GLP-1 is recognized as an important endogenous regulator of glucose and lipid homeostasis. Efforts to utilize GLP-1 analogs or GLP-1R agonists in the treatment of T2DM and obesity have lasted for decades. The successful clinical use of Exendin-4 and Liraglutide 
- injectable peptidic GLP-1R agonists and multiple ongoing human trials with other GLP-1 peptidomimetics all support the idea that GLP-1 analogs are probably the most beneficial therapeutic agents for T2DM in today's medical practices 
. The major pharmaceutical hurdle for an effective oral treatment has been the difficulty to find a non-peptidic GLP-1R agonist with a long half-life 
. Boc5 is one of the first such compounds that demonstrated an array of therapeutic actions in the treatment of diabetes and obesity in db/db
. Boc5 efficiently induced a durable restoration of glycemic control and its other dose-dependent effects include reduction in food intake, slowing of gastric emptying, stimulation of insulin secretion and elevation in insulin sensitivity following 4 weeks of daily administration. It also decreased body weight of diabetic db/db
mice but required a high dose (3 mg per day) 
. In the present study, we employed a rodent model of DIO to overcome the shortcomings of previously used genetic model (the db/db
mouse) and thus to provide data with more relevance to human diseases: obesity and T2DM 
. DIO model can in part mimic human energy consumption patterns and gives the possibility of studying the pathogenesis of obesity and related diseases (e.g.
, T2DM) and examining the consequences of therapeutic intervention 
. This model was originally introduced by Surwit et al
. in 1988 
and has been shown to be most efficient in C57 mice compared with other strains 
. Therefore, this was the rationale of utilizing HFD fed C57 mice to advance our understanding on the pharmacological properties of Boc5 in diet-induced obesity and related metabolic disorders.
During the 12-week induction period with HFD, energy consumption of C57 mice was significantly increased and their body weight gained progressively, leading to moderate hyperglycemia, glucose intolerance and hyperinsulinemia. A pilot experiment was performed to determine the treatment regimen with Boc5, in which drastic weight loss was observed following daily dosing especially at 3 mg (data not shown). In order to avoid possible adverse consequences 
, we applied an intermittent (tiw) dosing schedule to the present work. Our results clearly demonstrate that subchronic Boc5 treatment invoked marked suppression of food intake and sustained reduction of body weight in DIO mice with both 1 mg and 3 mg doses (). Although similar effects were seen in db/db
mice, Boc5 at a daily dose of 1 mg failed to induce marked weight loss 
. The anti-obesity action of Boc5 is in agreement with a recent study with Exendin-4 conducted in HFD fed C57 mice 
. While the weight-lowering effect of Exendin-4 mainly occurred in the first week of treatment 
, Boc5 seemed to manifest its regulatory role in a more sustainable manner, i.e.
, affecting the entire therapy period. As a result, a 30% loss from the initial body weight was achieved () and maintained for at least 7 weeks after the cessation of Boc5 therapy (3 mg; Figure S5
). This long-lasting benefit is consistent with that observed in rats following administration of GLP-1 
. Such a “memory effect” is probably attributable to its action on the transcription of key regulator genes controlling β-cell mass and function 
. BMI, an index reflecting severity of obesity derived from body weight and body length 
, was dose-dependently suppressed by Boc5 treatment, suggesting an ameliorated obese state in DIO mice (). Interestingly, despite equal energy intake, the pair-fed group showed body weight reductions only in the beginning of the 12-week treatment period and the effect was less pronounced than the Boc5-treated (3 mg, tiw) group. We presume that this phenomenon may have been caused by reduced body temperature and energy expenditure resulted from feeding suppression as a compensatory mechanism to conserve energy 
For an ideal anti-obesity therapy, it is preferable that the weight loss stems predominantly from fat. In the present study, the remarkable decrease in body weight was accompanied by a dose-dependent reduction in fat as a percentage of body weight (). Analysis of carcasses from Boc5-treated DIO mice indicates that the body mass was preferentially lost from fat. On the other hand, despite the observed fat diminishment, such regulated loss invoked by Boc5 did not reach the extent to bring adverse consequences that often occurs in adipose tissue ablated mice 
. It is known that increase in adipocyte size due to fat storage (adipocyte hypertrophy) plays a key role in the formation of adipose tissue mass, and such conversion from small into large adipocytes is closely related to common health risks including hyperlipidemia, diabetes, hypertension and cardiovascular diseases 
. Our histological examination suggests that increases of fat deposits in adipose tissue were accompanied by white and brown adipocyte hypertrophy in HFD fed obese mice (). In contrast, white adipocytes from Boc5-treated mice appeared to be smaller in size than that from vehicle-treated controls, an observation consistent with the low TG and NEFA levels in the circulation (, ). Boc5 treatment also led to a reduction of lipid content in brown adipocytes () thereby reflecting the adaptive status of BAT in regulating thermogenesis 
Adipose tissue has a substantial influence on systemic metabolic homeostasis via its role as an endocrine organ capable of secreting diverse adipocytokines 
. The dose-dependent reduction of serum leptin level towards the normal range observed in Boc5-treated mice () supports the existence of a correlation between circulating leptin and adiposity. This phenomenon was accompanied by simultaneous restoration of circulating adiponectin concentration to a level that is completely normal (). Such an altered secretion associated with changes in adiposity is suggestive of a potential role for adiponectin as an autocrine factor in WAT to modulate adipocyte size (secretion increases when fat mass is reduced but it decreases following fat deposition) 
. Different from Boc5 treatment, caloric restriction did not affect adiposity significantly and circulating adipocytokines levels remained abnormal ( and ). These apparent differences imply that the regulatory role exerted by Boc5 (and hence, incretin mimetics in general) on lipid metabolism is independent of its inhibition on food intake.
Numerous studies have suggested that obesity is accompanied by several related metabolic defects in adipocytes concerning glucose or free fatty acid uptake, lipolytic activity and lipid oxidation 
. Our results indicate that the basal glucose incorporation was significantly elevated in adipocytes isolated from obese mice while their response to insulin stimulation was severely impaired (). Although there are conflicting reports regarding the alteration in the basal glucose uptake, most previous findings demonstrate that insulin-induced glucose incorporation is suppressed in the obese state 
. Meanwhile, we observed that the basal lipolytic activity was increased in obese mouse adipocytes that also displayed evident resistance to NA stimulation (). This finding is consistent with earlier results obtained from human or animal adipocytes 
. There is evidence suggesting that increased cAMP and tumor necrosis factor α (TNF-α) production in fat cells from obese individuals may be responsible for the enhancement of basal lipolysis 
, whereas lipolytic resistance is probably due to decreased expression of β2
and hormone-sensitive lipase (HSL) capable of hydrolyzing TG 
. Even if Boc5 treatment did not normalize basal and stimulated glucose uptake and lipolysis in our ex vivo
experiments, the improvements were impressive (), especially in the context that both GLP-1 and Exendin-4 only exhibited modest effects on fat cell (rat or human) metabolism when introduced directly to culture medium 
. Similar to the data described elsewhere 
, the role of caloric restriction (pair-feeding) in modifying fat cell lipogenesis and lipolysis was minimal (), thereby further supporting the existence of an anorexia-independent pathway for Boc5 in modulating lipid metabolism.
It is well established in C57 mice that consumption of HFD results in both moderate hyperglycemia and progressive hyperinsulinemia, leading to eventual insulin resistance 
. In the present study, we observed that fasting glucose levels in untreated obese mice were deteriorating with time while Boc5 could reverse such a tendency (). Combined with the dose-dependent normalization of IPGTT, glucose-stimulated insulin release and pancreatic insulin content (), our data point to a Boc5-mediated glycemic control mechanism where restoration of insulin sensitivity as well as reduction of peripheral demand for insulin each plays its role. The increased rate of glucose clearance in response to a fixed (2 IU/kg) amount of insulin () provides additional independent evidence for an insulin sensitizing effect of Boc5 in DIO mice. We know that β-cells adapt to situations of chronic fuel over-supply and insulin resistance by increasing their mass 
, which in our hands was elevated 2.4-fold in untreated obese mice (). Although a non-biased “systematic uniform random sampling” method 
was not used in this study to estimate the β-cell area, our findings appear to be indicative of alterations in several parameters related to β-cell function after 12 weeks of Boc5 treatment: (i) complete normalization of β-cell mass; (ii) reduction in islet size; (iii) increase in small islets (); and (iv) suppression of pancreatic apoptosis. The latter was achieved by a significant decrease of caspase 3/7 activities in the pancreases of treated mice (Figure S6
). These morphological or biochemical changes are in agreement with the previous findings with Exendin-4 
, and may collectively be attributable to the functionality improvement reflected by enhanced insulin sensitivity, reduced insulin demand and optimized β-cell efficiency 
The subchronic (12 weeks) insulin-sensitizing effect of Boc5 observed here not only reproduces our earlier results generated in db/db
, but is also consistent with the responses to chronic GLP-1R agonist treatments in humans 
and rodents 
reported elsewhere. Moreover, since adipose tissue has a substantial influence on systemic glucose homeostasis through secretion of adipocytokines 
, reduced adiposity is likely to contribute to the preservation of insulin sensitivity as well. Recent studies have defined adiponectin as an insulin sensitivity mediator which stimulates tissue fatty acid oxidation and inhibits hepatic glucose production by activating AMP-activated protein kinase 
. Our results thus suggest that improvement of insulin resistance in DIO mice treated with Boc5 may be at least partially mediated by adiponectin. More importantly, unchanged hyperglycemia, hyperinsulinemia, pancreatic insulin content and ITT response in pair-fed mice clearly indicate an absence of effect by caloric restriction on insulin sensitivity, thereby implying a possible dissociation between anorexic and insulin-sensitizing effects of Boc5 
. Obviously, part of the glycemic benefits of Boc5 manifested in obesity is exerted through its potential regulation of adiposity.
A direct consequence of HFD consumption is increased fat deposition in both adipocyte stores and non-adipose tissues including liver and skeletal muscle 
. Our data confirm that liver and muscle weight gain is associated with TG content enhancement and hepatocyte fat infiltration (). In addition, serum ALT and AST concentrations (), as biomarkers of liver integrity, were either elevated or showed such a tendency in obese mice, suggesting that obesity and steatohepatitis co-exist in this animal model. Abolishment of liver/muscle fat accumulation and hepatocyte injury following Boc5 administration collaborates with the results obtained in ob/ob
obese mice and T2DM patients receiving mid- or long-term Exendin-4 treatment (60 days and 3 years, respectively) 
. Although insulin resistance is likely to account for liver and muscle damage 
, somewhat surprising is that the apparently less severe tissue fat infiltration in pair-fed mice occurred without concordant restoration in insulin sensitivity.
It should be noted that 12 weeks' Boc5 treatment was associated with remarkable improvements of several dyslipidemia-related circulating parameters such as elevated TC, TG and LDL/HDL ratio (). Although variations of these physiological measures are influenced by strain/race and diet ingredients 
, they are believed to be predictive of heart disease and atherosclerosis in the obese and diabetic populations 
. Therefore, intervention of obesity with incretin mimetics may have the potential to reduce the morbidity of cardiovascular diseases. Since NEFA released from adipose tissue has been proposed as a link between obesity and insulin resistance 
, the reduction of NEFA level resulted from Boc5 therapy () may therefore be one possible mechanism responsible for the restoration of insulin sensitivity in obese animals. This assumption is consistent with the low basal lipolytic activity detected in Boc5-treated obese mice, because circulating NEFA level is a main indicator of basal lipolysis 
. There were no significant differences in TC, TG and NEFA levels between obese and pair-fed animals, indicating that caloric restriction alone is not sufficient to correct the state of dyslipidemia in obesity.
In conclusion, this study using a rodent DIO model has confirmed the glycemic control and weight loss properties of Boc5, reported previously in db/db mice. Applying an intermittent dosing protocol, subchronic Boc5 exhibited typical dose-responses in regulating food intake, adiposity and glucose homeostasis. It is also efficacious in treating multiple conditions associated with obesity such as dyslipidemia, adipocytokines dysregulation, adipocyte malfunction and liver injury. These findings suggest that Boc5 may produce metabolic benefits via an array of synergistic mechanisms. Further investigations are required to expand our knowledge on this class of non-peptidic GLP-1R agonists aiming at pharmacotherapies for obesity and related metabolic diseases.