects were evaluated. RESULTS: WO95E binds to PPARγ with high affinity and acts as a PPARγ partial agonist. WO95E inhibits PPARγ phosphorylation and regulates PPARγ phosphorylation-dependent genes. WO95E ameliorates insulin resistance and glucose tolerance in mice of diet-induced obesity, with minimal TZD use-associated side effects. We found that WO95E promotes white-to-brown adipocyte conversion and energy expenditure and hence protects against diet-induced obesity. WO95E decreases the size of adipocytes and suppresses adipose tissue inflammation. WO95E also suppresses obesity-associated liver steatosis. CONCLUSIONS: WO95E improves insulin sensitivity and glucose homeostasis and promotes browning and energy expenditure by acting as a novel PPARγ phosphorylation inhibitor/partial agonist. Our findings suggest the potential of this compound or its derivative for the therapeutic treatment of insulin resistance and obesity.

Discovery of N-(2-(Benzylamino)-2-oxoethyl)benzamide analogs as a novel scaffold of pancreatic ß-cell protective agents against endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress-induced pancreatic ß-cell dysfunction and death play important roles in the development of diabetes. The 1,2,3-triazole derivative 1 is one of only a few structures that have thus far been identified that protect ß cells against ER stress. However, this compound has narrow activity range and limited aqueous solubility. To overcome these, we designed and synthesized a new scaffold in which the triazole pharmacophore was substituted with a glycine-like amino acid. Structure-activity relationship studies on this scaffold identified a N-(2-(Benzylamino)-2-oxoethyl)benzamide analog WO5m that possesses ß-cell protective activity against ER stress with much improved potency (maximal activity at 100% with EC50 at 0.1 ± 0.01 µm) and water solubility. Identification of this novel ß-cell protective scaffold thus provides a new promising modality for the treatment of diabetes.

Cardiac Snail family of transcription factors directs systemic lipid metabolism in Drosophila.

Maintenance of normal lipid homeostasis is crucial to heart function. On the other hand, the heart is now recognized to serve an important role in regulating systemic lipid metabolism; however, the molecular basis remains unclear. In this study, we identify the Drosophila Snail family of transcription factors (herein termed Sna TFs) as new mediators of the heart control of systemic lipid metabolism. Overexpression of Sna TF genes specifically in the heart promotes whole-body leanness whereas their knockdown in the heart promotes obesity. In addition, flies that are heterozygous for a snail deficiency chromosome also exhibit systemic obesity, and that cardiac-specific overexpression of Sna substantially reverses systemic obesity in these flies. We further show that genetically manipulating Sna TF levels in the fat body and intestine do not affect systemic lipid levels. Mechanistically, we find that flies bearing the overexpression or inhibition of Sna TFs in the postnatal heart only exhibit systemic lipid metabolic defects but not heart abnormalities. Cardiac-specific alterations of Sna TF levels also do not perturb cardiac morphology, viability, lipid metabolism or fly food intake. On the other hand, cardiac-specific manipulations of Sna TF levels alter lipogenesis and lipolysis gene expression, mitochondrial biogenesis and respiration, and lipid storage droplet 1 and 2 (Lsd-1 and Lsd-2) levels in the fat body. Together, our results reveal a novel and specific role of Sna TFs in the heart on systemic lipid homeostasis maintenance that is independent of cardiac development and function and involves the governance of triglyceride synthesis and breakdown, energy utilization, and lipid droplet dynamics in the fat body.

Design, synthesis, and evaluation of potent novel peroxisome proliferator-activated receptor γ indole partial agonists.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve insulin sensitivity by blocking PPARγ phosphorylation at S273; however, their full agonism on PPARγ also causes significant unwanted side effects. The indole derivative UHC1 displays insulin-sensitizing effect by acting as a partial agonist through the inhibition of PPARγ S273 phosphorylation, but without full agonist-associated side effects; however, its potency leaves much to be desired. Herein we report the design and synthesis of potent indole analogs as partial PPARγ agonists via the structure-activity relationship studies. Our studies revealed that vanillylamine and piperonyl benzylamine at Site 1 are favored to bind PPARγ with either biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, compound WO91A with vanillylamine at Site 1 displays highly potent PPARγ binding affinity (IC50 = 16.7 nM), over 30-fold more potent than the parental compound UHC1, yet with less side effect-associated transactivation activity.

Select Septate Junction Proteins Direct ROS-Mediated Paracrine Regulation of Drosophila Cardiac Function.

Septate junction (SJ) complex proteins act in unison to provide a paracellular barrier and maintain structural integrity. Here, we identify a non-barrier role of two individual SJ proteins, Coracle (Cora) and Kune-kune (Kune). Reactive oxygen species (ROS)-p38 MAPK signaling in non-myocytic pericardial cells (PCs) is important for maintaining normal cardiac physiology in Drosophila. However, the underlying mechanisms remain unknown. We find that in PCs, Cora and Kune are altered in abundance in response to manipulations of ROS-p38 signaling. Genetic analyses establish Cora and Kune as key effectors of ROS-p38 signaling in PCs on proper heart function. We further determine that Cora regulates normal Kune levels in PCs, which in turn modulates normal Kune levels in the cardiomyocytes essential for proper heart function. Our results thereby reveal select SJ proteins Cora and Kune as signaling mediators of the PC-derived ROS regulation of cardiac physiology.

Rapid and reliable identification of insulin 2 gene mutation in Akita diabetic mice by a tetra-primer-ARMS-PCR method.

The Akita mouse, one of the most frequently used animal models for the study of diabetes mellitus and its complications, carries a heterozygous missense mutation (C96Y) in the insulin 2 (Ins2) gene that results in proinsulin misfolding in the endoplasmic reticulum (ER), ER stress, pancreatic beta cell death and ultimately diabetes. Maintenance of Akita mice entails genotyping for the identification of the heterozygous Akita mutation. Current genotyping methods for the Akita mouse strain are time consuming, expensive, or needing special device. Here, we develop a simple, fast, cost-effective, and reliable genotyping methodology for the Akita mice. Utilizing the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) with primers that are specific for normal alleles or Akita mutant alleles, we obtained amplified PCR products that allowed us to distinguish between the wild-type (+/+), heterozygous (Ins2 Akita /+), and homozygous (Ins2 Akita /Ins2 Akita ) mice within 3 hours. These results present the ARMS-PCR analysis as highly desirable and suitable for the identification of the Akita mutation, which is expected to significantly facilitate and promote the Akita mouse-related studies.

Directional associations between memory impairment and depressive symptoms: data from a longitudinal sample and meta-analysis.

BACKGROUND: Previous cross-lagged studies on depression and memory impairment among the elderly have revealed conflicting findings relating to the direction of influence between depression and memory impairment. The current study aims to clarify this direction of influence by examining the cross-lagged relationships between memory impairment and depression in an Asian sample of elderly community dwellers, as well as synthesizing previous relevant cross-lagged findings via a meta-analysis. METHODS: A total of 160 participants (Mage = 68.14, s.d. = 5.34) were assessed across two time points (average of 1.9 years apart) on measures of memory and depressive symptoms. The data were then fitted to a structural equation model to examine two cross-lagged effects (i.e. depressive symptoms→memory; memory→depressive symptoms). A total of 14 effect-sizes for each of the two cross-lagged directions were extracted from six studies (including the present; total N = 8324). These effects were then meta-analyzed using a three-level mixed effects model. RESULTS: In the current sample, lower memory ability at baseline was associated with worse depressive symptoms levels at follow-up, after controlling for baseline depressive symptoms. However, the reverse effect was not significant; baseline depressive symptoms did not predict subsequent memory ability after controlling for baseline memory. The results of the meta-analysis revealed the same pattern of relationship between memory and depressive symptoms. CONCLUSIONS: These results provide robust evidence that the relationship between memory impairment and depressive symptoms is unidirectional; memory impairment predicts subsequent depressive symptoms but not vice-versa. The implications of these findings are discussed.

Discovery of a Benzamide Derivative That Protects Pancreatic ß-Cells against Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress-mediated pancreatic insulin-producing ß-cell dysfunction and death are critical elements in the onset and progression of both type 1 and type 2 diabetes. Here, through cell-based high throughput screening we identified benzamide derivatives as a novel class of ß-cell protective agents against ER stress-induced dysfunction and death. Through structure-activity relationship optimization, a 3-(N-piperidinyl)methylbenzamide derivative 13d markedly protects ß-cells against ER stress-induced dysfunction and death with near 100% maximum rescue activity and an EC50 of 0.032 µM. Compound 13d alleviates ER stress in ß-cells by suppressing ER stress-mediated activation of all three branches of unfolded protein response (UPR) and apoptotic genes. Finally, we show that 13d significantly lowers blood glucose levels and increases concomitant ß-cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of ß-cell-protective small molecules against ER stress provides a new promising modality for the treatment of diabetes.

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila.

The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Here, we show that Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a Drosophila genetic screen, we discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. We further showed that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, we determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. Our findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis.

Discovery, Synthesis, and Evaluation of 2,4-Diaminoquinazolines as a Novel Class of Pancreatic ß-Cell-Protective Agents against Endoplasmic Reticulum (ER) Stress.

Pancreatic insulin-producing ß-cell dysfunction and death plays central roles in the onset and progression of both type 1 and type 2 diabetes. Current antidiabetic drugs cannot halt the ongoing progression of ß-cell dysfunction and death. In diabetes, a major cause for the decline in ß-cell function and survival is endoplasmic reticulum (ER) stress. Here, we identified quinazoline derivatives as a novel class of ß-cell protective agents against ER stress-induced dysfunction and death. A series of quinazoline derivatives were synthesized from dichloroquiazoline utilizing a sequence of nucleophilic reactions. Through SAR optimization, 2,4-diaminoquinazoline compound 9c markedly protects ß-cells against ER stress-induced dysfunction and death with 80% maximum rescue activity and an EC50 value of 0.56 µM. Importantly, 9c restores the ER stress-impaired glucose-stimulated insulin secretion response and survival in primary human islet ß-cells. We showed that 9c protects ß-cells by alleviating ER stress through the suppression of the induction of key genes of the unfolded protein response and apoptosis.

Identification of 1,2,3-triazole derivatives that protect pancreatic ß cells against endoplasmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression.

The C/EBP-homologous protein (CHOP) acts as a mediator of endoplasmic reticulum (ER) stress-induced pancreatic insulin-producing ß cell death, a key element in the pathogenesis of diabetes. Chemicals that inhibit the expression of CHOP might therefore protect ß cells from ER stress-induced apoptosis and prevent or ameliorate diabetes. Here, we used high-throughput screening to identify a series of 1,2,3-triazole amide derivatives that inhibit ER stress-induced CHOP-luciferase reporter activity. Our SAR studies indicate that compounds with an N,1-diphenyl-5-methyl-1H-1,2,3-triazole-4-carboxamide backbone potently protect ß cell against ER stress. Several representative compounds inhibit ER stress-induced up-regulation of CHOP mRNA and protein, without affecting the basal level of CHOP expression. We further show that a 1,2,3-triazole derivative 4e protects ß cell function and survival against ER stress in a CHOP-dependent fashion, as it is inactive in CHOP-deficient ß cells. Finally, we show that 4e significantly lowers blood glucose levels and increases concomitant ß cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of small molecule inhibitors of CHOP expression that prevent ER stress-induced ß cell dysfunction and death may provide a new modality for the treatment of diabetes.

Nutrient sensing and utilization: Getting to the heart of metabolic flexibility.

A central feature of obesity-related cardiometabolic diseases is the impaired ability to transition between fatty acid and glucose metabolism. This impairment, referred to as "metabolic inflexibility", occurs in a number of tissues, including the heart. Although the heart normally prefers to metabolize fatty acids over glucose, the inability to upregulate glucose metabolism under energetically demanding conditions contributes to a pathological state involving energy imbalance, impaired contractility, and post-translational protein modifications. This review discusses pathophysiologic processes that contribute to cardiac metabolic inflexibility and speculates on the potential physiologic origins that lead to the current state of cardiometabolic disease in an obesogenic environment.

Identification of 5-nitrofuran-2-amide derivatives that induce apoptosis in triple negative breast cancer cells by activating C/EBP-homologous protein expression.

The transcription factor C/EBP-homologous protein (CHOP) is a key component of the terminal unfolded protein response (UPR) that mediates unresolvable endoplasmic reticulum stress-induced apoptosis. CHOP induction is known to cause cancer cell death. Chemicals that induce CHOP expression would thus be valuable as potential cancer therapeutics and as research tools. Here, we identified 5-nitrofuran-2-amide derivatives as small molecule activators of CHOP expression that induced apoptosis in triple negative breast cancer (TNBC) cells. Our preliminary structure-activity relationship studies indicated that compounds with an N-phenyl-5-nitrofuran-2-carboxamide skeleton were particularly potent inducers of TNBC cell apoptosis. The compounds activate CHOP expression via the PERK-eIF2α-ATF4 branch of the UPR. These results indicate that small molecule activators of CHOP expression may have therapeutic potential for TNBC.

Identification of small molecules that protect pancreatic ß cells against endoplasmic reticulum stress-induced cell death.

Endoplasmic reticulum (ER) stress plays an important role in the decline in pancreatic ß cell function and mass observed in type 2 diabetes. Here, we developed a novel ß cell-based high-throughput screening assay to identify small molecules that protect ß cells against ER stress-induced cell death. Mouse ßTC6 cells were treated with the ER stressor tunicamycin to induce ER stress, and cell death was measured as a reduction in cellular ATP. A collection of 17600 compounds was screened for molecules that promote ß cell survival. Of the approximately 80 positive hits, two selected compounds were able to increase the survival of human primary ß cells and rodent ß cell lines subjected to ER stressors including palmitate, a free fatty acid of pathological relevance to diabetes. These compounds also restored ER stress-impaired glucose-stimulated insulin secretion responses. We show that the compounds promote ß cell survival by reducing the expression of key genes of the unfolded protein response and apoptosis, thus alleviating ER stress. Identification of small molecules that prevent ER stress-induced ß cell dysfunction and death may provide a new modality for the treatment of diabetes.