The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.

Thirty-six patients with type 2 diabetes mellitus (T2DM) were randomized 1:1:1 to receive a once-daily oral dose of placebo or 150 or 300 mg of the dual SGLT1/SGLT2 inhibitor LX4211 for 28 days. Relative to placebo, LX4211 enhanced urinary glucose excretion by inhibiting SGLT2-mediated renal glucose reabsorption; markedly and significantly improved multiple measures of glycemic control, including fasting plasma glucose, oral glucose tolerance, and HbA1c; and significantly lowered serum triglycerides. LX4211 also mediated trends for lower weight, lower blood pressure, and higher glucagon-like peptide-1 levels. In a follow-up single-dose study in 12 patients with T2DM, LX4211 (300 mg) significantly increased glucagon-like peptide-1 and peptide YY levels relative to pretreatment values, probably by delaying SGLT1-mediated intestinal glucose absorption. In both studies, LX4211 was well tolerated without evidence of increased gastrointestinal side effects. These data support further study of LX4211-mediated dual SGLT1/SGLT2 inhibition as a novel mechanism of action in the treatment of T2DM.

.-In the kidney, the sodiumglucose cotransporters SGLT2 and SGLT1 are thought to account for Ͼ90 and ϳ3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40 -50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1Ϫ/Ϫ) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5-to 2-fold higher urine glucose/creatinine ratios in Sglt1Ϫ/Ϫ vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1Ϫ/Ϫ vs. WT after 24 h (Ϫ33 vs. Ϫ11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 Ϯ 2% in WT and 17 Ϯ 2% in Sglt1Ϫ/Ϫ. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ϳ10-fold and reduced FGR to 44 Ϯ 3% in WT and to Ϫ1 Ϯ 3% in Sglt1Ϫ/Ϫ. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50 -60% of filtered glucose is excreted. proximal tubule; glucose reabsorption; glucose transport; sodium glucose cotransport inhibitor; diabetes mellitus ABOUT 180 G OF GLUCOSE ARE filtered daily by the kidney and enter the renal tubular system in a healthy normoglycemic subject. Glucose in urine is absent or at very low concentrations in healthy adults due to near complete reabsorption along the nephron segments, primarily in the proximal tubule. The renal Na ϩ -glucose cotransporter SGLT2 (SLC5A2) is localized to the early proximal tubule and thought to mediate the bulk of tubular glucose uptake across the apical membrane of the kidney (14,17,20). Studies in mice lacking Sglt2 demonstrated that SGLT2 mediates all glucose reabsorption in the early proximal tubule and most of overall glucose reabsorption by the kidney (17). In comparison, low-capacity SGLT1 (SLC5A1) "cleans up" most of the remaining luminal glucose in further distal parts of the proximal tubule (1,3,14,20). Studies in mice lacking Sglt1 (Sglt1Ϫ/Ϫ) revealed a fractional glucose excretion of 3% compared with 0.2% in wild-type (WT) (3). Whether glucose transporters other than SGLT2 and SGLT1 contribute in a measurable extent to renal glucose reabsorption across the luminal membrane of the renal epithelia has never been tested. Potential candidates include a lowaffinity Na ϩ -D-glucose cotransporter cloned from the rat named NaGL...

Synopsis The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid 325 of human ZnT-8 are associated with altered susceptibility to type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analyzed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioral changes or differences in body weight were observed between wild type and ZnT-8 −/− mice and ZnT-8 −/− mouse islets were indistinguishable from wild type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8 −/− mouse islets, as evaluated both by Timm’s histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16 week old, 6 hr fasted animals of either gender, however, plasma insulin concentrations were reduced in both female (~31%) and male (~47%) ZnT-8 −/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8 −/− mice but glucose-stimulated insulin secretion from isolated islets was reduced ~33% relative to wild type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

LX4211 [(2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(methylthio)tetrahydro-2H-pyran-3,4,5-triol], a dual sodium/ glucose cotransporter 1 (SGLT1) and SGLT2 inhibitor, is thought to decrease both renal glucose reabsorption by inhibiting SGLT2 and intestinal glucose absorption by inhibiting SGLT1. In clinical trials in patients with type 2 diabetes mellitus (T2DM), LX4211 treatment improved glycemic control while increasing circulating levels of glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). To better understand how LX4211 increases GLP-1 and PYY levels, we challenged SGLT1 knockout (2/2) mice, SGLT22/2 mice, and LX4211-treated mice with oral glucose. LX4211-treated mice and SGLT12/2 mice had increased levels of plasma GLP-1, plasma PYY, and intestinal glucose during the 6 hours after a glucosecontaining meal, as reflected by area under the curve (AUC) values, whereas SGLT22/2 mice showed no response. LX4211-treated mice and SGLT12/2 mice also had increased GLP-1 AUC values, decreased glucose-dependent insulinotropic polypeptide (GIP) AUC values, and decreased blood glucose excursions during the 6 hours after a challenge with oral glucose alone. However, GLP-1 and GIP levels were not increased in LX4211-treated mice and were decreased in SGLT12/2 mice, 5 minutes after oral glucose, consistent with studies linking decreased intestinal SGLT1 activity with reduced GLP-1 and GIP levels 5 minutes after oral glucose. These data suggest that LX4211 reduces intestinal glucose absorption by inhibiting SGLT1, resulting in net increases in GLP-1 and PYY release and decreases in GIP release and blood glucose excursions. The ability to inhibit both intestinal SGLT1 and renal SGLT2 provides LX4211 with a novel dual mechanism of action for improving glycemic control in patients with T2DM.

Two-pore domain K+ (K2P) channels play an important role in tuning β-cell glucose-stimulated insulin secretion (GSIS). The K2P channel TWIK-related alkaline pH-activated K2P (TALK)-1 is linked to type 2 diabetes risk through a coding sequence polymorphism (rs1535500); however, its physiological function has remained elusive. Here, we show that TALK-1 channels are expressed in mouse and human β-cells, where they serve as key regulators of electrical excitability and GSIS. We find that the rs1535500 polymorphism, which results in an alanine-to-glutamate substitution in the C-terminus of human TALK-1, increases channel activity. Genetic ablation of TALK-1 results in β-cell membrane potential depolarization, increased islet Ca2+ influx, and enhanced second-phase GSIS. Moreover, mice lacking TALK-1 channels are resistant to high-fat diet–induced elevations in fasting glycemia. These findings reveal TALK-1 channels as important modulators of second-phase insulin secretion and suggest a clinically relevant mechanism for rs1535500, which may increase type 2 diabetes risk by limiting GSIS.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs <1% of the UGE of DKOs. Consistent with their increased UGE, DKOs had lower fasting blood glucose and improved intraperitoneal glucose tolerance than Sglt2 KOs. In conclusion, 1) Sglt2 is the major renal glucose transporter, but Sglt1 reabsorbs 70% of filtered glucose if Sglt2 is absent; 2) mice lacking Sglt2 display improved glucose tolerance despite UGE that is 30% of maximum; 3) Sglt1 KO mice respond to oral glucose with increased circulating GLP-1; and 4) DKO mice have improved glycemic control over mice lacking Sglt2 alone. These data suggest that, in patients with type 2 diabetes, combining pharmacological SGLT2 inhibition with complete renal and/or partial intestinal SGLT1 inhibition may improve glycemic control over that achieved by SGLT2 inhibition alone.

apeutic intervention for diseases such as atherosclerosis, pancreatitis, or dyslipidemia associated with metabolic syndrome or type II diabetes ( 1-3 ). Central to triglyceride metabolism is lipoprotein lipase (LPL), an extracellular enzyme primarily located in the vascular beds of many tissues ( 3,4 ). LPL catalyzes the hydrolysis of the triglyceride component of chylomicrons (CM) and VLDL, which constitute the major forms of triglycerides in plasma ( 3, 5 ). Although LPL is expressed in many different tissues, the enzyme is expressed at high levels in metabolically active tissues, such as adipose, cardiac muscle, and skeletal muscle, where fatty acids released by the action of LPL are stored or used ( 4 ).LPL appears to be regulated by a variety of mechanisms. Several apolipoproteins associated with CM and VLDL, including apolipoprotein CII (APOC2) and apolipoprotein AV (APOA5), stimulate LPL activity ( 6-9 ) apparently by increasing its V max ( 10,11 ). In contrast, apolipoproteins CI (APOC1) and CIII (APOC3) can inhibit LPL activity ( 7,12 ). LPL is inherently unstable and proteins or other factors that either stabilize or destabilize LPL are likely to play a role in regulating its in vivo activity ( 13 ). The active form of LPL exists as a head-totail homodimer, which dissociates into metastable monomers. These monomers can reassociate to form catalytically active LPL or they can undergo conformational changes, forming inactive, stable monomers. The spontaneous in- Our understanding of how triglyceride (TG) metabolism is regulated is essential for designing avenues of ther-