A Story of Serendipities: From Phlorizin to Gliflozins.

Diabetes has been acknowledged since ancient times. However, it was only during the late 1800s that we realized that the primary organ for blood glucose regulation was the pancreas. The 20th century witnessed insulin purification, which revolutionized the treatment of diabetes maigre; this was followed by the development of oral antidiabetic drugs. The sodium-glucose cotransporter 2 inhibitors or gliflozins are the latest class. Unique cardio- and renoprotective effects separate them from other oral antidiabetic drugs. Here, we present the history behind the development of these inhibitors, arguably the hottest and the most pleasant topic in nephrology. The first serendipity was Koninck and Stas (assistants to Prof. Van Mons, a renowned pomology expert); these researchers isolated a crystalline glycoside called phloridzin (phlorizin) from the bark of apple trees while working at their boss's nursery. Their discovery was published in German in 1835. The second serendipity, after a half century, was from Prof. von Mering, who decided to administer phlorizin to dogs. Oskar Minkowski initially observed polyuria than glucosuria. Insightfully, von Mering postulated that phlorizin affects kidneys. In 1887, they reported that phlorizin induced glucosuria in people with diabetes. The third serendipity was that phlorizin causes several gastrointestinal side effects and has poor oral bioavailability. The first phlorizin-based drug to enter trials was T-1095. The first clinically available gliflozin was dapagliflozin, receiving approval in Europe and the United States in 2012 and 2014, respectively. The 2015 EMPA-REG Outcome trial reported extremely satisfying results that no one expected. Subsequent trials and real-world data have resulted in changes in all impactful guidelines. The impact of these agents on heart failure and chronic kidney disease seems independent of their antidiabetic properties. More than 100 years after von Mering's original discovery, descendants of phlorizin are fast becoming the most inspiring medicine for the 21st century physician.

Pharmacological investigations on efficacy of Phlorizin a sodium-glucose co-transporter (SGLT) inhibitor in mouse model of intracerebroventricular streptozotocin induced dementia of AD type.

OBJECTIVES: The study has been commenced to discover the potential of Phlorizin (dual SGLT inhibitor) in streptozotocin induced dementia of Alzheimer's disease (AD) type. MATERIAL AND METHODS: Injection of Streptozotocin (STZ) was given via i.c.v. route (3 mg/kg) to induce dementia of Alzheimer's type. In these animals learning and memory was evaluated using Morris water maze (MWM) test. Glutathione (GSH) and thiobarbituric acid reactive species (TBARS) level was quantified to evaluate the oxidative stress; cholinergic activity of brain was estimated in term of acetylcholinesterase (AChE) activity; and the levels of myeloperoxidase (MPO) were measured as inflammation marker. RESULTS: The mice model had decreased performance in MWM, representing impairment of cognitive functions. Biochemical evaluation showed rise in TBARS level, MPO and AChE activity, and fall in GSH level. The histopathological study revealed severe infiltration of neutrophils. In the study, Phlorizin/Donepezil (serving as positive control) treatment mitigate streptozotocin induced cognitive decline, histopathological changes and biochemical alterations. CONCLUSIONS: The results suggest that Phlorizin decreased cognitive function via its anticholinesterase, antioxidative, antiinflammatory effects and probably through SGLT inhibitory action. It can be conferred that SGLTs can be an encouraging target for the treatment of dementia of AD.

Sodium-Glucose Cotransporter-2 Inhibitors: Lack of a Complete History Delays Diagnosis.

On 15 May 2015, the U.S. Food and Drug Administration (FDA) warned that administration of sodium-glucose cotransporter-2 (SGLT2) inhibitors could lead to ketoacidosis in patients with diabetes mellitus. This announcement came more than 2 years after the FDA's first approval of an SGLT2 inhibitor, although the phenomenon had been known for more than 125 years. Luminaries of diabetes research (including Josef von Mering, Frederick Allen, I. Arthur Mirsky, and George Cahill) had described ketosis and ketoacidosis induced by administration of the phytochemical phlorizin, the prototypical SGLT inhibitor, as well as in patients with familial renal glucosuria, a condition that is considered a natural model of SGLT2 inhibition. Neither government regulators nor manufacturers of SGLT2 inhibitors evinced an awareness of this extensive historical record. The absence of historical inquiry delayed notice of ketoacidosis as an adverse reaction, which could have reduced the burden of illness from these drugs.

Phloridzin, an Apple Polyphenol, Exerted Unfavorable Effects on Bone and Muscle in an Experimental Model of Type 2 Diabetes in Rats.

It is believed that apple fruits contain components with health-promoting effects, including some antidiabetic activity. One of the most known apple compounds is phloridzin, a glucoside of phloretin. Phloridzin and phloretin were reported to exert some favorable skeletal effects in estrogen-deficient rats and mice. The aim of the study was to investigate the effects of phloridzin on musculoskeletal system in rats with type 2 diabetes induced by a high-fat diet (HFD) and streptozotocin (STZ). The experiments were performed on mature female Wistar rats, divided into control rats (fed a standard laboratory diet), HFD/STZ control rats, and HFD/STZ rats receiving phloridzin (20 or 50 mg/kg/day per os) for four weeks. Serum biochemical parameters, muscle mass and strength, bone mass, density, histomorphometric parameters and mechanical properties were determined. The HFD/STZ rats developed hyperglycemia, with decreases in the muscle mass and strength and profound osteoporotic changes. Phloridzin at 20 mg/kg markedly augmented the unfavorable effects of diabetes on the muscle mass and strength and decreased growth of bones, whereas, at 50 mg/kg, it did not affect most of the investigated musculoskeletal parameters. Results of the study indicate the possibility of unfavorable effects of phloridzin on the musculoskeletal system in conditions of hyperglycemia.

Dietary phlorizin enhances osteoblastogenic bone formation through enhancing ß-catenin activity via GSK-3ß inhibition in a model of senile osteoporosis.

Osteoporosis is one of the most prevalent forms of age-related bone diseases. Increased bone loss with advancing age has become a grave public health concern. This study examined whether phlorizin and phloretin, dihydrochalcones in apple peels, inhibited senile osteoporosis through enhancing osteoblastogenic bone formation in cell-based and aged mouse models. Submicromolar phloretin and phlorizin markedly stimulated osteoblast differentiation of MC3T3-E1 cells with increased transcription of Runx2 and osteocalcin. Senescence-accelerated resistant mouse strain prone-6 (SAMP6) mice were orally supplemented with 10 mg/kg phlorizin and phloretin daily for 12 weeks. Male senescence-accelerated resistant mouse strain R1 mice were employed as a nonosteoporotic age-matched control. Oral administration of ploretin and phorizin boosted bone mineralization in all the bones of femur, tibia and vertebra of SAMP6. In particular, phlorizin reduced serum RANKL/OPG ratio and diminished TRAP-positive osteoclasts in trabecular bones of SAMP6. Additionally, treating phlorizin to SAMP6 inhibited the osteoporotic resorption in distal femoral bones through up-regulating expression of BMP-2 and collagen-1 and decreasing production of matrix-degrading cathepsin K and MMP-9. Finally, phlorizin and phloretin antagonized GSK-3ß induction and ß-catenin phosphorylation in osteoblasts and aged mouse bones. Therefore, phlorizin and phloretin were potential therapeutic agents encumbering senile osteoporosis through promoting bone-forming osteoblastogenesis via modulation of GSK-3ß/ß-catenin-dependent signaling.

Selective SGLT2 inhibition by tofogliflozin reduces renal glucose reabsorption under hyperglycemic but not under hypo- or euglycemic conditions in rats.

To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⁻¹·min⁻¹ and increased EGP by 1-2 mg·kg⁻¹·min⁻¹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⁻¹·min⁻¹ and increased EGP by about 4 mg·kg⁻¹·min⁻¹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.

[Sodium-glucose cotransporter type 2 inhibitors (SGLT2): from familial renal glucosuria to the treatment of type 2 diabetes mellitus]. / Inhibidores del cotransportador sodio-glucosa tipo 2 (SGLT2): de la glucosuria renal familiar al tratamiento de la diabetes mellitus tipo 2.

For centuries, the kidney has been considered primarily an organ of elimination and a regulator of salt and ion balance. Although once thought that the kidney was the structural cause of diabetes, which in recent years has been ignored as a regulator of glucose homeostasis, is now recognized as a major player in the field of metabolic regulation carbohydrate. During fasting, 55% of the glucose comes from gluconeogenesis. Only 2 organs have this capability: the liver and kidney. The latter is responsible for 20% of total glucose production and 40% of that produced by gluconeogenesis. Today we have a better understanding of the physiology of renal glucose transport via specific transporters, such as type 2 sodium-glucose cotransporter  (SGLT2). A natural compound, phlorizin, was isolated in early 1800 and for decades played an important role in diabetes and renal physiology research. Finally, at the nexus of these findings mentioned above, recognized the effect of phlorizin-like compounds in the renal glucose transporter, which has offered a new mechanism to treat hyperglycemia. This has led to the development of several potentially effective treatment modalities for the treatment of diabetes.

Plasma and liver metabolites and glucose kinetics as affected by prolonged ketonemia-glucosuria and fasting in steers.

For 28 days, four steers received 1,3-butanediol, which causes ketonemia, and phlorizin, which causes glucosuria. Steers also were fasted for 9 days. Effects of treatments on concentrations of metabolites in blood and liver and on kinetics of glucose metabolism were determined. Treatments were: control, control with dietary butanediol plus injected phlorizin, and fasting. Fasting caused hypoinsulinemia and decreased liver glycogen by 60%. Butanediol plus phlorizin and fasting caused 18 and 19% decreases of plasma glucose and 2.5- and 6-fold increases of free fatty acid concentrations in blood plasma. Glucose irreversible loss averaged 371, 541, and 182 g/day during control, butanediol plus phlorizin treatment, and fasting. Butanediol plus phlorizin increased liver ketone body concentrations, caused glucosuria, ketonuria, and ketonemia, but did not affect insulin, glucagon, or growth hormone concentrations in plasma or triglyceride and glycogen contents in liver. Steers given butanediol plus phlorizin did not show all the usual signs of lactation ketosis, but the treatment still offers promise for studying causes and effects of ketosis.

In vitro hepatic gluconeogenesis during experimental ketosis produced in steers by 1,3-butanediol and phlorizin.

Adaptations of in vitro incorporation of gluconeogenic substrates into glucose and adaptations of metabolite concentrations of liver to subcutaneous phlorizin and dietary 1,3-butanediol were examined for liver samples from dairy steers. Later, the same adaptations were examined after 6 days of feed restriction. Feeding 1,3-butanediol significantly decreased conversion of carbon-14 of lactate and propionate to glucose and to carbon dioxide. There were no changes of concentrations of hepatic glycogen or triglyceride, and increases were only minor for beta-hydroxybutyrate concentration. Both phlorizin, with or without 1,3-butanediol, and feed restriction significantly increased rates of carbon incorporation into glucose from aspartate, lactate, and propionate but did not change rates of oxidation to carbon dioxide. Phlorizin had no effect on hepatic glycogen or triglyceride concentrations, but feed restriction decreased liver glycogen and increased triglyceride concentrations. Changes associated with either phlorizin treatment or feed restriction are consistent with a decreased ratio of insulin to glucagon of blood plasma. When combined, phlorizin and 1,3-butanediol seem to have some utility for developing a ketosis model.

In vitro hepatic gluconeogenesis and ketogenesis as affected by prolonged ketonemia-glucosuria and fasting in steers.

Both 1,3-butanediol, which causes ketonemia, and phlorizin, which causes glucosuria, were given to four steers for 28 days to determine effects of prolonged ketonemia and glucosuria on in vitro hepatic gluconeogenesis and ketogenesis. Treatments were: control ration; control with butanediol plus phlorizin; and fasting for 9 days. Liver slices, obtained by biopsy, were incubated with carbon-14 substrates. Substrate converted to glucose [mumol/(h X g liver)] during control, butanediol plus phlorizin, and fasting averaged 2.34, 7.21, and 12.00 for propionate; .99, 3.80, and 12.26 for lactate; .30, .76, and 2.20 for alanine; and 2.06, 5.37, and 5.78 for glycerol. Omission of calcium++ eliminated increases of gluconeogenesis caused by butanediol plus phlorizin and by fasting. Ketone bodies, octanoate, and bovine serum albumin did not affect glucose production markedly. Stearate inhibited gluconeogenesis during all periods except fasting. Production of beta-hydroxybutyrate [mumol/(h X g liver)] during control, butanediol plus phlorizin, and fasting averaged 2.07, 4.27, and 3.25 from butyrate and .06, .27, and .02 from palmitate. Results demonstrate that the gluconeogenic capacity of bovine liver is responsive to physiological and nutritional status.