The assessment of macrophage function has been a topic of intense discussion due to multiple subtypes. This protocol describes the collection of bone marrow cells from the femur and tibia of mice, differentiation into bone marrow-derived macrophages (BMDM cells), and sampling from cultures. This protocol focuses on the efficient preparation of BMDM cells, providing a way to assess the function of macrophages.

OBJECTIVE: Randomized controlled trials have shown kidney-protective effects of sodium–glucose cotransporter 2 (SGLT2) inhibitors, and clinical practice databases have suggested that these effects translate to clinical practice. However, long-term efficacy, as well as whether the presence or absence of proteinuria and the rate of estimated glomerular filtration rates (eGFR) decline prior to SGLT2 inhibitor initiation modify treatment efficacy among type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) patients, is unknown. RESEARCH DESIGN AND METHODS: Using the Japan Chronic Kidney Disease Database (J-CKD-DB), a nationwide multicenter CKD registry, we developed propensity scores for SGLT2 inhibitor initiation, with 1:1 matching with patients who were initiated on other glucose-lowering drugs. The primary outcome included rate of eGFR decline, and the secondary outcomes included a composite outcome of 50% eGFR decline or end-stage kidney disease. RESULTS: At baseline, mean age at initiation of the SGLT2 inhibitor (n = 1,033) or other glucose-lowering drug (n = 1,033) was 64.4 years, mean eGFR was 68.1 mL/min per 1.73 m2, and proteinuria was apparent in 578 (28.0%) of included patients. During follow-up, SGLT2 inhibitor initiation was associated with reduced eGFR decline (difference in slope for SGLT2 inhibitors vs. other drugs 0.75 mL/min/1.73 m2 per year [0.51 to 1.00]). During a mean follow-up of 24 months, 103 composite kidney outcomes occurred: 30 (14 events per 1,000 patient-years) among the SGLT2 inhibitors group and 73 (36 events per 1,000 patient-years) among the other drugs group (hazard ratio 0.40, 95% CI 0.26–0.61). The benefit provided by SGLT2 inhibitors was consistent irrespective of proteinuria and rate of eGFR decline before initiation of SGLT2 inhibitors (Pheterogeneity ≥ 0.35). CONCLUSIONS: The benefits of SGLT2 inhibitors on kidney function as observed in clinical trials translate to patients treated in clinical practice with no evidence that the effects are modified by the underlying rate of kidney function decline or the presence of proteinuria.

Aging is considered to be accelerated by insulin signaling in lower organisms, but it remained unclear whether this could hold true for mammals. Here we show that mice with skeletal muscle-specific double knockout of Akt1/2, key downstream molecules of insulin signaling, serve as a model of premature sarcopenia with insulin resistance. The knockout mice exhibit a progressive reduction in skeletal muscle mass, impairment of motor function and systemic insulin sensitivity. They also show osteopenia, and reduced lifespan largely due to death from debilitation on normal chow and death from tumor on high-fat diet. These phenotypes are almost reversed by additional knocking out of Foxo1/4, but only partially by additional knocking out of Tsc2 to activate the mTOR pathway. Overall, our data suggest that, unlike in lower organisms, suppression of Akt activity in skeletal muscle of mammals associated with insulin resistance and aging could accelerate osteosarcopenia and consequently reduce lifespan.

In recent years, diabetes has been shown to be associated with cancer risk, and this has led to a joint committee being formed, enlisting experts from the Japan Diabetes Society and the Japanese Cancer Association to address this issue. Epidemiological data in Japan provides evidence to demonstrate that diabetes is associated with increased risk for cancers, especially colorectal, liver, and pancreatic cancers. The mechanisms through which diabetes is assumed to promote oncogenesis include insulin resistance and associated hyperinsulinemia, hyperglycemia, and inflammation. Common risk factors for type 2 diabetes and cancer include aging, male sex, obesity, physical inactivity, inappropriate diet (excessive red/processed meat intake, inadequate vegetable/fruit/dietary fiber intake), excessive alcohol drinking, and smoking. Given that inappropriate diet/exercise, smoking and excessive alcohol drinking are common risk factors for diabetes and cancer, diet/exercise therapy, smoking cessation and alcohol moderation may be associated with decreased risk for cancer in diabetic patients. There is as yet limited evidence as to whether any particular antidiabetic agents may influence cancer risk.

Significance: SLC15A4 is a lysosome-resident amino acid/oligopeptide transporter that is preferentially expressed in immune cells. SLC15A4 is required for TLR7/9-dependent type I interferon production and plays a critical role in the pathogenesis of lupus and colitis. However, the precise molecular mechanisms of SLC15A4-mediated inflammatory responses are still undetermined. We revealed that SLC15A4 loss disturbed the coupling of glycolysis and the TCA cycle, and SLC15A4-deficient macrophages preferred to use glutamine rather than glucose as a carbon source for the TCA cycle. SLC15A4-deficient macrophages produced low levels of itaconate and proinflammatory IL-12 cytokine members. Our results reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a guardian to allow macrophages to acquire an M1-prone metabolic phenotype.